EXS 39:

Experientia Supptementum
Vol. 39

Springer Basel AG
Jeremija Lj. Rasic
Joseph A. Kurmann
Blfldobacterla
and Thelr Role
Microbiological, Nutritionai-Physiological, Medical and
Technological Aspects and Bibliography

With 56 Illustrations and more than

1225 Bibliographie Data

1983 Springer Basel AG

This book is the next publication of the monographic series entitled:
"Fermented Fresh Milk Products and their Cultures"
Valurne I: J. l.J. RASu'; and J. A. KuRMANN: "Yoghurt. Scientific Grounds, Technology, Manu-
facture and Preparations" ( 1978-466 pp.) Distributed by Technical Dairy Publishing
Hause, Jyllingevej 39, DK-2720 Vanlese, Copenhagen, Denmark.
Valurne 2: J.l.J.RASu: andJ. A. KURMANN: "Bifidobacteria and their RoJe. Microbiological, Nutri-
tional-Physiological, Medical and Technological Aspects and Bibliography" ( 1983).
Ed. Birkhäuser, Basel, Boston, Stuttgart.
This series will be completed with other volumes.

Authors' addresses:

Dr. Joseph A. Kurmann Dr. Jeremija Lj. Ra5ic

Agricultural Institute Food Research Institute
CH-1725 Grangeneuve-Fribourg Rumenacka I 03
(Switzerland) Novi Sad (Yugoslavia)

Library of Congress Cataloging in Publication Data

Rasic, Jeremija Lj., 1922-

Bifidobacteria and their roJe.
(Fermented fresh milk products and their cultures
v. 2) (Experientia. Supplementum ; · v. 39)
Bibliography: p.
Includes indexes.
I. Bifidobacterium. 2. Bifidobacterium Industrial
applications I. Kurmann, Joseph A., 1931-
11. Title. III. Series IV. Series: Experientia.
Supplementum ; v. 39. [DNLM: I. Actinomycetaceae
Bibliography. Wl. EX 23 v. 39 I QW 125.5.A2 R224b]
QR 82. A35R37 1983 589.9'2 82-14781

CIP-Kurztitelaufnahme der Deutschen Bibliothek

Rasic, Jeremija Lj.:

Bifidobacteria and their roJe : microbiolog.,
nutritional-physiolog., med. and technolog.
aspects and bibliogr. I by Jeremija Lj. Rasic
and Joseph A. Kurmann. - Basel ; Boston
Stuttgart : Birkhäuser, 1983.
(Fermented fresh milk products and their
cultures ; Vol. 2) (Experientia : Suppl.
Vol. 39)

NE: Kurmann, Joseph A.:; Experientia I Supple-

mentum; I. GT

All rights reserved.

No part ofthis publication may be reproduced, stored in a retrieval system, ortransmitted in any form
or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior
permission ofthe copyright owner.

© 1983 Springer Basel AG

Originally published by Birkhäuser Verlag Basel in 1983.
Softcover reprint of the hardcover Ist edition 1983

ISBN 978-3-0348-5449-8 ISBN 978-3-0348-5448-1 (eBook)

DOI 10.1007/978-3-0348-5448-1
Preface

This book is the second volume ofthe monographs on "Fermented Fresh Milk Pro-
ducts and their Cultures ". It is the first book about bifidobacteria, the predominant
intestinal organisms ofbreast-fed infants and the major component ofthelarge intes-
tinal flora in human adults.
The significance of using bifidobacteria as dietary adjuncts is described; and
new trends for improving or supplementing the foods for different age groups of
humans are indicated. The role of bifidobacteria is considered from various Stand-
points. Whenever possible the papers and books have been studied in there different
originallanguages.
Every effort has been made to present the latest data and concepts in this growing
field, without ignoring the valuable contributions of the past.
Many years of scientific work on the microbiology and of fermented foods have
contributed in making this book possible. After an introductory chapter on growth of
the knowledge and uses of bifidobacteria, biology of these organisms, their growth-
promoting factors, and the fermentation ofhexoses are discussed in more detail.
Great advances in many aspects ofthe human gastrointestinal flora made in the
past two decades are outlined, and nutritional and health aspects of cultured dairy
foods containing bifidobacteria are discussed.
There are separate chapters on dairy preparations, their manufacture, technol-
ogy and starter cultures; and on the development of new cultured products contain-
ing bifidobacteria, including pharmaceutical preparations. A special chapter is
devoted to the techniques ofthe isolation, identification, and culturing ofbifidobac-
teria.
The bibliography included at the end ofthe book is intended to guide the reader,
not only to original data, but to many publications pretaining to the particular area of
interest. All papers dealing with bifidobacteria are included in the bibliographieallist
and only the ones charged most important by the authors have been cited in the text.
We are especially grateful to Mr. H.B. HAWLEY, a British scientist, who com-
mented on the manuscript the English text, and for his stimulating criticism and help-
ful suggestions.
We wish also to express our gratitude to many persans and organizations who
contributed to this book, and our thanks to the companies that kindly supplied illus-
trations and certain technical information. Their names are included under Acknow-
ledgments.
It is hoped this book will prove of value to microbiologists, nutritionists, medical
workers, scientists, technologists, engineers, students, and others interested in nutri-
tion and dairy foods.
We hope too that this Monograph will encourage a higher consumption of milk
and dairy products, and willlead to a wider study ofthe role ofbifidobacteria in rela-
tion to health.

J. A. KURMANN J.l.J. RAS1C:

Grangeneuve I Fribourg 1983 Novi Sad I Belgrade
Switzerland Yugoslavia
Contents

Preface . . . . . . V

Chapter 1: History . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Introduction- First Period (from 1899 to 1957)- Second Period (since 1957)

Chapter 2 : Biology of the Bifidobacteria . . . . . . . . . . . . . . . . . . . . 8

Introduction- Morphology- Physiology- Biochemical Characteristics- Ce II-Wall Char-
acteristics- Composition ofthe Lipidsand Phospholipids- Deoxyribonucleic Acid Base
Composition - Antigenicity - Taxonomy - ldentitication - Ecological Relationships -
Bacteriophages

Chapter 3: The Fermentation of Hexoses 35

Introduction- Fermentation of Hexoses- Enzymes

Chapter 4: Effect of substrate on the Growth of Bifidobacteria . . . . . . . . . 42

Introduction- Moditication of Cows' Milk Composition- Specitic Growth-Promoting
Factors- The Intrinsic Properties of Human Milk

Chapter 5: The Human Gastrointestinal Flora and Bifidobacteria . . . . . . . 51

Introduction- Some Characteristics ofthe Gut Micro-organisms- Normal Gastrointesti-
nal Flora in Man- Changes in the Human Gastrointestinal Flora- Intestinal Bacteria and
Soft-Tissue Infections- Enzymatic Activities of Intestinal Bacteria- Diet and Intestinal
Flora of Man- Other Environmental Factors and Intestinal Flora- The Signiticance of
Gut Micro-organisms

Chapter 6: Nutritive and Health Values of Dairy Foods Containing

Bifidobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Introduction- Infant Nutrition and Bitidobacteria- Dairy Foods Containing Bitidobac-
teria in the Diet of Adults- Therapeutic Applications

Chapter 7: Dairy Preparations, Mannfacture and Technology . . . . . . . . . 102

Basic Manufacture and Technology of Set and Stirred Bitidus Milk Products - Raw
Cows' Milk and Reconstituted Milk- Treatment of Milk in the Dairy- Cultures and Star-
ters- Fermentation in Incubation Vats or in Retail Containers- Cooling After lncubation
- Mechanical Treatment of the Gel- Packaging and Packaging Materials- Storage, Pro-
Ionging the Storage Life and Maintaining Quality - Defects of Bitidus Milk Products -
Model Processes - Equipment- Basic Manufacture and Technology of Dried Bitidus
Milk Products- A Survey of Various Milk and Food Preparations

Chapter 8: Pharmaceutical Preparations . . . . . . . . . . . . . . . . . . . . 134

Introduction- Preparations for Use in Foods for Infantsand Children- Therapeutic Pre-
parations Containing Viable Bitidobacteria for Human Use- Therapeutic Preparations
Containing Viable Bitidobacteria for Anima! Use
Chapter 9: Techniques for the Isolation, Culturing and
Characterization of Bifidobacteria . . . . . . . . . . . . 144
Original Technique of Tissier ( 1900 & 1905) - The Sampie Collection - Preparation and
Dilution of the Sampies- Media for Bifidobacteria Culturing, Differentiation and Isola-
tion - Use of Media - Reading of Colonies and Microscopic Smear of the Examined
Material- Identification ofthe Genus Bifidobacterium

Appendix 1: Glossary of Terms 159

Appendix 2: Colture Media . . 166

Appendix 3: Catalogue of the Supply Sources for Diverse Strains and

Coltures of Bifidobacteria . . . . . . . . . . . . . . . . . . . . . . . 191

Bifidobacteria Bibliography . . . . . . . . . . . . . . . . . . . . . . . 195

Introduction- Alphabetical List of Papers about Bifidobacteria 1899-1980/81

References . . . . . . . . . . . . 255
(which do not treat bifidobacteria)

Index of Organisms 260

Subject Index . 267

Advertising . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Advertising- List of the Advertisers- List of Equipment and Machinery

Acknowledgments . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 295

2
Chapter 1 : History

1.1 Introduction

Bifidobacteria were isolated and described in the period 1899 to 1900 by TISSIER who
named the type species Bacillus bifidus communisor B. bifidus. This organism was an
anaerobic, Gram-positive, curved-rod often occurring as bifurcated Y-forms. They
were the predominant organisms in the stools ofbreast-fed infants. About the same
time MoRo (1900 a,b) iso1ated from the stools ofbreast-fed infants facultative anae-
robic, Gram-positive, straight rods which he typified as Bacillus acidophilus. He
agreed with TISSIER that this organism was different from B. bifidus but believed that
B. acidophiluswas the predominant organism in the stools ofbottle-fed infants.
For taxonomical reasons, growth of the knowledge and uses of bifidobacteria
may be conveniently divided into two periods: from 1899 to 1957; and from 1957 to
the present. Table 1.1 surveys briefly these historical developments.

11 lll ll 11111 I I I\ 111111 I I\ I

tlllllttll.ll>~

11011! 1\TNI\\lf DL\ 111!1 Hl\\111~

... ----==.:..:.':...____ _

•-• t:A•
.,
•• ..,ur:
.. 41t
• • -.a

Fig. 1.1 The title-page of the first study of bifidobacteria

Table 1.1 A short historical survey ofthe knowledge and use ofbifidobacteria

I. Description, name, Bacillus bifidus TISSIER, 1899 & 1900.

taxonomy communis, B. bifidus
Bacteroides bifidus I-4th ed., Bergey's Manual, 1923-1934.
Lactobacil/us bifidus 5-7th ed., Bergey's Manual, 1939-1957.
Bifidobacterium 8th ed., Bergey's Manual, 1974.
II species

2. Occurrence
Human intestine Breast-fed infants TISSIER, 1899& 1900.
Bottle-fed infants ÜRLA-JENSEN et al., 1936; EGGERTH, 1935;
andadults ROUFOGALIS, 1941 a;ÜLSEN, 1949; ßOVEN-
TER, 1949; FRISELL, 1951; HAENEL, 1956.

3
 Species and biotypes Breast- and bottle-fed DEHNERT, l957b; WERNER&SEELIGER,
distribution infants and adults 1963 a; REUTER, 1963; PETUELY& LINDNER,
1965; MITSUOKA & KANEUCHI, 1977.
Animals (alimentary Adults MITSUOKA, 1969 b; SCARDOVI et a/.• 1969;
tract) SCARDOVI & TROV ATELL!, 1969.

3. Growth-promoting Moditications of cows' SITTLER, 1908; GERSTLEY et a/., 1932;

factors milk composition BESSAU, 1937 a;AoAM, 1949; MALYOTH,
1949, & others.
promotion Lactulose PETUELY & KRISTEN, 1949.
Bitidus factor I GYÖRGY et al., 1954 a, b; KUHN et al., 1953.
Bitidus factor 2 RAYNAUD, 1959.
Intrinsic properties of BuLLEN etal., 1973.
humanmilk
Uncharacterised factors SUZUKI eta/., 1966 a, b; BEZKOROVAINY et
al., 1979 (and see bibliography).

4. Physiology and Factors of anaerobiosis DE VRIES & STOUTHAMER, 1969.

biochemistry
Carbohydrate metab. SCARDOVI & TROV ATELLI, 1965; DE VRIES et
al., 1967.
DNA base composition SEBALD eta/., 1965; SCARDOVI eta/., 1970;
1971 b.
Ce II walland phos- CUMMINS eta/., 1957; KANDLER, 1970;
pholipid composition EXTERKA TE & VEERKAMP, 1969.

5. Culturemedia Semisynthetic mediulß NORRIS et a/., 1950.

Minimal medium HASSINEN et a/., 1951.

6. Bitidobacteria and the Not weil detined. Some

health ofthe newborn theories and reports:
"Self-cleaning" ofthe ADAM, 1925; BOVENTER, 1949; MA YER,
intestine 1956.
Reduction of putrefac- BESSAU, 1938; KLEINSCHMIDT, 1949.
tion in the intestine
The specitic rote of HAENEL, 1970.
humanmilk
Control of enteric KAwuo&STöGMANN, 1968; MATA etal.,
infection 1969b; MATA& URRUTIA, 1971; BULLEN &
WILLIS, 1971.
The influence offeed BULLEN eta/., 1976; BULLEN & TEARLE,
on the products of 1976.
bacterial metabolism
Aid in nutrition MANCIAUX, 1958; STENGER & WOLF, 1962;
POUPARD et a/., 1973.
Breast-fed infants and
human milk:
antibodies, Iysozyme, SUSSMAN, 1961; BRAUN, 1971; RENNER,
lactoferrin; 1974; BULLEN eta/., 1972.
the antistaphylococcus JELLIFE & JELLIFFE, 1981 ; S!MHON et a/.,
factor, antitoxins for 1979.
neutralising Vibrio
cholerae and E. coli
sialic acid-containing GYÖRGY eta/., 1974; NICOLAI, 1976.
oligosaccharides
Acid pH ofthe contents Ross & DA WES, 1954; BULLEN & WILLIS,
of the Iarge intestine 1971.
Cantamination of NETER, 1959.
artiticial feeds
4
7. Bifidobacteria in adults Treatmentof:
Adjuncts Intestinaldisorders MAYER, 1966; BAMBERG, 1966.
following antibiotic
therapy
Chronic liver disease MOTING eta/.,1968 a,b; MOTING, 1977.

8. Human intestinal flora Occurrence, numbers HOFFMANN, 1966; GORBACH et a[., 1967;
and bifidobacteria MATA et al., 1969 a; HAENEL, 1970; DRASAR
&HtLL, 1974.
Metabolie activities DRASAR&HILL, 1974.
Microbial ecology of SAVAGE, 1977; COATES & FULLER, 1977.
thegut

9. Theuseof
bifidobacteria
Infant foods Growth-promoting TOMARELLI et af., 1954 b; ÜYÖRGY et a[.,
factors with or without 1955 b ; PETUELY, 1957 a, b; LEVESQUE et af.,
a bifidus culture 1959;ZILLIKEN, 1966; MAYER, 1966; HAE-
NEL et al., 1970 (see bibliography).
Enriched with a culture MAYER, 1950; LEVESQUE eta[., 1959; SCHU-
incorporating LER, 1972; LANG&LANG, 1978; DOLEZA-
bifidobacteria LEK, 1979.
Pharmaceutical Freeze-dried cultures KLUDAS, 1959b; HAWLEY eta/.,1959;
preparations containing B. bifidum, MOTING eta/.,1968 a, b; REUTER, 1969;
or additionally L. acid- PREVOT, 1971; BRANCA et al., 1979, &
ophilus, or B. longum, others.
L. acidophilus, E. coli
Lactulose with or ßtRCHER et af., 1966; ÜRDNUNG & MOTING,
without bifidus milk 1973; MOTING, 1977; MENDEZ & ÜLANO,
preparations 1979.
Dehydrated prepara- MOTING&ÜRDNUNG, 1976;0GASA, 1979.
tions containing viable
bifidobacteria and
lactulose
Milk processing Cultured dairy foods SCHULER-MALYOTH eta[., 1968 b;
containing MOLHENS & STAMER, 1969; LANG & LANG,
bifidobacteria 1978;ANON, 1978.

1.2 First Period (from 1899 to 1957)

Investigations during this period were concerned with: the growth-promoting fac-
tors for bifidobacteria; the occurrence of these organisms in the human intestinal
tract; their significance in the health ofinfants; and the devising of culture media for
the isolation and maintenance of strains.
A preponderance ofbifidobacteria in the stools ofbreast-fed infants was thought
tobe due to the bifidus-stimulating properties ofhuman milk. Adjustments in the pro-
portians of carbohydrate, protein, minerals, and fat of cows' milk were made in order
to simulate the composition of human milk in an attempt to promote the growth of
intestinal bifidobacteria. Although such formula feeds improved the nutrition ofbot-
tle-fed infants, they generally failed to induce the growth ofintestinal organisms com-
parable to the flora of breast-fed infants (GERSTLEY et a/., 1932; BESSAU, 1937 a;
MALYOTH, 1949; ADAM, 1949 'and seealso bibliography).

5
 Specific growth-promoting factors were identified about 1950. Lactulose was
found to have a bifidogenic effect (PETUELY & KRISTEN, 1949), and N-acetylglucosa-
mine-containing saccharides (bifidus factor 1) present in human milk were identified
as essential growth factors for the human-milk requiring strain, Bifidobacterium bifi-
dum var. pennsylvanicus (GYöRGY et al., 1954 a,b).
The opinion of TissiER (1900) that bifidobacteria were confined to breast-fed
infants was generally accepted for a long time, but subsequently these organisms were
found in the stoo1s of adults and of bott1e-fed infants a1though in smaller numbers
than in breast-fed ones (Table 1.1 ). In young adults bifidobacteria constituted about
40% ofthe total faecal flora but their numbers were found to decrease significantly
withage.
The significance ofbifidobacteria in the health and nutrition of infants has been
overestimated. The proliferation of these organisms was regarded as a process of
"self-cleaning" ofthe intestine. The normal digestive processes occurring in the gas-
trointestinal tract were thought to be closely related to the predominance of bifido-
bacteria in the intestine and to their suppression of putrefactive organisms (Table
1.1). These theories were strengthened by clinica1 observations on the better resist-
ance ofbreast-fed infants, compared with bottle-fed ones, to infections gastroenteritis
{ALEXANDER, 1948; RoBINSON, 1951 ). However, the specific composition of human
milk was unknown at that time and the possibility of microbial contamination of for-
mula feeds during preparation was not appreciated.
These pioneers recommended the use ofbifidobacteria as dietary adjuncts. Tis-
SIER ( 1906 a) administered a culture of B. bifidum to infants sufferlog from diarrhoea,
and subsequently bifidus milk containing growth-promoting substances was used in
the feeding ofinfants sufferlog from nutritional disturbances (MAYER, 1948 c).
During much ofthe First period, bifidobacteria were included in the genus Lac-
tobacillus, although other generic names were proposed; and due to the limited diag-
nostic procedures these organisms were often regarded as variants ofthe better stud-
ied L. acidophi/us(WEISS & RETTGER, 1934 a,b & 1938 a,b).
Satisfactory culture media for the primary isolation and study of "bifids" were
lacking. An improved medium: cystine-lactose-liver agar described by BLAUROCK
(1937) was found tobe unsatisfactory for many strains, andin 1950 Norris and co-
workers devised a semisynthetic selective medium containing most of the known
growth factors for "bifids ". This medium was inhibitory to other intestinal organisms
except enterococci. Later a medium meeting the minimum nutritional requirements
of certain strains was described by HASSINEN and co-workers (1951). The addition of
defatted human milk to the NoRRIS medium facilitated the isolation of the milk-
requiring strain, B. bifidum var. pennsylvanicus, and this medium was used exten-
sively in subsequent studies (GYöRGY, 1953; GYöRGY et al., 1954 a).

1.3 Second Period (from 1957 to the present)

Know1edge concerning bifidobacteria advanced rapidly during this period. Studies

ofthe physiological and biochemical characteristics ofthese organisms have revealed
distinctive features such as, cell wall composition, deoxyribonucleic acid base com-

6
position, carbohydrate metabolism, factors determining anaerobic growth, and the
phospholipid composition of cells.
The first differentiation ofbifidobacteria into five groups (DEHNERT 1957 b) was
followed by the description of species from the human intestinal tract (REuTER,
1963), and additional species from the alimentary tracts of animals and bees (MITsu-
OKA, 1969 b; SCARDOVI et a/., 1969; SCARDOVI & TROVATELLI, 1969). In the eighth
edition of Bergey's Manual(BvcHANAN & GIBBONS, 1974) these organisms are desig-
nated as a separate genus: Bifidobacterium comprising eleven species.
The occurrence ofbifidobacteria in different age-groups of men and warnen has
been quantified using modern methods of analysis ofthe gut flora. The distribution of
species and biotypes has also been described for various age-groups and correlated
with host physiology and environmental factors.
Great progress has been made conceming gut bacteria: their distribution and
interrelationships; metabolic activities; and significance in health and disease.
Recent studies of the interactions between the host and its micro-organisms represent
a new approach to an understanding ofthe physiological role ofthe gut flora (DRA-
SAR & HILL, 1974; SAVAGE, 1977).
New growth-promoting factors for bifidobacteria such as: bifidus factor 2,
pantethine, muramidase, certain glycopeptides, and some chemically uncharacter-
ised factors have been demonstrated. In addition to these specific factors, the consti-
tuents and properties ofhuman milk have been shown to play a role in the growth of
organisms in the large intestine (Table l.l ).
The role of bifidobacteria in the health of newbom infants has not been weil
defined, thus indicating the need for further research. Some data on the antagonistic
effects ofthese organisms against harmful bacteria suggests that the nature ofthe feed
indirectly influences the growth of intestinal bifidobacteria and the Suppression of
undesirable organisms. There are many reports on the protective rotes of specific
antibodies, iron-binding proteins, and Iysozyme, present in human milk, against
enteric infections (Table l.l ). The possible aid of "bifids" in the nutrition of infants,
especially improved nitrogen retention, has been indicated (MANCIAUX, 1958;
STENGER & WOLF, 1962; POUPARD et a/., 1973).
Bifidobacteria, or their growth-promoting factors, are used in the manufacture
of infant foods and pharmaceutical preparations, andin the production offermented
dairy foods. Infant foods with or without bifidus growth-promoting factors, or
enriched with a culture of bifidobacteria, are manufactured on an industrial scale.
Freeze-dried preparations containing B. bifidum, or tagether with L. acidophilus,
have been used for the treatment of gastrointestinal disorders. Lactulose alone or
included in a bifidus-milk preparation has been reported to have beneficial effects in
the treatment of hepatic encephalopathy (BIRCHER et al., 1966; MüTING & ORD-
NUNG, 1976; MüTING, 1977). Numerous fermented dairy foods are produced on an
industrial scale in various countries using cultures incorporating bifidobacteria.
These products include: cultured milks, cultured-milk beverages, fresh cheese des-
serts, cultured buttermilk.
Although much has been elucidated about the bifidobacteria, there remain many
questions conceming the biological and biochemical characteristics of the various
species and biotypes, and the interactions ofthese organisms with their host and fur-
ther research is needed.

7
Chapter 2: Biology ofthe bifidobacteria

2.1 Introduction

Bifidobacteria have characteristic morphology, physiology, biochemical characteris-

tics, cell-wall constituents, and DNA (deoxyribonucleic acid) base composition.
They are found in the intestinal tract of human infants and adults, in the human
vagina and mouth, andin the alimentary tracts of various animals. There arehuman
and non-human species and biotypes.
Since in the past these organisms were regarded as one species: Lactobacillus
bifidus, the term bifidobacteria or bifids will be used when the identity ofthe species is
uncertain.

2.2 Morphology

Bifidobacteria are non-motile and non-sporeforming rods of variable appearance.

Freshly isolated strains may have forms ranging from uniform to branched, bifur-
cated Y and V forms, spatulate or club shapes; but on subculture they frequently
become straight or curved rods of variable width throughout their length and may

.'

I
~

•• ...
•
Fig. 2.1 Photomicrograph of the stool of a breast-fed infant. Magnilkation 1250 x
(MANCIAUX, 1958)

8
exhibit breaks resembling branching. In unfavourable culture media, bifidobacteria
showbrauehing and pleomorphism although they are mostly rod-shaped in their nat-
ural habitat.
Fig. 2.1, 2.2 and 2.3 show photomicrographs of bifidobacteria from various
sources.
The actual cause ofbranching and pleomorphism in these organisms is not weil
understood although some details are available. The first evidence was provided by
Glick and co-workers (1960) who demonstrated the formation of bizarre forms in
B. bifidum var. pennsylvanicus (L. bifidus var. pennsylvanicus) resulting from a defi-
ciency of an N-acetylamino-sugar, which is essential for growth and cell-wall synthe-
sis. A succession of morphological types occurs ranging from bulbous forms when
this strain is grown in the presence of only traces of this amino-sugar, through
knobbed forms, to simple rods without bifid structure in the presence of high Ievels.
Trials with Iahelied N-acetylamino-sugars have demonstrated their incorporation in
the peptidoglycan, which is an important component ofthe cell wall (O'BRIEN et al.,
1960; LAMBERT et a/., 1965).
N-acetylamino-sugars, however, fail to inhibit brauehing and pleomorphism in
most strains ofbifidobacteria, indicating that other factors are involved. According to
KOJIMA and co-workers ( 1968 & 1970 a,b) Ca-ions play a principal role in the preven-

Fig. 2.2 Photomicrograph of bifidobacteria grown on glucose-blood agar with china blue (incu-
bated anaerobically at 37°C for 72 h- isolated from an adult stool)
(KLUDAS & DoBBERSTEIN, 1959)

9
Fig. 2.3 Electronmicrograph of B. bifidum (TISSIER) grown in the BLAUROCK culture broth and
incubated anaerobically at 38° C for 48 h
Magnifiation 14000 x
(RA YNAUD & ÜUINTINI, 1959)

tion of pleomorphism and regulation of cell division. HUSAIN and co-workers ( 1972),
on the other hand, observed that when a strain of B. bifidum is grown in a minimal
medium, this organism shows profuse branching, but following the addition of cer-
tain amino acids (alanine, aspartic acid, glutamic acid, serine), it reverts to its rod-
shaped form. Further research is clearly needed to elucidate the mechanism of
brauehing in bifidobacteria.
Surface colonies grown on agar plates, incubated anaerobically, arevariable in
shape, surface appearance, and size depending on nutritional conditions and strain
characteristics. Their shape may be convex to lens-shaped, or convex to pulvinate;
and their colour and appearance may vary from opaque to shining, from porcelain
white through whitish to cream; with an undulating, or smooth to mucoid-soft sur-
face. In deep agar culture, colonies are of variable shape and no growth occurs near
the surface.
Bifidobacteria are Gram-positive, but often stain irregularly; occasionally inter-
nal granules may stain with methylene blue while the remainder of the cell may be
unstained.

10
2.3 Physiology

2.3.1 Atmospheric growth requirements

Bifidobacteria, although strictly anaerobic, may sometimes tolerate oxygen in the

presence ofCOz; and there are great differences in sensitivity to oxygen among differ-
ent strains. The anaerobic requirements ofbifidobacteria have different biochemical
bases forvarious strains (DE VRIES & STOUTHAMER, 1969). Some strains, less sensitive
to Oz, may possess a weak catalase activity which removes preformed traces of HzOz,
or it may be that the NADH oxidase (reduced form of nicotinamide adenine dinucle-
otide) of these strains does not form HzOz at all. Probably such strains only grow
below a certain oxidation-reduction potential. In some strains, moderately sensitive
to Oz, accumulation of HzOz is the principal reason for anaerobiosis since hydrogen
peroxide inactivates fructose 6-phosphate-phosphoketolase, a key enzyme of the fer-
mentation pathway ofbifidobacteria. Other strains, extremely sensitive to Oz, do not
accumulate HzOz since these strains apparently require a low oxidation-reduction
potential for growth and fermentation. Oxygen may prevent growth by establishing
too high an oxidation-reduction potential.

2.3.2 Temperature and pH requirements

Most human strains of bifidobacteria grow at an optimum temperature of 36-38° C

whereas animal strains have a somewhat higher optimum growth temperature and
some can grow at 46.5° C. Growth of almost all strains ceases at 20° C or below.
B. bifidum has an optimum initial pH of 6-7 for growth and at pH 5.5 or less no
growth occurs.

2.3.3 Nutrition

Bifidobacteria are nutritionally a fairly heterogeneous group and some strains use
ammonium salts as a source of nitrogen whereas others require organic nitrogen.
Lactobacilli are known to require complex media containing numerous amino
acids, vitamins and related growth factors, in addition to fermentable carbohydrate.
On the other hand bifidobacteria can be grown in a semisynthetic medium (NoRRIS et
al., 1950) containing only three amino acids, but including numerous vitamins and
nucleotides in addition to Iactose and some minerals. Many strains grow in a simple
medium containing ammoniumsaltsplus cystine (or cysteine) to lower the Eh, biotin,
pantothenate, fermentable carbohydrate, and minerals (HASSINEN et al., 1951).
Other strains cannot use pantothenic acid but require pantethine (GYöRGY & RosE,
1955 a; ÜYLLENBERG & NIEMELÄ, 1959). B. bifidum var. pennsylvanicus requires
pantethine and N-acetylglucosamine-containing saccharides for growth. Peptone
serves as a source ofpantethine and human milk provides N-acetylglucosamine-con-
taining saccharides but such milk-dependent strains can revert to the B. bifidum type
which does not require human milk (RosE & ÜYÖRGY, 1955). B. bifidum strains (TIS-

11
SIER) require peptides for growth but neither pantethine nor N-acetylglucosamine-
containing saccharideisessential (RA YNAUD, 1959).

2.4 Biochemical characteristics

2.4.1 Carbohydratefermentation

Bifidobacteria are saccharolytic organisms and all strains ferment glucose, galactose
and fructose. There are differences among various species and biotypes in the fermen-
tation of other carbohydrates and alcohols. Glucose is fermented via the fructose-6-
phosphate shunt to acetic and L( + )-lactic acids, generally in a molar ratio of 3 : 2.
Small amounts offormic acid, ethanol, and often succinic acid are produced. Carbon
dioxide, butyric and propionic acids are not produced. Gas (C02), however, is pro-
duced by B. adolescentis and strains from bees in the gluconate fermentation.
A pathway for the fermentation ofhexoses by bifidobacteria will be discussed in
Chapter3.

2.4.2 Other characteristics

Bifidobacteria are catalase negative, except B. indicum (from bees) which is positive
when grown in the presence of haemin.
Nitrate reduction, formation of indole and liquefaction of gelatin are negative.
Allstrains of B. infantisproduce urease (SuzuKI et a/., 1979) whereas some strains of
B. bifidum areureasepositive and others areureasenegative (TISSIER, 1900, DUMAS,
1951; PRt:voT, 1957; HosATTE, 1964).
Some strains ofbifidobacteria show a weak proteolytic activity in milk but there
are considerable differences in the activity of strains. B. bifidum is ab1e, according to
DITTMANN and co-workers (1965), to split amino acids from milk proteins (LAC-
TANA-B milk formula) when cultured at 37° C for 60 and 90 h. Six high acid-produ-
cing strains ofbifidobacteria (milk coagulated in 18-28 h) isolated from the human
mouth and intestine have been reported to show a proteolytic activity comparable to
that of lactic streptococci (BORISOVA & SuvKo, 1973).
The ability of some strains of bifidobacteria to produce amino acids has also
been observed; for example: L-iso1eucine production by bifidobacteria. Astrain of
B. thermophilum (B. ruminale) was selected as the best producer and, after treatment
of this strain with N-methyl-N'-nitro-N-nitrosoguanidine, a mutantwas obtained
capable of producing about 5 mg I ml of L-isoleucine in the presence of 1.5% DL-
a1pha-aminobutyric acid (MATTEUZZI et al., 1976).
Some strains of bifidobacteria produce the extracellular enzyme, dextranase
which hydrolyses dextran to a mixture of isomaltotriose, isotetraose, isopentaose,
and higher isomaltodextrins (BAILEY & CLARKE, 1959). Another the intracellular
enzyme, alpha-1~6 glucosidase (isomaltodextrinase), was found in extracts
obtained from a rumen strain of Bifidobacterium which was cultured on dextran
(ßAILEY & ROBERTON, 1962).

12
2.4.3 Polysaccharide production

Some strains ofbifidobacteria may produce polysaccharides of differing properties

and composition. The polysaccharides oftwo mucoid types are detailed in Table 2.1.

Table 2.1 Extracellular polysaccharides produced by mucoid types of bifidobacteria

Polysaccharide Chemical composition Some properties Reference

Capsular Glucose, xylose, uronic Colanies ofthe mucoid MALYOTH & BAUER,
acid, an unidentitied type incubated anaerobi- l950a; 1951
pentose, and an cally on solid media are VOGEL, 1952
unidentified hexose hygroscopic, and on expo-
sure to the atmosphere
become deliquescent
Non-encapsulated D-glucose, D-galactose, A highly polymerized poly- NORRIS eta/., 1954;
6-deoxy-L-talose and saccharide which on expo- WANG eta/., 1963
D-galacturonic acid sure to the atmosphere
undergoes depolymerisa-
tion with reduction in vis-
cosity

Globules, or aggregates of polysaccharides can also be demonstrated within the

cell ofboth mucoid and non-mucoid strains. The cultural requirements ofthe mucoid
strains are the same as those of the smooth parent type.

2.5 The cell wall characteristics

The cell walls of bifidobacteria contain peptidoglycan (murein) consisting of

muramic acid, glucosamine, alanine, glutamic acid, and ornithine or Iysine together
with either one or two of the amino acids glycine, serine, aspartic acid and threonine
(CuMMINS et al., 1957; VEERKAMP et a/., 1965). The cell wall polysaccharides consist
of g1ucose, galactose and rhamnose.
The amino acids content of some strains may be identical, but the amino acids
sequence, the basic amino acid components in a tetrapeptide and the types of cross-
linkage may be different. The basic amino acid component varies between ornithine
and Iysine in different strains; and the type of cross-linkage may be a sing1e amino-
acid residue, a dipeptide, or a even tripeptide (KANDLER et a/., 1968; KANDLER,
1970; HOLZAPFELet a/., 1969; KOCHet a/., 1970 a,b; VEERKAMP, 1971 a; KANDLER
& LAUER, 1974). Likewise the component sugars of the cell wall polysaccharides
show variations, particularly in the contents of rhamnose and glucose. Table 2.2
shows the cell wall composition of different species of Bifidobacterium.
The cell-wall peptidoglycan of B. bifidum contains glucosamine and muramic
acid, a tetrapeptide consisting of L-alanine, D-glutamic acid-amide, L-ornithine and
D-alanine; the cross-linking dipeptide bridge between the 5-amino group of ornithine
and the carboxyl group of D-alanine in the adjacent tetrapeptide consists of L-serine
and L-aspartic acid. B. adolescentis has Iysine or ornithine in the tetrapeptide and is

13
Table 2.2 The cell wall composition of different species of Bifidobacterium

Species lsolated Peptidoglycan type') Polysaccharide

from Gluc Ga I Rham

B. bifidum FA Orn-Ser-Asp + + +
B. infantis FB Lys-Giy + + +
B. breve FB Lys-Gly + + +
B.liberorum FB Lys-Gly + + +
B. parvulorum FB Lys-Giy + + +
B. asteroides Bees Lys-Giy + + -
B. suis Pig Orn(Lys)-Ser-Ala-Thr-Ala + + (+)
B.longum FA Orn(Lys)-Ser-Ala-Thr-Ala + + +
B. thermophiturn Pig Orn(Lys)-Giu + + +
B. adolescentis FA Lys(Orn)-Asp + + -
B. indicum Bees Lys-Asp - + +
B. pseudolongum Pig Orn(Lys)-Ala2-3 + + +
F = faeces; A =adult; B = infant
') The first amino acid is a basic amino acid component ofthe tetrapeptide, and subsequent ones are
single amino acids or cross-linking peptides.
Source: KANDLER & LAUER ( 1974).

cross-linked by a molecule of aspartic acid, or even by the tripeptide, L-serine-L-ala-

nine-L-alanine.
B. infantis, B. liberorum, B. parvulorum and B. breve contain Iysine in the tetra-
peptide and are cross-linked by a single glycine residue. B. /ongum contains an orni-
thine type oftetrapeptide and the cross-linking peptide is L-serine-L-alanine-L-thre-
onine-L-alanine. B. pseudolongum and B. thermophilum contain ornithine in the
tetrapeptide, but they differ in cross-linkage. Species, such as B. suis and B. aster-
oides, have the same peptidoglycan type as B. longum and B. infantis respectively.
Unlike Bifidobacterium, the cell-wall of Lactobacillus, particularly the group,
Thermobacterium has a very uniform peptidoglycan L-Lys-0-Asp type.

2.6 Composition of the Iipids and phospholipids

The principal fatty acids in Bifidobacterium strains are Cl4:o, Cl6:o, C1s:o, Cl6'h and
C1s:1, whereas Lactobacillus strains contain the same fatty acids, except C1s:o which is
only present in small amounts. Variations in fatty acid composition of the medium
and in the growth temperature considerably influence the fatty acid pattern of the
cells ofbifidobacteria.
The phospholipid composition of Bifidobacterium strains shows a marked dif-
ference from that of Lactobacillus strains. Diphosphatidylglycerol and phosphati-
dylglycerol are present in strains ofboth genera, except for polyglycerolphospholipid
and its lyso derivatives, alanyl phosphatidylglycerol, and lyso-derivatives of diphos-
phatidylglycerol which are present only in Bifidobacterium strains (VEERKAMP,
1971 b; EXTERKATE et al., 1971). The phospholipid composition of cells is, therefore,
a characteristic of the genus Bifidobacterium.

14
 Extensive sturlies on B. bifidum var. pennsylvanicus have shown that exclusion
of human milk from the growth medium causes numerous changes in the Iipids and
phospholipids as well as considerable morphological differences (ExTERKATE &
VEERKAMP, 1969; VEERKAMP, 1970; EXTERKATE et a/., 1970; EXTERKATE & VEER-
KAMP, 1971; VEERKAMP, 1972; VEERKAMP & SCHAlK, 1974; SCHAlK & VEERKAMP,
1975; VEERKAMP, 1976 a,b). Absence ofhuman milk in the growth mediumresults in
the following biochemical changes: (a) a high er percentage oflipids in both cell resi-
due and cell-membrane; (b) a decrease of carbohydrate in the whole cell, membrane
and cytoplasmic Iipids; (c) a shift in the fatty acid composition to shorter chain-length
and to unsaturation; (d) a decrease of the Iipid galactose content of cells and cell-
membranes; (e) a marked increase in the totallipid-phosphorus content of the cells,
together with a large increase ofthe relative amounts of diphosphatidylglycerol and
phosphatidylglycerol, and a decrease in phosphogalactolipids; (f) marked differ-
ences in the fatty acid composition of monogalactosyldiglyceride, phosphatidylgly-
cerol and the two main phosphogalactolipids.

2. 7 Deoxyribonucleic-acid base composition

Deoxyribonucleic acid (DNA) consists of two purines (adenine and guanine) and
two pyrimidines (cytosine and thymine ), the sugar (D-deoxyribose) and phosphoric
acid. It is a major component of the genes, which are located in the chromosomes of
the cell nucleus.
DNA is ofimportance in bacterial taxonomy. The mean base-composition ofthe
DNA, namely the guanineplus cytosine (G + C) content varies considerably among
differentgenera. The average G + C values are 60.1% (range 57.2-64.5 moles %) for
Bifidobacterium, and less than 50% for Lactobacillus(WERNER & SEELIGER, 1963 b;
SEBALD et a/., 1965; ÜASSER & MANDEL, 1968).
The narrow range of the D NA-base composition of Bifidobacterium species indi-
cates that it cannot be used forthe differentiation ofspecies in the genus, but the DNA
homology pattern is a reliable criterion. DNA-DNA homology studies (SCARDOVI et
al., 1971 b) have shown that the genus Bifidobacterium contains several distinct
groups. Some proposed species are genetically distinct, whereas others have homolo-
gaus DNA. This will be more extensively discussed in a Jater section.

2.8 Antigenicity

Strains of Bifidobacterium possess the antigenic properties, which are not serologi-
cally related to Lactobacillus or to any other related genera. However, the antigenic
composition of bifids is not homogeneaus (BOVENTER, 1949; RoBERT, 1971 ). Some
strains of bifidobacteria with identical biochemical characteristics may be serologi-
cally different, although they differ only in precipitating and immunofluorescent-
antibodies which arise in the host. ScHULERand SICKEL ( 1968) have conducted exper-
iments with two serologically different strains of B. bifidum (strains II A and B I) in
gnotobiotic and in conventional pigs.

15
2.9 Taxonomy

A historica1 survey ofthe taxonomy ofbifidobacteria is shown in Tab1e 2.3.

Table 2.3 A historical of the taxonomy of bifidobacteria

TISSIER Bacillus bifidus communis, B. bifidus 1899/1900

CASTELLANI and Bacteroides bifidus 1919
CHALMERS
Bergey's Manual
(lst-4theditions)
HOLLAND Lactobacillus bifidus 1920
ORLA-JENSEN Bifidobacterium bifidum 1924
LEHMANNand Bacterium bifidum 1927
NEUMANN
PRIBRAM TJSsieria bifida 1929
VUILLEMIN Nocardia bifida 1931
NANNIZZI Actinomyces bifidus 1934
PuNTONI Actinobacterium bifidum 1937
WEISSand REITGER L. bi.fidus(aerobic, unbranched-form) 1938 a, b
is a variant of L. acidophilus
L. parabifidus (anaerobic, branched-form)
PREVOT Bifidobacterium bifidum 1938
Bergeys Manual Lactobacillus bifidus 1939
(5thedition)
NEGRONI and FISCHER Cohnistreptothrix bifidus 1944
ÜLSEN Corynebacterium bifidum 1949
NORRIS et al. L. bifidus (branched-forrn) 1950
L. parabifidus (unbranched-form)
ÜYÖRGY L. bifidusvar. pennsylvanicus 1953
ÜYÖRGY et al. l954a
DEHNERT 5 groups or biotypes (1-V) l957b
REUTER Description of many species from the human intestinal tract 1963
MJTSUOKA New species from animals 1969b
SCARDOVI et al. New species from bees and animals 1969
HOLDEMAN and MOORE Additional species recognized 1972
Bergey's Manual A new genus: Bifidobacterium consisting of ll species 1974
(8thedition)

The designation Bacillus bifidus given by Tissier ( 1900) was changed to that of
Bacteroides bifidus by CASTELLANI and CHALMERS ( 1919). Other generic names were
proposed, but the designation Lactobacillus bifidus was common1y used.
Since 1957 a new phase in the deve1opment of the taxonomy of the bifidobacte-
ria began. The characteristic morphology physio1ogy, biochemical characteristics,
DNA-base composition, cell-wall constituents and phospholipid composition ofthe
organisms have been used for differentiation ofthe genus Bifidobacterium. Classifi-
cation schemes for the genus have been proposed on the basis of carbohydrate fer-
mentation, physiological characteristics, and DNA-homo1ogy patterns (Table 2.4).
DEHNERT (1957 b) was the first to present a scheme for the classification ofbi-
fidobacteria into five groups or biotypes (1-V) based on the fermentation of24 carbo-
hydrates. L. bifidus var. pennsylvanicus was placed in group II.

16
Table 2.4 The major c1assification schemes ofthe genus Bifidobacterium1)

EGGERTH, DEHNERT, REUTER, MITSUOKA, SCARDOVI et al., HOLDEMAN AND

M~ORE,
1935 1957b 1963 1969b 1971 b 1972

I I, II bifidum a, lila bifidum a bifidum bifidum

bifidum b, lllb bifidumb
111 infantisV infantisa infantis infantis SS
infantis
infantisb infantis SS
liberorum
IV liberorum IX liberorum infantisSS
/actentis
/actentis X lactentis infantis - other
111 brevea, VII brevea breve breve
breve b, VIII breveb
brevec
parvu/orum a, IV parvu/orum a
parvu/orum b, VI parvu/orum b

II V ado/escentis Ia ado/escentis a ado/escentis ado/escentis A

ado/escentis lb adolescentis b ado/escentis B
adolescentis lc ado/escentis c ado/escentis C
adolescentis ld ado/escentis d ado/escentis D
/ongum a longumvar. longumSS longumSS
/onguma /ongum
longumb /ongumvar.
longumb
longum Ila longumvar.
animalisa
/ongum Ilb /ongumvar.
animalisb
pseudo/ongum a pseudo/ongum pseudolongum
pseudo/ongum b
pseudo/ongum c
pseudolongum d
thermophiturn a thermophiturn thermophiturn
thermophiturn b rumina/e
thermophiturn c
thermophiturn d
asteroides asteroides
"dentium" "dentium"
"angutatum"
"catenutatum"
suis
globosum
indicum
coryneforme
comutum
eriksonii

1) SS, Subspecies.
Source: POUPARD et a/., (1973)

17
 REUTER (1963) introduced species designation for many ofthe organisms. He
proposed, on the basis of the carbohydrate fermentation of strains iso1ated from the
intestinal tract ofhuman infants and adults, that the genus Bifidobacterium should be
divided into the following eight species and several biotypes: B. bifidum a and b; B.
infantis; B. parvulorum a and b; B. brevea and b; B.lactentis; B. liberorum; B. ado-
lescentis a, b, c and d; and B. longum a and b.
MnsuoKA (1969 b) proposed a classification scheme which included animal
strains in addition to human strains. The former were isolated from the rumen of cat-
tle and sheep; and from the intestine of pigs, chickens, rats, mice, guinea pigs and
rabbits.
He differentiated non-human from human strains by their carbohydrate fermen-
tation patterns and their ability to grow at 46.SO C.
These new species and biotypes were recognized as: B. pseudolongum a, b, c and
d; B. thermophilum a, b, c and d; and a new variant, B. longum subsp. animalis was
described.
The next classification scheme (SCARDOVI et al., 1971 b) based mainly on D NA-
homology sturlies introduced the additional species B. asteroides, B. indicum, B. cor-
yneforme, B. suis, B. ruminale, and B. globosum. The first three ofthese species were
isolated from the alimentary tract ofbees (SCARDOVI & TROVATELLI, 1969), whilst B.
ruminale and B. globosum, described in 1969 (SCARDOVI et al., 1969) were isolated
from the bovine rumen. B. suiswas isolated from pig faeces in 1971 (MATTEUZZI et
al., 1971), and further new species were described (SCARDOVI & CROCIANI, 1974;
SCARDOVI & ZANI, 1974; TROVATELLI et a/., 1974) such as B. dentium (dental caries);
B. catenulatum (waste waters); B. angulatum (waste waters); B. magnum (rabbit
faeces); and B. pullorum (chicken faeces). The status ofthese organisms is not, how-
ever, weil established and further research is required.
DNA-DNA-homology studies have shown that B. infantis, B. liberorum and B.

Table 2.5 Differentiating characteristics ofthe genus Bifidobacterium

Morphology: Non-sporeforming and non-motile bacteria. Rods variable in appearance. Freshly

isolated strains have uniform or branched, bifurcated Y and V shapes, and spatulate
or club shapes. On subculture: straight or curved rods. Gram-positive but often stain
irregularly. Methylene blue may stain intemal granules, but not the entire cell.
Physiology: Anaerobic.
Biochemical Major products of glucose fermentation: acetic and L ( + )-lactic acids in a molar
characters: ratio of 3 : 2. Non-acid-fast. Gas (COz) not produced from glucose. Catalase nega-
tive, nitrate reduction, formation of indole, and Iiquefaction of gelatin negative.
Acid not produced from: rhamnose, sorbose, adonitol, dulcitol, erythritol, and
glycerol.
Cell wall The cell-wall peptidoglycan is not uniform. The basic amino acid in the tetrapeptide
composition: can be either omithine or Iysine; there are various types of cross-linkage (single
amino-acid residue or peptide ).
Phospholipid Specific phospholipids: polyglycerolphospholipid and its lyso derivatives, alanyl
composition: phosphatidylglycerol, and lyso-derivatives of diphosphatidylglycerol.
DNA-base Guanine plus cytosine (G + C) content: 57.2-64.5 moles %.
composition:

18
 lactentis; B. breve and B. parvulorum; as weil as B. thermophilum and B. ruminale,
are genetically homologous and should be merged into three single species: respec-
tively B. infantis, B. breve, and B. thermophilum.
The classification scheme proposed by HoLDEMAN and MoORE (1972) is based
on that given by SCARDOVI and co-workers ( 1971 b) with some alterations. B. infantis
is divided into three subspecies, and B. adolescentisis divided in groups A, B, C, and
D. Two additional species are described: B. comutum and B. eriksonii (syn. Actino-
myces eriksonil); the latter, named Actinomyces eriksonii by ÜEORG and co-workers
( !"965), has been isolated from human pleural fluid and from a Jung abscess.
In the eight edition of Bergey's Manual (BucHANAN & GIBBONS, 1974) bifido-
bacteria are classified in a separate genus, Bifidobacterium (family Actinomyceta-
ceae), as proposed by ÜRLA-JENSEN (1924). The diagnostic characteristics of the
genus are shown in Table 2.5.

Table 2.6 Fermentation reactions of named species of genus Bifidobacterium 1)

Previously named species Arabi· Xylose Ribose Gluco· Cello· Lactose Manni· Melezi· Salicin Stard Treha·
nose nate biose tot tose lose
I. B. bifidum ÜRLA-JENSEN - - - - + + - - - - -
2. B. adotescentis REUTER + + + + + + V V + V V
3. B. infantis REUTER - - + - V + - - V V V
B. liberorum REUTER - + + - V + - V V V V
B. tactentis REUTER - + + - V + + - - V V
4. B. breve REUTER - - + - + + + V V V V
B. parvutorum REUTER - - + - V + + - - + -
5. B. tongum REuTER + + + - - + - + - V V
B. tongum subsp.
animatis Mitsuoka + + + - - + - - V V V
6. B. pseudotongum
MITSUOKA + + + - V V - V V + -
7. B. thermophiturn
MITSUOKA - - - - V V - V V + V
8. B. suis MATTEUZZI et at. + + - - - + - - - - -
9. B. asteroides SCARDOVI
et at. + + + + + - - - + - -
10. B. indicum SCARDOVI et at. - - + + + - - - + - -
II. B. coryneforme SCARDOVI
et at. + + + + + - - - + - -
1) + =positive;- = negative;v = variable,slight,delayedorerraticreactionsbythesamestrainor

related strains (so-called biotypes).

All bifidobacteria ferment glucose, galactose and fructose (sometimes slowly). B. bifidum biotype a
(from adults) slowly ferments sucrose and melibiose; B. bifidum biotype b (from infants) does not
ferment sucrose but vigorously ferments melibiose; neither fermentsmaitose or raffinose. Other bif-
idobacteria (nearly 100%) ferment maltose, melibiose, raffinose and sucrose. Inositol is fermented
only by some strains of B. liberorum and B. tactentis. Inulin is fermented only by some strains of B.
adotescentis, B. infantis and B. tiberorum. Esculin and amygdalin are fermented by B. adotescentis
and B. breve; B. infantis, B. liberorum, B. tongum arenegative and B. thermophiturn is equivocal;
remaining have not been tested. Sorbitol is fermented only by certain strains of B. breve and B. ado-
tescentis. Mannose is fermented only by certain strains of B. breve, B. tongum, B. suis and generally
slowly, variably, or not at all by the others. Bifidobacteria do not ferment adonitol (ribitol), dulcitol,
erythritol, glycerol, rhamnose and a-methyl-o-mannoside.
Source: "Bergey's Manual of Determinative Bacteriology ( 1974)

19
Table 2.7 Morphological, cultural and physiological characteristics of human and some non-
human species of genus Bifidobacterium

B. bifidum (TISSIER) Rods highly variable in appearance. Surface colonies on agarmedia

0RLA-JENSEN incubated anaerobically are circular, convex or Iens-shaped; whitish,
opaque, with smooth to mucoid surface. In agardeeps no growth near
the surface. Gram-positive but may stain irregularly as the culture ages.
Methylene blue may stain internal granules but not the entire cell.
Organic nitrogen required for growth. Optimum growth at 36-3 8 o C.;
no growth at 20 o C or at 45 o C. Optimum pH 6-7; little orno growth at
pH 5.5 or less.
B. ado/escentis Short, curved, occasionally bifurcated rods. Optimum growth at
REUTER 35-37 °C.; nogrowthat20 °Corat46.5 oc.
B. infantis Rods small, thin, often spherical or bubble shaped, and often with cen-
REUTER trat granules; no branching tendency. No growth at 20 o C or at 46.5 o C.
B. breve Rods short, slender orthick, often club shapes. Colonies convex to pul-
REUTER vinate, 2-3 mm in diameter, smooth orundulatingsurface. No growth
at20 oc orat46.5 °C.
B.longum Rods long, curved, club shapes, swollen shaped. Gram-variable.
REUTER Colonies convex to pulvinate, 2-5 mm in diameter, shining orslimy. No
growth at 20 o C or at 46.5 o C.
B. pseudolongum Young cells have coryneform appearance, with short, coccoidal to
MITSUOKA curved cells. Colonies smooth, convex, cream to white. Ammonia satis-
fies nitrogen requirements. Optimum growth at 39-40 o C.; growth at
45 o C and no growth at 20 o C. Optimum pH 6.5-7 .0. No growth at
pH 8.0 and growth retarded at pH 6.0.
B. thermophiturn Slender rods, slightly curved, branching rare. Growth at 46.5 o C; pH
MITSUOKA optimum 6.5-7 .0. No growth below pH 5.0 or at 8.0.

Source: "Bergey's Manual of Determinative Bacteriology" (197 4)

Bifidobacterium is classified into eleven species on the basis offennentation pat-

terns, physiological characteristics, and DNA homology. Fennentation reactions of
named species; and morphological, cultural, and physiological characteristics of
human and some non-human species are shown respectively in Tables 2.6 and 2. 7.
All bifidobacteria fennent glucose, galactose, and fructose (sometimes slowly).
All human strains fennent Iactose, lactulose, and N-acetylglucosamine; except
strains of DEHNERT's group V ( B. adolescentis and B. longum) which do not fennent
N-acetyl-glucosamine.
B. bifidum has a limited fennentation ability; biotype a (from adults) fennents
slowly sucrose, and melibiose; whereas biotype b (from infants) does not fennent suc-
rose but fennents melibiose; B. bifidum does not fennent maitose and raffinose,
whereas other bifidobacteria fennent maltose, raffinose, sucrose, and melibiose.
B. infantis, B. liberornm and B. /actentis are genetically homologous and are
merged under B. infantis, but they differ in their ability to fennent xylose, inositol,
trehalose, mannitol, inulin and salicin.
B. breve and B. parvulornm have homologous DNA and are merged under B.

20
breve; they ferment ribose, mannitol, esculin, and amygdalin, but not arabinose or
xylose. Strains previously designated B. parvulorum ferment starch.
B. adolescentis is characterized by the fermentation of pentoses and gluconate
from which acids and carbon dioxide are produced.
Biotypes (a, b, c, d) proposed by REUTER (1963), differ in their fermentation of
mannitol and sorbitol, andin serological reactions. Biotype a ferments both mannitol
and sorbitol; biotype b ferments only mannitol and biotype c ferments only sorbitol;
neither mannitol nor sorbitol is fermented by biotype d. Strains previously designated
B. dentium, B. catenulatum and B. angulatum are included under B. adolescentis as
unassigned groups.
B.longum is distinguished by the fermentation of pentoses, but it does not fer-
ment gluconate and cellobiose. B. longum subsp. longum ferments melezitose, but
not B. longum subsp. animalis.
B. pseudolongum, B. thermophilum and B. suis are non-human species. Strains
previously designated B. pseudolongum and B. globosum, as well as B. thermophilum
and B. ruminale, have homologous DNA and are merged respectively under B. pseu-
dolongum and B. thermophilum. Animal strains are differentiated from human
strains by their fermentation pattems, ability to grow at 46.5° C., and by the electro-
phoretic mobilities of their enzyme, fructose 6-phosphate phosphoketolase (Table
3.2).
B. pseudolongum ferments pentoses, whereas B. thermophilum does not but
both ferment starch. B. suis ferments arabinose, xylose, and Iactose, but not starch.
B. asteroides, B. indicum and B. coryneforme (from bees) are characterized by
the fermentation of gluconate with the production of acids and carbon dioxide; also
by the electrophoretic mobilities of their fructose 6-phosphate phosphoketolases.
They do not ferment Iactose.

2.10 Identification

Identification of Bifidobacterium isolated from the stools or other materials may be

tentative or definitive. A tentative identification can be made on the basis of micro-
scopic appearance and the type of colony of organisms grown on a defined medium.
Some biochemical tests may also be made. Bifidobacteria have the characteristic bifid
forms when subcultured, after primary isolation, on to the NORRIS medium upon
which most strains produce porcelain white colonies (PouPARD et al., 1973).
A final identification of Bifidobacterium requires gas chromatography of the fer-
mentation products of glucose to supplement other tests (Table 2.5, p. 18, under
"Morphology, physiology and biochemical characters "). The presence of acetic and
L ( + )-lactic acids as the major fermentation products of glucose; and anaerobic
growth are typical properties ofthe genus Bifidobacterium. Other criteria arenegative
tests for catalase, nitrate reduction, formation of indole, gas production from glucose,
and liquefaction of gelatin. Acid not produced from: rhamnose, sorbose, adonitol,
dulcitol, erythritol, and glycerol (Table 2.5).
Identification of species within the genus is mainly based on fermentation pat-
tems (Table 2.6).

21
2.11 Ecological relationships

2.11.1 Occurrence of bifidobacteria in humans

2.11.1.1 The human intestinal tract

Bifidobacteria constitute a major part ofthe faecal flora ofhealthy humans. They are
the predominant organisms in the stools ofbreast-fed infants.
Durlog prenatallife the foetus Jives in a sterile en.vironment but theinfantat birth
is contaminated with the mother's vaginaland faecal flora; and subsequently with the
skin flora, and bacteria from the environment and food. This results in a rapid coloni-
sation of the intestinal tract with a diverse microbial flora consisting principally of
coliforms, enterococci, lactobacilli, and clostridia. Bifidobacteria appear in the stools
from 2 to 5 days after birth in breast-fed babies with the establishment of a relatively
stable microflora in the colon within several days (Fig. 2.4).
By the end of the first week, bifidobacteria become the predominant organisms
ofthe faecal flora and rise to about 99% (range 85 to more than 99 %), while other bac-
teria decrease more or less rapidly. Coliforms, lactobacilli, and enterococci comprise

"/.
100

,.... " ___ _

Bilidobacteria

80
I ,, .... --.....,,. ""
I
60 I
I
~)
/

20
E. coli
Enterococci
3 " 5 6 7 10 20 30
Age in days
Fig. 2.4 The proportians (in per cent) ofbifidobacteria, E. coli, and enterococci in the faecal flora of
breast-fed infants within 30 days ofbirth.
Clostridia often found in the faeces within the first 5 days, and thereafter nearly completely disap-
peared. Bacteroides found in each 5th sample only
(HOFFMANN, 1966)

22
about 1% ofthe faecal flora; and anaerobes such as bacteroides, clostridia and other
putrefactive organisms are drastically reduced and may disappear. The pH values of
the stools range from 5.0 to 5.5 or slightly high er. In premature infants, bifidobacteria
may proliferate more slowly than in full-term infants, and only become predominant
about 15 days after birth or even iater (FRISELL, 1951 ; MA YER, 1956; HOFFMANN,
1966). The counts of bifidobacteria range from 109 to 10 11 organisms per gram of
faeces (FRISELL, 1951; HAENEL, 1957 b; PETUELY, 1962; ZUBRZYCKI & SPAULDING,
1962; SEELIGER & WERNER, 1962; GORBACH et a/., 1967; MATA et a/., 1969 a; MATA
& URRUTIA, 1971).
Weaning ofthe infant causes a gradual change ofthe faecal flora. Counts ofbi-
fidobacteria often decrease by 1 log, and anaerobes, including bacteroides, pepto-
streptococci, eubacteria, fusobacteria increase significantly. The pH values of the
stools also increase. Bifidobacteria may reach numbers corresponding to those in
breast-fed infants provided that the amount ofbreast milk received daily is not less
than approximately 200 g (FRISELL, 1951; MAYER, 1956), orcomprise at least 50% of
the mixed diet (LEVESQUE, 1959 a; YOSHIOKA, 1971).
The faecal flora ofbottle-fed infants resembles that of older children and adults
and although bifidobacteria remain the principal part of the faecal flora, they
decrease in numbers by I to 2 log. They are approximately equal in numbers to, or

aerob. anaerob.
coli. enteroc. staphyl. fungi. putrides.
lactob. lactob.

: :
tl')
..
...
. ... ..
! I
~ 10
....
Cl>
... ..: ....
~
9
... .
:
....
tl')

c: 8
:J
I
I .. . . :
0
.... 7 ... !
.. :
E
.... 6
:
!
. . .....
. .
Cl>
0,
....0 5
0,
.2 4 ' ... I
......
' 1. .
3
'
2
'
I I
.
a b a
I
b a b a b a b a
..b a b
Fig. 2.5 Counts of different groups of micro-organisms, cultivated from the faeces ofthe same baby
during breast-feeding (a) and bottle-feeding (b). The main difference is in the number ofputrefactive
bacteria
(HAENEL, 1970)

23
Table 2.8 Approximate numbers of bifidobacteria and other organisms in the human stomach,
intestine and faeces

Mean log10 viable count per g wet-weight

Genus Stomaeh Uppersmall Lowersmall Large Faeces

Bacteroides N" 2.5 3.5 8.0 10.5

B!fidobacterium N 2.0 4.0 7.0 10.5
Lactobacillus N 1.0 N 6.5 4.0
Enterobacteria N N 3.3 7.0 6.0
Enterococci N N 2.3 7.0 3.5
Clostridium N N N N 3.0
Veillonella N N N 3.0 3.0
Yeasts N 1.0 2.3 N 1.0

N": less than I 0 per g

Source: HAWKSWORTH et a/., (1971)

may be outnumbered by bacteroides and most authors agree that the numbers of
organisms other than bifidobacteria are !arger in the stools ofbottle-fed infants than
in breast-fed infants (FRISELL, 1951; HOFFMANN, 1966; MATA et a/., 1969 a; HAENEL,
1970). The pH values ofthe stools range from 6.4to 7.0 orhigher, which indicates the
presence of putrefactive organisms (Fig. 2.5).
In the case ofpartly-breast and partly-bottle fed infants, and with lactulose-con-
taining diets, the faecal microflora may resemble the typical microflora ofbreast-fed
infants (HAENEL et al., 1970).
Bifidobacteria are reduced significantly in the stools of old people, but clostridia,
enterobacteria and streptococci are increased. This is usually due to a diminished
secretion of gastric juice (lower gastric acidity) in this age-group (ÜRLA-JENSEN et al.,
1945; ÜUTHOF, 1957; HAENEL, 1963; HOFFMANN, 1966; DRASAR & HILL, 1974).
Individualvariations in numbers may be found within each age-group and these
amount to about I log. The population ofbifidobacteria in the !arge intestine of adults
is much more stable than in infants, although changes in the gut flora, including bi-
fidobacteria, may be caused by various extemal and intemal factors. These will be
more fully discussed later.
Bifidobacteria are also present in the small intestine, but in much smaller num-
bers than in the !arge intestine and faeces (Table 2.8).
The host physiology, age, and diet influence the distribution ofthe various spe-
cies and biotypes ofbifidobacteria in the !arge intestine (Table 2.9).
lt may be concluded from Table 2.9 that strains of B. infantis are the predomi-
nant bifidobacteria in the stools ofbreast-fed infants, although other species such as
B. bifidum biotype b, B. breve, and B. longum subsp. longum may also occur in !arge
numbers in the stools of infants. Strains of B. /ongum and B. adolescentis are typical
of adults, but B. bifidum bio type a may also occur, whilst in the stools of old people,
B. adolescentis biotype b may occur in !arger numbers than in those of other age-
groups.

24
Table 2.9 Bifidobacteria: distribution of species and biotypes

Large intestine Predominant organisms Secondary Reference

organisms

Breast-fed infants Dehnert's group IV (B. infan- DEHNERT, 1957b; 1961 a;

tis) PETUELY & LINDNER, 1965;
WERNER, 1966; MÜLLER &
PECH, 1967; HAENEL, 1970.
Breast-fedinfants Dehnert's group III (B. infantis Dehnert's WERNER & SEELIGER, 1963
or B. breve) groupiV a; REUTER, 1963.
Breast-fed and bot- Dehnert'sgroup III and V (B. Dehnert's YOSHIOKA, 1971.
tle-fed infants adolescentis and B. longum) group 1/11 (B.
bifidum)
Infants B. infantis, B. breve, B. bifi- MITSUOKA & KANEUCHI,
dum biotype b, B.longum 1977.
subsp. longum
Adults B. adolescentis biotypes a-b,
B.longum
Oldpeople B. adolescentis biotype b (more
frequently), B.longum

2.11.1.2 Origin and transmission

The main source ofbifidobacteria for the initial colonisation of newbom infants has
not been determined. Most authors, however, consider that the mother's vaginaland
faecal microflora may be a primary source, followed contributions from the micro-
flora of skin and the environment (BLAUROCK, 1940, ßOVENTER, 1949; HARRISON et
a/., 1953; HOFFMANN, 1966; DRASAR & HILL, 1974).

2.11.1.3 The human vagina and mouth

The incidence and numbers ofbifidobacteria in the vagina are considerably less than
in the intestinal tract. Early workers reported the isolation of bifidobacteria from the
vagina (BERGHOLM, 1902; WEGELIUS, 1909; SALOMON, 1923 b). In subsequent stud-
ies an improved medium, cystine-lactose-liver agar, was used for the isolation of bi-
fidobacteria and these were found with greater frequency in the vagina (BLAUROCK,
1940; BovENTER, 1949). The frequency of bifidobacteria in the vagina of pregnant
women, particularly towards the end of pregnancy is significantly greater than in the
case of normal non-pregnant women (HARRISON et a/., 1953; WERNER & SEELIGER,
1963 a; MüLLER & PEcH, 1967). The mostfrequent strains ofbifidobacteria in the
human vagina are B. breve, B. /ongum and B. adolescentis.
The incidence ofbifidobacteria in the mouth also is considerably less than in the
intestinal tract. Early workers reported their isolation from the mouths of infants and
from carious teeth (BRAILOWSKY-LoUNKEVITCH, 1915; LAUTER, 1921; SALOMON,
1923 a; SEGUIN, 1931 ). Strains of bifidobacteria isolated from the mouth have dis-
tinctive fermentation characteristics (BEERENS et a/., 1957; MüLLER & PECH, 1967),
and the predominant strains are B. adolescentis biotypes a and b. Biotype b strains
occur very frequently in association with dental caries.

25
2.11.2 Bijidobacteria in animals

Early workers observed bifidobacteria in the faeces of rats (HULL & RETTGER, 1917;
DE ÜASPERI, 1911; ÜRLA-JENSEN eta/., 1936), a dog fed with human milk(JACOBSEN,
1909), calves and a pig (BREIT, 1935), dogs, rats, mice and monkeys (WEINBERG et al.,
193 7) but none mention the incidence of the strains isolated or their characteristics. In
subsequent studies these organisms were found to be the main microflora of the
bovine rumen (BAUMANN & FosTER, 1956), the predominant organisms in the faeces
of guinea pigs and rabbits, but smaller numbers were found in the faeces of chickens
and rats (HAENEL & MüLLER-BEUTHOW, 1956 a). Comparative studies on strains of
bifidobacteria from human faeces, from the alimentary tracts of animals, such as the
bovine and ovine rumens, and from the intestines of chickens, swine, rats, mice, gui-
nea pigs and rabbits, demonstrated distinctly different characteristics between ani-
mal strains and human strains (OCHI & MnsuoKA, 1958; ÜCHI et al., 1960; UcHIA et
a/., 1965; MITSUOKA, 1969 b).
The incidence and numbers of bifidobacteria in the alimentary tract of animals
vary according to the species of animals and their age and diet. Bifidobacteria have
been reported to outnumber lactobacilli in the faeces of monkeys and guinea pigs but
they are present in smaller numbers than lactobacilli in the faeces of chickens, swine,
dogs and rats, and are found only occasionally in horses, rabbits and mice (MITSUOKA
& KANEUCHI, 1977). The most frequently occurring bifidobacteria in animals are
shown in Table 2.1 0.

Table 2.10 Occurrence ofbifidobacteria in animals

Species ofbifidobacteria Animals

B. pseudolongum The intestine and faeces of swine, chickens, rats, mice, dogs,
ruminants, also in ovine and bovine rumens
B. thermophiturn The intestine and faeces of swine, chickens, andin the bovine
rumen
B. suis The faeces ofpiglets
B. longum subsp. animalis The intestine of calves, sheep, rats, mice, and guinea pigs
B. adolescentis biotypes a and b The intestine of monkeys
B. adolescentis biotypes c and d The intestine of dogs
B. asteroides The hind gut ofhoney bees
B. indicum The intestine ofbees
B. coryneforme The intestine ofhoney bees

Source: MITSUOKA ( 1969 b); SCARDOVI et a/., ( 1969); SCARDOVI & TROV ATELLI (1969); MITSUOKA &
KANEUCHI ( 1977).

2.11.3 Pathogenicity

Bifidobacteria present in the intestinal tract, the mouth, and the vagina (where they
normally occur) have not been reported tobe pathogenic to the host.
However, some strains ofbifidobacteria have been implicated in possible unde-
sirable effects for the host, when found in soft tissues. They usually occur in mixed
populations along with facultative anaerobic bacteria (BEERENS & TAHON-CASTEL,

26
1965; GEORG el al., 1964; 1965). Generally the access of intestinal micro-organisms
to soft tissues (which are not their native habitat) has harmful consequences. This will
be discussed in Chapter 5.

2.11. 4 Anlagonislic effecl ofbi.fidobacleria

2.11.4.1 Effect in vilro

TissiER (1906 a) was the first to report an antagonistic effect of B. bi.fidus (B. bi.fidum)
against B. coli, B. laclis aerogenes, R. ramosum and F.furcosa. This effect was also
observed against staphylococci and Proleus (KLING, 1914), Salmonella typhi
(BARKER, 1914; TORREY, 1915), and Shigella dysenleriae(DELBOVE, 1932).
Subsequent studies by Rose and GYöRGY (1955) showed that B. bi.fidum var.
pennsylvanicusinhibits the growth of E. coli and even other strains ofbifidobacteria.
Other workers have also reported an antagonistic effect of bifidobacteria against
enteropathogenic E. coli strains, Shigella dysenleriae, Salmonella typhi, Slaphylococ-
cus aureus, and Proleus (BIRKENBACH, 1957; MAYER, 1960), enteropathogenic E.
coli(RuscHMANN, 1958), E. coli, and S. aureus(SEMENIKHINA, 1967), enteropatho-
genic E. coli strain 0111 84 and Candida albicans (RADULOVIC, 1970; 1971 ).

2.11.4.2 Effect in vivo

A protective role of the gut microflora against enteric infections has been the subject
of a few investigations. For instance, studies on Shigella (agent of dysentery) in
breast-fed infants in Guatemala, demonstrated that the gut microflora enhances resist-
ance to the infection, or elimination ofthe invader (MATA el al., 1969 b). Other sturl-
ies demonstrated that the low incidence of enteropathogenic E. coli infections in
breast-fed infants was attributable to the predominance ofbifidobacteria (MATA &
URRUTIA, 1971).
A protective effect of bifidobacteria against enteropathogenic E. coli strain
011184 was also observed in infants which were either nursed or given fresh, raw,
bacteriologically-safe human milk (TASOVAC & Kocic, 1970).
The high er acidity ofthe lower intestinal contents may indirectly prevent the pro-
duction of toxic amines from the amino acids by the putrefactive bacteria (clostridia,
coliforms). The beneficial roles ofbifidobacteria and L. acidophilus in preventing the
formation of amines have been pointed out by NEGISHI and BILLY, ( 1962) and by PRE:-
voT(l971).
Bifidobacteria may not exert an inhibitory effect on enteric viruses, since in
enteric virus infections, most of the intestinal bacteria including bifid organisms
quickly disappear from the faeces (MAYER, 1965 b; 1969). However upon elimina-
tion of the infection, the dietary administration of bifidobacteria (preferably in con-
junction with L. acidophilus) may be used to re-establish a normal gut flora.
Comparative feeding studies oftwo groups of infants, one receiving a modified
cows' milk preparation containing bifidobacteria and growth-promoting substances
and the other receiving a buttermilk preparation, showed that enteric infections were

27
8 times morefrequent in the buttermilk group than in the modified cows' milk-prepa-
ration group (KAwuo & STÖGMANN, 1968).
Bifidobacteria, preferably in conjunction with L. acidophilus, may prevent the
overgrowth of harmful organisms in the intestine following antibiotic therapy. It has
been shown, that the oral administration of a bifidus milk preparation to an infant fol-
lowing the discontinuation of penicillin treatment, increased the population of bi-
fidobacteria in the faeces and suppressed the growth of Candida albicans (MA YER,
1966; 1969). (See Fig. 2.6, 2. 7, 2.8).
Likewise it has been shown that the oral administration of a freeze-dried culture
of B. bifidum to children with enteric infections, eradicated strains of enteropatho-
genic E. coli in about 60% of cases, and in more than 80% cases when supplemented
with lactulose (SCHNEEGANS et al., 1966). These workers concluded that there is no
correspondence between the implantation ofbifidobacteria and the disappearance of
pathogenic E. coli. Similarly, HEWITT and RIGBY (1976) did not find a consistent
inverse relationship between !arge numbers of bifidobacteria and small numbers of
E. coliin the faeces of newbom infants. They considered that implantation ofbifido-
bacteria in the I arge intestine of formula-fed infants may not be effective in reducing
the numbers of E. coli.
On the other band, bifidobacteria, when administered in conjunction with lactu-

Fig. 2.6 Photomicrograph ofthe stool of an infant following penicillin therapy (500000 IU/ day).
The growth of bifidobacteria is inhibited and that of Candida albicans promoted du ring 7 days of
treatment
(MA YER, 1969).

28
Fig. 2.7 Photomicrograph ofthe stool of an infant 3 days after discontinuation of penicillin therapy
and receiving a bifidus milk preparation
(MAYER, 1969)

Fig. 2.8 Photomicrograph ofthe stool of an infant 7 days after discontinuation of penicillin therapy
and receiving a bifidus milk preparation
(MAYER, 1969)

29
lose, have a relatively strong antagonistic effect against enteropathogenic E. coli
strains and this may be attributable to the lower pH in the Iarge intestine. It has been
suggested that the preponderance ofbifidobacteria in the large intestine ofbreast-fed
infants and the Iow pH of the contents of the large intestine are the main factors
responsible for natural resistance to gastroenteritis (Ross & DA WES, 1954; BULLEN &
WILLIS, 1971 ).

2.11.4.3 The nature of antagonisticeffect

Bifidobacteria have not been shown to produce antibiotic substances, but this area
requires further study. In contrast, Iactic acid bacteria have been shown to produce
various types of inhibitory substances. For example, L. acidophilus produces lacto-
cidin (VINCENT et al., 1959), acidophilin (VAHIL & SHAHANI, 1965) and acidolin
(HAMDAN & MIKOLAJCIK, 1974). The possibility oflysozyme production by bifido-
bacteria has been suggested on the grounds that egg-white Iysozyme and a metabolite
of bifidobacteria may have common antigenic determinants (MINI GA w A, 1970), but
more evidence is needed to confirm this observation.
Organic acids play a major role in the antagonistic effect of bifidobacteria
against many organisms both in vitro and in vivo. The increasing hydrogen-ion con-
centration ofthe growth medium, due to the production of acids, inhibits acid-sensi-
tive bacteria. Theseacids differ in their antimicrobial effects and acetic acid has a
stronger ability to inhibit microbial growth than lactic acid. It has been shown, for
example, that the minimum pH values at which salmonellae commence growth
(under laboratory conditions) are 5.40 for acetic acid, 4.40 for lactic acid, and 4.05 for
citric and hydrochloric acids (CHUNG & GoEPFERT, 1970).
Unlike the homofermentative lactic acid bacteria, bifidobacteria produce more
acetic than lactic acid from fermentable carbohydrate, and thus also produce small
amounts of formic acid. The acid-producing ability and the proportions of the fer-
mentation products vary among different strains even within the same species.
Organic acids present in their free (undissociated) forms may also have a direct
toxic effect on other organisms (GYÖRGY & RosE, 1955 a; PouPARD et al., 1973).
Acetic acid is present in both the free and combined forms in the large intestine con-
tent and it may exert a decisive inhibitory effect on undesirable bacteria. This is fur-
ther influenced by the nature of the food consumed.
The presence of a buffer consisting of acetic acid and acetatewas demonstrated
in the faeces ofbreast-fed infants, but not in those from bottle-fed infants. In vitro an
acetate-buffered medium was strongly bacteriostatic between pH 5.0 and 5.8 against
Gram-negative organisms, and at pH 5.4 and below against clostridia. An unbuffered
medium had no bacteriostatic effect on the growth of E. coli above pH 4.6, Salmon-
ella typhimurium above pH 5.0, Bacteroides above pH 5.6, Clostridium paraputrifi-
cumand Cl.peifringensrespectively above pH 4.8 and 5.6 (BULLEN & TEARLE, 1976;
BULLEN et al., 1976).
Moststrains ofbifidobacteria deconjugate bile salts to free bile acids which are
more inhibitory to susceptible bacteria than the conjugated forms. However, many
strains ofbacteroides, eubacteria, lactobacilli, enterococci, and clostridia can do the
same.

30
2.11.5 Sensitivity to unfavourable physiological conditions

2.11.5.1 Antibioticagents

Bifidobacteria have been reported to show variable sensitivity to antibiotics. There

are reports that bifidobacteria are sensitive in vivo to erythromycin, spiramycin, and
chloramphenicol; have moderate sensitivity to penicillin, tetracycline, neomycin,
and novabioein; and are resistant to streptomycin (FRISELL, 1951; MANCIAUX, 1958).
In contrast, other reports indicate that bifidobacteria are not inhibited by penicillin,
streptomycin, chlortetracycline, and chloramphenicol when administered at custom-
ary doses (PATZ, 1951; MAYER, 1956).
Various strains may differ significantly in sensitivity to a particular antibiotic
agent. Studies, in vitro, with 4 strains of B. bifidum have demonstrated marked differ-
ences among strains in sensitivity to penicillin, chloramphenicol, oxytetracycline,
neomycin, and Streptomycin (LIPINSKA, 1978).
Extensive sturlies on the effects of therapeutic antibiotics on the human faecal
flora have shown that some have relatively slight effects, whereas others more pro-
faund effects (FINEGOLD et a/., 1966; 1967; SUTTER & FINEGOLD, 1974). Oral tetra-
cyclines, after administration for about one week, may cause relatively little suppres-
sion of the normal faecal flora and only slight overgrowth of potential pathogens
(Kiebsiella-Enterobacter-Se"atia group, Proleus spp., Staphylococcus aureus, Pseu-
domonas spp., and Candida spp.). In contrast, broad-spectrum penicillins (ampicil-
lin and hetacillin) may cause significant depression ofthe normal flora thus going risk
of allowing morefrequent proliferation by potential pathogens.
The antibiotic cephalexin has effects comparable to those of the tetracyclines,
whereas cyclacillin has effects similar to the broad-spectrum penicillins.
Therapy with any antibiotic, particularly long-term and especially oral adminis-
tration, is liable to alter the balance of antibiotic-resistant to sensitive organisms in the
intestine (DRASAR & HILL, 197 4).
The powerful antibiotics: lincomycin, paromomycin, and clindamycin, cause
drastic changes in the normal faecal flora (FINEGOLD et al., 1966; SuTTER & FINE-
GOLD, 197 4). Therapy with clindamycin may eliminate most of the anaerobic organ-
isms (bifidobacteria, bacteroides, anaerobic cocci) and lactobacilli with the exception
of eubacteria and clostridia, while lincomycin eliminates completely the normal fae-
cal flora.
Changes in the normal intestinal flora resulting from antibiotic therapy empha-
size the importance of re-establishing the normal microbial balance. The dietary
administration ofbifidobacteria, preferably in conjunction with L. acidophilus, may
help in the regeneration ofthe normal gut flora. Many reports have demonstrated the
beneficial effects of using such cultures (HAWLEY et al., 1959; REuTER, 1969;
MA YER, 1969; SANDINE et a/., 1972; SPECK, 197 6; BRANCA et a/., 1979).

2.11.5.2 Bile acids

The bile acids synthesized in the Iiver from cholesterol are cholic acid and chenode-
oxycholic acid. Both are conjugated with the amino acids, glycine and taurine (3 : 1

31
ratio ). In this form, they facilitate digestion by the dispersion ofthe fat globules in the
gut and micelle formation.
The bile salts are stored in the gall bladder and pass into the duodenum in
response to a meal. Eventually about 80% of the bile salts are reabsorbed in the small
intestine and are returned to the liver, whilst the remainder reach the colon where they
are degraded by bacterial action (HAITER, 1978).
Bifidobacteria and bacteroides deconjugate bile salts, and the liberated bile
acids, cholic and chenodeoxycholic acids, are dehydroxylated to respectively, deoxy-
cholic and lithocholic acids. The greater part of the deoxycholic acid (the smaller part
is reabsorbed in the colon and returned to the liver) and all the lithocholic acid are
excreted in the faeces.
Bifidobacteria are sensitive to increased Ievels of faecal bile acids. It has been
shown that deoxycholic acid is bacteriostaticto bifidobacteria at Ievels of0.02-0.05%
and bactericidal at 0.2-0.5 %. The concentrations of deoxycholate in the normal
faeces may vary from 0.05 to 0.2% (CATTEAU et al., 1971).
The amount of faecal bile acids is influenced by the amount of fat in the diet
(DRASAR & HILL, 1974). Fatty diets may increase the amounts of bile acids in the
faeces, and consequently increase their inhibitory effect to bifidobacteria. The nature
of the diet may also influence indirectly the physiological conditions affecting the
activity ofbifidobacteria in the gut.

2.11.5.3 Gastric acidity

The sensitivity ofbifidobacteria to gastric acidity may be important when these organ-
isms are administered as dietary adjuncts. The gastric juice has a germicidal effect
mainly due to the presence offree hydrochloric acid. At a pH ofless than 4.0, 99.9%
of the bacteria may be killed within 30 min. (GIANELLA et al., 1972). In individuals
during normal fasting, the stomach is usually sterile or contains less than I 0 3 organ-
isms perml.
Acid-resistant or tolerant organisms such as lactobacilli, yeasts, streptococci, and
bifidobacteria may survive to a certain extent an acidity of the gastric juice and pass
into the small intestine. The administration of large numbers of bifidobacteria may
consequently increase their survival. Various strains of bifidobacteria may differ in
acid tolerance (LIPINSKA, 1978).
As recognized, the transit time of easily digestible fermented milk through the
stomach is relatively shorterthan that of ordinary food, and it has been shown that the
administration of serologically and biochemically defined strains of bifidobacteria
Iead to the appearance ofthe same strains in the faeces (SCHULER-MALYOTH et al.,
1968 b; REUTER, 1969).

2.12 Bacteriophages

A bacteriophage specific for some strains ofbifidobacteria was isolated in 1966 from
human faeces (YoussEF et al., 1966), and subsequent investigations resulted in the
isolation of further six phages from the stools of six persons aged from 14 to 57 years.

32
Fig. 2.9 Schematic drawing of the B. thermophiturn
(B. rurninate) bacteriophage
Head: octahedral, 600 Ain diameter
Tail: 2150 A long and II 0 A in diameter with a base plate
(DENTAN etat., 1970)

Fig. 2.10 Electronmicrograph of B. thermophiturn (B. rurninate) phage

Negative staining with 2% PTA (Phosphotungstic acid)
Magnification 216 000 x
(DENTAN etat., 1970)

33
These phages lyse B. adolescentis biotypes a and c ( B. adolescentis, types 1 a and 1 c,
REUTER), and B./ongum (B./ongum type 2a, REUTER) (YOUSSEF et a/., 1970).
These phages are characterized by a tadpole-like shape, and have a length of
2100 A(l Ais equiva1entto 0.0001 micron); ahead diameterof 400 A; a lenghtoftail
of 1700 A and width of 150 A. They were viable for at least 4 months at 4° C, and at
room temperature, but were inactivated at 70--80° C within 30 min; and quickly at pH
3.0. Two of the phages exhibited a bacteriocin-like effect on B. infantis (Dehnert's
group IV).
Other investigations have reported the isolation of a phage specific for B. therm-
ophilum (B. ruminale) from the calf rumen (DENTAN et al., 1970; MATTEUZZI &
Sozzi, 1971 ). This phage is characterized by an octahedral head, 600 A in diameter,
and a tail2150 A lang and 110 A wide with a base plate (Fig. 2.9 and 2.1 0). The tail is
non-contractile, slightly curved and has regular transverse striations.

34
Chapter 3: The fermentation of hexoses

3.1 Introduction

Lactic acid bacteria ferment glucose either, via the glycolytic system, or via the hex-
ose-monophosphate shunt. Glycolysis is the pathway of glucose fermentation in the
homofermentative lactic acid bacteria, which produce lactic acid and small amounts
of by-products (Fig. 3.2 and Table 3.1). The hexose-monophosphate shunt is the
pathway of glucose fermentation in the heterofermentative lactic acid bacteria, which
produce lactic acid, carbon dioxide, some acetic acid, and ethanol (Fig. 3.3). The
homofermentative and heterofermentative lactic acid bacteria are shown in Chapter
6 (Table 6.2).
Bifidobacteria ferment glucose via the fructose 6-phosphate shunt, which has
been reported by SCARDOVJ and TROV ATELLI ( 1965), DE VRIES and co-workers ( 1967),
and de VRIES and STOUTHAMER ( 1967 & 1968). This is a new pathway for the fermen-
ta,tion of hexoses.

3.2 Fermentation of hexoses

Fig. 3.1 shows a pathway for the fermentation of glucose in bifidobacteria.

Glucose is converted to fructose 6-phosphate by the action ofhexokinase and glucose
6-phosphate isomerase. Fructose 6-phosphate is cleaved by fructose 6-phosphate
phosphoketolase to yield erythrose 4-phosphate and acetyl-phosphate. The forma-
tion of acetic acid from acetyl-phosphate is catalyzed by acetate kinase.
Erythrose 4-phosphate and fructose 6-phosphate are converted to 2 moles of
pentose phosphate by the action of transaldolase and transketolase. Two moles of
xylulose 5-phosphate are cleaved by xylulose 5-phosphate phosphoketolase to yield 2
moles each of acetyl-phosphate and glyceraldehyde 3-phosphate. Acetic acid is
formed from acetyl-phosphate, and glyceraldehyde 3-phosphate is converted to pyr-
uvic acid through a reaction sequence occurring in glycolysis. Pyruvic acid is reduced
to L( +) -lactic acid by the action of Iactate dehydrogenase.
The bifids pathway yields 3 moles of acetic acid and 2 moles of lactic acid per 2
moles ofglucose. The ATPyield is 2.5 moles of ATPpermole ofglucose (Fig. 3.1). In
the homofermentative and heterofermentative pathways, the ATP yields are 2 ATP
per mole of glucose and one ATP per mole of glucose respectively (Fig. 3.2 and 3.3).
ATP (adenosine triphosphate) and ADP (adenosine diphosphate) are compounds
capable of storing or releasing energy.
Moststrains ofbifidobacteria, however, produce more acetic acid and less lactic
acid from glucose than is to be expected according to the breakdown of glucose via
the fructose 6-phosphate shunt (Fig. 3 .l ). The excess of acetic acid is the result of the
phosphoroclastic splitting of a part of pyruvic acid to acetic and formic acids, and
subsequent reduction of some of acetic acid to ethanol. The excess of acetic acid is
formed mainly in the log phase of growth. The proportion of the fermentation pro-
ducts varies considerably among different strains and even within the same species
(DE VRIES & STOUTHAMER, 1968; LAUER & KANDLER, 1976). Some strains reduce pyr-

35
 Fructose- 6- ®
fr;:-®
erythrose- I.-® acetyl -®
~
~
sedoheptulose-7-® acetate I I

ribose- 5- ®
~5
ribulose - 5- ®
~6
xylulose -5-®

2 glyceraldehyde-3-®
2NAD

2NADH2
!2 ac:etate j
2NAD

,2/actlJte I

Fig. 3.1 Formation ofacetate and Iactate from glucose by the bifidum pathway. I, hexokinase and
glucose-6-phosphate isomerase; 2, fructose-6-phosphate phosphoketolase; 3, transaldolase; 4, trans-
ketolase; 5, ribose-5-phosphate isomerase; 6, ribulose-5-phosphate 3-epimerase; 7, xylulose-5-phos-
phate phosphoketolase; 8, acetate kinase; 9, enzymes as in homofermentative pathway
(GOTTSCHALK, 1979)

36
 [ glucose]

2 Iactate 2 glyceraldehyde -3- ®

v:--
K___
2 NAD
2 NADH2 ..
------r~ -
2 P;

2pyruvate 2 1,3 - diphosphoglycerate

Fig. 3.2 Formation of Iactate from glucose by the homofermentative pathway. I, enzymes of the
Meyerhof-Embden pathway; 2, Iactate dehydrogenase
(GOTTSCHALK, 1979)

uvic acid to lactic acid, whereas others split all ofthe pyruvic acid to acetic and fonnie
acids. In most strains both routes are involved in the conversion of pyruvic acid.
The growth medium for bifidobacteria also influences the extent of the phos-
phoroclastic splitting of pyruvic acid. For example, in the fermentation of Iactose,
more lactic acid per mole oflactose is produced than from 2 moles of glucose or galac-
tose. The fermentation of galactose is close tothat of glucose, whereas in the fermen-
tation of mannitol more ethanol is formed than from glucose (DE VRIES & STOUT-
HAMER, 1968). Bifidobacteria preferentially metabolise glucose when both glucose
and fructose are present in the culture medium (SINTERHAUF et al., 1966).
Apparently, the proportians ofthe fermentation products influence the flavour
of a fermented product, which may vary from clean, mildly so ur to slightly acetic, to
slightly sharp.
Bifidobacteria are non-acid-fast organisms and their growth in culture media is
slow, but with repeated transfers their multiplication rate and acid production may

37
 lglucose I
Iethanol I L-®
~NAD fC®
f- NADH2 gluc~osetifö®
•cetaldehyde
NAD NAD~
6-®- gluconafe
NADH2 NAD
acetyi-CoA NADH2
~ P· .__ _ _ , co2l
CoAj 6
1
ribulose -5 -®
acetyl -® l4
;:>:::o--.....:5::,...__ _ _ _ xylu lose - 5 - ®
glyceraldehyde -3-®
NAD

NAD~

NAD

IIactate

Fig. 3.3 Formation of carbon dioxide, Iactate, and ethanol from glucose by the heterofermentative
pathway. 1, hexokinase; 2, glucose 6-phosphate dehydrogenase; 3, 6-phosphogluconate dehydroge-
nase; 4, ribulose 5-phosphate 3-epimerase; 5, phosphoketolase. The cleavage reaction yie1ds glycer-
aldehyde 3-phosphate and enzyme-bound a1pha, beta-dihydroxyethy1thiamin pyrophosphate. This
is converted to acetyl-TPP-E via the alpha-hydroxyvinyl derivative; phosphorylytic c1eavage results
in acetyl phosphate formation. 6, phosphotransacetylase; 7, acetaldehyde dehydrogenase; 8, alco-
hol-dehydrogenase; 9, enzymes as in the homofermentative pathway
(GOTTSCHALK, 1979)

38
be increased. A freshly prepared milk culture of B. bifidum is acidified and often
coagulated in 18-96 h. Acid production is increased particularly following the addi-
tion of growth-promoting substances (e.g., yeast extract or autolysate, pepsin or pep-
sin-digested milk, cysteine, peptone). The degree of acidity produced is dependent on
strain characteristics, and the titre may range from 0.1 to 1.4% titratable acidity.

3.3 Enzymes

Fructose I ,6-diphosphate aldolase, an enzyme unique to the glycolytic system (Fig.

3.2 and Table 3.1), is absent from bifidobacteria. This enzyme catalyzes the conver-
sion of fructose I ,6-diphosphate to glyceraldehyde 3-phosphate. The enzyme phos-
phofructokinase, responsible for the conversion of fructose 6-phosphate to fructose
I ,6-diphosphate in glycolysis, is present in low concentrations.

Table 3.1 The MEYERHOF-EMBDEN Pathway 1)

Reaction Enzyme

Glucose + ATP Hexakinase

!
Glucose I-phosphate

Glucose I-phosphate Phosphoglucomutase

!
Glucose 6-phosphate

Glucose 6-phosphate Glucose 6-phosphate

t! isomerase
Fructose 6-phosphate

Fructose6-phosphate + ATP Phosphofructokinase

t!
Fructose 1,6-diphosphate + ADP

Fructose I ,6-diphosphate Fructose 1,6-diphosphate

t! aldolase
Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate

Dihydroxyacetone phosphate Triasephosphate

t! isomerase
Glyceraldehyde 3-phosphate

Glyceraldehyde3-phosphate + inorganicphosphate + DPN Phosphoglyceraldehyde

t! dehydrogenase
I ,3-Diphosphoglyceric acid + DPN (H2)
1,3-Diphosphoglycericacid + ADP 3-phosphoglycerate
t! kinase
3-Phosphoglycericacid + ATP
3-Phosphoglycericacid Phosphoglyceromutase
t!
2- Phosphoglyceric acid

39
Reaction Enzyme

2-Phosphoglyceric acid Enolase

t!
2-Phosphoenolpyruvicacid

2-Phosphoenolpyruvicacid + ADP Pyruvate kinase

t!
Pyruvic acid + ATP
Pyruvicacid + DPN(H2) Lactate dehydrogenase
t!
Lacticacid

1) Abbreviations:

ATP and AD P = adenosine triphosphate and adenosine diphosphate

DPN = diphosphopyridine nucleotide, a coenzyme involved in hydrogen transfer. Also known as
nicotinamide adenine dinucleotide (NAD)
DPN (H2) = reduced DPN, i.e., DPN that has accepted hydrogen

Glucose 6-phosphate dehydrogenase, responsible for the hexose-monophos-

phate shunt (Fig. 3.3), is usually absent, but in B. adolescentis and strains from bees
both glucose 6-phosphate and 6-phosphogluconate dehydrogenases are present.
Two phosphoketolases are involved in hexosebreakdown by bifidobacteria: one
specific for fructose 6-phosphate and the other specific for xylulose 5-phosphate (Fig.
3.1 ). The electrophoretic behavior of fructose 6-phosphate phosphoketolase is typi-
cal ofthe host ofthe organism {Table 3.2). The enzymes found in various strains of
bifidobacteria demoostrate three types of electrophoretic mobility (SCARDOVI et al.,
1971 a).

Table 3.2 The electrophoretic mobilities of fructose 6-phosphate phosphoketolase and Iactate
dehydrogenase in bifidobacteria

Species lsolated from Electrophoretic mobility

L-LDH Phosphoketolase
Rkan5,0 (cm)

B. bi.fidum FA 4.5 15
B. infantis FB 4.5 15
B. breve FB 4.5 15
B. liberorum FB 4.5
B. parvulorum FB 4.5
B. asteroides Bees 4.5 16
B. suis Pig 4.5 10
B.longum FA 4.5 15
B. thermophiturn Pig 4.5 10
B. adolescentis FA 4.5 15
B. indicum Bees 5.6 16
B. pseudolongum Pig 5.6 10
B.pullorum Chicken 3.4 10

F = faeces; A = adult; B = infant

Source: KANDLER & LAUER (1974)

40
 The Iactate dehydrogenase (LOH) of bifidobacteria requires fructose I,6-di-
phosphate for activation. This is also the same for streptococci and L. casei, although
the latter requires additionally manganese ions. Recently, GARVIE (1978) has shown
that some strains of S. thermophilusdo not require fructose 1,6-diphosphate for acti-
vation.
Unlike lactobacilli, bifidobacteria have only a LOH stereospecific for L ( + )-Iac-
tate. The LOH has been found to have three types of electrophoretic mobility (Table
3.2).
Galactose is converted to glucose by the action of galactose I-phosphate uridyl-
transferase in the presence of an activating agentsuch as uridinediphosphate-glucose
(UPO-glucose).

Ga! I-phosphate
uridyltransferase
Galt-phosphate+ UOP-glu Glu I-phosphate+ UOP-gal

In addition, a pyrophosphorylase pathway in which gall-phosphate can be con-

verted to glu I-phosphate without the participation of hexose I-phosphate uridyl-
transferase has been proposed. Some properties of an enzyme from B. bifidum,
which has both UOP-glucose and UOP-galactose pyrophosphorylase activity, have
been determined (LEE et al., 1978 & 1979).
Qualitative and quantitative differences may occur among various species and
biotypes ofbifidobacteria in respect of carbohydrase activity (Table 2.6). It has been
found that a biotype, Oehnert's group IV (B. infantis) isolated from the faeces of
breast-fed infants, showshigh lactulase and alpha-galactosidase acitivities, and that it
readily hydrolyzes lactulose and raffinose. A further a biotype, Oehnert's group IV,
hydrolyzes lactulose faster than Iactose, whereas that of Oehnert's group II (B. bifi-
dum), isolated from bottle-fed infants and from adults, hydrolyzes lactulose more
slowly than Iactose (HAENEL et al., 1967; RuTTLOFF et al., 1967 a,b ). Strains of bi-
fidobacteria, which ferment Iactose, possess the enzyme, beta-galactosidase. It has
been shown that beta-galactosidase from a strain (A-3) of B. bifidum hydrolyzes Iac-
tose and Jactulose. The lacto-N-tetraose ofhuman milk issplitat the beta-(1-+3)-gal-
actosidic linkage with the Iiberation of galactose and the formation oflacto-N-triose
II (lwAKASI et al., 1971).

41
Chapter 4: Effect of substrate on the growth of
bifidobacteria

4.1 Introduction

Extensive studies on the growth-promoting factors for bifidobacteria have been con-
ducted on two lines:
(i) modifications of cows' milk composition; (ii) identification of "bifidus" and
bifidogenic factors.
Many modified cows' milk preparations for infant feeding have been developed.
Some of them contain "bifidus" or bifidogenic factors. The use of a factor, such as
lactulose for the treatment of chronic liver disease in adults is now recognized.

4.2 Modifications of cows' milk composition

4.2.1 Lactose and its derivatives

SITTLER (1908) was the first to claim that addition oflactose to cows' milk for infant
feeding promoted the growth of intestinal bifidobacteria. Subsequent studies have
shown that the addition of 12% Iactose (GERSTLEY et al., 1932), or at least 10%
according to BESSAU (1937 a; 1938), promoted the appearance of a Gram-positive
flora in the faeces. These effects were, however, inconsistent and indicated that other
factors may be involved. Likewise the oral administration of Iactose has been
reported to have only a partial and temporary effect in lowering the pH of the stools of
artificially-fed infants (Ross & DA WES, 1954); and apparently, this is because the Iac-
tose does not reach the !arge intestine in sufficient amount to promote the growth of
bifidobacteria.
KoKUBU (1960) has shown that a Ievel of2% Iactose plus certain essential gluco-
samines in the ileocaecum of infants promotes the multiplication of bifidobacteria,
whereas an excess of protein inhibits their growth.
The heating of cows' milk, containing additional Iactose, has been proposedas a
means of retarding its intestinal absorption (BESSAU, 1937 a & 1943; KLEINSCHMIDT,
1949). This method induces the formation of new bifidogenic compounds which are
attributable to modification of the Iactose molecule.
Two oligosaccharides, gynolactose and allolactose, isolated from human milk,
have been reported to have a stimulating effect on bifidobacteria (POLONOVSKI &
LESPAGNOL, 1930; 1931 ; 1933). Thesesugarsare not present as such in human milk,
but result from the transformation oflactose by certain bacteria, or digestive enzymes
of the calf (KuHN et a/., 1955).
A bifidogenic effect ofbeta-lactose has been reported by several workers (MALY-
OTH & KiRIMLIDIS, 1939; MALYOTH & STEIN, 1953; DüSTERWALD etal., 1950; DEL-
THIL, 1953), but this has not been confirmed by others (FRISELL, 1951; PETUELY,
1957 a; LEVESQUE et a/., 1959).
Artificially-fed infants receiving either a dextrin-maltose supplement (MALY-

42
OTH, 1949), or a dextrin-cystine supplement (BOEHM-AUST, 1952) has been reported
to supportintestinal bifidobacteria and to suppress coliforms. However, subsequent
studies using a dextrin-maltose supplement indicated that the effect is weak
(LEVESQUE et a/., 1959) or negligible (PETUELY, 1957 a).

4.2.2 Other milk constituents

Workers originally observed that the relatively high Iactose content and the low buf-
fering capacity of human milk favoured an acid pH in the faeces (ADAM, 1921 b;
1925; CRUICKSHANK, 1925). Subsequent studies showed that the relatively high con-
tents ofprotein and calciumphosphatein cows' milk inhibit the growth ofbifidobac-
teria, whereas the high cystine content of human milk-protein promotes it (ADAM,
1949; BLAUROCK, 1937).
This observation led to the development of modified cows' milk preparations of
reduced casein and calcium phosphate content and supplemented with Iactose and
cystine.
The fatty acid components of cows' milk have been reported tobe inhibitory to
bifidobacteria, but this effect was nullified by the milk proteins. Lauric and myristic
acids were the most inhibitory ofthose tested (TOMARELLI et al., 1950; GYLLENBERG
et al., 1954 & 1956). On the other hand, some studies have shown that certain fatty
acids have fairly strong bifidus growth-promoting effects in the order: propionic,
butyric, palmitic, and stearic acids, whereas the high er unsaturated fatty acids, oleic
and linoleic acids have very little growth-promoting effect (ÜISHI, 1966).
However, the relatively high content of essential fatty acids (linoleic and linol-
enic acids) in human milk has an important nutritional function, hence in modifica-
tion of cows' milk, the milk fat may be replaced by vegetable oil containing a higher
proportion ofthese fatty acids.

4.3 Specific growth-promoting factors

Early workers found that certain constituents of skimmed human milk, particularly
the whey promote the growth of intestinal bifidobacteria (MoRo, 1907 I 1908;
SCHÖNFELD, 1926), but the actual growth-promoting factors were only identified
more than twenty years later.

4.3 .1 Bifidogenic factors

Lactulose (beta-galactosidofructose) has been reported to be a bifidogenic factor

(PETUELY & KRISTEN, 1949; 1951; PETUELY, 1957 a,b).ltis adisaccharide composed
of one molecule each of galactose and fructose, but it is not present in a free state in
human milk.lt is only upon heating milkthatapart ofthe Iactose is converted to lactu-
lose, and in sterilized liquid formula feeds it amounts to 2-5% of the total carbohy-
drate content. Lactulose is ineffective in vitro on the growth of bifidobacteria, but its
inclusion in the diet of artificially-fed infants at a rate of 1.0-1.5 g per kg body-weight

43
a day, or 1-2% of the diet, i. e., 1.0-1.2 g per 293.08 kJ (70 kcal) a day, stimulates
growth of bifidobacteria in the large intestine within 24-96 h. The lactose: protein
ratio in the diet should be preferentially at least 2.6 : 1 (PETUELY, 1957 a).
The hydrogenation products of lactulose, lactite and lactulite, also have bifido-
genic effects. They may be added, either separately or in combination, to formula
feeds in amounts up to 3.0 g/293.08 kJ (70 kcal), and at a lactose: protein ratio of
between 2.5 and 5 (PETUELY, 1966).
The administration of lactulose to adults has been reported to promote the
growth of intestinal bifidobacteria after these organisms have been drastically
reduced and when the stools have an alkaline pH (HOFFMANN, 1966).
Lactulose is not hydrolyzed by the beta-galactosidase secreted by the mucosa of
the human small intestine, it is poorly absorbed, and reaches the colon where it is uti-
lised by the bacterial flora. Bifidobacteria, L. acidophilusand enterococci readily fer-
ment lactulose to lactic and acetic acids, thereby reducing the colonic pH. Bacteroides
,_....,
::....
~
"
~
Q)
II)

~ 0 ~~rM~TTTTTr~r.M~777TT77T~~r,r.n
E tÖ j~eLll{'Zll(/J/ßj;j!ljllllll{j//J/ß
~
.......
75 ~
~200
~
..,
::r::
<:
~ 100
E
11) ~----------~a.------~--
'll
CL

II) 7
Cll

2 6 8 10 12 14 16 Days
Fig. 4.1 The effect oflactulose on hyperammonaemia and the pH offaeces in liver cirrhosis
(MüTING, 1977)

44
spp. and Proteusspp. fail to ferment lactulose, but E. co/iproduces a little acid. It has
been postulated that bifidobacteria use lactulose preferentially in the colon as a
source of energy (HOFFMANN et al., 1964).
Although formula feeds containing lactulose have been reported to increase the
bifidobacterial population and to reduce the pH ofthe faeces; a lower pH was, how-
ever, attained in breast-fed infants and the intestinal microflora, although similar, was
not typical of the microflora ofbreast-fed infants (MACGILLIVRA Y et al., 1959; MEL-
CHIOR & BRAESTRUP, 1962; KASHKAREVA & BOREIKO, 1963; 1964; HORE6NY, 1964;
NEIMANN eta/., 1965; ÜRÜTTE& HAENEL, 1968; HAENEL eta/., 1970; BRAUN, 1971).
Lactulose alone, or as a supplement to bifidus-milk preparation is used in the
management ofliver cirrhosis, and especially in the accompanying hepatic encephal-
opathy in adults (BIRCHER et a/., 1966; ÜRDNUNG & MüTING, 1973; MüTING 1977).
Fig. 4.1 shows the effect oflactulose on hyperammonaemia and the pH values of
the faeces in livercirrhosis. This is more fully discussed in Chapter 6 (Section 6.4.2.2).

4.3.2 N-acety/glucosamine-containing saccharides (bifidusfactor 1)

N-acetylglucosamine-containing saccharides have been reported to be essential

growth factors for B. bifidum var. pennsylvanicus (GYöRGY et al., 1954 a-f; KuHN et
al., 1953). They are used by this organism for cell-wall synthesis, and are present in
human milk but lacking in cows' milk.
The most important N-acetylglucosamine-containing saccharides in human
milkare: lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I and II, lacto-
N-difucohexaose I and II, and lacto-N-difucodecaose.
N-acetylgalactosamine and N-acetylmannosamine also possess growth-promot-

Table 4.1 Bitidus factor I in milk ofvarious species andin human secretions')

Unit average ml Average mg dry weight In% activity of

humanmilk
Human, colostrum 0.02 1.8 300
Human, milk 0.06 5.5 100
Cow, colostrum 0.15 28
Cow,milk 2.5 250 2
Goat, colostrum 0.8 85
Goat,milk 1.3 130
Ewe, milk 2.3 250
Mare, milk 50
Meconium 3.0
Semen 0.07
Saliva 0.5 3.0 12
Gastric juice 0.5 12
Tears 1.0 6
Amniotic fluid 0.6-1.0 10.0 8
1) The unit of activity of bifidus factor I is the amount
of substance that is equivalent in growth-pro-
moting effect to 0.06 ml of average (bulked) human milk (equivalent to 5-6 mg dry weight) (GYÖRGY
et a/., 1954 a; RosE et al., 1954).
Source: GYöRGY et al., (1954b); TOMARELLI et a/., (1954 a).

45
ing activity for B. bifidum var. pennsylvanicus, but their activity is weaker than that of
N-acetylglucosamine.
Heat treatment of N-acetylglucosamine at 121 o C for periods of 15, 30 and 45
min decreases its bifidus activity in proportion to the heat degree oftreatment (JAo,
1975; JAo et al., 1978).
Milk from various species and human secretions are natural sources of bifidus
factor l (Table 4.1 ).
The activity of the bifidus factor is highest in human colostrum, followed by
human milk, and cows' colostrum. The milk of ruminants (cow, ewe, goat) hasslight
activity. The bifidus growth factor in human milk contains both dialysable and non-
dialysable fractions, 40-60% of the total activity being in the non-dialysable fraction
(GYÖRGY et a/., 1974).
The bifidus factor is present in high concentrations in human secretions, such as
saliva, semen, amniotic fluid, meconium, tears, and blood-group mucoids, but the
mechanism of distribution ofthese factors in the body is unknown.
YosHIOKA (1971) found a direct correlation between intestinal saccharides
derived from human milk and the growth ofbifidobacteria. Large amounts of oligo-
and polysaccharides were detected in the faeces of breast-fed infants, in contrast to
the small amounts found in the faeces ofbottle-fed infants. All bifidobacteria ofDeh-
nert's groups I I II and IV were able to utilise saccharides from faeces, but not all of
groups III and V (see Table 2.4).
N-acetylneuraminic acid (sialic acid, gynaminic acid, lactamin acid) is a natu-
rally occurring form of neuraminic acidinhuman milk, which is bound to the oligo-
saccharides in human milk and to the glycoproteins in body tluids. Non-dialysable
sialic acid-containing oligosaccharides and glycoproteins from human milk and
body fluids have been reported to be ineffective as growth-promoting agents for B.
bijidum var. pennsylvanicus unless they are pretreated with the enzyme neuramini-
dase, after which growth promotion is proportional to their N-acetylglucosamine
content (NICOLAI, 1971; NICOLAI & ZILLIKEN, 1972 & 1974; ÜYÖRGY et a/., 1974). B.
bifidum var. pennsylvanicus synthesizes a neuraminidase specific for the (2--+3)
linkage of sialic acid bound to carbohydrate chains. The enzyme effects a slow fission
of the (2--+6) linkage of sialic acid bound to the oligosaccharides of human milk; e. g.
di-neuraminyl-lacto-N-tetraose (shown below)

D-Gai-3D-GlcNAc-3D-Gal- 4D-Glc
3 6
I I
2 2
NeuN Ac NeuNAc
'
(Glc: glucose; Gal: galactose; GlcNAc: N-acetylglucosamine; NeuNAc: sialic
acid).
Neuraminidase production by B. lactentis (B. infantis) has been induced by
adding sialic acid or its precursor N-acetylmannosamine. Neuraminidase preferen-
tially cleaves low molecular-weight oligosaccharides (NI COLA!, 1976; NI CO LAI & ZIL-
LIKEN, 1976).
, The following derivatives of N-acetylglucosamine are obtained either by synthe-
sis or degradation of carbohydrates, and the most important are the following:

46
(a) 4-0-beta-D-ga/actopyranosyl-N-acety/glucosamine (beta-0-galactoside of N-
acetylglucosamine), or N-acetyl-lactosamine. This is a disaccharide composed of one
molecule each of galactose and N-acetylglucosamine, which is obtained by the incu-
bation of intact cells of B. bifidum var. pennsy/vanicus with Iactose and N-acetylglu-
cosamine. lt can be isolated from pigs' gastric mucin by mild acid hydrolysis. One
unit of activity is defined as 0.075 mg of the pure substance that is equivalent in
growth-promoting effect to 0.06 ml of averagehuman milk (equivalent to 5-6 mg dry
weight) (TOMARELLI et a/., 1954 a; ZILLIKEN et a/., 1954 a, b; ZILLIKEN et a/.,
1955 a,b).
(b) Crystalline N-N'-diacetylchitobioseis a partial breakdown product of chitin. lt is
hydrolyzed into two moles of N-acetylglucosamine by a crude enzyme preparation
from B. bifidumvar. pennsylvanicus. The unit of activity of crystalline N-N'-diacetyl-
chitobiose (added under sterile conditions) is contained in 2.2 mg (ZILLIKEN et a/.,
1955 c).
(c) N-beta-glucosides of N-acetylglucosamine (Alkyl N-acetyl-beta-glucosaminide)
is obtained by reacting N-acetylglucosamine with the corresponding alcohol in the
presence of a cation exchanger. Beta-methyl-N-acetyl-0-glucosaminide is a growth
factor for B. bifidum var. pennsylvanicus. The alpha-isomer is inactive, but when mix-
ture of the inactive alpha-methyl-N-acetyl-0-glucosaminide, and the active beta-
isomer used, the acitivity of the beta-isomer is enhanced as much as five-fold. The
ethyl- and n-propyl-N-acetyl-beta-0-glucosaminides are considerably more active
than the methyl-compound. The units of activity are: beta-methyl-N-acetyl-0-glucos-
aminide, 0.2 mg; ethyl- and n-propyl-N-acetyl-beta-0-glucosaminides, about 0.045
mg; N-acetylglucosamine, 2.5 mg (RosE et a/., 1954; ZILLIKEN et al., 1955 d).
(d) N-acyl derivatives of D-glucosamine. Theseare Novel growth factors such as: N-
caproyl-0-glucosamine; N-benzoyl-0-glucosamine; and N-carboethoxy-0-glucos-
amine. The most active is N-carboethoxy-0-glucosamine; one unit corresponding to
0.19 mcg M (LAMBERT et a/., 1964; LAMBERT & ZILLIKEN, 1965).
The growth-promoting activities of N-acetylglucosamine and its derivatives dif-
fer considerably. N-acetylglucosamine has the least activity and this has been
reported to be due to its rapid conversion to fructose 6-phosphate which is metabo-
lised to acetate and Iactate. B. bifidum var. pennsylvanicus produces considerably
more acid in the presence of N -acetylglucosamine than in the presence of beta-ethyl-
N -acety1-0-glucosaminide, N -caproyl-0-glucosamine, or N -benzoyl-0-glucos-
amine (VEERKAMP, 1969 a,b; YOSHIOKA, 1971 ).
The addition beta-ethyl-N-acety1-0-glucosaminide to a modified cows' milk
preparation (in the rate of 50 mg per 100 g milk) has been reported to promote the
establishment of a predominant bifidobacterial flora in most infants (WALCH,
1956 b; MüLLER & WALCH, 1960). Another report describes the effect of pigs' gastric
mucin upon the growth oflow birth-weight infants and their intestinal flora. The addi-
tion of 4.5 mg of purified pigs' gastric mucin to 100 ml of a modified cows' milk pre-
paration, enhanced the rate ofweight-gain ofthe infants, reduced the faecal pH in 14
out of 17 infants, and increased the counts ofbifidobacteria in the stools. When 5 of
the infants were fed with mucin-containing milk supplemented with 0.5 g lactulose
per 100 ml, there was a further reduction in faecal pH and an increased count of bi-
fidobacteria (INOUE & NAGA Y AMA, 1970).

47
4.3.3 Peptides derivedfrom the enzymatic hydrolysis ofproteins
(bifidus factor 2)

Some strains of B. bifidum var. pennsylvanicus require the supplementary growth fac-
tors present in human milk, cows' milk, pancreatic extract, insulin and casein hydro-
lysates prepared enzymatic or mild acid hydrolysis. These supplementary factors,
described by GYÖRGY and RosE ( 1955 a), are actually bifidus factor 2 (BFz). This fac-
tor has been reported tobe essential for the growth of B. bifidum (TISSIER), and it is
active bothin vitro andin vivo (RAYNAUD, 1959).
Natural sources of bifidus factor 2 are scarce and the best sources are enzyme
hydrolysates ofproteins. Table 4.2 shows titres ofbifidus factor 2 in various products.

Table 4.2 Titres of bifidus factor 2 (BF2) in various products

Substance Titre(A)

Humanmilk 1-20.
Human milk, freeze-dried 20-100
Pepsin, commercial raw 1250
Trypsin, commercial raw 85
Papain,raw I 000
Extract of Aspergillus oryzae 500-1 ooo•
Liver extract 20-660
Liverdigested by papain 2500
Mucin 100
Casein hydrolyzed enzymatically 50-15000

(A) Units per gram. This unit of activity of BF2 is the smallest amount of the factor which, under
defined conditions of growth gives a total acidity equivalent to 1 ml of NI I 0 acid.
a Units per ml.

Source: RAYNAUD(1959).

Bifidus factor 2 obtained by the action of enzymes, such as pepsin, trypsin, and
papain on proteins (particularly casein) has the characteristics of peptides (RosE &
GvöRGY, 1963; RA YNAUD & BIZZINI, 1971 ). This factor is also present in yeast extract
or autolysate.
The addition to cows' milk of 20% pepsin-digested milk has been reported to
influence significantly the in vitro growth and acid production of B. bifidum (ZIAJKA
et al., 197 4). The administration of 10 000 units I day of BFz is needed to promote in
vivo moderate growth of B. bifidum, and 20 000 are needed units I day for strong
growth (Table 4.2). The unit of activity of BFz is the smallest amount of the factor
which under defined conditions of growth gives a total acidity equivalent to 1 ml of
NI 10 acid (RA YNAUD, 1959).
In feeding trials with 4 infants receiving a modified cows' milk preparation con-
taining 3 to 5 g of enzymatically-hydrolysed casein (papain in conjunction with gas-
tric and intestinal mucosa) a marked effect was observed. The numbers ofbifidobac-
teria increased to 75% ofthe total faecal flora {LEVESQUE et al., 1959). Five g of this
casein hydrolysate contains 10 000-20 000 units ofbifidus factor 2 (BFz).

48
4.3.4 Other growth factors

4.3.4.1 Glycoproteins and glycopeptides

Glycopeptides from human colostrum and milk support the growth-promotion of B.

bifidum var. pennsylvanicus and B. bifidum (TISSIER). HIRANO and co-workers ( 1968)
isolated from human colostrum 5 fractions of glycopeptides with growth-promoting
activity, when present at a concentration of 2 mg/10 ml in a medium containing
cows' skimmed milk. Subsequently, NICHOLS and co-workers (1974) isolated 7 frac-
tions ofM-1 glycoprotein from human colostrum-whey and 2 fractions from bovine
colostrum. All fractions from human colostrum-whey had a marked growth-promot-
ing activity for B. bifidum var. pennsylvanicus, which was related to the carbohydrate
content ofthe fractions.
Glycopolypeptide fractions with B. bifidum var. pennsylvanicus growth-promot-
ing properties have been isolated from human milk-whey and from tryptic and
chymotryptic digests ofhuman milk casein. Both these fractions had similar propert-
ies and contained 60-70% carbohydrate consisting of galactose, glucosamine,
galactosamine, sialic acid, and fucose. It has been postulated that casein provides
slow-releasing growth-promoting factors for B. bifidum during its digestion in the
gastrointestinal tract of breast-fed infants (BEZKOROVAINY & NICHOLS, 1976; BEZ-
KOROVAINY et a/., 1979).

4.3.4.2 Pantethine

Some strains of bifidobacteria including B. bifidum var. pennsylvanicus require

pantethine for growth because they cannot use pantothenic acid. Peptoneis a good
source ofthe pantethine, 100 mg ofpeptone supplying 1 mcg ofpantethine (GYöRGY
& ROSE, 1955 a; ÜYLLENBERG & NIEMELÄ, 1959; RAYNAUD, 1959). The addition of
30 mg pantethine a day for 3 weeks to the diet of infants (2-4 months old) has been
reported to increase the population of bifidobacteria in the faeces of 7 out of 14
infants; in 2 infants there was no significant change, and 5 infants could not be eva1u-
ated (ÜTA et al., 1971).

4.3.4.3 Growth-promoting factors in plant extracts

The coenzyme-A precursor, pantetheine phosphate, has been identified in carrot-root

extracts as a growth-promoting factor for bifidobacteria (KANAO et al., 1965). In
addition to coenzyme A and its precursors, YosHIOKA and co-workers ( 1968 c) found
new growth factors in carrot extract. These factors are water-soluble, dialyzable and
heat-resistant.
Carrot extract is used in treatment ofinfant diarrhoea (KUROMIY A, 1960; MOMM-
SEN, 1968).
Potato extract has been reported to have a growth-promoting activity in vitrofor
bifidobacteria cultured in cows' milk (Morinaga Milk Industries Co., 1973).
Maize extract has been found to promote the growth and acid production ofbi-
fidobacteria in cows' milk. The rate of multiplication of bifidobacteria in sterile milk
containing 0.5-1.0% of maize extract, and incubated at 37° C for 16-20 h., was twice

49
to three tim es as much asthat in milk without maize extract. Moreover the pH values
of milk containing 0.25-l.O% maize-extract cultured at 37° C for 16 h were 4.5-4.6
compared with pH 5.4 for the control (SEMENIKHINA & SUNDUKOVA, 1980).

4.3.4.4 Muramidase

The enzyme muramidase (Iysozyme) occurs in human milk at a Ievel of 40 mg /I 00

ml, but in cows' milk it averages 13 mcg/100 ml (RENNER, 1974). Consequently
infants receiving human milk have a high er faecal muramidase activity than those on
modified cows' milk preparations.
The fortification of infantmilk formulae with 40 to I 00 mg of hen-egg murami-
dase I day for I 0 days increased faecal muramidase in 2 out of 6 infants (KoNDO,
1967 a-c). Muramidase activity was also detected when mixed diets containing lactu-
lose were given (BRAUN, 1965 a,b). The addition of muramidase and lactulose
increased faecal muramidase and decreased pH.
There are two methods of adding hen-egg muramidase: (a) direct addition of
muramidase to infant formulae at a rate of 130 mg/100 g; and (b) incubation of a
modified cows' milk-preparation with muramidase (50 mg/1 at 40° C for 2-3 h or
100 mg/1 at 30° C for 3 h) followed by heat inactivation ofthe enzyme. During incu-
bation the polysaccharides of glycoproteins are hydrolysed and their hydrolysis pro-
ducts exert a growth-promoting effect on B. bifidum (SuzuKI et al., 1966 a,b; KiTA-
BARA eta/., 1966; YAMADA eta/., 1967 a,b; NISHIHARA eta/., 1967).
In bottle-fed infants, the supplementation ofmodified cows' milk-preparations
with muramidase, may increase the population ofbifidobacteria in the faeces.
However, it has been shown recently that the direct addition of muramidase to
sterile cows' milk at a rate of 100 mg/l did not stimulate the growth of B. bifidum in
milk, whereas muramidase-treated milk (100 mg muramidase/1, incubated at 37° C
for 2 h and subsequently heated to 80° C for 15 min to inactivate the muramidase) sti-
mulated growth of B. bijidum (KiSZA & PANFIL-KUNCEWICZ, 1977).

4.4 The intrinsic properties of human milk

Breast-feeding influences the intestinal environment of the newbom in a direction

that is favourable to the development ofbifidobacteria and the production of an acid
pH. In addition to specific growth-promoting factors for bifidobacteria, the constitu-
ents and properties ofhuman milk play an important role. The most important factors
are: high Iactose, low protein particularly casein, and low calcium phosphate con-
tents, tagether with a low buffering capacity and a low residue (BULLEN et al., 1973;
BULLEN et a/., 1976; BULLEN & TEARLE, 1976).
The composition and properties ofhuman milk indirectly influence the nature of
the end-products ofmicrobial metabolism in the large intestine.

50
Chapter 5 : The human gastrointestinal flora and
bifidobacteria

5.1 lntroduction

Normal gut flora consists of micro-organisms which live in a stable relationship with
the host. Many factors control the size and composition of microbial populations in
different regions ofthe gastrointestinal tract. The stomach and small intestine, parti-
cularly its upper part, contain few micro-organisms, while in the large intestine mi-
crobial populations may be as high as 1010-10 11 I g.
The peculiar distribution of microbial florain the gastrointestinal tract is due to a
diminution in constraining factors throughout the alimentary tract (e. g. gastric acid-
ity and small intestinal motility).
The gut micro-organisms have a high metabolic activity. They breakdown some
dietary and endogenaus compounds, including amino acids, undigested carbohy-
drates, glucosides, biliary compounds and others, and they produce certain vitamins.
Some products ofbacterial metabolism may be a potential hazard to the health of the
host.
The gut bacteria have an effect on the intestinal structure (e. g. the intestinal vil-
lae, a connective tissue, and the reticulo-endothelial elements) ofthe host and on the
development of resistance factors (e. g. immune systems and microbial interference
with resistance to intestinal infections).

5.2 Some characteristics of the gut micro-organisms

Many bacterial species isolated from the human gut are indigenous gut inhabitants.
They include diverse bacterial genera comprising Gram-negative and Gram-positive,
anaerobic and facultatively anaerobic rods, and cocci. Some bacterial groups pre-
dominate in the intestine, e. g. bacteroides and bifidobacteria, while others occur in
only small numbers in the normal gut flora, e. g. enterococci and enterobacteria, or
are a minority, e. g. staphylococci, clostridia and aerobic sporeformers.
Some of the gut organisms may have important metabolic effects and interac-
tions with the host. The details of their taxonomic characteristics are given in Bergey 's
Manual of Determinative Bacteriology (BUCHANAN & GIBBONS, 1974).

Bacteria

5.2.1 Gram-positive non-sporing rods

5.2.1.1 Genus Bifidobacterium (Family Actinomycetaceae)

This genus of catalase-negative, anaerobic rods contains 11 species. All ofthem fer-
ment carbohydrates mainly to acetic and lactic acids. Some species use ammonia as
the sole source of nitrogen, while others require organic nitrogen.

51
 Bifidobacteria are the predominant organisms in the faecal flora of breast-fed
infants, and are major component ofthe faecal flora in adults. Numbers of 109-l 011
pergram faeces are common. They may occur insmall numbers in the small intestine
and often in the mouth and vagina.
B. infantis is the dominant organism in breast-fed infants, while B. adolescentis
and B. longum dominate in adults. B. bifidum and B. breve occur in both infants and
adults. Other species including B. thermophilum, B. pseudolongum and B. suis occur
in animals and B. asteroides, B. indicum and B. coryneforme occur in bees.

5.2.1.2 Genus Eubacterium (Familiy Propionibacteriaceae)

Anaerobic rods which are usually catalase-negative. This genusalso includes bacteria
originally placed in the genera:
Catenabacterium, Ramibacterium, Cillobacterium, and Butyribacterium. Sac-
charoclastic or non-saccharoclastic strains produce mixtures of organic acids from
carbohydrates or peptones. Ammonia is produced. Some species may occur in soft-
tissue infections.
Eubacterium aerofaciens, and frequently E. rectale, may be the major compo-
nents ofthe faecal flora in adults (GOSSLING & SLACK, 1974; MOORE & HOLDEMAN,
197 4). E. aerofaciens has also been found in chickens, pigs, and dogs (CLARKE, 1977).

5.2.1.3 Genus Lactobacillus (Family Lactobacillaceae)

These are facultatively anaerobic or anaerobic rods which occur in chains, and are
catalase-negative. They have complex nutritional requirements and are highly sac-
charoclastic. They occur in many carbohydrate-containing environments, and lactic
acid is the major product of fermentation.
Some members ofthe genus occur in the alimentary tract ofvarious animals. L.
acidophilus, L. salivarius, and L.fermentum are indigenous to the human intestinal
tract; also L. casei, L. plantarum and L. cellobiosus have been frequently isolated.
Some of these lactobacilli occur in the human mouth and vagina. The approximate
numbers of lactobacilli in the intestine and faeces are: N- 106 I g in the uppersmall
intestine (N = less than 10 per g); N- 108 I g in the lower small intestine; N- I 09 I g
in the large intestine; and 103·6-1 09·3 I g in the faeces (DRASAR, 1974; DRASAR & HILL,
1974; SANDINE, 1979).
Lactobacilli have been reported to colonise the epithelium ofthe alimentary tract
of mice and rats (SAVAGE, 1977); pigs (Dusos et al., 1965); chickens (COATES &
FuLLER, 1977); and probably the intestinal epithelium of humans (NELSON & MATA,
1970; SA VAGE, 1977; CLARKE, 1977).

5.2.1.4 Genus Propionibacterium (Family Propionibacteriaceae)

Theseare anaerobicto aerotolerant pleomorphic rods. P. acnesis an inhabitant ofthe

normal skin and intestinal tract of most animals including man, but it occurs in small
numbers. Moststrains produce propionate from Iactate.

52
5.2.2. Gram-negative anaerobic non-sporing rods

5.2.2.1 Family Bacteroidaceae

- Genus Bacteroides. These metabolize either peptones or carbohydrates which are

fermented to organic acids, alcohols, and hydrogen.
Bacteroides occur in the mouth and gut of man and many animals, and they are
the dominant organisms in the faecal flora of human adults, although in breast-fed
infants insignificant or absent.
B.fragilis is, the most numerous species, digests xylan (BRY ANT, 1974), metabo-
lizes hemicelluloses, pectin, and intact starch to succinate and hydrogen. Ammonia
can be used as the sole source of nitrogen, and this organism depends upon other bac-
teria that produce it from urea or amino acids. Ureolytic biotypes of B.fragilis prob-
ably exist in very small numbers (VAREL & BRYANT, 1974; VAREL et al., 1974).
B. oralis commonly occurs in the mouth (DRASAR & HILL, 1974). Bacteroides
spp. may be isolated from soft-tissue infections (HOFFMANN, 1966; SAVAGE, 1977).
- Genus Leptotrichia. May occur in the human mouth (CLARKE, 1977).
- Genus Fusobacterium. Commonly occur in the mouth and faecal flora of
human adults (DRASAR & HILL, 197 4), but are considered tobe only a minor compo-
nent ofthe intestinal flora (BRYANT, 1974).

5.2.3 Gram-positive cocci

5.2.3.1 Family Peptococcaceae

- Genus Peptostreptococcus. Anaerobic cocci, catalase-negative, which occur in

pairs, short or long chains. Ammonia is produced, and carbohydrates are fermented.
They occur in large numbers in the human large intestine and faeces, and may
also occur in the mouth. P. productus is the most numerous species which ferments
carbohydrates, including Iactose, cellobiose, raffinose, pectin, and intact starch with
the production of organic acids. This organism is the most numerous ureolytic species
so far isolated from human faeces (VAREL et al., 1974). Ammonia can be used as the
sole source of nitrogen.
- Genus Ruminococcus. They are anaerobic cocci, catalase-negative which
occur as single cells, pairs, or chains and usually digest cellulose. Carbohydrates are
fermented to acetic and formic acids, hydrogen and ethanol, or succinic acid. Ammo-
nia is used as the main source of nitrogen, and these organisms depend upon other
bacteria that produce it from urea or amino acids (HERBECK & BRYANT, 1974).
R. albus and R.flavefaciens are the dominant cellulose-decomposing bacteria in
the gut of herbivores, particularly ruminants (HUNGATE, 1966; PRINS, 1977). R.
bromii occurs in large numbers in the faecal flora of man and pigs (MOORE et al.,
1972; HERBECK & BRYANT, 1974).
- Genus Sarcina. Anaerobic cocci, catalase-negative, occuring in packets. S.
ventriculiis a common species occurring in the faeces ofvegetarian humans (CRow-
THER, 1971 ).
- Genus Peptococcus. Anaerobic cocci are not considered of great importance.

53
They can use protein decomposition products as energy source, but carbohydrates
not required. They may occur in the mouth and faeces of man (DRASAR & HILL,
1974).

5.2.3.2 Genus Streptococcus(Family Streptococcaceae)

Facultatively anaerobic cocci, catalase-negative, occurring in pairs or chains. They

commonly occur in the mouth and intestinal tract. S. salivarius, S. sanguis and S.
mitis occur mainly in the mouth, whereas the enterococci: S.faecalis, and S.faecium
commonly occur in the lower intestine and faeces. S. bovisnormally occurs in the ali-
mentary tract of ruminants, and it may be found occasionally in large numbers in
human faeces. Streptococci also occur in the small intestine of man.

5.2.4 Gram-negative cocci

5.2.4.1 Family Veillonel/aceae

- Genus Veil/onel/a. These anaerobic cocci occur in pairs or chains, and are usually
catalase-negative. They occur in small numbers in the mouth and intestinal tract of
man, also in animals including ruminants, pigs, and rodents (CLARKE, 1977).
These organisms ferment Iactate to acetate, propionate, carbon dioxide and
hydrogen, but they do not ferment carbohydrates. V. parvula and V. alca/escens are
common species.
- Acidaminococcus fermentans (genus Acidaminococcus) and Megasphaera els-
denii (genus Megasphaera) have been isolated from the intestinal tract and faeces of
man, and from the faeces ofpigs (SUGIHARA et al., 1974).

5.2.4.2 Family Neisseriaceae

- Genus Neisseria. These facultatively anaerobic cocci occur as single cells or pairs,
and may occur insmall numbers in the human mouth, intestine, and faeces (DRASAR
& HILL, 1974).

5.2.5 Gram-negative facu/tatively anaerobic rods

5.2.5.1 Family Enterobacteriaceae

It contains 12 genera and many species are pathogenic or potential pathogenic.

Escherichia co/i(genus Escherichia) occurs in the gut and faeces ofwarmblooded
animals and man. It is a minor component ofthe faecal flora ( 105-l 0 8 cells I g faeces)
of healthy humans, but it may also be found in the lower small intestine, and some-
times in saliva (DRASAR & HILL, 1974). The commensal E. coli are residents of the
large intestine, which may harbour several strains, albeit the majority ofthe dominant
coliform population is of a singletype (MASON & RICHARDSON, 1981 ). Volatile fatty
acids, especially butyric acid, are inhibitory to these organisms. Some strains of E. coli

54
produce the antibiotic substances colicins, which are in vivo probably inactivated by
proteases, while others (about 12% of intestinal strains of enterobacteria) produce the
broad-spectrum antibiotics microcins (BAQUERO et al., 1978).
Unlike the commensal E. coli, the pathogenic strains are able to produce entero-
toxin, to attach to the wall of the small intestine or to invade epithelial cells (MASON &
RICHARDSON, 1981 ).
Klebsiella spp., and Proteus spp., may also occur in very small numbers in the
human large intestine. K. pneumoniae from the gut of man, pig, and guinea pig may
fix small amounts of gaseous nitrogen (BERGERSEN & HtPSLEY, 1970).
E. coli, Klebsiella spp., and Proteus spp., may increase significantly during dis-
turbances of the human gastrointestinal flora.

5.2.6 Spore-forming rods

5.2.6.1 Genus Clostridium (Family Bacillaceae)

These Gram-positive, anaerobic bacteria occur in the gut and faeces of animals and
man. These organisms are a minor component of the faecal flora of healthy human
adults.
Cl. perfringens, Cl. sporogenes and Cl. bifermentans are common species (DRA-
SAR & HtLL, 1974). Cl. perfringens (Cl. welchii) is the most numerous clostridium
occurring in the human gut, and type A strains usually occur in numbers of 104 I g
wet-weight offaeces (COLLEE, 1974).
Clostridia may increase when the human gut flora is disturbed (HOFFMANN,
1966; HAENEL, 1970). Increased numbers of Cl. perfringens may have a possible role
in rheumatoid arthritis (MANSSON & ÜLHAGEN, 1969) and the colonic cancer (ALP-
ERN & COHN, 1969).
Cl. perfringens type-A strains may be a cause offood poisoning, when foods con-
taining large numbers are consumed (e. g., meat products). A high-ehallenge dose of
ingested vegetative cells may be ofthe order of 108-109 organisms, and the number of
clostridia in the faeces may be in the range 105-108 I g (COLLEE, 1974).

5.2.6.2 Genus Bacillus(Family Bacillaceae)

These Gram-positive, aerobic, or facu1tative1y anaerobic bacteria occur in soi1,

sewage, andin the gut and faeces of animals. They may occur in very small numbers in
the faecal flora of man. Their vegetative forms are sensitive to intestinal Iysozyme,
whereas their spores can survive the gastrointestinal transit (DRASAR & HtLL, 1974).
Strains of B. cereus may be a cause of food poisoning. The consumption of
foods, particularly starch-containing products (e. g., rice, pudding, legumes) contam-
inated with large numbers of B. cereus often causes food poisoning (PANTALEON,
1965; DRASAR & HILL, 197 4).

55
5.2. 7 Other bacterial groups

- Genus Staphylococcus (Family Micrococcaceae). These Gram-positive, catalase-

positive and facultatively anaerobic cocci occur in clusters. They normally occur in
very small numbers in the human mouth and intestine. S. aureusis a common, poten-
tially pathogenic species. Under abnormal conditions staphylococci in the gut flora
may reach larger numbers in the lower small intestine than in other regions ofthe gas-
trointestinal tract (HAENEL, 1970).
S. aureus may cause a severe enterocolitis after broad-spectrum antibiotic ther-
apy (HAWLEY et al., 1959; DRASAR & HILL, 1974; HAFfER, 1978). This organism is
sometimes implicated in food poisoning. The consumption of food containing
enterotoxin produced by S. aureusoften causes acute vomiting and diarrhoea.
- Genus Pseudomonas (Family Pseudomonadaceae). These Gram-negative,
aerobic non-sporing rods, may occur in very small numbers in the human intestinal
tract. P. aeruginosa, possibly of intestinal origin, has been implicated in urinary tract
infections (BUCHANAN & GIBBONS, 1974).
- Genus Corynebacterium. These facultatively anaerobic bacteria producing
catalase, may occur in many habitats. C. pseudodiphtheriticum has been isolated from
the human nasopharyngeal mucosa, and C. xerosis from the human skin and mucous
membranes. Both strains are non-pathogenic (BuCHANAN & GIBBONS, 1974).

Other micro-organisms

5.2.8 Yeasts

These usually occur insmall numbers in the alimentary tract of animals, such as rumi-
nants, horses, and pigs (CLARKE, 1977); also in the saliva and intestine of man.
Candida albicans is often present in the human gastrointestinal tract, whereas
Candida tropicalis and Torufopsis glabra occur less frequently (HAFTER, 1978).
Yeasts which are regarded to be potentially pathogenic, such as C. albicans, C.
tropicalis and C. paropsilosis, have been found to predominate in malnutrition rather
than those, such as C. krusei, which are thought tobe saprophytic (GRACEY, 1981 ).
Yeasts may be found in 29-60% of salivary samp1es (HANDLEMAN & MILLS,
1965), in 25% of duodenal samples (in numbers less than 103 I ml) (BERNHARDT &
KNoKE, 1976), andin 50% offaecal samples (Paris et al., 1967). Yeasts may increase
during disturbances of the human gut flora following gastric Operations, after anti-
biotic therapy, and particularly in some intestinal diseases; for example: moniliasis
due to C. albicans "overgrowth" of the intestine after antibiotic therapy.

5.3 Normal gastrointestinal flora in man

5.3.1 The microbialjlora in different age groups

Many factors control the size and composition of microbial populations in different
regions of the gastrointestinal tract. The starnach and small intestine, particularly the

56
 Germ Counts(log)
1 2 3 4 5 6
f Feces
8 9 10
c::
Fecal Microflora in 265 School ....
-S2
Children (6-16 Years) 432 Sampies cp 11:1
AnG 100 ....
::..
·-
a
.._

c:p Bi 100 ......

0

c::p Ba 96 G-
c::Q)
:::,
t::J-
AG 100 ~
u::
Co 99

ALS 100

Pro 61

Zi 73

Ma 43 Klebs-
Enterob.
Jno 41

c:::t=:J Sta 35

c:::P Spo 25

~ Yea 38

I Clo 48

Fig. 5.1 Mean values (horizontallines) and standard deviations (bars) of different groups offaecal
microflora, cultivated from 432 stool samples of265 school children, 6-16 years old. AnG: total ana-
erobes; Bi: Bifidus group, Gram-positive anaerobes; Ba: Bacteroides group, Gram-negative anaer-
obes; AG: total aerobes; Co: coliforms; ALS: aerobic lactobacilli and streptococci; Zi, Ma, Ino:
Klebsiella-Enterobacter group, multiplying on citrate, malonate and inositol agar, respectively; Pro:
Proteus group; Sta: staphylococci; Spo: aerobic sporeformers; Yea: yeasts; Clo: clostridia
(HAENEL, 1970)

57
 upper part, harbour few micro-organisms, whereas the large intestine has a dense
microbial population.
The compositions ofthe microbial flora in the large intestine and faeces of differ-
ent age groups may differ. This is particularly true for breast-fed infants and adults.
However, anaerobic bacteria comprise more than 90% of the faecal flora in all age
groups.
1he faecal flora of breast-fed infants is relatively simple.
Bifidobacteria are the dominant organisms, accounting for about 99% (range 85% to
more than 99 %) of the cultivable flora. Strains of B. infantis usually dominate.
Enterococci, coliforms, and lactobacilli comprise about I % (range 1-15 %) ofthe fae-
cal flora (HOFFMANN, 1966; HAENEL, 1970), while bacteroides, clostridia and other
organisms may be absent or insignificant. The stools have an acid pH (5.0-5.5). The
flora ofthelarge intestine in breast-fed infants is relatively unstable; small influences
may upset the normal microbial balance (HAENEL, 1970).
Thefaecaljlora ofweanlingsrepresents a transient phase to a more stable adult
flora. Various other organisms, especially bacteroides, peptostreptococci, eubacteria,
fusobacteria, appear in increasing numbers in the faeces; bifidobacteria gradually
decrease and the pH ofthe stools increases.
Thefaecaljlora ofadolescents and adultsis more complex than that ofbreast-
fed infants. Bifidobacteria remain a major component of the cultivable flora (Fig.
5.1), but they are usually outnumbered by bacteroides (including eubacteria, pepto-
streptococci and ruminococci) although they may reach approximately equal propor-
tions.
Other organisms, including facultative anaerobes, form a minor component of
the faecal flora. In adults, the flora of the large intestine is more stable than that of
breast-fed infants. Variations in the flora, particularly the minor components, may be
present in different individuals. Drastic influences such as, for example: disorders of
gastric function; or of intestinal motility, may disturb the normal microbial balance.
Diminished gastric acidity in old people usually Ieads to a decrease ofbifidobacteria
and an increase in enterobacteria, enterococci, and clostridia (ORLA-JENSEN et al.,
1945; ÜUTHOF, 1957; HAENEL, 1963; HOFFMANN, 1966).
The stools of adults have a low Eh, a neutral or slightly alkaHne pH (6.4-7.0 or
above), a typical odour and colour, and they contain relatively large amounts of
ammonia, amines, and degraded bile acids.

5.3.2 The microbialjlora of different regions in the gastrointestinal tract

The saliva contains diverse microbial populations consisting principally of strepto-

cocci, veillonella, lactobacilli, bacteroides, bifidobacteria, fusobacteria, staphylo-
cocci, corynebacteria (DRASAR & HILL, 1974). The range of bacterial counts is
104-109 per ml of contents (HAFTER, 1978).
The saliva may be important source of micro-organisms entering the stomach.
Fig. 5.2 shows a schematic representation ofthe human gastrointestinal tract.
The stomachisapart of the alimentary canal in which food is stored immediately
after swallowing and is partially digested by the gastric juice, which contains
hydrochloric acid, the enzyme pepsin, mucin, andin infants the enzyme rennin.

58
 Oesophagus

Cardia

-Jejunum
tte-JIH--+--- Descending colon
"Y::::::\7:~/,J-/M-t--- Ileum
Caecum ---t---\-l.-...
'.l-..tf51J'--I'----t-- Sigmoid colon
...__~t::::-"...,::;.7'"----t--- Reet um
The vermiform

Fig. 5.2 A schematic representation of the human gastrointestinal tract

Legend. - The stomach is continuous at the cardiac end with the oesophagus, at the pyloric end with
the duodenum. The sma/1 intestine consists of the duodenum, the jejunum and the ileum. The /arge
intestineconsists ofthe caecum (separated from the small intestine by the ileo-colic sphincter), the
colon (ascending colon, transverse colon, descending colon and sigmoid colon) and the rectum; the
anus is the outlet ofthe alimentary canal

The stomach of normal fasting individuals is either sterile or contains less than
I 03 organisms per ml of contents. Organisms swallowed from the mouth (usually dur-
ing a meal) are either killed by the free hydrochloric acid in gastric juice, or pass
quickly into the small intestine where peristalsis is of primary importance in controll-
ing the distribution of micro-organisms. The germicidal effect of the gastric juice is
mainly attributable to the pH ofthe hydrochloric acid (Fig. 5.3). No germicidal effect
has been observed at a pH greater than 4.0 (GIANELLA et al., 1972).
The pH ofthe normal stomach is less than 3 .0, while in the achlorhydric stomach,
associated with pernicious anaemia or the effect of aging, it is greater than 6.0.
The germicidal effect of gastric juice may also be influenced by the following fac-
tors: a) the number ofmicro-organisms ingested; b) the rate of gastric emptying; c)
the physical protection ofmicro-organisms by ingested food; d) buffering ofthe sto-
mach contents (GIANELLA et al., 1972). For example, a slow rate of gastric emptying

59
 !(J)

9~------~------~--------r--------
PERNIC/OUS ANAEMIA

NORMALS ----9,
\
\
\
\
\
\
\
\
\
\
\
\
\
\
'o
0 10 20 30 40 50 60
Minutes
Fig. 5.3 lntragastric survival of Serratia marcescens in three normal subjects and three patients
with pernicious anaemia. Ten ml of a 3% PEG (polyethylene glycol solution) suspension of I x I 09
organisms/ml was instilled as a bolus. Results areexpressedas the number of surviving bacteria per
ml of gastric contents. In patients with pernicious anaemia the pH never feil below 6.8; in normal sub-
jects, pH was 3.0 or less
(GIANELLA et. a/., 1972)

or ingestion of small numbers of micro-organisms, favour the germicidal effect of

normal gastric juice. Few micro-organisms, however, can talerate the acidic condi-
tions of the stomach.
The small intestine functions in the digestion and absorption of food. It extends
from the starnach to the !arge intestine, and consists ofthe duodenum (with the open-
ings of the pancreatic duct or ducts and the bile duct), the jejunum, and the ileum (Fig.
5.2). The small intestine of normal fasting individuals has in the upper section only a
sparse microbial population, but the lower part has a richer and morepermanent pop-
ulation. The uppersmall intestine may contain up to I 04 organisms per ml of contents
(DRASAR & HILL, 1974). Organisms include streptococci, lactobacilli, yeasts, and
sometimes small numbers of bifidobacteria and bacteroides (HAWKSWORTH et a/.,
1971 ; HAFTER, 1978).
The 1ower small intestine harbours richer microbial populations consisting of
streptococci, 1actobacilli, bifidobacteria, bacteroides, enterobacteria, and yeasts. The

60
 c:.
c:
0
Gasfraintestinal microecology of an infant with eubiotic re/ations
"
~
~
.!!!
.5;
...
0

~ 10
" ~
_..:::::::::s::::=::~ Bacleroides- group
Bitidus- group

. .,
8
/
,~o
//,~--::
, .... . .o----
__
..o Aerobic Lactobacilli
anS Streptococci
," ," -- .... Coliforms
6
,_..". ... _,
,0/

o....... ,o"" Staphylococci

......... 'o ____ -o............... (mostly Staph.aureus)
'
....0

Fig. 5.4 Normal, eubiotic gastrointestinal microflora of an infant, aged three months. Death
occurred because of toxic dyspepsia and endocardial fibrosis. Gastrointestinal contents were
examined 7.5 hours post mortem
(HAENEL, 197la)

distal ileum may contain 105-107 organisms per ml of contents (GORBACH et al.,
1967; DRASAR et al., 1969). The distribution of microbial flora in the lower ileum is
similarto that ofthelarge intestine but the counts are lower (Fig. 5.4).
The small intestine oftropical residents (e.g. India and Guatemala) has richer
microbial populations than that of residents in W. Europe and N. America. This is
probably influenced by the nutritional status oftropical residents and by their greater
exposure to microbial infections (GoRBACH et al., 1970; DRASAR, 1974; GRACEY,
1981).
The distribution of the bacterial flora in the small intestine is influenced by the
intestinal motility in addition to the effect of gastric acidity. The movement of the
intestinal contents (forward peristalsis) is much faster in the jejunum and upper ileum
than in the distal and terminal ileum, and accordingly the lower intestine contains a
richer bacterial population than its upper part. Disorders affecting intestinal motility
usually Ieads to bacterial "overgrowth" of the small intestine. Other factors, which
may also influence, to certain extent the distribution of intestinal flora, are the type of
diet, and the rate of gastric emptying (DRASAR, 1974).
Intestinal immunoglobulins, particularly lgA, Iysozyme, and bile salts may
selectively affect the intestinal bacteria (DRASAR & HILL, 197 4; DRASAR, 197 4).
1he /arge intestine extends from the ileum to the anus, and consists of the cae-
cum, the colon, and the rectum (Fig. 5.2). Food residues take 18-68 hours to pass
through it. The large intestine is the major reservoir of micro-organisms in the body.

61
The microbial population of the large intestine is qualitatively similar to that of the
faeces, upon which most investigations have been conducted for practical reasons.
App~oximately 10--30% ofthe faecal mass consists ofbacteria, and the number of cul-
tivable organisms range from 1010 to 10u pergram wet-weight (LucKEY, 1965;
ZINSSER, 1976; HAFfER, 1978).
Anaerobic bacteria are the major components ofthe faecal flora and comprise
more than 90% of all the organisms. In adults, these include bacteroides and bifido-
bacteria, followed by eubacteria, peptostreptococci, and ruminococci. The most
numerous bacterial species include: Bifidobacterium adolescentis and Bifidobacte-
rium /ongum (MITSUOKA & KANEUCHI, 1977; MCBEE, 1977), Bacteroides fragi/is
(BRYANT, 1974; MOORE& HOLDEMAN, 1974), Peptostreptococcusproductus(BRYANT,
1974; ÜOSSLING & SLACK, 1974), Eubacterium aerofaciens (GOSSLING & SLACK,
1974; MOORE& HOLDEMAN, 1974), and Ruminococcusbromii(BRYANT, 1974; ÜOSS-
LING & SLACK, 1974).
The minor components ofthe faecal flora include mainly facultative anaerobes,
such as Iactobacilli, enterococci, and coliforms. Theserange from less than 1% to 5%
of the cultivable flora (HAENEL, I 970).
Many other organisms occur insmall numbers, principally: clostridia, staphylo-
cocci, aerobic sporeformeres, yeasts and proteus.
Interactions bacteria in the large intestine may affect the stability of the bacterial
populations.
The transient occurrence of viruses in healthy individuals, particularly early in
life, has been reported (REEo & TYRRELL, 1974). These include enteroviruses (echovi-
ruses) found in the faeces of healthy infants and children. Transient asymptomatic
infections may be caused by certain serotypes, which are subsequently eliminated in
the faeces within a few weeks. The human organism acquires immunity to most of
these serotypes, which are not considered tobe inhabitants of the normal gut.

5.3.3 Indigenous (autochthonous) and a/lochthonous micro-organisms ofthe

gut

The gut micro-organisms colonise available habitats (physica1 spaces) in the gastroin-
testinal tract. They receive shelter and nutriments from the host, which does not elimi-
nate them by immunological activity. Each species occupies a niche in a particular
habitat. Such organisms are called indigenous (autochthonous) micro-organisms.
According to ALEXANDER ( 1971) and SA VAGE ( 1977) they are always present in nor-
mal adults, they can grow anaerobically, colonise particular regions of the tract, colo-
nise these habitats in succession durlog infancy, maintain stable population Ievels in
normal adults, and they may adhere to the mucosal epithelium of the tract.
On the other band, transient organisms derived from food and water, or another
habitat in the intestinal tract are called allochthonous micro-organisms, and normally
are in transit through the gut. Nevertheless, allochthonous micro-organisms may col-
onise vacated habitats in a disturbed intestinal ecosystem (SA vAGE, 1977). Probably
many ofthese situations can be extrapolated to man.
Experiments on gnotobiotes (animals which are either germ-free or infected with
one or more known species of micro-organisms) have shown that the total number of

62
bacteria in the large intestine is relatively constant irrespective of whether one, two, or
a mixture of bacteria are present (LuCKEY, 1965). There is a biodynamic balance
between the reproduction and death of bacteria, and overgrowth of bacteria in the
large intestine is prevented by transit during intestinal peristalsis, followed by their
removal in the faeces.

5.4 Changes in the human gastrointestinal flora

5.4.1 Abnormal gutjlora

An abnormal distribution of the bacterial flora in the gastrointestinal tract leads to

bacterial "overgrowth" ofthe stomach and small intestine. The balance ofthe faecal
floraisalso disturbed (Fig. 5.5).
The abnormal gut flora is similarin infants and adults, butthe causes may differ.
In infants, even small influences, such as: a sudden change in nutrition, common
infections, vaccinations, convalescence, sudden changes ofthe weather, and possibly
other factors, may upset the microbial balance in the gastrointestinal tract.
In adults, more drastic influences, such as: disturbances of gastric function, dis-
orders of intestinal motility, and some diseases, cause changes in the gut flora. Exam-
ples include: (a) gastric achlorhydria resulting from aging, or associated with pemi-

Gastrointestinal microeco/ogy of 21 infants with dysbiotic

c: relations (average counts of microorganisms)
c:
~

8 10
_________. Bacteroides- group

·---<>-----.
~.~ Kfebsiefla -Enterobacter- group

.. ___. ,-- Sfaphylococct

(mostly Staph_ aureus J

o, o3 o. c, c1
.;a
fvf 01 c3
Sto l up.Jej low ~;..ej__:.up_J_I_lo_w_.J_t.Jl Cae. Co Co. sigm"/

Small mtestine Large intestine

Fig. 5.5 Gastrointestinal disturbed (dysbiotic) microflora of 21 infants, 1/2-22 Months old. Diag-
nosis: sudden and unexpected death during infancy; gastrointestinal contents taken between 6 and
24 hr post martern
(HAENEL, 1971a)

63
cious anaemia; (b) stagnation ofthe intestinal contents due toblind loops; (c) disor-
ders and diseases impairing intestinal motility, e. g. diverticulosis of the small intes-
tine, regional enteritis (Crohn's disease), scleroderma, x-ray irradiation ofthe abdo-
men, intestinal strictures; (d) gastro-colic and entero-colic fistulae; (e) liver cirrhosis;
and (f) disorders of immunological systems, e. g. hypogammaglobulinaemia (HoFF-
MANN, 1966; HAENEL, 1970 & 1971 a,b; DRASAR & HILL, 197 4; SAVAGE, 1977; ßERN-
HARDT & KNOKE, 1980).
Changes in the gut flora are non-specific, and the starnach and small intestine
harbour !arge numbers of bacteria, particularly facultative anaerobes. The starnach
of fasting individuals subject to achlorhydria or hypochlorhydria may contain
105-107 organisms per ml of contents whereas the normal starnach is either sterile or
contains no more than I 0 3 organisms per ml (GIANELLA et al., 1972; ZINSSER, 1976).
Achlorhydria also causes an increase in the number ofbacteria entering the small
intestine (DRASAR & HILL, 1974).
Bifidobacteria may disappear or be considerably reduced in the faecal flora, but
facultative anaerobes (mostly enterobacteria and enterococci) increase significantly.
There are also increases in staphylococci, proteus, clostridia, the klebsiella-entero-
bacter group, and yeasts. Various other factors cause similar changes in the gut flora.
Bacterial "overgrowth" of the starnach and small intestine has harmful conse-
quences. Organisms compete with the hast for nutrients and frequently degrade intes-
tinal enzymes and other secretions ( e. g. bile salts). In the blind-loop syndrome, bacte-
ria consume vitamin B12 resulting in megaloblastic anaemia, and they deconjugate
bile salts leading to fat malabsorption and steatorrhea (HAENEL, 1971 a,b; DRASAR &
HILL, 1974; HAFTER, 1978).
The normal gastrointestinal flora may be re-established when influences causing
abnormal distribution of intestinal micro-organisms are eliminated, and re-establish-
ment of normal flora may be accelerated by the dietary administration of lactobacilli
(e. g. L. acidophilus) and bifidobacteria.

5.4.2 Changes in the gutflora accompanying gastroenteritis

The appearance ofthe particular pathogen in !arge numbers is the major characteris-
tic of these changes in the gut flora.
Intestinal pathogens cause acute diarrhoea, and their location is dependent on
the species. Thus, salmonellae grow in the small and !arge intestine, shigellae in the
!arge intestine, and enteropathogenic E. coli strains may be located in either small or
!arge intestine. In children, the pathogenic strains of E. coli probably grow and pro-
duce enterotoxin in the small intestine. Vibrio cholerae and Clostridium perfringens
also grow in the small intestine (PANTALEON, 1965; DRASAR & HILL, 1974; HAFTER,
1978), and its microecology is disturbed.
The small intestine is found to contain an increased number of normal intestinal
bacteria, while the faeces contain, in addition to large numbers of pathogens
( 10 6-108 I g), an increased number of enterobacteria, and a reduced number of anaer-
obes including bifidobacteria.
The protective mechanisms of the hast include the gastric-acid barrier (ORLA-
JENSEN et al., 1945; GIANELLA et al., 1972), the motility ofthe small intestine, campe-

64
tition of the normal intestinal flora, and the immune systems (LucKEY, 1965; HoFF-
MANN, 1966; DRASAR & HILL, 197 4; SA VAGE, 1977; COA TES & FULLER, 1977).

5.5 Intestinal bacteria and soft-tissue infections

The access of intestinal micro-organisms to soft-tissues (which arenot their native

habitat) has pathological consequences. Most ofthe factors restricting the growth and
distribution of micro-organisms in the gastrointestinal tract (e. g., gastric acidity and
intestinal motility) are absent in soft-tissues.
The mostfrequent sources of infection areintestinal perforation, injuries to soft-
tissue and postoperative contamination. Anaerobic organisms such as Bacteroides
spp., Peptococcus spp., Peptostreptococcus spp., Clostridium spp., Propionibacterium
spp., and Eubacterium spp. participate in soft-tissue infections (MooRE & HOLDE-
MANN, 1972), comprising abdominal and liver abscesses, septic abortion, and inva-
sions of the urinary tract and pleuropulmonary spaces. Such infections are mostly
attributable to mixed populations of facultative anaerobes (mainly enterobacteria)
and anaerobic bacteria (SAVAGE, 1977).
Some strains ofbifidobacteria have also been reported to participate in soft-tis-
sue infections initiated by mixed populations. These include some strains of bifido-
bacteria associated with peritonitis, dental pyorrhoea and various abscesses (BEER-
ENS & TAHON-CASTEL, 1965). B. eriksonii (syn. B. adolescentis biotype b) has been
isolated from human pleural fluid and a lung abscess (GEORG et al., 1964).
Recently BROOK (1981) has indicated anaerobes, such as Bacteroidesfragilis, B.
melaninogenicus, Peptococcus asaccharolyticus, and Bifidobacterium adolescentis as
a cause ofurinary tract infection in five children-girls; three children had mixed infec-
tions (B.fragiliswith E. co/i in two andin one case two anaerobes were found).
Soft-tissue infections are treated with broad-spectrum antimicrobial agents
effective against both facultative anaerobes and other anaerobic organisms (SA VAGE,
1977). The undesirable side-effects of antibiotic therapy on the indigenous intestinal
flora may be prevented by treating patients with preparations of lactobacilli.

5.6 Enzymatic activities of intestinal bacteria

The intestinal bacteria possess diverse constitutive and inducible enzyme systems,
capable of many metabolic reactions. According to DRASAR & HILL (1974): "The
metabolic activity of the gut bacteria is potentially equal tothat of the liver".

5.6.1 Reactions involving carbohydrates, glycosides and glucuronides

Glycosidases produced by the intestinal bacteria include beta-glucuronidase, beta-

glucosidase, beta-galactosidase and various other carbohydrases which hydrolyze
carbohydrates, glycosides and glucuronides excreted in the bile. The main dietary
polysaccharides (gelatinised starch, dextrins, glycogen) and disaccharides (sucrose,

65
Table S.l Common Food Carbohydrates: Types, Composition, and Sources

Type Component monosaccharides Most common food sources

Polysaccharides
Starch, dextrins D-Glucose Cereals, roots, tubers, legumes
Cellulose D-Glucose Plant cell walls and fiber
Glycogen D-Glucose Liver, animal tissue, sweet corn
Hemicelluloses L-Arabinose, D-xylose, L-rham- Plant cell walls and fiber; cereals,
nose, D-galactose, D-mannose, legumes, and nuts; flour, bran
D-glucose, D-glucuronic, and
D-galacturonic acids
Pentosans L-Arabinose, D-xylose With hemicelluloses and pectic
substances
Pectic substances D-Galacturonicacid, L-arabi- Fruits, especially citrus and apples;
nose, D-galactose, L-rhamnose, sugarbeet, vegetables
L-fucose
Oligosaccharides
Raffinose, stachyose D-Galactose, D-glucose, Legurne seeds, cereals, tubers
D-fructose
Fructosylsucroses D-Fructose, D-glucose Cereals, onion, leek
Maltooligosaccharides D-Glucose Starch syrups, malt, amylodextrins
Disaccharides
Sucrose D-Glucose, D-fructose Sugar cane, sugar beets, fruits, and
vegetables; sweetened foods
Maltose, isomaitose D-Glucose Starch syrups, malt, honey
Lactose D-Galactose, D-glucose Milk, cheese, dairy products

Source: HODGE & ÜSMAN ( 1976)

maltose, isomaltose, Iactose) are hydrolyzed by intestinal enzymes (Tables 5.1 and
5.2).
A deficiency of intestinal beta-galactosidase, prevalent in more than 70% of
some human populations, Ieads to fermentation of dietary Iactose by the colonic
flora, resulting in flatulence, abdominal discomfort and diarrhoea.
Human digestive enzymes have little effect on raw starch and such plant polysac-
charides (constituting 10-20% or more ofthe plant foods consumed by man) as: cel-
lulose, pectin, hemicelluloses, and pentosans; and oligosaccharides such as meli-
biose, raffinose and stachyose (Tables 5.1 and 5.2). These substances are hydrolyzed
to varying degrees and digested by the colonic bacteria with the production of organic
acids, mainly volatile fatty acids (acetate, propionate and butyrate), gas (carbon diox-
ide and hydrogen) and energy; methane may also be produced in some people (CuM-
MINGS, 1981 ). Small amounts of lactic, formic and succinic acids arealso produced.
For example: Bacteroides fragi/is is able to utilise hemicelluloses, pectin, xylan and
raw starch; Peptostreptococcus productus pectin, raw starch, and raffinose; Rumino-
coccus bromii raw starch; and R. a/bus cellulose, and xylan (MACBEE, 1977). BETIAN

66
Table 5.2 Approximate Carbohydrate Composition of Selected Food Typesa

Cereal grains, Legumes, mature, dry Fruits, edible portion Vegetables

whole

Corn Wheat Navy Soy- Pea- Apples Or- Prunes White Toma-
beans beans nuts anges pota- toes
toes
(%) (%) (%) (%) (%) (%) (%) (%) (%) (%)

Monosaccharides
D-Fructose 0.1-0.4 0.1 Trace 5 1.5 30 0.1 1.6
D-Glucose 0.2-0.5 <0.1 Trace 2 2.5 15 0.1 1.2
Reducing sugars 0-2 0.2 8 5 47 I (var.) 3.4
Oligosaccharides
Sucrose 1-2 I 3 5-7 4-5 3 4.6 2 Var. <1
Raffinose 0.1-0.3 0.3 o.8b 1-2
Stachyose 2.5b 3-5
Other 1-2° 3.8d +d
Polysaccharides
Starch, d.b. e 64-78 59-61 35-40 0-2 4 0.6 0.7 75
Dextrins 6-7 4 1-3 3
Pectin + 0.6 1.3 0.9 + 0.3
Pentasans 6 5-6 8 4-5 0.3 2.0
Hemicelluloses 5-6 6 6-7 5-7 4 0.7 0.3 10.7 0.3 0.3
Cellulose I 2 3 2-3 2-3 0.4 0.3 2.8 0.4 0.2
Water(%) 16 14 11 10 2 84 86 3 80 93

a HARDINGE et a/., ( 1965), KAW AMURA ( 1967), and D' APPOLONIA et a/., ( 1971)
b Green mung bean data
c Fructosylsucroses
d Other = verbascose; + = present but not quantitated
e d.b. = dry basis
Source: HoDGE & ÜSMAN (1976)

and co-workers ( 1977) have iso1ated a cellu1ose-splitting Bacteroides from faeces,

while SALYERS and co-workers (1978) have shown that several Bacteroides species
and Bifidobacterium species are ab1e to utilise a wide range of non-starch polysaccha-
rides.
The polysaccharide-degrading enzymes of intestinal bacteria are inducible.
Non-cellulosic polysaccharides have better digestibility (ab out 80% on average)
than cellulose (about 50% on average). Pectin is, for example, almost completely
digested in the gut, while the extent of breakdown of cellulose varies with its source.
Cellulose ofwheat bran (including all bran) is broken down to a much lesser extent
than that in fruit and vegetables (CUMMINGS, 1981).
Human strains of bifidobacteria metabolize indigestible raffinose (except B.
bifidum), lactulose, and N-acetylglucosamine-containing saccharides present in
human milk to acetic and lactic acids. Lactulose is also metabolized by enterococci
and lactobacilli.
The monosaccharides: D-mannose, L-sorbose, L-arabinose, D-xylose; and the
alditols: dulcitol, D-mannitol, and L-arabinitol are poorly absorbed, but they are fer-

67
mented by the colanie bacteria. Bifidobacteria are unable to ferment rhamnose, sor-
bose and dulcitol.
The production of glucuronides in the liver (foreign compounds are conjugated
with glucuronic acid) has an important detoxication function.
However, the glucuronides produced are excreted in the bile and may be subse-
quently hydrolyzed by intestinal bacteria, including bacteroides, bifidobacteria, lac-
tobacilli, E. coli, S. faecalis, and clostridia with the release a potentially toxic moiety.

5.6.2 Reuetions involving nitrogen compounds

These reactions include ureolysis, deamination and decarboxylation of amino acids,

N-dealkylation, nitrosation, N-esterification, and reduction of diazo-compounds.
Such reactions are shown in Fig. 5.6.
Intestinal bacteria such as enterobacteria, clostridia, peptostreptococci, eubacte-
ria and some strains of bifidobacteria produce the enzyme urease which hydrolyses
urea to ammonia and carbon dioxide. About 20-25% of the total urea pool is hydro-
lysed each day (VAREL & BRY ANT, 197 4), and urea hydrolysis is the major source of
ammonia produced in the gut. The released ammonia is normally detoxified in the

\GUT LUMEN\: \TISSUES\

I
PROTEINS
( Dietary or endogenaus ) I
Gut pro~eases _;;:::.:::-------------:-DIGESTIVE ENZYMES

UNDIGESTED
PROTEIN - - - - - Microbial _ _ _ --AMINO ACIDS ---+PROTEIN SYNTHESIS
proteases / 1 \ \\
ENERGY
PEPTIDES ~ / I \ \\
~ / / \ Transam(~ases 1
/i , \
1 Decarboxyla ses \\ 1
Microbial and
/
1 /1
CO I
\ \ cX.-KETO\ ACIDS
I
gut proteases I 2 I Oxidases \
~ 11 I dehydrogenases 1 I
I /
I AMifY...ES
Amine'oxidases
\....
\ _ _..
/ II
MICROBIAL .... , , \
PROITEIN ______________.... N f ! 3 - r ~g~;ffSSENTIAL AMINO

: L-. UREA,URIC ACID

UREA_.....,.-~

FAECES - - - - - - - - : URINE

Fig. 5.6 Outline scheme of some posssible activities of the micro-organisms ofthe gut which might
influence the course of digestion and utilization of proteins. The broken lines show suggested or
known microbial pathways
(SALTER, 1973)

68
Iiver by the synthesis of urea and by incorporation into non-essential amino acids, but
the cirrhotic Iiver is unable to do this, so ammonia accumulates the general circulation
resulting in hyperammonaemia and encephalopathy.
The bacterial enzymes decarboxylases convert amino acids to amines; for exam-
ple, histamine is produced from histidine, tyramine from tyrosine, tryptamine from
tryptophan, phenylethylamine from phenylalanine, cadaverine and putrescine from
Iysine and ornithine respectively.
The major producers of amines include E. coli and anaerobes, particularly clos-
tridia (PREVOT, 1971; DRASAR & HILL, 1974). Amines have undesirable effects in
man, but those produced in the large intestine are normally detoxified in the liver by
various amine-oxidases.
The intestinal clostridia and E. coli can convert enzymatically tyrosine to phe-
nols, ammonia, and pyruvate; and enterobacteria (E. coliand P. vulgaris), and some
strains of B.fragilis and Fusarium spp. produce indole, ammonia, and pyruvate from
tryptophan (DRASAR & HILL, 1974). Many workers have drawn attention to the pot-
entially harmful effects of phenols and indole (ÜRLA-JENSEN et al., 1945; HoFF-
MANN, 1966; DRASAR & HILL, 197 4; HAFTER, 1978), but normally phenols and indole
are detoxified in the liver by conjugation with sulphuric acid to yield respectively the
sulphate esters of phenol and p-cresol, and indole (indican-indoxyl potassium sul-
phate) which are excreted in the urine.
Intestinal bacteria such as E. coli, S. faecalis, clostridia and bacteroides are able
to nitrosate secondary amines using nitrite as the nitrosating agent. This reaction
Ieads to the formation of nitrosamines, which have been shown to be potent carcino-
gens. The amount of ingested nitrite is, however, much smaller than that of nitrate,
which occurs naturally in many foodstuffs (e. g., root vegetables) or as a food additive
(e.g., meat products) and it is often a contaminant of drinking water. Many bacteria
(except bifidobacteria and lactobacilli) can reduce nitrate to nitrite, although much
ingested nitrate is absorbed from the uppersmall intestine and excreted by the kidney
in the urine.
Secondary amines such as dimethylamine, piperidine and pyrrolidine are pro-
duced in the large intestine from Iecithin or choline by the bacterial action, and these
too are excreted in the urine. Secondary aminesarealso found in foodstuffs, such as,
for example, fish, cooked meats and tea.
Nitrosamines may be synthesized from nitrate and secondary amines in the pres-
ence oflarge bacterial counts but these conditions do not occur in healthy individuals,
even though secondary amines are present in large amounts in the large intestine and
bladder and nitrate is present in the stomach, uppersmall intestine and the bladder,
because the bacterial population is confined to the large intestine.
However, in certain disorders, such as gastric achlorhydria and especially blad-
der infection (often due to E. colr), nitrosamines may be produced because of inva-
sion ofthe stomach, small intestine, and bladder by large numbers ofbacteria (DRA-
SAR & HILL, 1974). However, the net synthesis ofnitrosamines, under conditions in
which bacterial synthesis is possible, may be reduced by simultaneaus enzymatic
degradation. Lactobacilli and E. coli, followed by bifidobacteria and bacteroides, are
the most active in the degradation of nitrosamines (ROWLAND & GRAsso, 197 5).
The hydrolysis and reduction of diazo compounds. The gut flora may degrade
ingested foreign compounds, such as sulphonamides and azo-dyes (food colours),

69
and anaerobic bacteria are more active in these circumstances than facultative organ-
isms. It is postulated that some dyes (e.g., orange G, orange RN, ponceau MX, red
6 B, red 10 Band red 2 G) may undergo reduction ofthe diazo-linkages to yield poten-
tially toxic products (DRASAR & HILL, 197 4; SANDINE, 1979).

5.6.3 The metabolism of endogenous compounds

Bile acids, bile pigments, and cholesterol undergo degradation by bacterial enzymes
(Fig. 5.7).

I LIVE;~-~

Cho/estero/
+
I

i i
Cholesteryl ester I
t
..__1---'B~'';.;;..e acids
I

- 8/LE oucr: I~ . . --~·~-------.------------,

~Bile sa/ts ..fl-:;_oßl.'!i!'!!2,.Bi/e acids- -0.ga}{if,'l;?/-:!Jc"fk~Lithocholic ac1d
1 e.g. Cholic acid and other denvatJVes
GUT j FAECES
Cholesterol ____ f!!~c1J..of1.Q!1~@'L ________ +Coprostanol ar:d
other metabolites
--------------------------------------------------------~

Fig. 5.7 Outline scheme ofthe enterohepatic cycle ofbile acids and sterols. The broken lines show
microbial pathways
(Adapted from CüATES & FULLER, 1977)

Bile acids (cholic and chenodeoxycholic acids) are secreted into the duodenum
in the conjugated form (with glycine and taurine), and they subsequently undergo
deconjugation, oxido-reduction and dehydroxylation in the colon. Many colonic
bacteria including bacteroides, bifidobacteria, eubacteria, lactobacilli, enterococci,
clostridia, and veillonella are able to deconjugate bile salts. The hydrolases are consti-
tutive enzymes- extracellular in bifidobacteria and intracellular in others. Their spe-
cificity for glycine ortaurine conjugates varies; some bacteria deconjugate both while
others deconjugate only one.
The freed bile acids- cholic and chenodeoxycholic acids- are dehydroxylated
to deoxycholic and lithocholic acids respectively. The colonic bacteria including bac-
teroides, bifidobacteria, clostridia, and to a lesser extent enterococci and veillonella,
participate in this reaction. The enzyme 7 alpha-dehydroxylase is inducible, but it is
not produced when the pH is below 6.5, but the dehydroxylating ability ofthe bacte-
ria increases with the concentration of the bile acids (DRASAR & HILL, 197 4). The
amount ofbile acidslost in faeces may be 200-600 mg/ day or 15-20% ofthe total
quantity produced.
Cholesterol is reduced in the colon to coprostanol and other compounds, in
which reaction bacteroides, clostridia, and bifidobacteria participate.
Bilirubin, produced from the catabolism of haemoglobin, is metabolized by E.

70
coli and clostridia to urobilinogen, which is mainly excreted in the faeces, although
some is reabsorbed from the intestine and excreted in the urine and bile.

5.6.4 Reactions occurring with antibiolies

Many antibiotics are inactivated by the intestinal flora either by enzymatic break-
down of the antibiotic molecule or in various other ways, and consequently many
intestinal organisms become resistant.
The intestinal flora, especially enterobacteria, may serve as the reservoir for the
accumulation of antibiotic resistance-transfer factors. R ( = resistance) factors in bac-
teria are carried on plasmids (extrachromosomal DNA fragments) which may be
transferred from cells to cells during physical their contact in a mixed population.
Multiple resistance to several antibiotics may be transferred from members of the
enterobacteria (e. g., E. coli), inhabiting the intestinal tract of man and animals, to
antibiotic sensitive strains of salmonellae, shigellae, S. aureus and other pathogenic
or potentially pathogenic micro-organisms (ANDERSON, 1968; MEYNELL et al., 1968;
MITSUHASHI, 1969).
Organisms such as enterobacteria carrying R factors occur in a small proportion
in the human intestinal tract, but they can outgrow other organisms in the presence of
selective concentrations of antibiotic. However, sodium deoxycholate, present in the
large intestine, reduces the rate of transfer of antibiotic resistance (MASON &
RICHARDSON, 1981 ), and this may contribute to the low transfer rates observed in the
intestine (WIEDEMANN, 1972; WILLIAMS, 1977).
The indiscriminate use of antibiotics in agriculture (animal busbandry and veter-
inary medicine), Ieads to a strong selective pressure on the R factors, and conse-
quently the proportion of antibiotic-resistant organisms carrying transmissible R fac-
tors shows a trendtobe reduced (SMITH, 1977; MASON & RICHARDSON, 1981); prob-
ably due to the incorporation of R factor into the main chromosome (ANDERSON,
1974).

5.6.5 Synthesis ofvitamins

Intestinal bacteria (e.g. some strains ofbifidobacteria, and coliforms) synthesize B-

group vitamins, including B1, B2, B6, B12, folic acid, biotin, niacin, and pantothenic
acid, and vitamin K (REICHELT, 1935 & 1939; ÜRLA-JENSEN et al., 1939; PETER,
1960; LIEBSCHER, 1961 ), whilst others may require them for growth (e. g. other strains
ofbifidobacteria, bacteroides, lactobacilli, enterococci).
The major synthesis of vitamins occurs in the large intestine which has a low
absorptive capacity, and the small amounts absorbed make only an insignificant con-
tribution to the nutritional requirements of man. Organisms in the small intestine are
also able to synthesize vitamins (DISRALY et al., 1959; BAKER, 1981).

71
5. 7 Diet and intestinal flora of man

5. 7.1 Dietary effects of the jlora in the small intestine

Older investigations have shown that a high carbohydrate diet increased the numbers
of Gram-positive bacteria in the small intestine, whereas a high-meat diet favoured
enterobacteria particularly E. co/i(ßoGENDÖRFER, 1924; VAN DER REis, 1925). How-
ever, recent reports have indicated that the type of diet and frequency offeeding influ-
ence the rate of gastric emptying and thereby influence indirectly the distribution of
bacteria in the small intestine (DRASAR, 197 4; DRASAR & HILL, 197 4 ).

5. 7.2 Dietary effects on the composition ofthefaecaljlora

The composition ofthe flora ofthe human !arge intestine is not easily changed by the
diet, except in the case ofbreast feeding which has a significant influence on the gut
flora.
Extreme diets including: a predominantly meat and egg diet, a strict vegetarian
diet, or a predominantly milk and vegetable diet (each of 14 days duration) did not
changesuch a major component as the bifidobacteria ofthe faecal flora (HAENEL et
al., 1957 a,b ). Similar results have been observed following the consumption oflarge
amounts of fermented milks, for example: yoghurt up to I kg I day (HAENEL et al.,
1963; HoFFMANN, 1966), or 40 g I day or more of Iactose; although frequently an
increase in the microscopic counts ofGram-positive bacteria was observed in the lat-
ter case (HAENEL et al., 1958).
A residue-free or completely soluble diet did not substantially change the faecal
flora; only the bulk ofthe faeces was considerably reduced (ATTEBERY et al., 1972;
DRASAR & HILL, 1974; HAFTER, 1978).
In a Iang-term study in which one personwas fed during successive periods of
many weeks over a period of 328 days: a high-carbohydrate, a high-fat, and a high-
protein diet; it was observed that the high-carbohydrate diet increased the numbers of
bifidobacteria, the high-fat diet favoured the bacteroides, and the high-protein diet
was without effect on the faecal flora (HOFFMANN, 1964).
HILL and co-workers (1971) compared the faeca1 floras of peop1e 1iving in Bri-
tain and the U. S. A. on a mixed "western" type of diet with those living in India,
Uganda and Japan on a largely vegetarian diet, and found some differences in the rel-
ative counts of the organisms of various bacterial groups. A high-carbohydrate diet
decreased bacteroides and increased enterococci with S. faecium predominating
over S. faecalis in this group. A high animal-protein and fat diet increased bactero-
ides and decreased enterococci, whilst a change to a vegetarian diet did not cause any
detectable change in the predominant faecal flora (MooRE et al., 1969). Similar
results have been reported for the faecal flora of Japanese-Americans consuming
either a "western" or a traditional Japanese type of diet (FINEGOLD et al., 197 4).
Non-digestib1e carbohydrates such as lactulose and cellu1ose produce relatively
small changes in the faecal flora, although addition of lactulose to the diet may pro-
mote a relative increase in Gram-positive bacteria, including bifidobacteria, lactoba-
cilli, and enterococci, and an accompanying reduction in coliforms and proteolytic

72
bacteria attributable to a reduction in the pH of the gut contents (HAENEL et al.,
1958). The effect of lactulose was pronounced in cases where the proportion of bi-
fidobacteria in the florawas considerably reduced and the faeces had an alkaHne pH
(HOFFMANN, 1966), but the gross composition ofthe faeca1 florawas not substantially
altered.
The addition of 40-60 g I day of cellulose to anormal mixed diet induced, in 4
out of 6 persons, small changes in the faecal flora; there was a small reduction of such
minor components as clostridia, staphylococci, and proteus but the major compo-
nents were unchanged (HAENEL et al., 1964; HAENEL 1971 b).
Apparently, it is difficult to induce major changes in the human faecal flora by
alterations in the diet. Dietary effects on the composition of the faecal flora are
demonstrably slow and their long-term effects may be variable.
The stability of the human faecal flora is dependent on many factors. The diges-
tion and absorption of food occur in the starnach and small intestine; non-digestible
and unabsorbed dietary constituents enter the !arge intestine tagether with bile salts
(secreted in response to a meal), neutral steroids, intestinal mucosal cells, mucus, and
biliary excretions. The amounts of indigestible dietary constituents, mostly dietary
fibre (consisting of cellulose, hemicellulose, pectin, inulin, the plant gums, and pento-
sans ), and steroids entering the !arge intestine depend on the type of diet. A high-fibre
diet provides the colanie flora with !arger amounts of residue, while a high-fat diet
provides indirectly !arger amounts of bile salts and neutral steroids.
Concerning the protein constituents, HAENEL ( 1971 b) has stated that 1 g of die-
tary protein is diluted with 6-7 g of endogenaus proteins (digestive enzymes and
mucosal cells) and consequently nitrogen compounds entering the colon may not ref-
lect differences in dietary protein intake. Experiments on gnotobiotes have shown
that germ-free chicks consistently excrete more endogenaus nitrogen than their con-
ventional Counterparts, which indicates that the gut micro-organisms may degrade
endogenaus proteins (COATES & FULLER, 1977).

5. 7.3 Dietary effects on the metabolic characteristics of the faecal jlora

The metabolic effects ofthe faecal flora are more easily changed by alterations in the
diet than are the bacterial components, and especially extreme diets induce great
changes in the biochemical action ofthe gut flora. For example, bile-salt degradation
by the gut bacteria in persans living respectively on high or low-fat diets; also the
excretion of indican in the urine of persans receiving various types of diet.
Bacteria isolated from people living on a high-fat diet were more active in the
degradation of bile salts than were similar bacteria isolated from persans living on a
low-fat diet (HILL et al., 1971 ). Indican excretion reached its highest concentration in
persans receiving a meat and egg diet, and the 1owest concentration in persans receiv-
ing a vegetable diet (Fig. 5.8).
The metabolic activity of the faecal flora depends on the biochemical milieu in
the 1arge intestine, and a diet rich in dietary fibre, but 1ow in animal protein and satu-
rated fats, provides the colanie flora with relatively !arge amounts of non-starch poly-
saccharides which are fermented to volatile fatty acids and gas. Since the enzymes cat-
alysing polysaccharide-degradation are inducible, this suggests that an increase in the

73
 60

50

Meat- Veget. Veget. Common Lac to - individ.

egg raw mixed veget. mixed

Fig. 5.8 The 24 hr indicanuria (mg) in eight subjects during 14 days on different diets. Mean values
(bars) with standard deviation (verticallines)
(HAENEL, 1970)

provision of substrate resulting from a high-dietary fibre intake would increase the
prevalence and concentration of these enzymes within the colanie flora.
On the other band, a diet high in meat products (animal protein and fat), but low
in dietary fibre, provides indirectly the flora with comparatively !arge amounts of
amino acids, urea, bile salts and neutral steroids, resulting in the production of ammo-
nia, amines, phenols, indole, faecal bile acids, and the degradation products of cho-
Iesterol. Many ofthese compounds are absorbed and normally detoxified in the liver,
but the long-term effects such diets resulting in the production ofvarious potentially
harmful substances may contribute to the development of degenerative diseases;
such as: ulcerative colitis, diverticulosis of the colon, haemorrhoids, and the colanie
Cancer (DRASAR & HILL, 197 4; SAVAGE, 1977).
Recently THORNTON ( 1981) postulated that a high colanie pH promotes colorec-
tal cancer. Acidification of the colon, either by dietary fibre (following its bacterial
digestion to volatile fatty acids) or by milk (in lactose-intolerant persons), may pre-
vent the formation of carcinogens or co-carcinogens, such as bacterially degraded
bile acids or cholesterol metabolites. Acidification of the colon after the administra-
tion oflactulose inhibited 7 alpha-dehydroxylation ofbile acids.

74
5.8 Other environmental factors and the intestinal flora

5.8.1 Microbial contamination

The microbial contamination ofthe infantbegins at birth with colonisation ofthe gas-
trointestinal tract, and it persists throughout adult life during which new strains of
micro-organisms are contributed.
PETUELY and LINDNER ( 1957) found that the faeces ofbottle-fed infants, occupy-
ing the same room with breast-fed infants, contained 25% of the strains of bifido-
bacteria typical of breast-fed infants. Likewise, MnsuoKA and KANEUCHI (1977)
observed variations in the incidence of species and types of bifidobacteria in the
faeces ofinfants at different hospitals, although infants in the same hospital had simi-
lar strains ofbifidobacteria in their faeces.
Patients in hospital may also acquire strains of micro-organisms typical of the
environment. It has been shown that oral tetracycline therapy frequently increases the
incidence of facultative pathogens, including Proteus spp., Klebsiella spp., S. aureus
and C. a/bicans, in the stools of hospitalized patients but not in case of non-hospital-
ized ones (KNOTHE, 1963).
Humans are usually exposed to a variety of micro-organisms in their diet. Most
of these ingested micro-organisms are destroyed in the stomach, but some, such as
acid-tolerant organisms and sporeformers, may escape the gastric-acid barrier and
enter the intestinal tract as transient strains. KAuFFMANN ( 1954) found, for example,
the same E. coliserotypes in the stools ofpersons feeding at the same communal kit-
chen, and other workers have reported frequent great contamination of food and eat-
ing utensils in canteens with E. coli (CooKE et a/., 1970; SHOOTER et al., 1970).
In gastric achlorhydria or hypochlorhydria the ingested micro-organisms,
including pathogenic bacteria, may represent a potential hazard to health (ÜRLA-JEN-
SEN et a/., 1945; GtANELLA et a/., 1972).
The malnutrition of tropical residents together with a greater exposure to micro-
organisms in a diet can result in an increased bacterial population in the small intes-
tine (GORBACH eta/., 1970; DRASAR & HILL, 1974; ÜRACEY, 1981).

5.8.2 Antibiolies and irradiation

Several antibiotics affect the facultatively-anaerobic flora of the intestine, and such
antibiotics as clindamycin, and lincomycin ( FINEGOLD et al., 1966 & 1967; SuTTER &
FtNEGOLD, 197 4) have drastic effects on the indigenous gut bacteria, including bacter-
oides, bifidobacteria, anaerobic cocci, and lactobacilli (see Chapter 2).
Irradiation ofthe abdomen with gamma or x-rays may also upset the normal ~i­
crobial balance and give rise to an abnormal gut flora (HoFFMANN, 1966; HAENEL,
1970).

75
5.9 The significance of gut micro-organisms

5.9.1 Gnotobiotes in research

Research on gnotobiotes has provided new data on the significance of the microflora
in many aspects oflife. Gnotobiotes areexperimental animals which are either germ-
free (sterile, axenic) or have been inoculated with one or more known species of
micro-organisms (associated, gnotophoric), whereas conventional (classic, holo-
xenic) animals retain their normal gut flora.
Experiments on gnotobiotes have shown that the gut micro-organisms, although
not essential for life, continue to live in a stable relationship with their host. Their pres-
ence is manifested by their effects on intestinal structure and function, in the develop-
ment of defence mechanisms, and the production of many bacterial metabolites.

5.9.2 Ihe function ofgut micro-organisms under normal conditions

5.9.2.1 Intestinal structure and function

The conventional animal has a greater weight and surface area of intestine relative to
body size, and the small intestine contains more connective tissue (particularly the
Iamina propria), and less epithelial tissue than is the case with germ-free animals. In
germ-free animals the reticulo-endothelial elements in the gut are reduced; also cell
tumover in the intestine, whilst the villus structure is more regular (LUCKEY, 1965;
COATES & FULLER, 1977).
In germ-free rodents and rabbits, the caecum is considerably enlarged.

5.9.2.2 Defence systems

The gut micro-organisms contribute to production of resistance factors, including the

humoral and cellular defence systems ofthe host, and the indigenous flora has its own
protective function.

5.9.2.2.1 Humoraland cellular defence systems

The lymph nodes (masses oflymphoid tissue) are larger and more numerous and the
number of lymphocytes within the nodes is greater in conventional than in germ-free
animals. Lymphocytes (spherical cells) are the major constituents ofthe lymphoid tis-
sues, and are associated with specific immunity. B lymphocytes (made in the hone
marrow) are associated with humoral immunity, and T lymphocytes (derived from
the thymus gland) with cell-mediated immunity. Both types of lymphocytes, how-
ever, participate in the production of antibodies (Fig. 5.9).
T lymphocyte and B lymphocyte attach simultaneously to different determi-
nants on the antigen, which is previously fixed on the surface ofthe macrophage. The
antigen interacts with a specific receptor on the T cell and induces this cell torelease a
signal totheB cell. In response to this signal the B cell differentiates and proliferates
into plasma cells, that actually produce the specific antibodies. They include gam-

76
 Thymus cel/s Bone marrow cells

@ 'i' (!!) @
@> @ ~ Antigen @) .
@@~.-~ ®~@)
1.
•
@@ ~ ~ ~
r;::.r.. t:i\&J ~b
~ ~ @;
Macraphage

~ Thymus Sone marrow

@ ~ ce/1 ce/1 ~ ~

2. @@ ~&,~@ ·. ~
@ ~ ~
@~(!)~ ® ®
Macraphage

3.

Fig. 5.9 Thymus-derived cell and bone marrow cell interact synergistically in the development of an
antibody response. As schematized (I), the macrophages firstfix antigen on their surface membranes.
Specifically committed cells ofboth lymphoid populations then attach to different determinants on
the aggregated antigen. As a result of this interaction with antigen, the hone marrow and thymic cells
are brought into close proximity for a sufficient period oftime to permit passage ofinformation from
the thymic to the hone marrow cell (2). This interaction provides a signal to the hone marrow cell,
probably in the form of a change in the surface membrane configuration. In response to this signal,
the synthetic mechanisms ofthe marrow cell are activated and the cell differentiates and proliferates
into plasma cells that elaborate the specific antiborlies (3)
(FUDENBERG eta/., 1972)

ma-A globulin (IgA), gamma-D globulin (lgD), gamma-E globulin (lgE), garnma-G
globulin (IgG) and gamma-M globulin (IgM).
IgA comprises 13% of serum immunoglobulins where it occurs as a monomer. It
constitutes the major immunoglobulin in mucosal secretions especially from the gut
as weil as in colostrum, milk and saliva where it occurs as a dimer (possessing a diva-
lent junctional or J chain which binds the IgA associated peptide chains into dimers
or polymers) linked to epithelial cell surface receptors, the secretory piece which gives
lgA resistance to proteolytic enzymes (Wright, 1981 ).
lgG is the major immunoglobulin in the human serum (comprising about 80% of

77
theserum immunoglobulins ofthe adult), crosses human placenta and fixes comple-
ment, while IgM comprises 5-l 0% of theserum immunoglobulins and fixes comple-
ment. IgD occurs in small amounts, and lgE is associated with reaginic antibodies.
The formation of each type of immunoglobulin is dependent on clones. Genes
occurring in different celllines are called clones, and each clone makes a singletype
of antibody. A specific antigen, introduced into a vertebrate, stimulates one specific
clone of lymphocytes to proliferate, while other clones are repressed (LEHNINGER,
1979).
The Ievels of plasma cells and serum immunoglobulins are much less in germ-
free animals than in conventional animals, but when germ-free animals are exposed
to micro-organisms, the lymph nodes rapidly enlarge and there is a gradual increase
of plasma cells and serum immunoglobulins (GusTAFSSON & LAURELL, 1959;
LuCKEY, 1965; CoATES & FuLLER, 1977). Consequently micro-organisms are appar-
ently the major source of antigens in normal animals, with dietary constituents mak-
ing a secondary contribution. As is known, an antigen is any substance eliciting an
immunological response, such as the formation of antibody specific for a particular
substance.
Germ-free animals are able to produce interferon (a proteinthat prevents viral
reproduction) afterviral challenge (CONSIDINE & STARR, 1965; COATES & FULLER,
1977).
Serum immunoglobulins act synergistically in immunological reactions with
complement (an enzymatic system of serum proteins that is activated by many anti-
gen/ antibody reactions). Complement Ievels may be less in germ-free than in conven-
tional animals (NEWTON et al., 1960), and often properdin (a macroglobulin of nor-
mal plasma involved in the alternative pathway of complement activation) titres are
also reduced in germ-free rats (GuSTAFSSON & LAURELL, 1960).
The phagocytic activity of cells im germ-free animals seems to be unimpaired,
but the macrophages from germ-free animals digest bacteria more slowly than those
from conventional animals.
Recent evidence suggests that indigenous (autochthonous) micro-organisms are
less immunogenic in their hosts than allochthonaus micro-organisms of similar types
(BERG & SA vAGE, 1972; Foo & LEE, 1972). Some indigenous micro-organisms have
antigens that are chemically similar to antigens oftheir host's tissues or even seem to
have antigens in common with their host. For instance: some strains of E. coli have
antigens in their cell-envelopes which are chemically related to antigens ofthe colanie
tissue of humans (MAIS & FINK, 1971 ). Likewise Bacteroides spp. fail to induce anti-
hodies on parenteral injection into its native host, the mause (Foo & LEE, 1972 &
1974).
Other indigenous micro-organisms have antigens chemically different from the
antigens of their host's tissues, but they fail to induce antiborlies if they reach to high
population Ievels early in the animal's life (SAVAGE, 1977). For instance, a strain of
Lactobacillus, isolated from rats, did not induce antiborlies in baby rats which were
colonized immediately after birth by !arge numbers of the lactobacilli, but they
induced antiborlies when monoassociated with adult gnotobiotic rats (WAGNER &
WosTMANN, 1961 ). The explanation of this phenomenon may be that the bacterial
antigens entered the animals early in life and in sufficient amount to inactivate their
immunological mechanisms (KAGNOFF, 1974).

78
 The relatively stronger immunogenical activity of allochthonaus micro-organ-
isms compared with indigenous ones may be important when extrapolated to
humans.
The consumption of fermented milks provides the human gut with large num-
bers of lactic acid bacteria and I or bifidobacteria, which are transient or allochtho-
naus organisms, even though they are of species commonly encountered in the intes-
tine. These observations may perhaps explain some of the beneficial effects of fer-
mented milks.

5.9.2.2.2 Protective function ofthe gut flora

The gut micro-organisms increase an animal's resistance to intestinal infectious dis-

eases. They may prevent colonisation of an area in the intestinal tract by invading
pathogens by competition foressential nutrients orfor attachment sites on the epithel-
ium (ßROCK, 1966; SA VAGE, 1977).
The gut micro-organisms may inhibit the growth of invading pathogens by the
production of organic acids, particularly volatile fatty acids, by the deconjugation of
bile salts and by the production ofbacteriocins. Bifidobacteria, bacteroides, eubacte-
ria, peptostreptococci and ruminococci produce volatile fatty acids from carbohy-
drates, and many of them deconjugate bile salts releasing free bile acids which are
more inhibitoryto susceptible bacteria than theirconjugated forms. Bacteriocins may
be produced by some members of the intestinal flora, including some strains of L.
acidophilus which produce inhibitory substances, such as: "lactocidin" (VINCENT et
a/., 1959), "acidophilin" (VAKIL & SHAHANI, 1965) and "acidolin" (HAMDAN &
MIKOLAJCIK, 1974); also many strains of E. co/iproduce "colicins" and "microcins"
(BAQUERO et a/., 1978; MASON & RICHARDSON, 1981), and 5 different "enterocines"
may be produced by enterococci (BROCKet al., 1963).
Direct intervention ofthe indigenous gut flora in protecting against infection by
intestinal pathogens has been proved experimentally. Shigellaspp. are lethal to germ-
free guinea pigs, whereas they cannot be established in conventional guinea pigs
(LUCKEY, 1965). Treatment of mice and guinea pigs with antibiotics increases their
sensitivity to infection by Shigella spp., and to Vibrio choleraein the case of guinea
pigs (FRETER, 197 4). Mice treated with streptomycin show increased sensitivity to sal-
monellae (MEYNELL, 1963).
Organic acids produced during the growth of some gut micro-organisms (e. g.
bifidobacteria, lactobacilli, peptostreptococci, ruminococci, bacteroides) stimulate
intestinal peristalsis and indirectly the removal of invading pathogens from the intes-
tinal tract.
Normally the indigenous gut flora acts synergistically with its host's immunolog-
ical system (e. g. IgA) in protecting against infections by intestinal pathogens.

5.9.2.3 Nutrition and digestion

In ruminants, the micro-organisms in the rumen ferment the ingested food (hydrolyze
cellulose, other plant polysaccharides, and fermentsoluble sugars) and thus facilitate
its utilisation (HuNGATE, 1966; BAUCHOP, 1977). On the other band, the digestion
and absorption ofingested food in man occurs in areas ofthe gut which have sparse

79
microbial populations. The dense microbial populations in the large intestine may
utilize undigested plant polysaccharides and oligosaccharides, and some of these
products of fermentations may be absorbed from the intestine (McBEE, 1977; CuM-
MINGS, 1981 ).
The main microbial synthesis of vitamins occurs in the large intestine which has a
low absorptive capacity, but the vitamins may be utilized by some micro-organisms
and destroyed by others.

5.9.2.4 Microbial metabolic products

The micro-organisms in the human gut metabolize body secretions and ingested for-
eign compounds, in addition to undigested or unabsorbed dietary constituents
(mostly non-starch polysaccharides). Consequently numerous metabolic products
occur in the intestinal tract. These include a variety of organic acids and gas (pro-
duced from carbohydrates), dehydroxylated bile acids (such as deoxycholic and lith-
ocholic acids ), hydrogenated derivatives of cholesterol (such as coprostanol), protein
degradation products (e. g. phenols, indole, many amines, large amounts of ammo-
nia) and urobilinogens (products ofbilirubin metabolism).
Many such microbial metabolites are potentially harmful to humans, but most of
them are excreted in the faeces and urine. Other toxic metabolites, such as ammonia,
phenols, indole, and amines are detoxified in the liver before excretion. However,
high Ievels of some products (e. g. dehydroxylated bile acids, cholesterol metabolites,
phenols, indole and amines) found in the intestinal tract, due to the effect oflong-term
changes in diet on the metabolic activities of the indigenous flora, may be implicated
in the aetiology of some degenerative diseases and cancerous conditions (HILL et al.,
1971; SAVAGE, 1977; THORNTON, 1981).

5.9.3 1he gut micro-organisms in abnormal conditions

An abnormal distribution of the microbial flora in the human gastrointestinal tract

can have harmful consequences. Large microbial populations in the stomach and
small intestine compete with the host for nutrients and they may degrade the digestive
enzymes and bile salts. Moreover, a disturbed microbial balance in the large intestine
(a reduction of bifidobacteria and an increase of facultative anaerobes and poten-
tially pathogenic organisms) affects the normal microbial metabolism.
The microbial balance in the intestines may be disturbed by antibiotic therapy,
and cause overgrowth of potentially harmful micro-organisms with pathological con-
sequences.
If the detoxication function of the Iiver is impaired, then the metabolic activities
of some gut micro-organisms may present a serious hazard. In cirrhosis, the liver is
unable to detoxify ammonia, amines, and phenols which re-enter the general circula-
tion and cause intoxication.

80
Chapter 6: Nutritive and health values of dairy foods
containing bifidobacteria

6.1 Introduction

Bifidobacteria may play significant roles in the intestinal tract of infants. They pro-
duce organic acids which inhibit the growth of undesirable bacteria, and stimulate
intestinal peristalsis. Their consumption also influences the metabolism of the gut
bacteria, and some reports have indicated the possible value of bifidobacteria in
improving the nutrition of infants.
This potentially beneficial role of bifidobacteria in the intestinal tract of babies
and children has led to their suggested use as dietary adjuncts in combination with
their growth-promoting substances. Consequently cultured milk products containing
B. bifidum (L. bifidus)may improve the nutritional and health values ofthe weaning
diet.
The use of B. bifidum tagether with L. acidophilus for the treatment of the side-
effects of antibiotic therapy has shown beneficial results.
Many reports have indicated the role of lactulose and I or B. bifidum in the
compensational detoxication of subjects with chronic liver disease.

6.2 Infant nutrition and bifidobacteria

6.2.1 Roles ofbifidobacteria in the intestinal tract

This section considers the potentially beneficial roles ofbifidobacteria in the intesti-
nal tract of infants. The most important are: production of organic acids and interre-
lationships between bifidobacteria and the host.

6.2.1.1 Production of organic acids

Bifidobacteria produce acetic, lactic and formic acids which lower the pH in the large
intestine and thereby inhibit the growth of undesirable bacteria. In addition, these
acids even in their buffered condition have a direct destructive effect on many organ-
isms. Acetic acid has a stronger antagonistic effect against Gram-negative bacteria
than lactic acid, and the form er is produced in greater amounts by bifidobacteria.
Organic acids stimulate intestinal peristalsis which helps in removing invading
pathogens from the intestinal tract (MA YER, 1969; SA VAGE, 1977).

6.2.1.1.1 Influence of diet

The nature of the feed influences the metabolism of the gut bacteria. Feeding with
breast milk (high Iactose, low protein particularly casein, low minerals content and
with small amounts of oligosaccharides) favours a predominance ofbifidobacteria,
an absence of putrefactive organisms and a lower pH in the large intestine. Breast-fed

81
Table 6.1 Composition of mature human milk and cows' milk

Composition Humanmilk Cows'milk

Water(mi/IOOml) 87.1 87.2

Energy (kcal/ I 00 ml) 71 66
Total so Iids (g/ I 00 ml) 12.9 12.8
Protein (g/ I 00 ml) 1.1 3.5
Fat(g/IOOml) 3.8 3.7
Lactose (g/ I 00 ml) 6.8 4.9
Ash(g/IOOml) 0.2 0.7
Proteins (% oftotal protein)
Casein 40 82
Whey proteins 60 18
Non-protein nitrogen (mg/ I 00 ml) 32 32
(% oftotal protein) 15 6

Source: FOMON (1974)

infants have acidic stools (pH 5.0-5.5) containing both free and combined forms of
acetic acid, also lactic and fonnie acids. Table 6.1 shows the composition of mature
human milk and cows' milk.
Studies on Shige//ainfections (MATA et a/., 1969 b) and on enteropathogenic E.
coli(MATA & URRUTIA, 1971) in breast-fed infants in Guatemala have shown the low
incidence of both infections due to the predominance of bifidobacteria in the gut
flora.
However, feeding with breast milk supplies the newbom with protective factors,
including high Ievels of antiborlies to enteropathogenic E. co/i and rotavirus in colos-
trum (SUSSMAN, 1961; MICHAEL et a/., 1971; JELLIFFE & JELLIFFE, 1981); lactoferrin
in human colostrum and firstmilk active against E. co/i infections (BuLLEN et al.,
1972); large numbers ofmacrophages and lymphocytes (KLAUS & DIAs-RosSELLO,
1980); Iysozyme active against Gram-positive bacteria and viruses (BRAUN, 1971;
HAMBRAEUS, 1978); the antistaphylOCOCCUS factor (JELLIFFE & JELLIFFE, 1981); anti-
toxins for neutralising Vibrio cholerae and E. co/i (SIMHON et a/., 1979), and sialic
acid-containing oligosaccharides possibly active against bacterial infections (Nico-
LAI, 1971; GYöRGY et al., 1974). Lactoperoxidase and volatile fatty acids in breast
milk may also play protective roles. Many such protective factors are present only in
trace amounts in cows' milk.
According to BuLLEN and co-workers ( 1976) the initial protective mechanisms of
the newbom against the growth of Gram-negative bacteria depend upon the activity
ofboth specific antiborlies and lactoferrin, but subsequently protection is dominated
by the acidic intestinal environment.
Feeding infants with modified cows' milk preparations gives rise to a faecal flora
resembling that of adults, and the stools have a neutral to alkaline pH (HAENEL et al.,
1970; MAYER & KESCHAWARZI, 1970; BULLEN et a/., 1977).
Growth-promoting factors for bifidobacteria (lactulose, N-acetylglucosamine-
containing saccharides and their derivatives, casein hydrolyzed enzymatically) may
be added to modified cows' milk preparations for the purpose of promoting a faecal
flora in artificially-fed infants nearest tothat ofbreast-fed infants (see Chapter 4).

82
 The addition of, for example, lactulose to the diet of artificially-fed infants at a
rate of 1-1.5% increased the viable count ofbifidus bacteria in the faeces and reduced
their pH although not so low at that of breast-fed infants (MACÜILLIVRAY et al.,
1959; MELCHIOR & BRAESTRUP, 1962; KASHKAREVA & ßOREIKO, 1964; NEIMANN et
a/., 1965; ÜRÜTTE & HAENEL, 1968). Studies of HAENEL and co-workers (1970)
showed that the faecal flora ofinfants receiving a lactulose-containing diet (3.4% fat
incorporating v; of vegetable oil, 1.9% protein, 1.2% lactulose, 6.6% Iactose, 0.1 %
galactose and 0.4% minerals) approached that of breast-fed infants, and the stools
had a pH 5.52 ± 0.28 compared with 5.05 ± 0.28 for breast-fed infants.
The increased acidity ofthe lower intestinal contents indirectly prevents the pro-
duction ofharmful amines from amino acids by the putrefactive bacteria (NEGISHI &
BILLY, 1962; PREVOT, 1971).

6.2.1.1.2 Diet and faecallysozyme activity

Feeding infants with lactulose-containing diets promotes high faecallysozyme activ-

ity, which corresponds tothat offeeding with heated human milk (Fig. 6.1 ). The high-
est concentration oflysozyme occurs in the faeces ofbreast-fed infants, although it is
not present in the faeces of older children and adults (BRAUN, 1971 ).
The intestinal transit of Iysozyme is controlled by an inhibitor present in the
duodenal secretion which inactivates it at a pH above 6.0 (Fig. 6.2).
Although Iysozyme per se is not a growth-promoting factor for bifidobacteria, it
always occurs in high concentrations in the faeces ofinfants fed a bifidogenic diet and
coincides with a reduction in faecal pH.

6.2.1.2 Interrelationships between bifidobacteria and the host

The possible value of bifidobacteria in the nutrition of infants, particularly in

nitrogen retention has been suggested (MANCIAUX, 1958; STENGER & WoLF, 1962;
PouPARD et al., 1973) and this is supported by the following observations.

6.2.1.2.1 Nitrogenretention and weight gain

Comparative studies on 8 infants fed modified cows' milk preparations with and with-
out lactulose showed greater (5.26 %) N retention upon the lactulose-containing diet
(STENGER & WOLF, 1962). Similar studies with three different types of modified cows'
milk preparations showed the best N retention on the lactulose-containing diet on
which infants also gained the most weight and performed best (HoREtNY, 1964).
Low birth-weight infants fed with a reconstituted dried cows' milk formula (sup-
plemented with 2% carbohydrate and 4.5 mg% of purified pigs' gastric-mucin)
gained weight at an increased rate and had a lower faecal pH (INOUE & NAGAYAMA,
1970).
According to MA YER (1969 & 1971) bifidobacteria may influence the metabo-
lism of amino acids in a host. Feedingexperiments on white rats with B. bifidum cells
Iabelied with 32 P (SEIDEL, 1966) and P5 S) L-methionine (WUNDERW ALD, 1968)
showed that up to 61 % of the Iabelied phosphorus was retained in the body of rats
(liver, bones, musdes and blood), and even more of the Iabelied methionine was

83
 Lysozyme
200 tlg faeces

-
16~

150

100

;!..§.
1-P.

50

11.

n
!.!.. ,!.§.

~~ ~
OE .....
Qj
111
~

·-·o
-
Qj
111 'i:..
111
c:
....
"i::
f1
·-E
~
n
c:
0
111'- ....0tJ E .... E 111 111
...1:11
CIJ1:1 1::1
~ E CIJ
o..CIJ
10.!! c: CIJ .!! CIJ
I tJ
...,.!! ::l
::l
:r:: ....
CIJ ....
I
.!!
'""
~~~~ 111 CIJ
Eg ~
~ ~~
10
~ ll..

-.
::l tJ lll
:r:: ...... ~· ....::l lll "'{

.E .!!tJ
~
""

Fig. 6.1 Lysozyme excretion in the faeces ofinfants receiving various foods. (Average values in y I g
faeces)
(BRAUN, 1971)

84
 Breast -fed infants Bottle- fed infants

j Brea~t mit~ Cows' milk

Lysozyme: Safiva Lysozyme : rJ
40 -100mg/ f
Intestinal
Lysozyme Lysozyme
Epithefia

Leukocytes

Duodena{ juice:
pH<6 f---- pH>6
Lysozyme inhibitor

Lysozyme IFaeces Lysozyme

+++ -e-
Fig. 6.2 Mechanism of Iysozyme excretion in the faeces
(BRAUN, 1971)

retained in the liver and serum. However, it is unknown whether this is the same for
human beings.

6.2.1.2.2 Vitamins

Bifidobacteria synthesize vitamins, including thiamine, riboflavin, B6, and K, but

these are absorbed only slowly from the human colon, and the extent oftheir contri-
bution to nutrition of infants is uncertain. Moreover, some gut micro-organisms
require vitamins for their growth.
Studies on 8 infants fed a range of modified cows' milk preparations showed a
reduction in urinary excretion ofvitamin B6 only during the period on the lactulose-
containing diet (STENGER & WoLF, 1962). Also, the oral administration of B. bifidum
to 1-2 months old infants has been reported to produce higher blood and urine Ievels
of thiamine at intakes exceeding I 09 cells I day (ÜYAKE, 1967 a,b,c)

6.2.1.2.3 Inhibition of nitrate reduction

Bifidobacteria and lactobacilli do not reduce nitrate to nitrite; they produce organic
acids which inhibit the growth of many nitrate-reducing bacteria in the intestinal tract
infants.

85
 Some strains ofbifidobacteria may partially or completely inhibit the reduction
of nitrate to nitrite by other intestinal organisms (DoLE:~ALEK, 1979). Nitrite-ions
oxidize the ferrous-ions of haemoglobin to ferric ions, and the resulting compound,
methaemoglobin, is incapable ofbinding molecular oxygen.
Infants are more susceptible to development ofmethaemoglobinaemia than are
older children and adults. The reduction of ingested nitrate to nitrite is favoured by
gastric juice with a pH greater than 4.0 and by the presence of nitrate-reducing bacte-
ria in the intestinal tract. Methaemoglobinaemia usually occurs in infants receiving
weil water (i. e. used to reconstitute formula feeds) of high nitrate content (FOMON,
1974).

6.2.2 Dietary adjuncts

The potentially beneficia1 role of bifidobacteria in the intestinal tract of babies and
children has led to their proposed use as dietary adjuncts as such or tagether with their
appropriate growth-promoting substances. B. bifidum is the species most often used.

6.2.2.1 Formula feeds (modified cows' milk preparations) and cultures of B. bifi-
dum

Cultures of B. bifidum have been added to the diet of artificially-fed infants to modify
their gut flora. LEVESQUE and co-workers ( 1959) have shown that by adding a freeze-
dried culture of B. bifidum (I 07 cells I day) to the diet of healthy bottle-fed infants,
bifidobacteria appear in the faeces, after 2-3 days, at 70% ofthe Ievel found in breast-
fed infants, and persisted after their administration is terminated. Subsequent sturlies
by ScHNEEGANS and co-workers ( 1966) conducted on both healthy and sick children
showed that the implantation of B. bifidum, as a freeze-dried cu1ture to the diet, was
effective in 75% ofthe cases, but at Ievels below those found in breast-fed infants.
A protective role of B. bifidum cultures against some enteric infections in infants
has been demonstrated. The feeding of 114 infants with a modified cows' milk pre-
paration, 7j LACTAN A-B, (containing a culture of B. bifidum with its growth factors,
Iactose, ascorbic acid, and Fe) has been compared with 78 infants receiving
ELEDON buttermilk. Those receiving LACTANA-B had an average of 2.5 stools I
day and those on ELEDON had 1.8. Enteric infections occurred 8 tim es in the butter-
milk group and once in the LACTANA-B group (KAwuo & STöGMANN, 1968).
Some commercial infant foods incorporating bifidobacteria include: (a) the
dried formula feed Lactana-B, containing lactulose and viable B. bifidum (MA YER,
1966); and (b) a dried milk formula, containing B. bifidum, L. acidophilus and Pedio-
coccus acidi/actici (PECH & HRABOVA, 1975; DEDICOVA & BURDOVA, 1977). Concen-
trated (20-25% total solids) whole milk is fermented with a mixed culture of B. bifi-
dum, L. acidophilus and P. acidilactici (ratio 1 : 0.1 : 1) at 31 o C to the desired acid-
ity, and then cooled; heat-treated vegetable oil, malto-dextrin and Iactose are added;
and the mixture is finally homogenized and spray-dried.
Cultures of S.lactis arealso used in the manufacture of dried-milk feeds, includ-
ing: (a) ha1f-skimmed milk powders produced from fermented milk, with or without
added carbohydrates; (b) a whole-milk powder, PELARGON, produced from fer-

86
mented milk containing added carbohydrates and usually vegetable oil. Their manu-
facture involves fermentation of concentrated milk with a culture of S. lactis to the
desired acidity, followed by cooling, mixing with carbohydrates (malto-dextrin, suc-
rose and maize starch), homogenisation and spray-drying. Some 20% ofthe S.lactis
cells survive the drying (GRIEDER, 1969).
Industrial processes for the production of cultured acidophilus products for
feeding infants under 12 months of age have also been developed (SUKHOVA et al.,
1978 a,b; ÜORSHKOV et a/., 1978; KOROLEVA et a/., 1980).
Selected strains of L. acidophilusofhigh proteolytic and antibiotic activity have
been used for the preparation of modified cows' milk products. Fermentation with L.
acidophilusincreases the biological value ofthe protein; and these products contain
108-109 L. acidophilusperml. Their acidityranges from 0.45 to 0.72% lacticacid (50°
to 80° Th).

6.2.2.2 The prophylactic and therapeutic use of B. bifidum cultures

B. bifidum may be used to correct a disturbed microbial balance in the large intestine
after antibiotic therapy. For example, feeding the infant with a milk formula, LAC-
TANA-B, which contains viable B. bifidum with its growth factors, prevented the
over-growth of Candida albicans in the intestine following penicillin therapy
(MA YER, 1966 & 1969).
A recent report indicates the protective roles of B. bifidum and L. acidophilus
bacteria in dyspeptic conditions attributable to antibiotic therapy. The administra-
tion of a preparation containing antibiotic-resistant B. bifidum and L. acidophilus to
children (between 13 days and 8 years of age) during long-term broad-spectrum
antibiotic therapy for a variety of conditions prevented the occurrence of dyspeptic
disturbances, and these organisms were detected in the faeces of 50% of the patients
even several days after termination of their administration (BRANCA et al., 1979). A
disturbed intestinal flora in premature infants with septicaemia has been corrected,
according to Kozwv A ( 197 6), by administering cultures of bifidobacteria.
According to FoMON ( 197 4), acute diarrhoea caused by enteropathogenic bacte-
ria requires appropriate treatment with antibiotics, and the temporary reduction or
withholding of food, while mild diarrhoea of short duration may be treated with spe-
cial preparations made from strained carrots. However, B. bifidum may be used as
supplementary treatment in some enteric infections.
The oral administration of a culture of B. bifidum in conjunction with a dietetic
regimen has been reported to produce beneficial effects in infants with bacterial
enterocolitis. The feed consisted of a high-fluid diet, followed by cream of rice and
human milk (TASOVAC, 1964). In contrast, ScHNEEGANS and co-workers ( 1966) have
shown that the oral administration of a freeze-dried culture of B. bifidum to children
with enteric infections, could eradicate enteropathogenic E. coli strains in about 60%
ofthe cases, andin more than 80% when lactulose is included. These authors did not,
however, observe a parallelism between the implantation of B. bifidum and disap-
pearance of enteropathogenic E. coli, but the rather strong antagonistic effect ofbifi-
dus bacteria, when used in conjunction with lactulose (or comparable dietetic feed-
ing), against enteric infections indicates the significance of a low pH in the large intes-
tine, and the influence ofthe feed.

87
 Many enteric infections and dyspeptic phenomena are located in the small intes-
tine, where lactobacilli occur in larger numbers than bifidobacteria; consequently the
use of B. bifidum tagether with L. acidophilus may be justified. Hence the impor-
tance of microbiological control of pharmaceutical bacterial preparations containing
viable germs which has been pointed out particularly by REUTER ( 1969) and BAJARD
(1977).

6.2.2.3 Cultured dairy foods in the mixed feeding ofbabies and children

The feeding of infants in the first 4-6 months of life consists of breast milk or a for-
mula feed (modified cows' milk preparations, adapted milks). An infantmilk for-
mula should have a composition which is near tothat ofbreast milk.
The fat content should be 2.0-4.0 gl 100 ml incorporating appr. ~ ofvegetable
oil, and the contents of Iactose, protein, minerals and vitamins should approximate to
those ofbreast milk.
Some formulae have an adapted ratio of whey proteins: casein between 40 : 60
and 60 : 40 vs. 18 : 82 for cows' milk. However, the whey protein composition differs
markedly between human and cows' milk (Fig. 6.3), and consequently formula feeds
have low lactoferrin and Iysozyme contents and a high content ofbeta-lactoglobulin
in comparison with human milk.
Some formulae are supplemented with growth-promoting substances for bifido-
bacteria, including lactulose, mucin, and enzymatically hydrolyzed casein (see Chap-
ter 4).
The mixed (weaning) feeding of infants begins after 4-6 months of life with the
introduction of solid foods, such as fruits, cereals, vegetables, meat and eggs.
Nevertheless, milk still makes the greater contribution to infants' diet during the
first year oflife and at least 500 ml I day should be consumed (LEVI, 1978; REY, 1978).
Milk is a rich source of calcium compared with such supplementary foods. Modified
cows' milk preparations may not be recommended for infants after 4-6 months of
age, because many have a reduced concentration of calcium and protein. Hence,
cows' milk of normal composition (e.g. at least 80 mgl 100 ml calcium); and
enriched with iron is preferable, since after 6 months of age, infants have increasing
requirements for iron ( 10-15 mg I day).
The use oftbis milk has also been recommended as dietary supplement for preg-
nant and lactating warnen, convalescents and the elderly (REY, 1978).
Cultured milk products (e. g. yoghurt and buttermilk) are more easily digested
than the original milk, because modifications of the main constituents by the starter
cultures improve their digestibility (SHAHANI & CHANDAN, 1978; RASIC & KURMANN,
1978; SPECK & KA TZ, 1980). The partial hydrolysis of the milk protein in cultured
milk products increases the content offree amino acids and peptides and this partial
disruption of the protein structure facilitates the action of the digestive enzymes.
Sturlies on yoghurt have shown that the biological value of its protein is superior to
that ofthe original unfermented milk protein (RASIC et al., 1971; BRESLAW & KLEYN,
1973; SIMHAEE & KESHAVARZ, 1974).
Recently, Hargrove and Alford (1978 & 1980) observed that rats fed yoghurt
have improved feed efficiency and gained weight faster than those fed unfermented

88
 g nitrogen
per fitre
~ non-protein nitrogen

IT]. Serumalbumin

[[I]] immunoglobufins

5 ~ f:.ctoferrin

D Iysozyme
~ oL-factafbumin

II ~ -fac togfobu fin

' ~ casein

Cow's Humanized Human

milk breast milk milk
substifute
Fig. 6.3 The protein composition of cows' milk, human milk and humanized breast milk substitute
(HAMBRAEUS, 1978)

milk. These authors postulated that the yoghurt culture produces a "growth stimu-
lant" (present in high molecularweight components) of, as yet, undetermined nature.
A large number of starter organisms may also .have nutritional significance, since
the constituent enzymes and single-cell protein of bacterial cultures possibly contri-

89
bute to the overall nutritional profile of the fermented product (SHAHANI & CHAN-
DAN, 1978).
Some strains of B. bijidum have a proteolytic activity in milk comparable tothat
of lactic streptococci. The use of B. bijidum together with a yoghurt culture (ratio
2 : I) in the manufacture of yoghurt has shown that the changes in the nitrogen com-
pounds were greater than those in the control yoghurt. The extent of these changes
was dependent upon the presence of bifidobacteria (KiszA et al., 1978), and it is
probable that the synergistic action of B. bijidum and yoghurt bacteria contributes
beneficially to this effect.
Bijidobacterium produces L ( + )-lactic acid in addition to acetic acid from Iac-
tose in milk; whereas L. acidophilus produces D L-lactic acid, and L. bulgaricus pro-
duces D (- )-lactic acid (Table 6.2).

Table 6.2 Lactic acid bacteria and bifidobacteria used in the manufacture of cultured milk products

Culture Optimum Optical Fermen- Product

growthtem- rotationof tation')
perature lacticacid

Bifidobacterium bifidum, 36-38 oc L(+) - Cultured milk products

longum
Lactobacil/us acidophilus 35-38 oc DL Homof. Acidophilus milk, koumiss,
BIOGARDE
L. casei 2) 37°C L(+) Homof. YAKULT
L. /actis 40-43 oc D(-) Homof. Kefir
L. bu/garicus 40-45 oc D(-) Homof. Yoghurt, koumiss
L. brevis 30°C DL Heter. Kefir
L. plantarum 30-35°C DL Homof. Fermented foods
Streptococcus 40-45 oc L(+) Homof. Yoghurt, BIOGARDE,
thermophilus cultured milk products
S. lactis 30°C L(+) Homof. Buttermilk, kefir
S. cremoris 30°C L(+) Homof. Buttermilk, kefir
Leuconostoc cremoris 18-25 oc D(-) Heter. Buttermilk, kefir
L. dextranicum 20-30°C D(-) Heter. Buttermilk, kefir
Pediococcus acidilactici 40°C DL Homof. Cultured milk products

') Homof. = homofermentative; Heter. = heterofermentative

2) L. casei strain Shirota (Yakult Institute, Yakult Honsha Co., Ltd., 1978)

L ( + )-lactic acid is completely metabolized by the infant either in the respiratory

process or in the synthesis of glucose or glycogen. In contrast, D (- )-lactic acid is
only metabolized to a limited extent and at a slower rate; consequently its excretion in
the urine is increased. It is important in feeding infants, particularly during the first
weeks oflife, to use cultured milks which contain nearly exclusively L ( + )-lactic acid.
The objective is to prevent the appearance of metabolic acidosis due to the inability of
the body to transform the sufficient amount of D (- )-lactic acid (DRURY & WICK,
1965; ßRIN, 1965; MEDZIHRADSKY & LAMPRECHT, 1966; ßALLABRIGA et a/., 1970;
ßALLARIN, 1971 ).
Calcium is better absorbed from cultured milk than from unfermented milk,
since the presence of acid and the improved digestibility of the protein enhances cal-
cium absorption.

90
 Starter bacteria are a significant source of antigens which may induce the forma-
tion of antibodies in their hosts. Recent evidence suggests that allochthonous micro-
organisms (transient organisms derived from food or other extraneous sources) are
more immunogenic to their hosts than are indigenous micro-organisms of similar
types in the gastrointestinal tract (BERG & SAVAGE, 1972; SAVAGE, 1977; MOREAU et
al., 1978).
A large proportion ofthe starter organisms in cultured milks may survive the gas-
tric transit (SIEGENTHALER et a/., 1960; SCHULER-MALYOTH et a/., 1968 b; REUTER,
1969; LIPINSKA, 1978) and may thus compete beneficially with other gut bacteria.
Some organisms (L. acidophilus, B. bifidum, L. casei, L. fermentum) are better able
to survive in the intestinal tract than such others as L. bulgaricus, S. thermophilus,
S. lactis, and L. cremoris. The former are species commonly encountered in the intes-
tine, whereas the latterare associated with milk products.
Although the consumption of starter organisms may not induce major changes
of the gut flora, metabolic activity may be altered beneficially, particularly when cul-
tured milks are consumed regularly (cf. Chapter 5).
Industrial processes for the production of cultured dairy foods containing bi-
fidobacteria have been developed; examples include the following:
- Cultured milk fermented with B. bifidum, L. acidophilus, and S. thermophilus.
Mother culture of B. bifidum prepared from sterile milk with a large inoculum of
10% of B. bifidum(ScHULER-MALYOTH etal., 1968 b).
- Liquid food for infants prepared from sterile milk containing yeast autolysate, and
fermented to pH 5.9 with bifidus strains typical of breast-fed infants (SCHULER,
1972).
- Cultured milk, BIOGARDE, prepared with B. bifidum, L. acidophilus, and S.
thermophilus. Mother culture of B. bifidum, and bulk starter prepared from heat-
treated milk containing yeast extract as a growth-promoting substance (KLUPSCH,
1968).
- Supplementation of a yoghurt culture with L. acidophilus and B. bifidum in the
manufacture of a special yoghurt (MüLHENS & STAMER, 1969).
- Apreparation of B. bifidum together with a cream culture (ratio I : 1) in the pro-
duction of fresh cheese desserts for children (HRABOVA, 1975), and B. bifidum
together with L. acidophilus and Pediococcus acidilactici (ratio 45 : 10 : 45) in the
manufacture of dried milks forfeeding infants and children (DOLE:hLEK, 1979).
- Cultured beverage, Mil-Mil, produced using B. bifidum, B. breveand L. acidophi-
lus. This product also contains carrot juice and is sweetened with a small amount of
dextrose and I or fructose (YosHIOKA, 1980).
- Cultured milk produced using B. longum, L. bulgaricus and S. thermophilus
(YOSHIOKA, 1980).

6.3 Dairy foods containing bifidobacteria in the diet of adults

6.3.1 Nutritional significance

As mentioned already, cultured milks are more easily digested by humans than the
milk from which they are manufactured.

91
 Lactose-intolerant individuals may consume cultured milks without the gas-
trointestinal disturbances (abdominal discomfort, flatulence, diarrhoea). This is attri-
buted to the starter culture which reduces the Iactose content during fermentation.
The production of acids from Iactose contributes to the organoleptic quality of
the finished product and to its superior shelf-life. Each type of fermentedmilk; such
as, yoghurt, buttermilk, acidophilus milk, kefir, koumiss has its specific flavour. Milk
fermented with a culture of B. bifidum or other Bifidobacterium species has also a spe-
cific flavour, which can be described as clean, and mildly sour to slightly acetic. The
organoleptic qualities of cultured milks can be changed to some extent by including
such ingredients as fruit or fruit flavourings.
During the manufacture of cultured milks, changes in the vitamin content, parti-
cularly those of the B-complex, may occur depending upon such features as the type
ofmicrobial inoculum, incubation conditions, and processing. Some organisms con-
sume vitamins during growth, while others synthesize them. The folic acid content of
milk, for example, has been reported to increase significantly during fermentation
with streptococcal cultures admixed with and yoghurt cultures ( S. thermophilus and
L. bulgaricus) and to diminish during fermentation with L. acidophilus, with B. bifi-
dum (SHAHANI & CHANDAN, 1978; DREWEK & ROCZNIAK, 1978).
Biologie enrichment of cultured milks with vitamin B12 and folic acid using Prop-
ionibacterium freudenreichii subsp. shermanii showed highest degree of enrichment
with kefir, followed by streptococci cultured milks, and milks fermented with
L. acidophi/us and B. bifidum (CERNA & HRABOVA, 1977).
Milk and milk products may contribute about 74.6% of the calcium, 39.3 % of the
riboflavin, 35.0% ofthe phosphorus, 22.1% ofthe protein, 21.7% ofthe magnesium,

;:....
......
V; 150
<: MALES - 1036
Lu-... FEMALES c=J '280
ClE TOTAL 5316
Lu :::I
<: .!;: 125
OE
Q)-2

.•••
ll..cu
0 .....
0 100
'-<ll
<:=-:::
~E
(..)'-
~ 75
Lu
0
(..)

15 25 35 '5 55 65 75 85 95
AGE IN YEARS
Fig. 6.4 Effect of age on the bone density of "normal healthy" subjects
(ALBANESE eta/., 1978)

92
....c:
....CIJ
c:
0
(..)01

E ·~200
:::s ...
·- CIJ
~Cl)
111 ...
(..)Cb

~ ~100
EE
-~
a..
°Food
Group
Milk-
Dairy
Bread-
Cereals
Fruits-
Vegetables
Products White

Fig. 6.5 Calcium content ofthe four food groups

(ALBANESE eta/., 1978)

20.1% of the vitamin B12, 13.3% of the vitamin A, and significant amounts of other
constituents in the mixed diet (Milk Industry Foundation, 1979).
Nutritional deficits of calcium and iron have been reported to occur among old
people (SHANK, 1976), but clinical studies have shown that hone loss (osteoporosis), a
characteristic of the aging process, can be retarded by increasing the calcium intake
(Fig. 6.4). A calcium intake of at least one gram daily is recommended to maintain
normal bone density (ALBANESE et al., 1978), and the consumption ofmilk and dairy
products (particularly cultured milks) makes an important contribution of calcium in
a readily assimilable form (Fig. 6.5).
Cultured milks supplemented with iron arealsotobe recommended for the
elderly as weil as durlog pregnancy and convalescense.

6.3.2 Injluence on the gutflora

The composition of the intestinal flora is very stable in healthy adults. Current data
indicates that a dietary supplement of intestinal strains of lactobacilli or bifidobacte-
ria does not replace other intestinal bacteria, but it hel ps to maintain a proper balance
ofthe resident flora. These organisms have certain characteristics (resistance to unfa-
vourable conditions in the gastrointestinal tract) which enable them to survive for
some time, and possibly some multiplication, in the intestinal tract before excretion in
the faeces. Regular consumption of fermented milks containing these organisms
ensures that they are continually in.passage through the gut.

93
 ÜRLA-JENSEN and co-workers ( 1945) tried to establish bifidobacteria in the !arge
intestine ofthree elderly persons by feeding them with milk cultures of arabinose-fer-
menting strains of bifidobacteria. Each person consumed daily 400 ml of this milk
culture containing I 09 bacteria per ml.
Although no bifidobacteria were detected in the faeces before treatment, they
were found thereafter in increasing numbers. The bifidobacteria rapidly disappeared
from the faeces when the administration ofthe culture was discontinued.
The metabolic properties ofthe gut flora may be readily changed by the regular
consumption of dietary Supplements oflactobacilli and bifidobacteria, and there are
indications that various strains oflactobacilli exhibit some degree ofhost specificity.
L. acidophilus isolated from the human intestinal tract could not be established in the
intestines of chickens; only biotypes specific to a particular animal species could be
established (MITSUOKA, 1969 a; MORISHITA et a/., 1971).
Subsequent studies have revealed differences in the biochemica1 characteristics
of strains of L. acidophilus iso1ated from humans, pigs, and chickens. The guanine
plus cytosine (G + C) ratio for DNA of L. acidophilus strains isolated from humans
was lower than that of pigs or chickens ( GtLLILAND et al., 197 5 ; GtLLILAND, 1979).
Similarly, the biotypes of bifidobacteria differ between human infants and
adults, which suggests that the biotypes occurring in adults (e. g. B. bifidum biotype a,
B. longum biotype a, etc.) should preferably be used in dietary adjuncts for this age
group (MITSUOKA, 1972).
The regular consumption of dietary Supplements ofbifidobacteria and lactoba-
cilli may mitigate certain undesirable effects of some components ofthe gut flora. For
instance, the excretion of biogenic amines was less in weanling piglets receiving a
milk diet supplemented with Lactobacillus acidophilus than in the controls in which
diarrhoea was also more severe and prolonged (HILL et a/., 1970). Bifidobacteria also
prevented the formation of amines in the gut.
Experiments on rats have shown that supplementing the feed with L. acidophilus
reduced significantly the activity of the faecal bacterial enzymes, azoreductase and
nitroreductase, in animals on a high meat diet (GOLDIN & GoRBACH, 1977). These
enzymes are capable of reducing certain azo and aromatic nitrogen compounds to
potentially carcinogenic substances.

6.3.3 Effect on the immunosystem ofthe host

Current evidence suggests that micro-organisms indigenous to the gastrointestinal

tract are less immunogenic in their hosts than allochthonous micro-organisms of simi-
lar types (BERG & SAVAGE, 1972; SAVAGE, 1977; MOREAU et a/., 1978).
Administration of viable and non-viable mixtures of various bacterial strains of
intestinal origin to germ-free mice has shown that intestinal production of immuno-
globulin A (lgA) plasmacytes was greater than that following the implantation of a
single strain or species. Lactic acid bacteria capable of establishment in the intestinal
tract of mice were less immunogenic than the transitory strains (MOREAU et al., 1978).
A recent study of the comparative effects of diets, enriched respectively with
"live" and heat-treated yoghurt, on the response of the murine immunosystem has
shown that !arge inocula stimulate the immunological activity of the spieen and thy-

94
mus gland. Live bacteria were more effective than dead ones in increasing theserum
immunoglobulin G (IgGz.) (CONGE et a/., 1980).
Experiments on animals strengthen the supposition that regular ingestion of star-
ter bacteria (viable lactic acid bacteria and bifidobacteria) may induce an immuno-
genic response in humans.
However, the effect of starter bacteria on the immunosystems of their hosts
requires further investigation.

6.4 Therapeutic applications

6.4.1 Gastrointestinal disturbances

The use of L. acidophilus cultures, either in liquid ( acidophilus milk) or freeze-dried

forms (e. g. ENPAC, ACIDOPHILUS ZYMA), in the treatment ofthe side-effects of
oral antibiotics therapy and therapeutic irradiation has been weil documented (GoR-
DON et a/., 1957; MARGET, 1957; HAWLEY et a/., 1959; REUTER, 1969; SANDINE et
al., 1972).
Subsequently, B. bifidum has been used for the same purposes, but use of this
organism together with L. acidophilus seems preferable, since many intestinal dis-
turbances occur in the small intestine where lactobacilli are more numerous than
bifidobacteria. However, other lactic acid bacteria have also been used.
Beneficial results have been reported in treatment of intestinal disturbances after
antibiotic therapy (ßAMBERG, 1966), irradiation therapy (HALLER & KRÄUBIG, 1960;
NEUMEISTER & SCHMIDT, 1963 a), with the freeze-dried preparation OMNIFLORA,
containing viable B. longum, L. acidophilus and a saprophytic E. coli. Similar claims
have been made for a preparation containing L. acidophilus, B. bifidum, L. bulgari-
cus, S. thermophilus and S. lactis, in admixture with kaolin and aluminium hydrox-
ide (LEPERCHEY & FROTTIER, 1965).
Other intestinal disturbances have also been treated with preparations contain-
ing a culture of Bifidobacterium (STRASSBURG & Pooszus, 1960; HIRTZMANN, 1964;
LEPERCHEY & FROTTIER, 1965).
Bacterial preparations for therapeutic use should be administrated in appropri-
ate concentrations. For instance, the common dose of L. acidophilus ranges from 108
to 109 viable cells per day (GORDON et a/., 1957; REUTER, 1969; SPECK, 1976).
The significance of an appropriate dietetic regimen during the period of admini-
stration ofbacterial cultures has been pointed out by DEMOLE (1951 & 1965), and by
Vu~:EK & KNEIFL ( 1964).
Digestivetroublesare more apt to occur in elderly people. Theseare accompan-
ied by alterations in the gastrointestinal flora, resulting in diminution or disappear-
ance ofbifidobacteria from the faecal flora; and an increase of enterobacteria, clostri-
dia and enterococci.
ÜRLA-JENSEN and co-wokers (1945) have reported that the consumption offer-
mented milks containing acidophilus and bifidus bacteria may assist in re-establish-
ing the normal microbial balance, provided that the diet is not too rich in meat pro-
teins.
The administration ofbifidus milk to 12 patients (aged 67 to 92) with dyspepsia

95
has been reported to give good results (SAMEC, 1969). They were treated for 3 weeks (3
times daily for l 0 days, and thereafter once daily) with 150 ml portions of a reconsti-
tuted EU GALAN preparation containing viable B. bifidum (L. bifidus) bacteria in a
base consisting of 1.5% fat, 11.5% carbohydrate, 2.1 % protein, and 0.6% minerals.
Although no bifidobacteria were detected in the faeces before treatment, they were
detected thereafter.
In general, the nature of diet influences the intestinal health and the rate of aging.
High fat diets, forinstance, appearto be correlated with a decrease in life span, and an
increased incidence of colon cancer (HILL et al., 1971 ; STUNKARD, 1976).

6.4.2 Hepatic disease

6.4.2.1 Gut bacteria and portal encephalopathy

Gut bacteria produce potentially toxic products; such as ammonia, phenols, phar-
macologically active amines, and indole, which are normally detoxified in the liver
before excretion in the urine and faeces. In hepatic disease, the liver fails to detoxify
these products, which enterthe systemic circulation and may reach Ievels toxic forthe
brain. Consequently in advanced hepatic cirrhosis the derangement of the central
nervous system may range from mild mental aberration to coma. This is also a symp-
tom of portal systemic encephalopathy (KOSMAN, 1976), which may occur after por-
tal-systemic anastomosis, whereby ammonia and other toxic products escape detoxi-
cation in the Iiver and thus enter the systemic circulation.
Ammonia produced in the gut is the main contributor to hepatic coma; its main
source is from the hydrolysis of urea and, to a less extent, from the deamination of
amino acids.
Gut bacteria such as: enterobacteria, peptostreptococci, clostridia, and eubacte-
ria produce urease which hydrolyzes urea to ammonia and carbon dioxide. About
20-25% of the total urea production each day is thus hydrolyzed (VAREL & BRYANT,
1974). The ammonia formed in the gut thus passes viathe portal blood system to the
Ii ver where it is either converted into non-essential amino acids, or detoxified by the
resynthesis ofurea. The diseased liver, especially in cirrhosis, is unable to do this and
ammonia enters the systemic venous system resulting in hyperammonaemia and
intoxication.
The production of ammonia by the deamination of amino acids derived from
milk proteins is small compared with that produced from the blood proteins con-
tained in meat, and also meat proteins.

6.4.2.2 Lactulose and bifidus milk in treatment of chronic Iiver disease

Portal encephalopathy control requires measures which help to reduce the produc-
tion and absorption of intestinal ammonia and other potentially toxic nitrogenous
substances (DRASAR & HILL, 1974). The following measures are the more important:
- Dietary protein restriction
- Bowel cleansing including the use of enemas and purgatives

96
- The administration of a broad-spectrum antibiotic, e. g. neomycin
- The administration of lactulose and bifidus milk
The purpose of dietary protein restriction is to reduce the production of ammo-
nia and other nitrogenaus compounds in the intestine. However, patients are usually
very sensitive to a reduction of protein intake, but in hyperammonaemia the protein
intake may be reduced temporarily to 30 g I day (MüTING, 1977).
Bowel cleansing is used in cases of intestinal bleeding to remove rapidly blood
from the gut, and the administration of neomycin is recommended in acute exacerba-
tion of hepatic encephalopathy to reduce the number of ureolytic organisms in the
gut. However, the long-term use of a broad-spectrum antibiotic causes side-effects
due to disturbance of the intestinal flora and diarrhoea.
Lactulose is preferable for long-term use because of its non-toxicity.
Lactulose (4-0-ß-D-galactopyranosyl-D-fructose) is a ketose produced from Iactose at
an alkaline pH and in heated milk. Although the preparation of lactulose was first
described by MoNTGOMERY and HuosoN (1930), its bifidogenic effect was reported
only much later by PETUELY ( 1957 a,b) and others (see Chapter 4).
BIRCHER and co-workers ( 1966) were the first to use lactulose in the control of
portal systemic encephalopathy. These authors observed that lactulose administered
in two patients produced an improvement in their condition, with a significant
decrease in blood ammonia and a reduction in the pH ofthe faeces.
Subsequent investigations confirmed the beneficial effects of lactulose in the
treatment of liver cirrhosis with or without portal encephalopathy (MünNG et al.,
1967 & 1972; AVERY et a/., 1972; FESSEL & CONN, 1973; ÜRDNUNG & MüTING,
1973; MüTING, 1977).
Since lactulose is not hydrolyzed by the beta-galactosidase ofthe small intestinal
mucosa, and is poorly absorbed from the small intestine, it reaches the colon where it
undergoes bacterial degradation. Bifidobacteria, lactobacilli, and enterococci fer-
ment this sugar to lactic, and acetic acids, thereby reducing the colonic pH. At a low
colonic pH, much ofthe ammonia is in the form ofthe ammonium ion (NH/) which
is not absorbed as is its non-ionized form (NH3) by passive diffusion. The altered
equilibrium ofthe non-ionized and ionized forms of ammonia may influence moving
of ammonia from the blood to the colon, thereby reducing the blood ammonia Ievel.
Organic acids produced in the colon stimulate intestinal peristalsis and induce soft
stools.
The administration oflactulose may assist in re-establishing the normal gut flora,
which is usually disturbed in advanced cirrhosis. The number ofGram-positive bacte-
ria, including bifidobacteria, lactobacilli, and enterococci may be increased, while
other as enterobacteria, clostridia, and staphylococci may be reduced.
The re-establishment ofthe normal microbial balance is acompanied by a reduc-
tion of ammonia and free phenols in the blood (Fig. 6.6).
Another characteristic of this regimen isthat it allows the patient to increase pro-
tein intake (to 70 g per day), which is normally restricted.
Lactulose is administered as a 50% (w I w) syrup after meals. The dose is
adjusted so as to induce two soft stools a day, under which conditions the faecal pH
reaches 5-6 or less. It is important to commence treatment with small doses, e. g.
3 x I 0 g per day, followed by a gradual increase to 3 x I 00 gor better 5 x 60 g per
day (MÜTING, 1977).

97
 I" gl
100m/ Before During ' Weeks after mg%
LIJctulose Lactulose Lactulose
200
Ammonia
I 2.0
Free Serum
Phenols 7. 6

7.2

0.8

~0 0.1.

Coli-
Group

1d Lactobacl7/i

1cf

Fig. 6.6 The effect of lactulose on the gut bacteria and toxic nitrogenaus substances in liver cirrho-
sis (average values for 12 patients)
(ORDNUNG & MüTING, 1973)

Bitidus milk may prove beneficial for protein metabolism in liver cirrhosis
(M ÜTING et al., 1968 a,b ). Thirty-three patients (3 older children and 30 adults) with
various degrees of liver cirrhosis were given a reconstituted bifidus milk preparation
EUGALAN FORTE during an average period of 100 days, but some received the
preparation for up to 2 years. The dehydrated preparation contained 10% protein,
84% carbohydrate including lactulose, and 3% minerals, also viable B. bifidum bac-
teria. Treatment began with an initial dose of 3 x 10 g per day, which was subse-
quently increased to 3 x l 00 g daily. The authors observed that this bifidus milk pre-
paration produced an improvement in the patients, and decreased the ammonia, free
phenols, and indican in the blood; also it reduced faecal pH, and increased.the bifi-
dus content (Fig. 6.7 and 6.8).

98
 Blood Ammonia
3x70 311.100 3~t70 3-70 3 1'100g
!"91 ~
10 0 m I
250
illillllillllff llllllllllllllllllll 1I1 llllllllllllllllllllllllll
A bifidus-milk preparation

200

100

50

Free Serum Phenols

mg/100ml
2.0

8. bifidum in the faeces

+++
++
+
21.12 26.1 7.3 11.5 21.7 10.3 23.5
13.12 5.1 10.2 23.3 2.6 30.8 M.2 18., 20.7
1965 1966 1967

Fig. 6.7 The long-term treatment of decompensated Iiver cirrhosis with a bifidus-milk preparation
EUGALAN FORTE (12 years old patient)
(MÜTING eta/., 1968b)

99
 'Y'!~ ~ngir;_a!} _
,.,g/ 1n 2'h - - -
IOOml Urine

"
0
t30 ~~~
110 ~
ammonia
90

70

so ............ ""."' "' "

............._,"' ml Urine
30 1500
wo
10 500
3QB ~.9 18.9 20.9 26.9 ~.10. {)10 17.10
Fig. 6.8 The influence of a bifidus-milk preparation EU GALAN FORTE on the blood ammonia
and indicanuria in decompensated Iiver cirrhosis (46 years old patient)
(MÜTING eta/., 1968b)

A combination oflactulose and bifidus milk for the dietary management of Iiver
cirrhosis has been proposed (ORDNUNG & MOTING, 1973; MüTING & ORDNUNG,
1976). It is important to use non-urease producing strains of B. bifidum in the manu-
facture of bifidus milk preparations. The superior digestibility of bifidus milk com-
pared with unfermented milk from which it is made, enables the patient to maintain
bis protein intake.
However, neomycin should temporarily be administered to patients, iflactulose
and I or bifidus milk fails to reduce blood ammonia in portal encephalopathy (Fig.
6.9).
The initial dose of neomycin, 6-8 g per day, is reduced upon clinical improve-
mentto 2-3 g per day; and simultaneously lactulose (and I or a bifidus-milk prepara-
tion) is administered in increasing amount. Underthis regimen dietary protein intake
is restricted during the administration of neomycin, but restored upon the introduc-
tion of lactulose or a bifidus-milk preparation.
A recent report shows that the urease inhibitor isolated from Cucumis melo
seeds decreases the urease activity of intact rumen microbes in vitro, and inhibition
reached 50% at an inhibitor concentration of 3.0 mg I ml (MAKKAR et al., 1980). The
inhibitor appeared to be only slightly sensitive to hydrolysis by pepsin, trypsin, and
other proteases. MALHOTRA and RANI ( 1978) have reported that the inhibitor is a pep-
tide.
It may be worth to investigate the possible effect of this inhibitor on the urease of
human-gut bacteria, particularly in subjects with Iiver cirrhosis.

100
 Faeces- pH 9
...
8 ~8--~8--~--7~---
8 10 12
7 ~__5.-~_
1t
5 ___ ,_5--~~~~~
16 20 2t
0 2 5 ?2 26 Days

Fig. 6.9 Scheme of portal encephalopathy therapy

(MÜTING, 1977)

6.4.3 Prophylaxis and therapy of intestinal disturbances in domestic animals

The administration of lactic acid bacteria and bifidobacteria to domestic animals,

especially pigs, for the prophylaxis and therapy of intestinal disturbances has been
the subject of some reports.
L. acidophilus preparations have been reported to reduce scours and enteritis in
pigs (PASICNYJ, 1959; REDMOND & MOORE, 1965; STERN & STORRS, 1975) and to
counteract diarrhoea in weaning pigs (50 g of a freeze-dried L. acidophilus prepara-
tion given daily for l 0 days) (OLSSON, 1961 ).
Cultures of B. bifidum have also been used in the treatment of enteric disturb-
ances in pigs, calves, and dogs (ROBERT, 1971 ). The results indicated that the oral
administration of a freeze-dried culture of B. bifidum may correct a disturbed intesti-
nal flora attributable to a faulty dietary regimen rather than disturbances due to infec-
tion or functional disorders, although B. bifidum may be used as supportive treat-
ment in the last two conditions. The author concluded that the prophylactic efficacy
ofbifidobacteria may be superior to their therapeutic value.
Moreover, the prophylactic administration of a preparation containing anti-
biotic-restistant lactobacilli, such as L. acidophilus, L. lactis and L. salivarius, also
Bifidobacterium spp., has been reported to reduce considerably the mortality of
treated animals (VARGA & MESZAROS, 1974).
The use of animal strains of bifidobacteria for the prophylaxis and therapy of
intestinal disturbances in domestic animals may be preferable.

101
Chapter 7: Dairy preparations, manufacture and
technology

Bifidobacteria were used the first in 1949 by Mayer in the making of baby foods.
Later, SCHULER-MALYOTH and co-workers (1968 b) showed that bifidobacteria also
can be cultivated in the dairy.
Cultured milks containing bifidobacteria possess the following nutritional and
technological advantages: a) A mildsourtaste; b) The limited ability of after-acidifi-
cation, including that occurring during breaks in the refrigeration chain; c) An occur-
rence ofbitter taste more rare than in other cultured milks; d) As intestinal organisms,
bifidobacteria may be used in manufacturing both dairy foods and pharmaceutical
preparations; e) The formation ofphysiological L ( + )-lactic acid occurs; f) A parti-
cular suitability for manufacturing fermented beverages; g) The nutritional-physio-
logical properties typical for cultured milks (RAS1c & KURMANN, 1978).

7.1 Basic manufacture and technology of set and stirred bifidus milk
products

Fig. 7.1 gives a review of the various steps of the manufacture and technology of cul-
tured milks including bifidus cultured products. There are specific procedures within
each of these generat steps in using bifidobacteria.
In order to avoid overlapping with our Yoghurt monography (RASIC & KuR-
MANN, 1978), the following reference will be made in discussing the technology:
"Yoghurt monography (page ... ) ".
The major differences and problems of manufacturing cultured milks contain-
ing bifidobacteria compared with those of yoghurt may be summarised as follows: a)
The cultivation of bifidobacteria in milk, und er routine conditions in the dairy, is
more difficult; b) The inoculum is much larger and accordingly the amount of bulk
starter is larger; c) An altered milder taste is present; d) Non-acid fast bacterium
results in slow acidification and weak consistency and viscosity of the finished pro-
duct; e) The bacteria dies more quickly at lower pH values during storage; f) The total
solids content including buffering capacity (points d and e) increases; g) Aseptic
working is preferred because of Ionger incubation time and slower acidification.
Bifidobacteria alone have been used comparatively rarely in making cultured
milks. They are often used together with L. acidophilus and S. thermophilus and
accordingly called a mixed flora containing bifidobacteria or a mixed culture, type
BAT (bifidus, acidophilus, thermophilus ).

7.1.1 Raw cow's milk and reconstituted milk

General requirements of the quality of raw milk, including reconstituted milk, have
been thoroughly discussed in the Yoghurt monography (pp. 140--147 and pp.
326-328). The influence of the milk flora on the growth of bifidobacteria has been

102
 Pre- treated milk

Starter culture
preparation

Set Stirred

Flavouring lncubation

Fit/ ing Cooling

lncubation Flavouring

Cooling Filling

Chili
Fig. 7.1 A review ofthe various steps in the manufacture and technology of cultured milk products,
including bifidus cultured milks.

discussed by MERGEL (1978) and KosiKOWSKA (1979). Fig. 7.2 gives schematic dia-
gram of the recombined milk process which occurs in many countries. The milk
stored in vats should be tested for the presence of inhibiting substances (antibiotic res-
idues, etc.) before it is used in this manufacturing process.

7.1.2 Treatment of milk in the dairy

Treatment of milk in the dairy is similiar tothat used in manufacture of yoghurt.

103
 1

DRY AREA. WET AAEA.

WALL

Fig. 7.2 Recombined milk process. Equipment for milk powder reconstitution.
(FIL/IDF, 1979)

7 .1.2.1 Clarification of milk

This process has been described in Yoghurt monography (pp. 149; 177-178).

7.1.2.2 Adjusting the milk composition

In order to avoid defects in the consistency and viscosity and to increase the buffering
capacity of the finished product, the total solids content is increased to 15-20% (cf.
MüLLER, 1976); however, the total solids content of 30% is inhibitory to bifidobacte-
ria (PEcH et al., 1974 a). Evaporation ofthe milk isthebest method ofincreasing the
solids content (see Yoghurt monography, pp. 160-162, 181 and 326). Evaporation of
the milk by 10-20% increases the so1ids content by 1.5-3.0%.
The adjustment of the non-fatty solids content may also be done with concen-
trated skim milk or by adding skim milk powder (See Yoghurt monography, pp.
154-156 and 178-180). One per cent of skim milk powder contains 0.92% oftotal so!-

104
ids. According to MüLLER ( 1976) skim milk powder is usually added to whole milk at
a rateof2%.
The fat content in whole milk which is used in processing should be at least 3.5%
andin skim milk not more than 0.5% with a corresponding increase in non-fatty sol-
ids. The influence ofthe fat content on the taste and consistency ofthe finished pro-
duct has been discussed in the Yoghurt monography (pp. 327-328).

7.1.2.3 Additions before heat-treatment ofthe milk

Various additions do not affect flavour and colour ofthe milk. The use ofmilk pow-
der or skim milk concentrate has been mentioned. The additions of caseinates, co-pre-
cipitates or whey protein preparations to improve consistency have been treated in
the Yoghurt monography (pp. 156-157 and 337-339). The use of sugar and sweeten-
ers has been discussed in the Yoghurt monography (pp. 102-103; 157-158 and
328-329).

Fig. 7.3 APV Gaulin homogenizer Type MC 18. Aseptic applications

(APV Ltd., Sussex, Eng/and)

105
Fig. 7.4 Milk processing plant. Background right: H & K-Junior evaporating plant with automatic
control. Foreground, left to the right: H & K-plate heat-exchanger for pasteurization of milk, isolated
tubular holding unit, and homogenizer
( Holstein & Kappert GmbH, Dortmund, W. Gerrnany)

The addition of growth-promoting substances (cf. Chapter 4 and 7 .1.3 .4.1)

should be avoided if possible and is dependent upon the following conditions: a) The
selected strain of bifidobacteria; b) The desired rate of acid production within the
defined incubation time; c) The eventual necessity of preventing the growth of con-
taminants by faster growth of bifidobacteria; d) The addition of !arge enough
amounts of growth substances during the preparation ofbulk starter, since they are in
turn transferred to the production milk; e) The intensity ofthe heat-treatment ofthe
milk; f) Large enough amounts ofinoculum; g) The kind ofproduct which is manu-
factured (e. g. baby food).

7.1.2.4 Homogenization of milk

Consistency, taste and digestibility ofthe finished product are improved by the hom-
ogenization of milk, e. g. 150-200 bar at 60° C (MüLLER, 1976) (cf. Yoghurt mono-
graphy, pp. 163-170; 181-183 and Fig. 7.3).

7.1.2.5 Heat treatment of milk

Milk is heated to90-120° C (MüLLER, 1976)to destroymostofthe germs as weil as to

denature whey proteins, thus improving consistency and viscosity of the product (cf.
Fig. 7.4).

106
 According to MAYER ( 1948) heat-resistant germs proliferate more quickly than
bifidobacteria. Heat treatment of the milk has been discussed in more detail in the
Yoghurt monography (pp. 170-176). UHTtreatment ofthe milk or Sterilisation may
also be applied (Evoa, 1965 a).

7.1.2.6 Additions to milk after heat treatment

Since the flavour of milk products containing bifidobacteria is not always acceptable
to consumers, various flavours, e. g. strawberry, raspberry, vanilla and apricot may be
added (cf. Fig. 7.5).
Sterile flavour concentrates may be added·either before or after heat treatment of
the milk(See Yoghurt monography, pp. 176-177 and 330-331).

Fig. 7.5 Aavour development laboratory for milk products and foods. Development of flavours
using the exactly dosed mixture of more than 50 chemical basic substances for each flavour
(Givaudan Dübendorf AG, Dübendorf, Switzerland)

107
7.1.3 Cultures and starters

The selection of a proper culture ofbifidobacteria as weil as its cultivation and main-
tenance is especially important (WENGER, 1976).

7.1.3.1 Criteria for the examination and selection of a pure bifidus culture.

Strains of the species B. bi.fidum have been those most often used. In some cases,
those of B. longum have been used as well.

7.1.3 .1.1 Selection and examination according to technological criteria

This heading deals with the examination ofbifidobacteria strains as regards the most
important technological characteristics.

7.1.3 .l.l.l Acid production

The rate of acid production (1actic and acetic acids, cf. Chap. 3.2) is an important
characteristic of bifidobacteria strains. There are considerable differences among
various strains in acid production (ßROWN & TOWNSLEY, 1970; ßORISOV A & SLIVKO,
1973; KlSZA & ZIAJKA, 1973; KOSIKOWSKA, 1978) as shown in Fig. 7.6.

.'\
... ~
~

~.~
·~:\
'·
5.0
'·,..,'~~'".-- .
.,
4.5 A --·----491
'·~··-.............. 961
""' ~··-· ·-1060
··-""-·
~~--.557 ·~3

12 24 36 1,8
Hours
Fig. 7.6 Alteration of the pH value of sterilized cows' milk by different stains of B. bifidum during
48 hat 37° C. Inoculum I 0%.
Origin of strains (designation):
491,557,961 and 1060: PZH Warsaw
J: Institute of Immunology and Virology, Belgrade
(K.!SZA & ZIAJKA, 1973)

108
 The examination of acid production of different strains is usually carried out by
consulting the acid curves, i. e. the titratable acidity detennined during incubation at
different time intervals to detennine the rate of acid production (cf. Yoghurt mono-
graphy, pp. 188-189).

7 .1.3.1.1.2 After-acidification (Over-ripening)

After-acidification refers to acid production during cooling and cold storage. The fol-
lowing factors influence after-acidification of cultures: a) The final pH value of acid-
ified milk may considerably vary, depending on the strain used, e.g. pH 4.0-3.7
(WEINBERG et al., 1937), pH 3.5 (KiszA & ZIAJKA, 1973), pH 3.3 (ÜRLA-JENSEN,
1943). In order to prevent over-acidification of a cultured milk during insufficient
cooling and short-tenn storage, the final pH of selected strains should not be below
4.3; b) According to SCHULER-MALYOTH and co-workers ( 1968 b) bifidobacteria do
not show any metabolic activity below 15° C.

7.1.3.1.1.3 Taste and aroma

Cultures ofbifidobacteria in the milk give rise to a typical aroma and taste (mild, aro-
matic, slightly spicy and distinct from yoghurt flavour according to SCHULER-MALY-
OTH et al., 1968 b; cf. Chap. 3.2), not comparable with other types of cultures. Strains
used in manufacturing cultured milks do not produce diacetyl, acetaldehyde and ace-
toin ( Laboratory Wiesby Niebüll, 1980). The organoleptic testing of strains is neces-
sary in order to exclude strains with undesirable characteristics.

7.1.3.1.1.4 Requirements for growth substances

It is desirable to use strains that are able to proliferate nonnally in cow's milk without
adding growth substances. This is controlled by the comparative cu1tivation of
examined strains in cow's milk with and without growth substances added.

7.1.3.1.1.5 Sensitivityto oxygen

The sensitivity to oxygen varies from not very sensitive to strictly anaerobic ( cf. Chap-
ter 2.3.1). Freshly iso1ated strains of B. bifidum may be anaerobic but after severa1
transfers in the milk they may show certain oxygen tolerance (ORLA-JENSEN, 1943;
HRABOVA, 1975). lt is desirable to select strains capable of growing without special
anaerobic cultivation, e.g. strains used by SCHULER-MALYOTH and co-workers
(1968 b).

7.1.3.1.1.6 Mucoid strains

Some strains ofbifidobacteria produce mucus substances which may improve consis-
tency and viscosity ofthe finished product (cf. Chapter 2.4.3). Mucoid strains may be
incorporated in a culture.

7.1.3.1.1. 7 Biotechnogram (Biotechnological characteristics)

The selected strains may be additionally examined for the following characteristics:
minimum, optimum and maximum growth temperature; minimum, optimum and
maximum pH value f or growth; minimum inoculum tobe used in processing; the rate
of survival during drying at different temperatures and pH values for different inter-
vals oftime; tenfold reduction ofthe thennal death time at different temperatures and

109
pH values for different intervals oftime in buffer solutions and skim milk powder (cf.
Table 7.6); and the production ofvolatile fatty acids.
The tabulated results of these examinations (average values of at least three tests)
represent the so-called biotechnogram. This should be consulted by technicians
engaged in the manufacture of cultured milks containing bifidobacteria.

7.1.3.1.2 Selection and examination according to dietetic-therapeutic criteria

This heading deals with the possible selections ofbifidobacteria strains according to
dietetic-therapeutic criteria.

7.1.3.1.2.1 Microecological parameter of selection

The isolation ofbifidobacteria strains from the faeces ofhealthy milk-fed babies or
small children is an important criterion of selection and frequently the only one possi-
ble. Examples of such isolation for dairy use may be found in papers by SEMENIKHINA
(1967); KYOWA HAKKO KOGYO(l970); KtszA&ZIAJKA(l973); ZIAJKAandco-work-
ers (1974).

7 .1.3.1.2.2 Examination in-vivo

For therapeutic use etc., the isolated strains are often examined in-vivo to note the
effect either in humans or in animals.

7 .1.3.1.2.3 pH-resistance in gastricjuices

Methods oftesting bacterial resistance in gastric juices at different pH values and dur-
ing different intervals oftime (up to 120 min) have been described by Gianella and co-
workers (1972) and subsequently applied by LIPINSKA (1978). Table 7.1 shows a dis-
tinct effect at pH 4.0 and a strong bactericidal effect at pH 2.0 on the four strains of
bifidobacteria investigated.
A simple resistance test may be made in artificial gastric juices at pH 3.0 and
maintained at 37° C through the test period (Yakult Honsha Co. Ltd., 1971).

7 .1.3.1.2.4 Biliary salts resistance (sodium deoxycholate)

Determination of the survival rate in the presence of 0.05, 0.2 and 0.3% of sodium
deoxycholate has been made by CATTEAU and co-workers (1971). LtPINSKA (1978)
examined different strains of bifidobacteria using this method. Higher concentra-
tions of deoxycholate (0.2; 0.3 %) bad a bactericidal effect on the investigated bifido-
bacteria. In the normal caecum, the concentration of deoxycholate varies between 0.5
and 0.2% (CATTEAU et a/., 1971).

7 .1.3.1.2.5 Antibiotic sensitivity

Sensitivity to antibiotics is variable. LtPINSKA ( 1978) recommended testing the sensi-
tivity to therapeutic antibiotics by using methods proposed by theSerum and Vaccine
Institute Warsaw (1976).

7 .1.3.1.2.6 Antagonistic effect

The antagonistic effect ofbifidobacteria has been discussed in Chapter 2.11.4. REHM
(1970) patented a method of bacterial inoculation and cultivation to maintain the
antagonistic effects of B. bifidum and L. acidophilus. This method consists in culti-

110
Table 7.1 Survival ofbifidobacteria in solutions buffered at different pH

Lbc. Temp Number and percentage of surviving bacteria per I cm 3

bifidus of of solutionbuffered at pH
strains action
min. 6.5 4.0 2.0

number % number % number %

X !0 7 X 107

0 5.3 100.0 5.3 100.0 5.3. 107 100.0

15 5.2 98.0 3.3 62.7 1.4. 107 27.0
J 30 5.3 100.0 2.9 54.1 1.2. 1o•
60 5.4 101.8 2.5 46.4 2.3. 102
120 5.3 100.0 2.2 40.9 2.0 • 10 1

0 4.6 100.0 4.6 100.0 4.6. 107 100.0

15 4.3 93.5 2.9 62.5 5.5. 106 12.0
62 30 4.1 89.1 3.3 70.8 0
60 4.5 97.8 2.8 60.9 0
120 4.5 97.8 2.9 63.4 0

0 3.8 100.0 3.8 100.0 3.8. J01 100.0

15 3.5 92.1 2.7 70.3 1.2. 106 3.1
6459 30 3.8 100.0 2.6 68.0 7.6. 103
60 3.7 97.4 3.0 80.1 0
120 3.0 79.0 3.1 82.1 0

0 1.8 100.0 1.8 100.0 1.8. 107 100.0

15 1.9 105.6 1.3 70.2 3.4 • 105 1.9
567 30 1.8 100.0 1.3 69.2 3.0 • 10 1
60 1.8 100.0 1.7 94.4 1.8. JOI
120 2.0 111.1 1.4 80.0 10 1

0 5.0 100.0 5.0 100.0 5.0. 107 100.0

Mixed 15 5.1 102.0 3.6 70.9 7.5. 106 15.0
culture 30 4.9 98.0 3.6 71.4 5.0 • 10 1
60 5.0 100.0 3.6 71.4 0
120 5.1 102.0 3.4 68.0

Origin of strains (designation):

567 and 6459: Pasteur Institute Paris
J: Institute of Immunology and Virology, Belgrade
62: Institute ofFood and Nutrition, Warsaw
Mixed culture: Mixture of the above four strains
Source: LIPINSKA (1978

vating acidophilus and bifidus strains in culture media containing living or dead cul-
tures of pathogenic bacteria.

7.1.3.1.2. 7 Urease production

There areureasepositive and negative strains of B. bifidum;it is important to select
only urease-negative strains for therapeutic use (see Chap. 6, sect. 6.4.2.2).

111
Fig. 7.7 Photomicrograph of Lactobacil/us acidophilus. The intestinal bacterium, often incorpo-
rated into mixed cultures containing bifidus bacteria. Its acid production is slowerthan that of L. bul-
garicus, but it grows better in milk than bifidobacteria. It is used in the therapy like bifidobacteria
(Chr. Hansen's Laboratory A/S, Copenhagen, Denmark)

7.1.3.2 Selection of mixed and complementary cultures containing

bifidobacteria

Non-bifidobacteria strains present in mixed and complementary cultures containing

bifidobacteria should not lower pH value of the milk below 4.3 (see Chapter
7.l.3.l.2).

7.1.3 .2.1 Mixed cultures containing bifidobacteria

It is advantageaus to cultivate bifidobacteria tagether with other intestinal organisms
such as L. acidophilus (Fig. 7. 7).
S. thermophilus (Fig. 7.8) or P. acidilactici (HYLMAR, 1978) is usually incorpo-
rated into a mixed culture to help acidification.
There are various types of mixed cultures containing bifidobacteria. A type BAT
(bifidobacteria, L. acidophilus and S. thermophilus) is most often used. Separate and
individual cultivation of selected strains as monoculture is the best.

112
Fig. 7.8 Photomicrograph of Streptococcus thermophilus. lt is often incorporated into mixed cul-
tures containing bifidus and acidophilus bacteria to enhance acidification and toshorten the produc-
tion time for cultured milk products. It is non-intestinal bacterium, grows fast in cows' milk, but its
acid production is weaker than that of other lactobacilli (e. g. L. bu/garicus, L. lactis and L. he/veticus)
(Federal Dairy Research Institute, Liebefeld/ Bern, Switzerland)

7.1.3.2.2 Complementary cultures (without bifidobacteria)

The mucoid strain of S. thermophilus is occasionally added to a mixed culture to
improve the consistency of cultured milks containing bifidobacteria. As mentioned it
is possible to use mucoid strains ofbifidobacteria too. Furthermore, butter culture ( S.
cremoris and L. cremoris) may be occasionally added to improve flavour; or small
amounts ofyoghurt culture (0.01 to 0.1 %), to increase acid production.

7 .1.3.3 Source of supply and maintenance of strain- or mother culture

Various strains ofbifidobacteria may be mixed according to the following criteria: a)

MUTAI and co-workers (1978 c) mixed obligate anaerobic and oxygen-resistant
strains at a ratio of 1:1 to 3 :1, thus making it possible to cultivate oxygen-sensitive
strains; b) Mixing strains of different biotypes (MüLLER, 1976).

7.1.3.3.1 Sourceofsupply
Purestrains ofbifidobacteria may be obtained either from institutes, etc., or by isola-
tion and selection from suitable materials (See Appendix 3). A photomicrograph of a
culture ofbifidobacteria is shown in Fig. 7.9.

113
 f

Fig. 7.9 Photomicrograph of a culture ofbifidobacteria in cows' milk

(SCHULER-MALYOTH eta/., 1968b)

7.1.3.3.2 Culture medium for propagation

Reconstituted sk.im milkpowdersterilized at 110-115° C for 15 min. maybe used as a
medium forthe transfer of cultures. Yeast extract (or other growth substances) may be
added at a rate of0.5% to promote the growth ofstrains (see Chapter 7.1.3.4.1).

7.1.3.3.3 Conditionsfortransfer
Coltures should be aseptically transferred. MA YER (1948) recommended transfer
every three days.
Freeze-dried cultures should be transferred at least once before use. Fig. 7.l 0
shows an example of the rate of acidification of such a culture.

7.1.3.4 Bulkstarter

lt is desirable to use the aseptic preparation ofbulk starter (cf. Fig. 7.ll ). lt is also rec-
ommended to avoid extremely low pH values during culture preparation.

7 .1.3 .4.1 Culture medium for propagation

The milk ofuniform composition (high total solids) is heated at 90-95° C for 15-30
min., followed by cooling to the incubation temperature. The addition of growth sub-
stances may be used to promote the growth of bifidobacteria and acid production
(EvoG, 1965 b; MüLLER, 1976). Growth substances not used by the bifidobacteria

114
during the preparation of bulk starter are transferred to the production milk (see
Chapter 7.1.2.3).
Growth-promoting substances for different types of bifidobacteria have been
described in Chapter 4. In many cases, a mixture of growth-promoting substances is
used, e. g. a mixture suggested by the Labaratory Wiesby Niehüll ( 1980).
Yeast extract and yeast autolysate have been most often used; however, they do
not always demoostrate desirable growth-promoting effect. The amount added varies
between 0.1 and 0.5 %, and no adverse effect on the flavour of cultured milks has been
noted (EvoG, 1965 a). The addition of glucose at a rate of 1-5% in conjunction with
yeast extract may further shorten the coagulation time (EvoG, 1965 a). The addition
of 20% of pepsin-digested milk promoted the proliferation of bifidobacteria isolated

pH

60
.......
',
''
''
' '\
\
\\
50 \
''
' ',
''
'
' ' ,,
' ....... ........

40•-------.------.- -----.,------,------ T------,--

---- -- ---
o 2 3 5 6
Hours
Fig. 7.10 Acidification of bulk starter B. bifidum after direct inoculation of a freeze-dried culture
VISBYVAC (2.5 g/500 ml ofmilk, after6 h at42°C, pH 4.60) and aftertransfer(IO% ofinoculum, 6
hat 42°C, pH 4.60). Culture medium is skim milk (II% total solids, heated to 95°C for40 min) with
the addition 1.5% of Bios 2000 (mixture of growth substances, Wiesby)
Legend: _ _ Directset
_____ Firstpropagation
(Laboratory Wiesby Niebü/1, 1980)

115
from the faeces ofinfants (ZIAJKA et a/., 1974; KiszA et a/., 1974). Various mixtures
of growth substances for milk cultures (EvoG, 1965 b; ScHULER, 1971 ; 1972), includ-
ing one on the basis of amino-carbonyl reactions products (Kyowa Hakko Kogyo Co.
Ltd., 1970) have been described.

7.1.3.4.2 A culture ofbifidobacteria

lt is important to obtain a large number of bacterial cells and a good rate of acid pro-
duction. In order to prevent darnage to bacterial cells and acid production, pH values
should not be below 5.0-4.7 and the numberofbacteria should be at least 108 perml.
Fig. 7.10 shows an example of acidification of a freeze-dried culture cultivated in the
laboratory.

CIP

Sterileair

Milk
Culture
Fig. 7.11 Equipment and simplified flowchart for aseptic preparation of starter production
I lncubator
2 Culture Container
3 Bulk startertank
4 Sterile air filter
5 Air/CIPvalve
(Aifa-Laval, Tumba, Sweden)

116
7.1.3 .4.3 The mixed cu1ture, typeBAT containing bifidobacteria

Fig. 7.12 gives a photomicrograph of a mixed culture containing bifidobacteria.

Changes of acid and bacterial counts are shown in Fig. 7 .15. Mter four hours of incu-
bation B. bifidum shows the lowest concentration when compared with S. thermophi-
lus and L. acidophi/us (SCHULER-MALYOTH et a/., 1968 b).
A possible growth suppression of the bifidobacteria due to the different rate of
multiplication ofvarious strains represents the greatest problern in the cultivation of
bifidobacteria in a mixed culture. There are two possibilities to ensure the presence of
bifidobacteria in a mixed culture: a) The use of monocultures (inoculation of the pro-
duction milk with monocultures ( cf. Fig. 7.13); b) The use of a concentrated deep-
frozen culture, which may be applied in the direct preparation 500-1000 I ofbulk star-
ter. Thus, the preparation ofa culture is simplified (see Fig. 7.14 and a model process
plan in Table 7 .2).

Fig. 7.12 Microflora of BIOGARDEsour milk containing

Streptococcus thermophilus, Lactobacillus acidophilus and Bijidobacterium bijidum
(Biogarde-Company, c/o evog, Balzers, Principality Liechtenstein)

117
 Streptococci 7h at 42°C

Acidophilus 24 h at 42°C ~!,hat 42°C 3 h at !,2 oc

Bitidus !, h at 42 oc /
(1) ( 2) (3)

Fig. 7.13 Scheme of the manufacture of a cultured milk containing bifidobacteria, type BAT (B.
bifidum, Lb. acidophilus, S. thermophilus) using monocultures of different bacterial species, mixing
them at a ratio I: I: I in the preparation ofbulk starter, followed by manufacture of a cultured milk.
Legend: (I )Monocultures
(2) Bulk starter(mixed culture)
(3) Production
(SCHULER-MALYOTH et a/., 1968b)

Table 7.2 Model processing plan for the preparation of bulk starter using direct inoculation of a
concentrated deep-frozen mixed culture of B. bifidum, Lb. acidophilus and S. thermophilus1)

Timetahle Processing parameter Product Desired production

recommended parameter

Culture milk pH6,8-6,6

Skimmilk + 2%non-fat SH6,6-7,0
milk powder + 1-1 ,5 % void of antibiotics
Bios2000
Pasteurization 90-95 oC
Keeping warm
30-45 minutes
Cooling44-42 oc
Culture milk
Ohs. lnoculation with 70 ml
MSKFERMOVAC®
7-Shs lncubation, temperature
44-42°C
lncubation, finish pH4,8-4,5
Bulkstarter
Cooling to 4 oc
Bulkstarter pH4,6-4,4

') called MSK, FERMOVAC. 70 gofFERMOVAC per 500 I ofskim milk (II %oftotal solids) with
added 1.5% of growth activator Bios 2000
Source: Laboratory Wiesby Niehüll ( 1980)

118
 1

§25'C
0.5% Ch/orine
·n 15 Min.
2 5

3
Fig. 7.14 The use of concentrated and deep-frozen bacterial cultures for preparation of different
bulk starters.
(Pallasdies, 1976; Labaratory Wiesby, Niebü/1, 1980)
I. A box containing 70 ml of a concentrated deep-frozen culture, e. g. mild acidifying culture (B. bifi-
dum, Lh. acidophilus, S. thermophilus} is stored for long-term in a special container in fluid
nitrogen at - 195.8° C and stored for short-term at - 25° C to -40° C (Deep-freeze chamber) and
in carbon dioxide ice during transport (- 78.8 o C, working with gloves ).
2. Carefully take the box out of the container and thaw in a 0.5% sodium hypochlorite solution for
5-10 min. at 25° C forsterilisation.
3. Cautiously raise the pull ring and open the box under sterile conditions. It is desirable to work with
sterile gloves.
4. Pourthe content into the prepared milk in bulk starter vat.
5. Stir for 15 min. to distribute the added bacteria.
6. lncubation and cooling.

119
7.1.4 Fermentation in incubation vats or in retail containers

The technical basis of conducting fermentation yields the already mentioned biotech-
nogram.

7.1.4. I Inoculation in vat

The production of pure bifidus cultured milk is carried out using a !arge inoculum of
approximately 10% (EVOG, 1965 a; SCHULER, 1969 a,b,c; KURMANN, 1977, 1981)
and in some cases 20% (MAYER, I 948). The technological significance of using the
!arge inoculum may be evidenced as follows: a) Promotion of the rate of acidifica-
tion; b) Approximately tenfold smaller numbers of bifidobacteria compared with
yoghurt bacteria; c) Overcoming the lag phase more quickly; d) Transfer of enzymes
in !arger amounts; e) A desirable short-time fermentation.
Table 7.3 shows the influence of different amounts of inoculum on the Iowering
the pH values of the production milk. Lowering the pH of the production milk to
6.0-6.35 by inoculum of I 0% is not significant forthe start of multiplication ofbifido-
bacteria. Inoculation is followed by stirring using a high-speed turbine agitator in
order to prevent possible defects of consistency.

Table 7.3 Influence of different amounts of inoculum, different acidified cultures on the lowering
pH ofthe production milk

Amountof pH value ofthe production milk afterinoculation of culture

inoculum withdifferentpH value(4.71 ;4.32; 3,76)

4.71 4.32 3.76

5% 6.48 6.43 6.22

10% 6.35 6.28 5.94
15% 6.23 6.12 5.72
20% 6.14 6.02 5.50

Source: KURMANN (1980)

It was shown that a !arge inoculum of bifidobacteria may result in a concentra-

tion ofbacteria similar tothat in manufacturing yoghurt (approximately 1-5 x 10 7
cells per ml). A !arger inoculum of 6-8-10% is also used in manufacturing a cultured
milk type BAT incorporating bifidobacteria (SCHULER-MAL YOTH et al., 1968 b;
MüLLER, 1976; Laboratory Wiesby Niebüll, 1980). The ratio of bifidobacteria, S.
thermophilus and L. acidophilus may be variable.
In direct inoculation of milk with a concentrated deep-frozen culture, 500 I of
milk may be inoculated with 70 g of culture (cf. Fig. 7.14 and Table 7.2). Direct
inoculation has the following advantages: a) There is no preparation of a mother cul-
ture or intermediate culture; b) No transfer and maintenance of mother cultures in the
Iabaratory is necessary; c) The growing out of various strains in a mixed culture is pre-
vented; d) The culture may thus be free of contaminants; e) The introduction of a sys-
tem of rotation of strains against attack by bacteriophages is simpler; f) The activity of
the culture is much more constantly maintained.

120
7.1.4.2 Filling the inoculated milk into retail containers for the manufacture of
set bifidus milk

During the filling process, the temperature of the milk can be maintained if necessary,
by warming up. Temperature during filling should not exceed 50° C. The tempera-
ture ofinoculation must therefore be increased, e. g. for an incubation at 43° C, cool-
ing during filling at 40° C gives a temperature of inoculation of 47° C (MüLLER,
1976).

7.1.4.3 Incubation in vat or in retail container

During incubation the concentration ofbacteria should be 108 cells permland the pH
value should be 4.7.

7 .1.4.3.1 Temperature ofincubation

The choice ofthe incubation temperature is dependent upon the following factors: a)
The optimum temperature for growth ofbifidobacteria; b) The time desirable for fer-
mentation; c) The intensity of fermentation; d) The rate of proliferation; e) The
equipment used for cooling; f) The temperature favourable for growth of different
strains in mixed cultures. The temperature of incubation for bifidobacteria ranges
between 36 and 42° C (EVOG, 1965 a; KYOWA HAKKO KOGYO, 1970; Laboratory
Wiesby Niebüll, 1980). The temperature of incubation for mixed cultures, type BAT
is 38-42° C (SCHULER-MALYOTH et al., 1968 b; EvoG, 1965 b; MüLLER, 1976;
Laboratory Wiesby Niebüll, 1980).

7 .1.4.3 .2 Time ofincubation

The choice of incubation time is dependent upon the following factors: a) The
amount of inoculum; b) The temperature of incubation; c) The rate and intensity of
acidification; d) The ability ofproliferation in cow's milk; e) The lag and generation
time (the time needed to obtain a minimum number ofbacteria from 107 to 108 per
ml); f) The eventual need toshorten the incubation time in orderto prevent the growth
of contaminants.

7 .1.4.3.2.1 Method offast acidification

In fast acidification with pure cultures ofbifidobacteria, the time of incubation is 6-8
hrs (Evoo, 1965 a; SCHULER, 1969 a,c). It is 2 ~-4 hrs when using mixed cultures,
type BAT incorporating bifidobacteria (Evoo, 1965 b, Laboratory Wiesby Niebüll,
1980). The time of incubation may be prolonged to 7-8 hrs.

7 .1.4.3.2.2 Method of slow acidification

Slow acidification (time ofincubation 12 hrs or more) may be used forthe following
purposes: a) To utilize the capacity offermentation vats during the night; b) To utilize
the capacity of packaging machin es at the beginning of the working day; c) To incor-
porate non-acidfast physiologically valuable strains in a mixed culture. The amount
of inoculum and temperature of incubation may be reduced if over-acidification
occurs.

121
7.1.4.3.3 The proliferation of culture bacteria during incubation
During the manufacture of yoghurt, the number of bacteria reaches 109 cells per ml,
while in manufacturing cultured milks using either pure culture ofbifidobacteria or a
mixed culture type BAT (method offast acidification) the number ofbifidobacteria
reaches 107-108 cells per ml (cf. Fig. 7.15). The relatively small numbers ofbifidobac-
teria may be explained as follows: a) Non-fast growth; b) Non-optimum conditions
for the growth ofbifidobacteria in the dairy; c) The relatively short time of incubation
forthistype of culture; d) The lack of symbiosis in mixed cultures like that in yoghurt
culture.
SASAKI and co-workers ( 1968) reported the following observations on the prolif-
eration of B. bifidum in mixed cultures: a) The growth rate of S. faeca/is was not
affected by the presence of L. acidophilus, but that of the latter was initially
repressed; b)The growth rate of L. acidophilus was not affected by B. bifidum, but
that of B. bifidum was repressed unless the initial inoculum was in the ratio of
104 : 103 (B. bifidum: L. acidophilus);c) Growth of S.faecaliswas abundant in the
presence of B. bifidum, butthat ofthe latter was poor, comparable growth only result-
ing from an initial inoculum of I 06 : I 0 1•

7.1.4.4 Kinetics ofthe product formation

The production of acid is the most important fermentation product (minimum pH 4. 7

and maximum pH 4.3 ofthe coagulum afterthe completed incubation). After-acidifi-
cation is dealt with in heading 7 .1.8.2.

7.1.4.4.1 Pure bifidus milk

Some examples of acidification during the manufacture ofbifidus milk are shown in
Fig. 7.6 and 7.10, andin Table 7.4.
Table 7.4 The count of different strains of bifidobacteria and the amount of acid produced after
different time of incubation 1)

Time of culturing 0 17 24 41
in hrs.

Determination Acid Countof Acid Countof Acid Countof Acid Countof

0
SH viable bact., 0 SH viable bact., osH viable bact., osH viable bact.,
Strain ml ml ml ml

YIT-4002 14.0 4.2 X 107 57.6 4.9 X 109 84.4 4.8xl09 108.8 7.3 X 108
YIT-4006 13.6 7.0 X 107 40.0 2.3 X 109 54.0 8.9 X 108 68.0 5.6 X 108
YIT-4005 14.0 4.5 X 107 55.6 5.0xl09 78.4 4.6 X 109 100.4 3.1 X 109

B.longum 14.4 5.7xl07 20.8 6.2 X 107 26.6 5.4 X 107 25.2 1.0 X 107
B.breve 14.4 4.0 X 107 26.8 5.1 X 107 31.2 3.6 X 10 7 36.8 1.1 X 107
B.adoles. 13.6 3.4xl07 17.6 1.8 X 107 18.4 1.2 X 107 20.4 6.4 X 106
B.infantis 14.0 6.0 X 107 18.0 4.6 X 106 19.2 1.2 X 106 21.2 1.0 X 105
B.bifidum 13.6 4.0 X 107 23.6 6.0 X 107 26.4 3.9 X 107 30.0 2.4 X 107

') Reconstituted skim milk powder (16% powder). Inoculum 2%, incubation temperature 37 oc,
time of incubation 0, 17 and 41 hours
2 ) YIT-4002 -4005 oxygen-resistant mutant strain

Source: MUTAI et al.. (1978 c)

122
 The rate of acidification and the number ofbacteria should be determined if dif-
ficulties in acid production occur, and yoghurt culture may be simultaneously added
at a rate of 0.01-0.1% to improve acidification. The final pH value should be rela-
tively high, because bifidobacteria rapidly die out at pH below 4.6 (cf. Section
7.1.8.3).

7 .1.4.4.2 Cultured milk type BAT containing bifidobacteria

Fig. 7.15 shows the curve of acidification of a cultured milk containing bifidobacte-
ria.

pH
6

~,
Streptococcl

Bitidus
3

0 1 2 3 4
Hours
Fig. 7.15 Proliferation of B. bifidum, I.b. acidophilus and S. thermophilus du ring the manufacture
of a cultured milk. Inoculum 6-10%, incubation at 42° C for 2-3 h to a pH 4.6-4.9. I06-10 8 bifidus
bacteria after incubation
(SCHULER-MALYOTH eta/., 1968b)

7.1.5 Coo/ing after incubation

The following factors influence cooling and equipment for cooling: a) Acidification
of bifidus milk is slower than that of yoghurt, and accordingly over-acidification
occurs with lesser frequency; b) Over-acidification does not occur in properly
selected strains (minimum pH 4.3); c) Equipment for cooling may be simplified thus
sparing the consumption of energy. For instance, the stirred products may be cooled
directly in their packages. Cooling has been discussed in more details in the Yoghurt
monography (pp. 231-241) and cf. Fig. 7.16).

7.1.6 Mechanical treatment ofthe gel

The manufacture of stirred cultured milks requires mechanical treatment of the gel;
however, the mechanical treatment ofthe gel during stirring in the fermentation vat,

123
Fig. 7.16 Incubation chambers line with vertical operating doors, and with two incubation and
cooling chambers. Cooling from the incubation temperature to 20°C within 20 min., following by
conveying the palettes with cultured milk cups to a tunnel for the continuous cooling within one hour,
until temperature of about r C is achieved
(Jöler AG, Hauptwil, Switzerland)

pumping out, flowing through pipes and filling into retail Containers should not be
too excessive (see Yoghurt monography pp. 242-252 and Fig. 7.17).
A minimum content oftotal solids is needed to maintain the right consistency in
the finished product. The manufacture ofbifidus milk beverages requires dilution of
the coagulum using either a turbine stirrer or the homogenization process.

7.1. 7 Packaging and packaging materials

The packaging process (packaging materials and machines) is the sameasthat used
for yoghurt (see Yoghurt monography, pp. 255-272). Fig. 7.18 shows an example of
the ingredients labe! on a package.

7.1.8 Storage, proionging the storage life and maintaining quality

The storage ability ofbifidus milk is a function ofthe cultures used, storage tempera-
tures, eventual contaminations, the growth of contaminants, and the quality of pack-
aging (closure, migration of soluble matter, etc.).

124
Fig. 7.17 Metering pump NORMADOS P for dosing starter cultures and similar smallest
amounts. The adjustement of dosing flow is carried out using specially designed devices
(Bran & Lübbe, Norderstedt, W. Germany)

7.1.8.1 Hygienic production

Hygienic production is in direct relation with good manufacturing practices (Food

and Drug Administration, 1969). More details have been outlined in the Yoghurt
monography (pp. 283-288 and 320-322).

7.1.8.2 After-acidification and final pH value ofthe finished product

After-acidification is very weak in pure bifidus cultures and in mixed cultures (type
BA1) containing bifidus bacteria due to the high pH and temperature minimum of
enzymes (see Fig. 7.19; Yoghurt monography, p. 277 and MüLLER, 1976).

7.1.8.3 The survival rate during storage

The survival rate ofbifidobacteria during storage is dependent upon different factors.
Requirements for the survival rate and the number of viable bacteria vary depending
on the use. If a cultured milk is consumed as a foodstuff, variations in the number of
viable bacteria have no essential role. If, however a cultured milk is intended for die-
tetic-therapeutic use, a large number of viable bacteria may be important. According
to REuTER ( 1969) "High doses should be used and the administration so adjusted as

125
Fig. 7.18 Advertising on the cup for a cultured milk LÜNEBESTcontaining bifidus, acidophilus,
and yoghurt bacteria. The presence of acidophilus and bifidus bacteria has been indicated on the cup.
Since most ofthe consumers did not understand the significance of printed latin names, the advertis-
ing has been changed to: "with LÜNEBEST special cultures"
(Dairy Lüneburg Hans Stamer KG, Lüneburg, W. Germany)

126
 5. pH

4.5
.. ........ ,_..............
4.6to.:---
'---------4.5
\\
\
----,......3.9
3.5~-~7~--,~4----~2,~----~30
Days
Fig. 7.19 After-acidification of skim milk yoghurt containing bifidobacteria during storage at
20-22°C and l0-l2°C. A cultured milk is made using a mixed culture called MSK (B. bifidum, Lb.
acidophilus and S. thermophilus) with the addition of a yoghurt culture (0.1-0.25 %).
Legend: _ _ l0-l2°C
_ _ 20-22°C
(PALLASDIES, 1976)

to ensure rapid transit through the stomach, thus reducing the rate of dying out. Data
vary in various reports conceming the optimum doses. The largest doses are 10 10-10 11
bacteria per day. The common doses may be 108-109 bacteria per day, andin some
cases 106-107 ".
As regards the value of dehydrated and fresh bacterial preparations, ScHULER-
MALYOTH and co-workers (1968 a) stated the following: "Despite allpositive find-
ings, the question arises as to whether the administration of dehydrated cultures is the
optimal method of substitution therapy or whether the choice be made to use fresh
cultures. In our opinion, the latter should be the method of preference ". These work-
ers consider that a good cultured milk should contain 106-108 bifidobacteria per ml.
The number ofbifidobacteria in cultured milks (pH 4.3-4. 7) may diminish by 2
Iogs during storage for 1-2 weeks, i. e., tenfold reduction ofbacterial numbers occurs
during 'li-1 week (cf. Table 7.5 and Fig. 7.20).

Table 7.5 Time for the decimal reduction ofbifidobacteria numbers in cultured milks with variable
initial number, pH value, final number and time of storage to reach the minimum bacterial number of
105 to I 06 cells per mP)

pH Initial number Timeforthe Calculation of coldstoragetime at 4-6

value start of storage decimal reduction o C to reach minimum bacterial number
ofnumber (N)of:
(D-value)
I 05 germs/ml l 06 germs/ ml

4.70 108 /ml I week 3weeks 2weeks

4.70 J01/ml I week 2weeks I week

4.30 108 /ml ~week l~weeks I week

4.30 107 /ml ~week I week ~week

1) By consuming one portion of cultured milk l 00 g containing I 05- I 06 bacteria per ml, the number of

bifidobacteria introduced will be I 07- I 08

127
 Possible variations may occurin, for example: a) The pH value of cultured milks.
MUTAI and co-workers (1976) showed that the number ofviable bifidobacteria rap-
idly declines at pH 4.3 and below, while the number declines more slowly at pH 4.6 or

·-..... 10
tU
I..
~
............. ······· ..............~·.ft!~f.'!!.9.P.Q!{f!~... ~ ...
8 -------...
.........
L.
. <iic'cto
.
.
•...........
•.••
u
tU 10 7 ......
... ...
',01}1/v_
s
..()
........
..... 10
0 105
6
Therapeutic
m~ntmum
.. .... ..
I.. 10.: pH
~
..()
E 103
:::s
:<: 10 2
10 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days
Fig. 7.20 Decrease of numbers of B. bifidum, Lb. acidophilus and S. thermophilus in a cuJtured
milk at pH 4.5 during storage for J4 days
(SCHULER-MALYOTH, 1968)

Table 7.6 Changes of counts oftwo different bifidobacteria strains in soJutions with different pH
vaJues during storage at 5 °C for I 0 days 1)

pH Days of storage
Strain
value 0 3 5 7 10

V 4.7 X JOB 3.8x lOB 3.7 X lOB 4.0x JOB 3.0X JOB
6.0
s 4.5 X lOB 3.1 X lOB 6.8 X 107 4.2 X J07 2.1 X 107

V 5.0 X JOB 3.8 X JOB 2.4x lOB 3.0X lOB 3.3 X JOB
5.0
s 4.9 X JOB 3.1 X lOB 5.5 X 107 1.9x 107 2.J X 106

V 4.2 X JOB 5.1 X lOB 4.2 X JOB 2.0x JOB 9.3 X 107
4.6
s 4.5 X JOB 1.0 X J0 7 3.6 x tos 4.0 X J0 3 < J02
V 4.6 X lOB 4.0 X lOB 2.J X lOB 6.9 X J07 3.0x 107
4.3
s 4.2 X JOB 3.9 X 106 2.1 x Jo• < 102 < 102
V 4.7 X lOB 9.5 X 107 3.4 X J07 5.6x J06 4.3 x tos
4.1
s 4.5 X lOB 4.J x to• 1.0 X 103 < 102 < 102
V 5.0 X JOB 7.0 X 106 8.0 X JOS 2.8 X JOS 2.9 x to•
4.0
s 4.9 X JOB 1.2 X J0 3 < 102 < 102 < 102
1) Transferofsuspension ofwashed cells (opticaJ density 660 nm = 1.5)
V = B. bifidum YIT-4005, acid-resistant at pH 4.0
S = B. bifidum E 3J9, standard strain (comparative strain)

Source: MUTAI et a/., (1976)

128
above (cf. Table 7.6); b) Temperature of storage; c) The initial number of bacteria;
d) Time of storage; e) Buffering capacity and the composition ofthe culture medium.
TAKATA ( 1966) found, forinstance that bifidobacteria survived for 5 days in a culture
medium containing Iactose, and survived much Ionger in the presence of aspergillus
and saccharomycetae; f) The kind ofbifidobacteria strain used.
Table 7.7 shows the number ofbacteria ingested according to consumption of
different portions, i. e. 100 to 180 g. For instance, by consuming 100 g of cu1tured milk
containing 106 bacteria per ml, a total number of 108 bifidöbacteria is ingested; and by
consuming 100 g of cultured milk containing 105 bacteria per ml, a total number of 107
bifidobacteria is ingested. The minimum number ofbifidobacteria should therefore
be 105-1 06 per ml if 100 g cultured milk is consumed.

Table 7. 7 Number of bifidobacteria introduced during consumption of different portions of

cultured milk containing different number ofbifidobacteria

Number ofbifidobacteria during Number ofbifidobacteria introduced per portion

consumption per ml lOOg 120g 180g

IX 102 1 X 104 1.2 X 104 1.8 X 1Q4

IX 103 1 X lOs 1.2 X lOS l.8x lOs
1X 104 I X 106 1.2 X 106 1.8 X 106
1X lOs*) I X 107 l.2x 107 1.8 X 107
1X 106 I X 108 l.2x 108 1.8 X 108
1X 107 I X 109 1.2 X 109 l.8x 109
1X 108 1 X 1010 l.2x 1010 1.8 X 1010
1X 109 1 X 1011 1.2 X 1011 1.8 X 10 11

*) Therapeutic minimal bacterial number according to ScHULER-MALYOTH (1968)

7.1.9 Defects ofbifidus milk products

Defects of cultured milks containing bifidobacteria may be divided into the following
groups:

7.1.9.1 Defects of appearance:

settled, fermented, unclean, formation of colonies or film on the surface, aged non-
fresh, formation of condensed water.

7.1.9.2 Defects offlavour and aroma

Too sour or too mild, yeasty, cheesy, yoghurty, rancid, bitter, vinegary, too little
aroma. Taste may be affected by the addition of some growth factors (e. g. a bitter
taste results from the addition of casein-peptone).

7.1.9.3 Defects of consistency

(whey separation; soupy, sandy or slimy liquid).

129
7.1.9.4 Defects ofhygiene

(large numbers of contaminants, coliforms, yeasts, moulds, etc.).

7.1.10 Model processes

Different model processes for the manufacture of cultured milks containing bifido-
bacteria (type BAT) are surveyed in Tables 7.2 and 7.8 as weil as in Fig. 7.I and 7.13.
They may also be app1ied in manufacturing pure bifidus milk.

Table 7.8 Model processing scheme for the production of a mildy acid yoghurt with a mildy acidify-
ing culture (containing bifidobacteria), with yoghurt-culture (altematively) and S.filant-culture*)

Timetahle Recommended processing Product Desired production

parameter parameter

Standardized pH6.8-6.6
processing SH6.5-7.0
milk void of antibiotics
Addition ofhydrocolloids
Pasteurization
80-l20°C
5-30minutes
Homogenizing
l50-l75atm
5o.:-60°C
Cooling 46-44 o C
Ohs. Inoculation**)
8%MSK
l-1,5%
Sc. filant
Filling (ortank incubation) pH 4.65-4.40
2.5-4hs. Incubation at 42 o C pH 4.65-4.40
Cooling to 2-4 o C Cultured milk pH4,30-4,10
Storage
Distribution

*) Without any notable postacidification or gas development, firm or stirred, with orwithout fruits
**) Preparation of bulk starters with deep-frozen bacterium concentrates for mildly acidifying cul-
ture (B. bifidum, Lb. acidophilus, S. thermophilus), S.filant-culture and eventually for yoghurt-
culture
Source: PALLASDIES (1976) and Labaratory Wiesby Niehüll (1980)

7.1.11 Equipment

For the manufacture of cultured milks containing bifidobacteria approximately the

same machines and equipment are used as those for yoghurt (see Yoghurt monogra-
phy,pp.139-323 and Fig. 7.3; 7.4; 7.11; 7.16; 7.17). Asepticorhygienicmanufacture
ofthese cu1tured milks is sometimes especially important.

130
7.2 Basic manufacture and technology of dried bifidus milk products

Strains ofbifidobacteria should be pH- and heat-resistant because ofthe high drying
temperature used in processing. In order to counteract possible darnage to the cells of
bifidobacteria during drying, the pH value ofthe coagulum should be relatively high
and the number ofbacteria large. For drying, HINTERW ALDNER ( 1971) recommended
the addition of 0.1-0.5% yeast extract to the production milk.
In the optimal process of spray-drying, numbers of bacteria my be obtained as
high as 107 -10 9 per g (cf. Yoghurt monography, pp. 294-29 5 and Chapter 8).

7.3 A survey of various milk and food preparations

This heading considers reports dealing with various milk and food preparations con-
taining bifidobacteria and the manufacture thereof. This survey is not intended tobe
complete. There are many possibilities for making various products and preparations
containing bifidobacteria (See Yoghurt monography, pp. 325-362).

7.3.1 Baby foods

The manufacture of foods of a milk base for infants and children is mentioned in
Chapter8.

7.3.2 Bifidus sweet milk

In the U. S. A., a culture concentrate of lactic acid bacteria is added to heat-treated

sweet milk (SHAHANI, 1979). Similar preparations containing bifidobacteria have
been described. ScHULER & HINTER w ALDNER ( 1971) mentioned a preparation made
by inoculating dried sweet milk with cultures ofbifidobacteria. In Japan, a sweet milk
containing B. longum and L. acidophilus is manufactured by the Morinaga Milk
Prod. Co. and bythe Meiji MilkProd. Co. (YosHIOKA, 1980). Inaddition, MuTAI and
co-workers ( 197 6) described a freeze-dried powder containing bifidobacteria, which
is added to sterilized milk at rate of 2 %, and then packaged.

7.3.3 Cultured milk preparations

A short survey of cultured milk preparations containing bifidobacteria and the manu-
facture ofthese products in various countries is given as follows.

7.3.3.1 Canada

BROWN & TowNSLEY (1970) described the manufacture of a cultured milk containing
bifidobacteria. At present, small amounts of a cultured milk containing bifidobacte-
ria (type BAT) are produced (cf. Section 7.3.3.4).

131
7.3.3.2 Chile

SCHACHT & SYRAZYNSKI ( 197 5) developed a cultured milk enriched with protein and
Iactase, and manufactured using cu1tures of S.lactissubsp. diacetilactis, S. cremoris,
L. acidophilus and B. bifidum. This product, called PROGURT, is made semi-indus-
trially in Valdivia (ESPINA, 1980).

7.3.3.3 Czechoslovakia

A dairy in Prague manufactures a cultured milk called BIOKYS, containing B. bifi-

dum, L. acidophilusand Pediococcusacidi/actici(CERNA, 1977; HYLMAR, 1978, MER-
GEL, 1978; BREZINA et al., 1979). A dried milk preparation for feeding infants and
chi1dren is also industrially produced as weil (PEcH et al., 1974 a; PEcH & HRABOVA,
1975; CERNA, 1977; DEDICOV A& BURDOVA, 1977).

7.3.3.4 Germany

The principal investigations as to the use ofbifidus culture in the dairy were made in
1966/67 (SCHULER-MALYOTH etal., 1968 b; MüLLER, 1976). Subsequent1y, the first
industrial production of a cu1tured milk containing bifidobacteria, type BAT, was
undertaken by the Biogarde Company in Muni eh. This product is now manufactured
in more than 50 dairies in Germany, France, Argentina, etc.
More than 400 million cups of BIOGARDEare produced in Germany per year
(MÜLLER, 1976).
Collaborators at the BIOGARDE Company have authored various publications
and taken out patents, e.g. SCHULER (1969 a,b,c; 1971; 1972), SCHULER & HINTER-
WALDNER ( 1971 ), HINTERW ALDNER ( 1971 ). The manufacture of a cultured milk con-
taining bifidobacteria (B. bifidum, L. acidophilus and a yoghurt culture) has been
undertaken by the dairy Lünebest in 1969; and subsequently a licence was given to
the USA (ANON, 1969; MüLHENS & STAMER, 1969; Dairy Lünebest Hans Stamer
KG, 1980).
Since 1977 a mixed cu1ture containing bifidobacteria, type BAT, is availab1e.
This type of culture is now distributed to more than 45 dairies in Germany, Austria,
France, Switzerland, Canada, etc. ( Laboratory Wiesby Niebü/1, 1980). Frequently a
product simi1ar to yoghurt is made using a mixed culture, type BAT, with the addition
0.01-0.1% of a yoghurt culture. Dairy Südmilch AG, Stuttgart, has produced a cul-
tured milk containing bifidobacteria, called BIOBEST since 1980 (Südmilch AG
Stuttgart, 1980).

7.3.3.5 Italy

Rossi et al., (1978) described a cu1tured milk made using cultures of L. acidophilus,
B. bifidum, L. bulgaricus and S. thermophilus.

7.3.3.6 Japan

A cultured milk made using cultures of B. longum, L. bulgaricus and S. thermophilus

is produced by the Marinaga Milk Prod. Co. (Yoshioka, 1980). Another product,

132
called MIL-MIL, containing B. bifidum, B. breve and L. acidophilus, is produced by
the Yakult Honsha Co. Ltd. Tokyo. The product may be used as a beverage or as a
soup (ANON, 1978). In addition, various preparations and methods of manufacture
have been described by the following authors: Taisho Pharmaceutical Co. Ltd., 1964;
Takata, 1966; SASAKI etal., 1968; KOYOWA, HAKKO KOGYO, 1970; Marinaga Milk
/ndustries, 1974; MUTAI et a/., 1976; MUT AI et al., 1978 c, 1980.

7.3.3.7 Po land

Bifidobacteria has been incorporated into yoghurt starter (KiszA et al., 1978).
Smaller quantities ofbifidus milk have been produced (LIPINSKA, 1980). In addition,
important reports have been made by KiszA & ZIAJKA, 1973; ZIAJKA and co-workers
(197 4); KiSZA and co-workers ( 197 4); KOSIKOWSKA (1978) and LIPINSKA ( 1978;
1979).

7.3.3.8 United States of America

RoBERTS (1977 a,b) patented a method forthe manufacture (in three stages) of a cul-
tured milk enriched with Iactase. Cultures of S.lactis and L. citrovorum (L. cremoris)
are used in the first stage, those of L. bulgaricus and S. thermophilus in the second
stage, and L. acidophilus and B. bifidum in the third stage. Cultured milks containing
bifidobacteria have been produced in smaller quantities (see Chapter 7.3.3.4).

7.3.3.9 Union ofSoviet Socialist Republics

There are reports by SEMENIKHINA (1967) and BORISOVA & SLIVKO (1973) on milk
products containing bifidobacteria.

7.3.3.10 Variouscountries

The production of smaller quantities of cultured milks, type BAT, often distributed
under the name yoghurt, e. g. Argentina, France, Austria, Switzerland is to be noted
(see Chapter 7.3.3.4).

7.3 .4 Various milk products and foods

Bifidobacteria have also been used in the manufacture of the following products:
sour milk, kefir, buttermilk, soups, cheese, fresh cheese, butter, sour cream, quark
creme (bificreme), ice-cream, cottage cheese, tvarog (KLuPSCH, 1968; EvoG, 1965 b;
HRABOVA, 1975; LANG & LANG, 1978; KRUK et al., 1976; HYLMAR, 1978, 1980;
ANON, 1978; LIPINSKA, 1980; Biogarde Company, 1980; Labaratory Wiesby Niebüll,
1980). The use of mixed cu1tures containing bifidobacteria (type BAT) for the ther-
moquark method has been elaborated by Westfalia (Laboratory Wiesby Niebüll,
1980). The manufacture of vegetable juices (MUTAI et al., 1976) and the incorpora-
tion of bifidobacteria into refrigerated foods can also be found (Morinaga Milk
Industries, 1974).

133
Chapter 8: Pharmaceutical preparations

8.1 Introduction

The manufacture of a dry pharmaceutical preparation containing sufficient number

of viable bifidobacteria was dependent upon advances in the technique of bacterial
cultivation, the knowledge of the physiological-therapeutical significance for
humans and animals, as weil as advances in the technique of drying. Some important
milestones to this were:
1956 Töpfer, GmbH., Dietmannsried, W. Germany, introduced on the market the
first bifidobacteria preparation EUGALAN in powder form for the regenera-
tionofthegutflora(Töpfer, GmbH, 1980).
1962 As aresult ofworkreported by KLUDAS (1959 b), Med. J. Carl Pflüger, Berlin,
W. Germany introduced on the market a freeze- dried preparation containing
bifidobacteria for the regeneration of the gut flora (Süss, 1977; Med. J. Carl
Pflüger, 1980).
1964 Töpfer, GmbH, Dietmannsried, W. Germany, introduced on the market the
first infants preparation containing bifidobacteria LACTANA B-milk). The
preparation represented a further development of the basic work of MA YER in
1948 (Töpfer, GmbH, 1980).
1968 Nisshin Flour Milling Co. Ltd., Tokyo, Japan: Probably the first utilization of
animal strains and first commercial bifidobacteria preparation for veterinary
use (KOBA YASHI, 1980).

8.2 Preparations for use in foods for infants and children

There aredifferent (as regards chemical and biological composition) infants prepara-
tions on the market. They are designated by MAYER and KEscHA w ARZI ( 1970) as bi-
fidogenic provided that the faeces ofhealthy infants microscopically demoostrate in
2-4 days' timeapredominant B. bifidum population.

8.2.1 Food preparations infants without addition ofbifidobacteria

A number of preparations added have been investigated in view of a bifidogenic

effect. These investigations are partly out of date, too little informative, or difficult to
control due to the Iack of parallel investigations. lt is therefore difficult to compare
different preparations (humanized, partly humanized or non humanized).

8.2.1.1 Humanized milk preparations

Human milk isthebest infant food and is also bifidogenic. Investigations have been
made, therefore, to adapt the chemical composition of cow's milk to that of human
milk (called humanization or adaptation). Table 8.1 gives the composition ofhuman
milk, humanized milk and cow's milk. Fig. 8.1 showsdifferent steps in the production

134
 Cheesewhey Skim milk
(Lactalbumine + Iactose) + Milk tat
.j.
a) ion-exchange + Fat rich in linoleic acid
or b) electrodialyse + Fat-soluble vitamins
or c) Ultrafiltration + Water-soluble vitamins
.j. + Trace elements

I
a) and b) Deminera/ized whey
or c) Protein concentrate
+ Iactose crystallized

Milk adapted for infants

or
Humanized milk

Deficiency: - Immunologie factors

(lmmunoglobulines, Iysozymes, etc.)

- 0/igosaccharides
(mucopolysaccharides)

- Biofactars unknown
(peptides, enzymes, etc.)

Source: Werner (1978)

Fig. 8.1 Scheme of the manufacture of humanized (adapted) milk for the artificial feeding of
infants
Source: WERNER (1978)

of a humanized milk preparation. Tahle 8.2 gives a survey of some preparations

whose effects were testedas to the growth ofhilidohacteria in the intestines of infants.
The hilidogenie effect ofthese humanized milk preparations may he interpreted tak-
ing into consideration the ahove-mentioned ohjections. Depending on the prepara-
tion fed, the following effects may he ohserved (when compared with human milk):
irregular, helated or Iack of hilidohacteria in the faeces; a lesser proportion of hif-
idohacteria in the total count of gut hacteria; in the most favourahle cases the hilido-
genie effect, however, not as strong asthat ofhuman milk.

8.2.1.2 Partly or non humanized preparations

The following authors investigated the hilidogenie effect of partly or non humanized
preparations: MAYER & KESCHAWARZI (1970) the preparations APONTI, POMIL,
ALETE, ELEDON, HIPPON, HONEY milk, MILUMIL, PELARGON, PRODIE-
TON; GROSS & MACHO (1958) the preparation ALETE; BERGER (1957) the prepara-

135
Table 8.1 Average chemical composition ofhuman milk, humanized milk and cow's milk

Constituents (g/1) Humanmilk H umanized milk Cow'smilk

Total so Iids 125 125 125

Lipides 40 35 38
Nitrogen substances 12 16 35
(N X 6.4)
-Casein 4 6 26
-Lactalbumine 6 7 5
Lactose anhydrous 70 67 45
Mineral substances 2.0 3.0 7.0
-Na 0.2 0.2 0.4
-K 0.5 0.75 1.6
-Ca 0.3 0.32 1.2
-P 0.15 0.30 1.0
-CI 0.40 0.40 0.9
Citrates 0.54 0.80 1.7

Calories/1*) 720 645 634

*) Lipides= 9.3 kcal/g; Nitrogensubst. = 4.1 kcal/g; Carbohydrates = 4.2kcal/g

Source: WERNER ( 1978)

tion ELEDO N; EscuoRo and co-workers ( 1944) the preparation ESCUD RO milk;
KELLER (1961) condensed milk and acidified whole milk; LEUTERER (1963) and
SCHNEEGANS and co-workers (1966) cows' milk.
Depending on the preparation, the intestinal co1onization of bifidobacteria was
very variable, and even in the mostfavorable cases incomparable with that ofhuman
milk.

8.2.2 Infant preparations containing viable bifidobacteria

Food preparations containing viab1e bifidobacteria are listed below. MAYER (1969)
described the stages of development for a preparation containing bifidobacteria as
follows: "Further study ofthe effect of Bifidobacterium bifidum required the creation
of an infant food capable of producing a bifidus flora like that ofbreast milk. Ressau
worked on this problern 20 years and Adam 25 years. Both cooperated with DR.
SrEUBE, from the Töpfer Company. The research Ievel at that time was not adequate
for the manufacture of a bifidus milk. The knowledge of numerous biological pro-
perties of Bifidobacterium bifidum, which I somewhat described, was needed as was
the identification of numerous growth factors, in order to develop tagether with DR.
SrEUBE, the LACTAN A-B-bifidus food, which produced apredominant bifidum flora
within 2-4 days in healthy infants as did breast milk. It was possible then to conduct
in-vivo experiments to demoostrate the antibiotic effect of bifidum flora in the gut".
(See Fig. 2.6; 2. 7 and 2.8 about moniliasis)

136
Table 8.2 A survey of works on the hilidogenie effect of humanized food preparations

Designation of Author Bifidogeniceffect (analysis ofinfants' faeces)

preparation

CORRELA GAU PP ( 1953) Bifidobacteria found in the infant faeces, but with
lesser frequency than with HUMANA feeding.
HUMANA ROHR & KIRSCH ( 1953) Failed to establish a bifidus flora in the !arge intes-
tine of II out of 12 healthy infants.
BERG ER et a/ (1956) Bifidobacteria frequently but not regularly detected
in the stools.
BERG ER ( 1957) Bifidobacteria detected in 80% ofthe stools ana-
lyzed; and of that, only in 3 5% of the cases in such
purity as in human milk feeding.
KELLER(i96J) Variations found in the content ofbifidobacteria.
Bifidobacteria comprised 70-7 5% ofthe total count
ofbacteria. The stools void ofbifidobacteria were
rare and occasional.
MA YER & KEscHA w ARZI Observed belated, occasionally only after4 days, an
(1970) occurrence ofbifidobacteria in all three HUMANA
milktypes (HUMANA 0, early feeding;
HUMANA I, beginning feeding; HUMANA 2,
permanent feeding).
LAKTON HORECNY(l966) Feeding infants 1-3 months old with humanized
milk preparations resulted in a proportion of
bifidobacteria in the stools of 30 to 80 %.
MULTIVAL MA YER & KEscHA wARZI Bifidobacteria promoting, but not as distinct as with
(1970) human milk.
NAN MA YER & KEscHA w ARZI Bifidobacteria promoting, but not as strong as with
( 1970) human milk.
GOLDCAPMILK BuLLEN et a/(!977) The counts of coliforms and putrefactive bacteria in
faeces high, while counts ofbifidobacteria low.
PREMIUM MILK BULLEN et al ( 1977) Faeces with relatively high bifidobacteria count in
the Ist week, thereafter no bacterial species predom-
inated.
S-14 LEUTERER ( 1963) In approximately 50% of cases, apredominant
bifidobacteria florawas established, butthiswas a
belated and temporary occurrence.

8.2.2.1 Dried preparations

The following dried preparations have been produced: LACTANA-B and LAC-
TANA-strainedfruit byTöpfer, Dietmannsried, Allgäu, W. Germany. LACTANA-B
(since 1964) is a partly adapted bifidus food for infants and produces a bifidum tlora
within 2-4 days similar to that produced by human milk feeding. I 00 g of powder
contains: fat 22 g, protein 13.3 g, carbohydrate 60 g, mineral salts 2.90 g, enriched
with vitamins A, 03 and C as weil as iron. kJ 2082 (491 kcal). Packages of 125,400
and 1000 g. References: HUSCHKE (1968), KALOUD & STÖGMANN (1968), HEYNEN
( 1969), MA YER ( 1969), MAYER & KESCHA W ARZI ( 1970), MA YER ( 1973; 1977).

137
 LACTANA-strained fruit is used laterthan LACTANA-B milk, i.e. for infants
who have attained 3-4 months of age (Bifidus strained food). I 00 g of powder con-
tains: fat 9.10 g, protein 12.50 g, carbohydrate 70.00 g, minerat matters 2.80 g, fibre
0.90 g, an enrichment consisting ofvitamins A, D 3, E, B~, B6, C, Ca-D-pantothenate,
niacin; kJ 17 41 (422 kcal), gluten free. Package 400 g, powder, instant. Reference:
HARMS ( 197 4).
Various dried preparations containing viable bifidobacteria have been described
byCHAPPAZ (1966), SCHULER& HINTERWALDNER(l971), PECH eta/.,(1974 a), PECH
& HRABOVA (1975), CERNA (1977), DEDICOVA & ßURDOVA (1977).

8.2.2.2 Fresh preparations

ScHULER-MALYOTH and co-workers ( 1968 a) have shown a preference forusing fresh

bifidobacteria cultivated in milk. They write as follows: "Despite allpositive find-
ings, the question arises as to whether the administration of dehydrated cultures is the
optimal method of substitution therapy or whether the choice should be made of
using fresh cultures. In our opinion, the latter should be the method preferred, but
technical difficulties made it impossible to advance our research".
The following problems may arise in fresh bifidobacteria preparations: a) Qual-
ity is maintained for a short time due to the relatively short decimal bacterial nurnber
reduction time; b) Coldstorage is needed (without breaks in cooling-chain) to slow
down dying ofbifidobacteria cultures; c) Some defects may occur (See Chapter 7).
Such fresh preparations have been mentioned by ScHULER-MALYOTH et al., ( 1968 b ),
VLtEK & KNEIFL (1972), SCHULER (1972) SCHACHT & SYRAZINSKI (1975) and Bio-
garde Company ( 1980).
Furthermore, there are two patents on baby foods containing bifidobacteria
(Morinaga Milk Inc.Co.Ltd., 1970; 1974).

8.3 Therapeutic preparations containing viab1e bifidobacteria for human use

Preparations described below refer to either pure bifidobacteria preparations or

rnixed preparations containing bifidobacteria tagether with other intestinal bacteria.

8.3.1 The preparation INFLORAN BERNA

(Swiss Serum and Vaccine Institute, Bern, Switzerland and Istituto Sieroterapico
Berne, Corno, Italy)

It has been on the market and for export since 1977. Freeze-dried, containing I 0 9 cells
of L. acidophilus and 109 cells of B. bifidum (since 1980 B. infantis). Intestinal spe-
cific eubiotic: digestive disturbances in artificially fed infants, acute unspecified
enterocolitis, chronic enterocolitis, acute enterocolitis after antibiotic therapy,
chronic constipation. - Packages of 20 capsules. - References: SANNA & SEGNI
(1977), GERMANIER (1977), BRANCA et a/., (1979), SATTA et a/., (1980). See Fig. 8.2
(Example of an advertising for a bifidobacteria preparation).

138
Fig. 8.2 Picture ofthe advertisement prospectus for the child's preparation INFLORAN BERNA.
Romantic representation oflively, healthy and vigorous looking children during play. The picture is
taken from: J. STELLA "Play and the Pleasures ofChildhood", Paris, 1657
(lstituto Sieroterapico Berna, Corno, Italy; Swiss Serum and Vaccine Institute, Bern, Switzerland)

8.3.2 The preparation EUGA-LEIN

(Töpfer, GmbH, Dietmannsried, Allgäu, W. Germany)

The preparation has been produced since 1973, and contains a mixture of various
strains of bifidobacteria. The chemical composition is as follows: fat 1.80 g, protein
15.10 g, carbohydrate 71.10 g, minerals 3.10 g, fibre 4.80 g, vitamin C 0.20 g, gluten
free. kJ 1432 (337 kcal).- Availab1e in packages containing 400 g powder.- Used for
chronic constipation (instead of purgatives). - References: MA YER ( 1971 ), DoERBECK
(1972), DOERBECK & TOBIASCH (1973), RIETH (1974), TOBIASCH (1975).

8.3.3 The preparation EUGALAN Töpfer FORTE

(has been produced since 1963; Töpfer, GmbH, Dietmannsried, Allgäu, W. Ger-
many)

The preparation contains bifidobacteria. I 00 g ofbifidus-milk powder contains : milk

protein 9.40 g, plant protein 0.70 g, Iactose 62.60 g, lactulose 5.90 g, salts 3.00 g, fat
free, gluten free; kJ appr. 1498 (350 kcal). - Available in packages containing 400 or
1200 g of powder.- Indication: liver cirrhosis, chronic constipation, disturbed bal-

139
ance of the intestinal flora after irradiation, sulphonamides or antibiotics. - Refer-
ences: EISENBURG (1969), SAMEC (1969), LINDNER (1970), MÜTING (1970), OHREN-
DERGER (1970), KAUTZSCH (1971), MAYER (1971), MüTING (1972 a,b), MÜTING
(1973), ORDNUNG & MüTING (1973), KREMER (1974), MÜTING (1974), MüTING &
ORDNUNG (1976), MÜTING et a/., (1976 a), UNGEHEUER & PEGLOW (1976), MüTING
(1977).

8.3.4 1he preparation LACTOPRIV

(Töpfer, GmbH, Dietmannsried, Allgäu, W. Germany)

The preparation contains bifidobacteria. I 00 g of powder contains : plant protein

17.35 g, plant fat 12.30 g, carbohydrate 64.00 g, Iactose free, salts 4.00 g, fibre 1.19 g,
saccharin 6.70 mg, enriched with vitamins A, D3, C; and with iron; kJ 1850 (436
kcal).- Available in packages containing 400 g powder. -lndication: a disturbed bal-
ance of the gut flora after antibiotic therapy, irradiation or sulphonamides therapy;
milk intolerance in persons of any age group. Reference: no data.

8.3.5 1he preparation "Milk sugar Töpfer"

(Töpfer, GmbH, Dietmannsried, Allgäu, W. Germany)

Contains B. bifidum, vitamin B2, carbohydrate (milk sugar) 97.10 g, fat 0.05 g, protein
1.90 g, minerat matters 0.40 g, gluten free; kJ 1683 (396 kcal).- Available in packages
containing 250 g of powder. - lndication: normalization of disordered intestinal
flora; undesirable putrefactive organisms ofthe digestive tract; supporting therapy in
constipation; promoting intestinal peristalsis. - References: no data.

8.3.6 1he preparation OMNIFLORA

(Med. J. Carl Pflüger, Berlin, W. Germany). Introduced on the market in 1962

Export. 75 mg of a freeze-dried product contains L. acidophilus, B. longum REuTER

1963 and E. coli. lndication: normalization of disordered intestinal flora.- Available
in capsule form.- References: HALLER & KRÄUBIG (1960), HALLER et al. (1960),
HüBNER (1960), K.LUDAS & ZSCHUNKE (1960), STIEVE (1960), STRASSBURG & PODS-
ZUS ( 1960), ZIRNER ( 1960), BERNDT & KLUDAS ( 1961 ), CAMERER ( 1961 a,b), KLUDAS
(1961), MEYER-ROHN (1961 SCHWARZ (1961), DORN (1962), HIRTZMANN & KLUDAS
( 1962), ZINZIUS ( 1962), KEPP (1963), NEUMEISTER & SCHMIDT (1963 a,b ), SCHMIDT &
NEUMEISTER (1963), SCHEIDERHAHN (1963), HIRTZMANN (1964), VLCEK & KNEIFL
( 1964), KUEMMERLE ( 1965), NEUMEISTER & PfEIFFER ( 1966), BAMBERG ( 1966), KUEM-
MERLE (1967), SCHMIDT (1967), BERNDT (1967), REUTER (1969).

140
8.3.7 The preparation SYNERLAC
(Laboratory Roger Bellon, Neuilly I Seine, France)

A freeze-dried product containing B. bifidum, L. acidophilus and L. bulgaricus. 100 g

of powder contains alumini um hydr. coll. sterile 54.00 g, silicate of alumini um hydr.
sterile (kaolin) 18.00 g, Iactose 18.00 g.- Packages contain 50 g ofpowder.- Indica-
tion: diarrhoea (acute and chronic enterocolitis, diarrhoea, intestinal infections coli-
bacillose).- References:ÜARZONlS et a/. (1963), MALAMOS et a/. (1963), LEPERCHEY
et a/. ( 1965), PlNTER ( 1968), REUTER ( 1969).

8.3.8 The preparation J

(Serum and Vaccine Institute, "Torlak", Belgrade, Yugoslavia)

A freeze-dried product containing B. bifidum. Very high activity in acid production

slightly acetic flavoured. High survival at gastric pH. Resistance to antibiotics (in vitro
test): resistant to penicillin and Streptomycin, average resistance to chloramphenicol
and oxytetracycline, and susceptibility to neomycin. Packaged in glass ampoules as a
powder.- Indication:treatment of enteric infections in infants.- References: DAMJA-
Novic & RADULOVIt ( 1967; 1968), LIPINSKA ( 1978), KOSIKOWSKA ( 1978).

8.3.9 The preparation LYOBIFIDUS

(developed by the Laboratory Etienne, Rumilly, now manufactured by the Labora-
tory Galler, S. A., 7 5007, Paris, France)

Contains 109 I g bifidobacteria and of that, a minimum of 106 living organisms ( B.

bifidum TISSIER according to SCHNEEGANS, 1966). Available in powder form (modi-
fied milk powder).- Indication: diarrhoea. References: LEvY-MATE et a/. (1963),
TASOVAC (1964), PERRIN (1964), ROUANE-CREPAUX (1964), SCHMITTBÜHL (1965),
BERNARD (1965), SCHNEEGANS et a/. (1966), DISSARD (1967), ÜRSINI et a/. (1968),
CASANOVA ( 197 5).

8.3.10 Various preparations

The following preparations mentioned in the Iiterature may be listed:

8.3.1 0.1 The preparation ORTHOBACTER

(French origin)
Contains L. acidophilus, B. bifidum and L. casei. - Declared species not dernon-
strahle according to investigations by REUTER ( 1969).

8.3.10.2 ThepreparationBIFIDER
(Japanese origin)

Contains B. bifidum (REUTER, 1969).

141
8.3.1 0.3 The preparation BIFIDOGENE
(French origin)

In drinkable ampoule doses (DoRosz, 1980). Present on the market. Indication: after
antibiotic therapy (see also BAJARD, 1977).

8.3 .1 0.4 Apreparation containing lactic acid bacteria and bifidobacteria, accord-
ing to a patent ( Taisho Pharmaceutical Co. Ltd., 1960).

8.3.1 0.5 Apreparation with high contents ofviable bifidobacteria and lactulose;
favourable influence on human gut flora. Patented (ÜGASA, 1979).

8.4 Therapeutic preparations containing viable bifidobacteria for animal use

There are several preparations containing viable bifidobacteria. Their effect is influ-
enced by the extremely different conditions in which animals live.

8.4.1 The preparation KOROLAC B

(Nisshin Flour Milling Co. Ltd., 19-12 Nihonbashi-Koami-Cho, Chuo-Ku, Tokyo,
Japan)

On the market since 1968. - Containing I 08 viable cells I g of B. thermophilum. 1t

comes in granulated particles which are prepared by vacuum drying.lt is not made by
conventionallyophilization, which itself is not good for stability of viable cells. The
preparative method is given in the German Patent (Nisshin Flour Milling Co. Ltd.,
1969 b ). - Indication: Prophylaxis and therapy of diarrhoea of pig1ets. Anti-scouring
(suppressing deteriorative proliferation ofhaemolytic bacteria) and growth stimulant
for swine during weaning period. Prevention of diarrhoea in early weaned Holstein
bull calves. It is administered orally at I 07 to I 0 8 B. thermophilum per piglet per day
from birth up to 40 to 60 days old. References: Mostly publications in Japanese. Nis-
shin F/our Mil/ing Co. Ltd. (1969 a,b), FUJJWARA et a/. (1977), KOBAYASHI (1978),
KJMURA (1979), TANAKA et a/. (1979).

8.4.2 The preparation KOROLAC D

(Nisshin Flour Milling Co. Ltd., 19-12 Nihonbashi-Koami-Cho, Chuo-Ku, Tokyo,
Japan)

109 viable cells I g of B. pseudolongum- Fine dried granules. The preparation was
developed during the years 1976-1980 and marketed in 1980.- Prepared by the same
procedure asthat used for Korolac B.- Indication: Anti-scouring agent for dogs. For
some types of canine diarrhoea. Prevention and therapy of diarrhoea for dogs and
calves (5 days after birth).- References: Mostly pub1ications in Japanese. TAKATORI
et a/. (1975), KoGURE et al. (1976), KAWASE et a/. (1977), Nisshin Flour Milling Co.
Ltd. (1976).

142
8.4.3 Various products

8.4.3.1 Freeze-dried and liquid cultures of B. bifidum

A studywas made ofthe therapeutic effect on sick calves and no difference was found
between the two cultures (RADULOVIC et al., (1970).

8.4.3.2 Culture of B. bifidumTISSIER

Good results in treatment and prophylaxis of gastrointestinal disturbances occurring

in dogs, pigs and calves (young animals). The administered doses were 109 bacteria
per animal (RoBERT, 1971).

8.4.3.3 B. bifidum!Enterococcus spray

After prophylactic treatment the mortality in piglets and calves was reduced (VARGA
& M~SZAROS, 1974).

8.4.3.4 Bitidus activated without bitidobacteria

Cattle feed containing lactuloseincreased the bitidobacteria population in the faeces

of 71 calves and reduced the counts of P. aeruginosa, Proteus spp. and Clostridium
spp. (GEDEK, 1969). Bitidus activated animal milk products orthose produced by the
addition of sulphonic acid derivative, such as pantetheine-S-sulphonic acid are also
tobe considered (Daiichi Seiyaku Co. Ltd., 1972).

143
Chapter9: Techniques forthe isolation, culturing and
characterization ofbifidobacteria

Two factors are important in the cultivation of bifidobacteria: an adequate culture

medium and anaerobic conditions.
General questions about culture media and their application are considered in
this chapter, while details on their preparation are presented in Appendix 2.
There are three procedures (a, band c) used to create anaerobic growth condi-
tions. In procedure a, all the manipulations are carried out under a continual flow of
oxygen-free carbon dioxide (Hungate technique used since 1950) or are effected
using an anaerobic chamber taking the necessary precautions (see HoLDEMAN et al.,
1977). A number ofthe panel routinely used this method for all their anaerobic isola-
tions and hence isolated bifidobacteria at the same time as the more oxygen sensitive
anaerobic bacteria (SYKES & SKINNER, 1973). Since this laborious and complicated
anaerobe technique is not absolutely necessary, weshall describe procedure b, a sim-
plified anaerobe technique and procedure c, a traditional technique employed with-
out any special precautions.

9.1 Original technique of TISSIER (1900; 1905)

Tissier applied a I% dextrose deep-agar for cultivation and isolation ofbifidobacte-

ria from the faeces of infants. Higher dilutions were investigated under a microscope
to detect B. bifidum (branched forms). Positive findings were followed up by transfer-
ring the same type of colonies to dextrose agar to obtain pure cultures. Subsequent
authors tried to change or improve upon this technique; however, essential advances
were not obtained. In contrast, the application of inadequate anaerobe technique of
cultivation resulted in many drawbacks, e. g., confusion about the identity of B. bifi-
dumand L. acidophilus(BovENTER, 1949). WEINBERG and co-workers (1937) consid-
ered, before the introduction of liver-cystine-milk sugar-agar by BLAUROCK (1937)
was made known, that there was no simple, fast and reliable method for bifidobacte-
ria cultivation. lt would be superfluous to consider in more detail the techniques of
isolation and cultivation which were used before 1937. Some culture media used in
this period will be mentioned later in Appendix 2.

9.2 The sample collection

Theseare the most important points to follow to obtain unmodified samples (HoLDE-
MAN et a/., 1977):
A) Use sterile techniques to obtain specimen (syringe aspirates, swabs, etc.),
without contamination by micro-organisms or possible residues from cleaning and
desinfection; b) Sampies for quantitative sturlies should be representative of the
region being studied (e.g. the gut flora ofruminats); c) All samples must be fully pro-
tected from exposure to oxygen if sturlies aretobe made on oxygen sensitive bacteria;

144
d) In all cases, the most satisfactory procedure is to take the samples only when the
study can be commenced immediately and culture specimen as soon as possible after
collection; e) Hold specimen at room temperature (HoLDEMAN et al., 1977). - When
the study cannot be commenced immediately, the sample should be cooledas soon as
it is removed from the animal. Freezing at - 75° Cis widely used for samples ofthe
rumen contents and has proved satisfactory for human faeces and intestinal content
(CLARKE, 1977); f) Transport specimen to Iabaratory for culture as soon as possible
after collection; g) Do not use enrichment or transpoft media; rather streak directly
from the specimen (overgrowth of facultative bacteria and less anaerobes result
because even pre-reduced mediaarenot as reduced as many infection sites).- For fur-
ther details the MANUAL of HoLDEMAN and co-workers ( 1977) is an excellent guide
on the treatment of samples.

9.3 Preparation and dilution of the samples

As a rule, 1 gofmaterialtobe investigated (faeces, saliva, human milk, etc.) is taken

and suspended in 100 ml of dilution solution. HAENEL ( 1960) diluted (from 1o-2 ) the
faeces samples with double quantities of diluent (2 ml to 18 ml of dilution solution).
Sometimes one y;o ml is inoculated in place of 1 ml.

9.3.1 Simp/ified anaerobe technique

Most of the panel used the more rigorous anaerobe technique: preparing dilutions in
a reducing medium which immediately prior to use has been held in a boiling water
bath for at least 20 min. to expel any oxygen (SYKES & SKINNER, panel discussion,
1973). The sample can be diluted without harm in saline solution (buffered or non-
buffered) containing reducing substances; phosphate buffer solution according to
HAENEL ( 1960) and ÜCHI and co-workers ( 1964) (see Appendix 2. 7); saline water
with 0.3% of cysteine (WERNER, 1966); or saline water with 0.05% of cysteine
hydrochloride, RCM or LV (SYKES & SKINNER, 1973).

9.3.2 Traditional technique

The traditional technique can be used without any special precautions. A solution
containing 0.1 % of yeast extract or corn steap Iiquor yielded viable cell counts of
bifidobacteria in fermentedmilk products superiorto the physiological saline as dilu-
ents of samples for serial dilution (SHIMADA et al., 1977).

9.4 Media for bifidobacteria culturing, differentiation and isolation

Very different culture media for bifidobacteria may be divided into comp1ex (natu-
ral), selective, semisynthetic, synthetic, as weil as pre-fabricated dried culture media.
HoFFMANN ( 1966) considers that investigations of gut flora should be begun with sim-

145
ple culture and culturing procedure, since too complicated a procedure may render
progress more difficult. According to WERNER ( 1964) the simultaneaus use of a num-
ber of suitable culture media may be the optimal procedure for detection of bifido-
bacteria.

9.4.1 General directions

lt is especially important that a culture medium for bifidobacteria fill all requirements
for nutrients, buffering capacity and pH value.

9.4.1.1 Supply of nutritive and growth substances

Requirements for nutritive substances have been considered in Chapter 2.3.3 and for
growth-promoting factors in Chapter 4. Culture media containing many constituents
may be enriched with Tween 80 to improve the emulsion of particular constituents.
Examples include culture media used by ÜYLLENBERG & NIEMELÄ ( 1959), MITSUOKA
and co-workers ( 1965 b ), HAENEL and co-workers (1970) and TERAGUCHI and co-
workers ( 1978).

9.4.1.2 Low oxidation-reduction potential

Reducing substances are added to most culture media to lower oxidation-reduction

potential (Table 9.1). The substances include ascorbic acid, calcium sulphide, cys-
teine hydrochloride, fresh fragments of intemal organs, glucose, glutathione, pyruvic
acid, sodium sulphite and sodium thioglycollate. These additions make possible a
more or less anaerobic work and technique, e. g., in deep-agar or liquid culture media,
but not in surface culturing. According to ÜRLA-JENSEN ( 1943), ascorbic acid shows a
lesser reducing effect than cysteine. Ascorbic acid is filtrated sterile separately and
added to culture medium previously sterilized. The prepared culture medium may be
kept up to one week. - Culture media containing cysteine should be used quickly
because cysteine quickly oxidizes to cystine. Cysteine is filtrated sterile and added to
the culture medium previously sterilized. Li ver or liver extract is successfully used for
culturing bifidobacteria and other anaerobes, because liver contains cysteine in addi-
tion to certain growth substances (ÜRLA-JENSEN, 1943). - Heating of glucose (e. g.
during sterilization of a culture medium) in the alkaline pH range considerably
increases its reducing capacity (HALLMANN, 1953). However, during fermentation the
gas production may act as an inhibitor; 0.1% of sodium thioglycollate may eventually
show a growth-inhibiting effect (ÜRLA-JENSEN, 1943). - 0.5% of agar (minimum) in
liquid culture medium or in deep-culture prevents free circulation of air and helps to
preserve anaerobic conditions.

9.4.1.3 Maintenance ofthe pH value during growth and the buffering capacity

The maintenance of a defined pH value during growth is important for acid-produ-

cing organisms, but not for acid-resistant bifidobacteria. The destructive effect of acid
on bacteria (e. g. durable cultures) may be prevented as follows: a) using non-fermen-

146
table substrate; b) using a culture medium containing an exactly added carbohydrate
(thioglycollate medium without added glucose); or using selective agar according to
HAENEL and co-workers ( 1970), etc.; c) an eventual very small amount of sugar may
be added to commonly used culture medium; d) the addition of calcium carbonate to
neutralize produced acids; and if insoluble ingredients are undesirable in a culture
medium, sodium carbonate may be added (see KL-G medium, MurAI, 1976). The
addition of substances for acid neutralization may be important for mixed cultures,
for culture media containing high concentration of carbohydrates and for culture

Table 9.1 Kind and amount of various additives which lower oxidation-reduction potential in
culture media for bifidobacteria

.1. Ascorbic acid and its sodium satt (sodium ascorbate)

0.005% L-ascorbicacid in ascorbic-acid-amino-lactose-agar(PLoTHO, 1947)
0.1 % ascorbic acid Tornato juice-yeast autolysate-agar ÜRLA-JENSEN ( 1943);
Culture medium ofNoRRIS etal., (1950)
1.0% ascorbic acid M.R.S. broth(KocH etal.. 1970b)
1.0% sodium ascorbate Bifidobacteria agar (see 2.1.6 Appendix)
1.0% sodium ascorbate Culture medium ofGYLLENBERG & NIEMELÄ (1959).
2. Calcium sulphide
Addition according to DELBOVE ( 1932).
3. Cysteine and Cysteine hydrochloride
0.02% Cysteine Tornato-casein peptone-yeast autolysate-agar
(ÜRLA-JENSEN, 1943)
Cysteine hydrochloride M.R.S. Broth(KocH etal.,l970a)
0.03% Cysteine M.R.S. Broth (CATTEAU etal.. 1973)
0.05% Cysteine hydrochloride Bifidobacteriaagar(see 2.1.6 Appendix)
NPNL-agar (TERAGUCHI et al., 1978)
Selective agar I (HAENEL et al., 1970)
0.2% Cysteine Tornatojuice agar (DE VRIES & STOUTHAMMER, 1967).
4. Fragments fresh of organs
Brain: Calf or sheep's brain in Rosenow medium (see 2.6.12 Appendix); Calfbrain infusion and
brain-heart-infusion, BBL, Difco and Oxoid.
Heart: Beefheart infusion and brain heart infusion, BBL, Difco and Oxoid.
Kidney: Rahbit kidney in dextrose or Iactose broth (CRUIKSHANK, 1925).
Liver: Beefliverinfusion in liver-cystine-milk sugar-agar(BLAUROCK, 1937); Liverextract solu-
tion in NPNL-agar (TERAGUCHI et al.. 1978).
5. Glucose (Dextrose)
I% Bifidobacteria agar (see 2.1.6 Appendix)
2% Tornatojuice-casein peptone-yeast autolysate-agar(ÜRLA-JENSEN, 1943).
6. Glutathione
0.02% LBSagar(MUTAI eta/.,1977)
7. Pyruvic acid
I ml/1 LBSagar(MUTAI eta/.,1976)
8. Sodium sulphite (DELBOVE, 1932)
9. Sodium thioglycollate
0.03% Buffersolution (HAENEL, 1960); Thioglycollate medium Difco;
0.1 % Thioglycollate medium Brewer Modilied BBL;
0.11% ThioglycollatemediumOxoid;
0.5% VL(Meat-yeast-agar)(CATTEAU etal., 1971).

147
media used for transfers of bifidobacteria strains, etc. Marble, a form of natural cal-
cium carbonate, may also be added (as small peas-like particules) to culture medium
(See Terrazzibouillon, ADAM, 1927; Rosenow medium, Appendix 2.6.12); e) buffer-
ing of a culture medium may contribute to a limited pH-stability. Inorganic phospate
compounds, for instance, are often added to culture media and diluents for bifido-
bacteria (Dilution solutions, see Appendix 2. 7; bifidobacteria agar, culture media
according to TOMARELLI and co-workers (1949 a) and HASSINEN and co-workers
(1951 ), etc.
When the metabolic activity ofbifidobacteria has been established (acid produc-
tion, final pH value ), buffering ofthe substrate and various additives for acid neutrali-
zation should be undertaken.

9.4.1.4 Final pH ofthe prepared medium

The pH value is adjusted to 6.6-7.2 (in some cases to pH 7 .5) before the sterilization of
the culture medium. If additions are made after sterilization of the culture medium,
the final pH value should be checked. The addition of sorbic acid lowers the pH value
to 5.0 and shows a growth-inhibiting effect (LERCHE & REuTER, 1961 ).

9.4.2 Optimal growth media

lt is generally considered that bifidobacteria have complex growth requirements and

for this reason rieb media are frequently used (SYKES & SKINNER, 1973). Self-pre-

Table 9.2 Optimal culture media used for culturing bifidobacteria

Optimal culture media Reference

Appendix
9.2.1 Freshly prepared media
Liver-cystine-milk sugar-agar I pH 7.4
(BLAUROCK, 1937) 2.1.1
Tornato-casein peptone-yeast autolysate-agar I pH 6.4-6.5
(ÜRLA-JENSEN, 1943) 2.1.2
Alkaline blood agarwith dextrose according to HACKENTHAL und BIERKOWSKI I
pH7.5
(BIERKOWSKI, 1956) 2.1.3
VL-agar(meat-yeast-agar) I pH 7.4-7.5
(BEERENS et af.• 1963) 2.1.4
BL-agar (glucose-blood-liver-agar) pH 7.2
(ÜCHI eta/., 1964) 2.1.5
Semisynthetic culture medium I pH 6.8
(TOMARELLI et af., 1949a) 2.3.1

9.2.2 Dehydrated commercial culture media

Brain-heart infusion agar I pH 7.4 2.6.3
Eugon agar I pH 7.0 2.6.5
M. R. S. broth with cysteine I pH 6.2 2.6.8
Trypticase-soy-broth-agar I pH 7.3 2.6.16

148
pared culture media are often of a complicated composition and some ingredients
such as fresh liver, human milk, etc., are not always availabe. The success of bifido-
bacteria detection in an optimal culture media is mainly dependent upon the follow-
ing factors: a) If culture media have no selective effect, non-bifidobacteria may out-
grow bifidobacteria; b) the macroscopic identification of bifidobacteria colanies
may be facilitated using differential colouring (see 9.4.3.1 ); c) the optimal growth of
bifidobacteria is dependent upon the freshness ofthe ingredients ofthe medium; d)
the composition ofthe culture medium should make possible the growth of different
biotypes present in the investigated material.
The optimal culture media (fresh and dehydrated) used for bifidobacteria are
shown in Table 9.2. Dehydrated culture mediapromote the growth ofvarious bacte-
rial species including bifidobacteria.

9.4.3 Media with aidsfor selective reading (identi.fication) ofbi.fidobacteria

colonies in a mixed popu/ation

The selective differentiation of colanies permits better distinction of bifidobacteria

from other organisms by using the following measures:

Table 9.3 Survey ofthe colour differentiation of colonies grown in culture media containing
different indicators

Indicator Culture medium Colour of the bifidobacteria

colonies

Anilin blue R.C.M.agar pale blue; first isolation

(Cotton blue, watersoluble,
0.1 %)
Anilin blue
(Biuish, 6 B extra, Gübler,
Stuttgart, W. Germany)
3 ml of I % solution per I
Bromacresol green
and Neutral red
Chinese blue
(Ferri ferrocyanide)
0.03%
0.40%
2,3 ,5- Triphenyltetrazolium
chloride (TTC), 0.01%
BULLEN etal. ( 1973); see
Appendix 2.6.1 0
Alkaline blood agar brown
BIERKOWSKI ( 1956); see
Appendix 2.1.3
Selective agar I nodata
HAENEL et aJ. ( 1970); see
Appendix 2.2.1
Chinese blue-lactose-agar darkblue
(HENNEBERG, 1934)
R.C.M.agar brown; first isolation
WIEL-KORSTANJE & WINKLER
( 1970); see Appendix 2.6.1 0
Liver-cystine-milk sugar-agar intensive blue colour
according to BLAUROCK (Bov-
enter, 1949)
Synthetic agar ÜYLLENBERG & red precipitation appears in the
NIEMELÄ ( 1959); see Appendix colonies
2.4.2.3

149
9.4.3.1 Colour differentiation of colanies

BovENTER ( 1949) tested many indicators which were unsuitable due to the growth-
inhibiting effect or weak colour change. It was especially difficult to obtain a colour
change vis-ä-vis non bifidobacteria, e. g. E. coli. Table 9.3 shows a survey of the
colour differentiation ofbacterial colanies in various culture media.

9.4.3.2 Exhibition of the bifid cell morphology

It is important in the microscopic work to distinguish bifidobacteria by their typical

cell morphology. According to a proposal by KALz (1961 a), the addition of 0.0325
lU Im! of penicillin-G did not affect the growth of bifidobacteria, but promoted the
appearance oftypical cell morphology. (HoFFMANN, 1966).
When bifidobacteria grow in a medium that contains an excess of specific
growth factors, the cell structure becomes much more regular. The branched, swollen
rods considered characteristic of this organism, may therefore, reflect a deficiency of
an essential murein precursor in the media normally used for its cultivation (STANIER
et al., 1971 ). For more details see Chapter 2.2 (morphology) an 2.11.5.1 (antibiotic
agents).

9.4.4 Mediafor selective growth (isolation)

The selective isolation ofthe bifidobacteria when they are considerably outnumbered
by other anaerobes has not yet been satisfactorily solved (SYKES and SKINNER, panel
discussion, 1973).
For the selective isolation of bifidobacteria from the faeces, MtTSUOKA and co-
workers ( 1965 b) developed BS-agar I and II and HAENEL and co-workers ( 1970)
selective agar I. The NPNL-agar according to TERAGUCHI and co-workers ( 1978) and
the modified selective Rogosa's agar (SHIMADA and co-workers (1977) are used for
the selective isolation ofbifidobacteria from milk products.
Selective substances have been added to various dehydrated commercial media.
Table 9.4 gives a survey of various selective substances added to various culture
media. These substances (kanamycin sulphate, neomycin sulphate, Iithium chloride,
nalidixic acid, sodium azide, sorbic acid) mainly inhibit the growth of E. coli (gas-
producing enterobacteria). Relatively littleis known about the selective effect of con-
centrations used on particular culture media.
The effect of selective substances depends upon the following factors: a) The
dominant bacterial species which may vary depending on the material examined; b)
The number ofbifidobacteria; c) The kind and concentration of selective substance
being used as weil as the additional effect of a possible mixture of selective sub-
stances; d) Incubation technique (whether the growth of aerobes is inhibited so that
only facultative anaerobesandanaerobes may grow); The kind of culture medium
(e. g. optimal culture medium, minimal culture medium).

150
Table 9.4 Survey on various selective substances added to various cu1ture media

Se1ective substance and its Cu1ture medium Reference

effect

9.4.1 Antibiolies
Kanamycin sulphate Standard B1ood Agar FINEGOLD et a/., ( 1971)
75 ).l.g/m1, poorse1ective
Neomycin su/phate (NBC)
200).l.g/m1 BS Agar I and II MITSUOKA eta/., (1965b)
200).l.g/m1 EG-Agar ÜCHI et a/., ( 1964)
100).l.g/m1 Co1umbia-Agar-Base SYKES & SKINNER, pane1
discussion ( 1973)
lOO).l.g/m1 NPNL-Agar TERAGUCHI et a/., ( 1978)
30).l.g/m1 NPNL-Agar TERAGUCHI et a/., (1978)
IOO).l.g/m1 LBS-Agar ßRANCA eta/., (1979)
200).l.g/m1 Rogosa's Agar modif. SHIMADA et a/., ( 1977)
Paromomycin sulphate
200 ).l.g/m1 and 50 ).l.g/m1 NPNL-Agar TERAGUCHI et a/., (1978)
50).l.g/m1 BS-Agari MITSUOKA et a/., (1965b)
20).l.g/m1 BS-Agarll MITSUOKA et a/., (1965b)
50).l.g/m1 Rogosa's Agarmodif. SHIMADA et a/., ( 1977)

9.4.2 Other bacteriostatic agents

Lithium ch/oride
3mg/ml BS-Agar I and II MITSUOKA et a/., ( 1965b)
3mg/m1 NPNL-Agar TERAGUCHI et a/., ( 1978)
3mg/m1 Rogosa 's Agar modif. SHIMADA et a/., ( 1977)
Nalidixic acid
80).l.g/m1 Rosenow ßEERENS & TAHON-CASTEL
(1965)
15 ).l.g/m1 NPNL-Agar TERAGUCHI et a/., (1978)

Sodium azide (NaN 3)

0.01% Tornato Agar Or1a-Jensen FRISELL (1951)
modif. Frisell; Selective Agar I HAENEL et al., (1970)
Sodium propionate
1.5% of a 40% so1ution BS-Agari MITSUOKA eta[., (1965b)
1.0% BS-Agarll MITSUOKA et a/., (1965b)
1.5% Rogosa 's Agar modif. SHIMADA et a/., (1977)
Sorbic acid
0.04% Sorbic acid culture medium HAENEL & M0LLER-8EUTHOW
I,llandiii (1957c)
0.04% Tomato-AgarOrla-Jensen, HAENEL & M0LLER-8EUTHOW
modif. Frisell (1957c)
9.4.3 Mixtures
Lithiumchloride (3 mg/ml),
Nalidixicacid(15 mg/ml), NPNL-Agar TERAGUCHI eta/.,(1978)
Neomycin su1phate
(I 00 ).l.g/ m1), Paromomycin sul-
phate (200 ).l.g/ml)
Kanamycin and Neomycin FINEGOLD ( 1965)
(each 100).l.g/ml)

151
Table 9.4 (cont.)

Selective substance and its Culture medium Reference

effect
Sodium propionate (0.0 15 g/
ml), Paromomycin sulphate SHIMADA et a[., ( 1977)
(0.050mg/ml), Neomycin-
sulphate (0.200 mg/ml),
Lithiumchloride (0.003 g/ml)

9.4.5 Media with selective growth ofdifferent biotypes ofbifidobacteria

The kind of culture medium may show a selective effect on certain strains ofbifido-
bacteria. The culture medium used should be suitable for detection and isolation of
the bifidobacteria, as weil as for their cultivation. Depending on the culture medium,
the selection of various mutants of the original strain may be possible, with minimal
nutritional requirements (RAYNAUD, 1959; DEHNERT, 1961 a).
Biotypes with minimal requirements (RA YNAUD, 1959) may grow on the full-syn-
thetic medium of HASSINEN and co-workers ( 1951 ). Bifidobacteria, type A according
to Dehnert, may grow on the full-synthetic medium of Petuely. They predominate in
the faeces ofbreast-fed infants (PETUELY, 1956 c; ÜYLLENBERG & NIEMELÄ, 1959).
Bifidobacteria, subtype B1 according to DEHNERT, may grow on the full-synthetic
medium modified by GYLLENBERG & NIEMELÄ (1959). They are regularly found in
the faeces ofhuman adults.

9.4.6 Semi-synthetic andfull-synthetic culture media

Various semi-synthetic and more comp1ex related media have been developed for use
in the physiological studies and detection of bifidobacteria. The culture media of
TOMARELLI and co-workers (1949 a), NORRIS and co-workers (1950), GvöRGY and
co-workers ( 1954 a) were developed on the basis of the semi-synthetic medium of
TEPLY & ELVENHJEM (1945). The latter was originally designed for growth experi-
ments with folic acid with Lb. casei.- The ascorbic-acid-amino-lactose agar of PLo-
THO (1947) contains growth-promoting substances present in Bessau's infant food
(BOVENTER, 1949). Further cu1ture media were described by MALYOTH & BAUER
(1953) and RA YNAUD ( 1959). The medium of Raynaudwas devfloped on the basis of
the media of PLOTHO (1947), HASSINEN and co-workers (1951), and MALYOTH &
BAUER (1953), on which the growth of Tissier-strain was not observed (RAYNAUD,
. 1959).
The following full-synthetic culture media have heen developed for physiologi-
cal studies and for detection of the bifidobacteria with agar added. The minimal
medium of HASSINEN and co-workers ( 1951) was developed on the basis of the cul-
ture media of TOMARELLI and co-workers (1949 a) and NoRRIS and co-workers
( 1950). The culture medium of PETUELY & LYNAU ( 1954) represents a modification of
the medium of HASSINEN and co-workers (1951) which was subsequently modified
by PETUELY (1956 c) and ÜYLLENBERG & NIEMELÄ (1959). The agar medium of

152
PETUELY (1956 c) permits the growth only oftypical "breast milk bifidus" due to its
poverty in growth substances (PETUELY, 1956 c; ÜYLLENBERG & NIEMELÄ, 1959).
LIEBSCHER (1964 b), however, showed that bifidobacteria isolated from the faeces of
infants as weil as adults may grow on the above medium.

9.4. 7 Dehydrated commercial culture media

Dehydrated culture media are manufactured for culturing various bacteria, including
anaerobes, lactobacilli, pathogenic organisms, antibiotic-resistant, as weil as bifido-
bacteria. Furtherdetails are given in Appendix 2.6.

9.4.8 Jhe Iist of culture medianot obtainable in commercial Irade

Appendix 2.5 presents culture media which are used for culturing bifidobacteria.
They are not obtainable in commercial trade as dehydrated media.

9.5 Use of media

The choice of culture media and their preparation for examination, incubation and
storage are briefly outlined below.

9.5.1 1he choice ofmediafor isolation and enumeration ofbifidobacteria and

their preparation

Special attention should be devoted to the choice of a culture medium for isolation
and enumeration ofthe bifidobacteria from materials where they may not be the dom-
inant organisms. As mentioned above, there is no optimal selective culture medium
for the various materials examined. The nutritional requirements of the various bio-
types ofbifidobacteria should also be considered. Moredetails on the preparation of
culture media for detection of bifidobacteria are outlined in Appendix 2. Fig. 9.1
shows an analytical balance for weighing of culture media ingredients.

9.5.2 Preparation of media for inoculation

The growth conditions ofbifidobacteria in a culture medium may be improved using

suitable techniques.

9.5.2.1 Simplified anaerobe technique

After pouring, the plates are dried for 1 %hat 52° C for ~hin a laminar flow cabinet.
The plates are then pre-reduced by at least overnight storage under Hz : COz (90 : 10)
in a cold catalyst anaerobic jar in order to remove the last traces of oxygen. An indica-

153
Fig. 9.1 Electronic analytical balance with 0. 1 mg readability. Weighing range : 0-160 g. Different
peripheral instrumentssuch as printers can be directly connected to the balance. lt is used for weigh-
ing ingredients of culture media
(Mettler Instrumente AG, CH-8606 Greifensee, Switzerland)

tor strip (BBL Cat.No. 70504) is used to check anaerobiosis. The liquid media or
liquefied agar in tubes should be held in a water bath for at least 20 min to remove oxy-
gen, then cooled and inoculated immediatly (SYKES & SKINNER, panel discussion,
1973).

154
9.5.2.2 Traditional technique

In the traditional technique, no special precautions are taken to remove oxygen. It is

not considered necessary to store the plates anaerobically before use (SYKES & SKIN-
NER, panel discussion, 1973).

9.5.3 Incubation technique

Immediately after inoculation most of the panel used a simplified anaerobe tech-
nique and incubated their plates in an anaerobic jar containing H2 + C02 (90 : I 0)
with a catalyst; one used Gaspack (BBL) (SYKES & SKINNER, panel discussion, 1973).
BROWN & TowNSLEY ( 1970) and SHIMADA and co-workers ( 1977) used aGaspack an-
aerobic jar too.
All samples are incubated at 35° C or 37° C (S_YKES & SKINNER, 1973). Incuba-
tion time is 48 h or more until growth is observed.

9.5.4 Storage time ofprepared media

Most prepared media should be used in a fresh state and stored not Ionger than one
week (see 9.4.1.2).

9.6 Reading of colonies and microscopic smear of the examined material

Reading of a culture medium covers the macro- and microscopic reading of colonies.

9.6.1 Appearance of colonies on solid media

The colonies of bifidobacteria should be distinguished on a culture medium from

colonies of other organisms. Often it is not very easy to do this. The following factors
beyond a wealth of experience in working with bifidobacteria may influence observa-
tion results: a) Correct preparation, inoculation and incubation of a culture medium
to obtain a reasonably good growth of bifidobacteria. The exclusion of 02 during
incubation may have a pre-selective effect (the growth of aerobes is prevented); b)
The visual differentiation oftypical colonies (size, colour, height) and good knowl-
edge about the characteristics of other bacterial colonies. The latter depends a Iot on
the examined material (faeces, cultured milk, saliva, etc.) as weil as the numberofbif-
idobacteria present in lower and high er dilutions. Table 2. 7 shows cultural character-
istics of human and some non-human species of the genus Bifidobacterium (see also
BuCHANAN & GIBBONS, 1974). Some bifidobacteria strains may show improved
growth ("nursing growth ")in association of instance with coli bacteria on blood agar
(KLUDAS, 1959 b; HAENEL, 1960).- Examine incubated plates every 12 hours until
growth appears. As soon as growth appears, examine (upper and lower side of colon-
ies) under 10 x stereomicroscope and mark isolated colonies ofvarious typestobe

155
picked (HOLDEMAN et al., 1977); c) Many colonies (at least I 0-15 per plate according
to HoFFMANN, 1966) can be examined under a microscope, as to colour and morpho-
1ogical characteristics. - Great difficulty has been encountered in observing the typi-
cal branching ofthe cells. This tendency is more often observed in primary isolation
than in later stages (SYKES & SKINNER, panel discussion, 1973).

9.6.2 Microscopic examination

The technique of microscopic examination used may be especially important for

detection (lacking selectivity ofmost culture media) and study ofthe bifidobacteria
biotypes.

9.6.2.1 Smear for enumeration

Make a smear of specimen (a loop of the material in a small amount of water), Gram
stain and record all morphotypes present and the relative numbers of each. This is
important (HOLDEMAN et al., 1977). Gram staining the samples of examined material
may belong to cu1tural detection because a certain correspondance should exist
between cultural detection and microscopic preparation (BRAUN, l959).1t is possible
that many bifidobacteria colonies grown on a defined agar medium may be enumer-
ated following Gram staining to obtain distribution in percentage of Gram positive
and Gram negative rods and cocci. These percentage numbers must correspond to
those obtained by direct microscopic countafterGram staining of the original smear
(HOFFMANN, 1966).

9.6.2.2 Bacteriological picture ofinfant stools

A crushed sample is prepared from the stool, fixed, Gram stained and then observed
under an optical microscope and photo-micrographed. As a resu1t of this examina-
tion, it may be determined whether the food is bifidogenic or not, i. e. whether any
foodstuff influences a strong increase of B. bifidum population in the stools follow-
ing its consumption by at least 10 days (MA YER & KESCHA W ARZI, 1970). MA YER
(1971) considers that the Gram preparation offresh infant stools gives as much evi-
dence as the quantitative analysis ofthe gut flora. The latter has used this method for
28 years.

9.6.2.3 Staining procedure

Gram staining is the procedure most often used. ScHULER-MALYOTH (1968) pro-
posed the following staining procedure for fermentedmilk products: a) Preparation
ofa thin, even smear; b) Air-drying and then heat fixation; c) Staining with Loefflers
methylene blue stain, 5 min.; d) Careful rinsing with tap water, drying between filter
paper; e) Differentiation in 96% ethyl alcohol until the preparation is stained a very
weak blue; f) Careful drying between filter paper.- Alcohol differentiation results in
decolourizing mik protein. Streptococci and lactobacilli appear dark blue, while bi-
fidobacteria are more delicately coloured due to weaker colour intensity. Because of

156
this property as weil as morphological characteristics, the bifidobacteria may be rela-
tively easily differentiated from the lactobacilli. The metachromatic reactions ofthe
bifidobacteria cells may be established with careful.analysis (red-violet colour).

9.7 ldentification of the genus Bifidobacterium

According to BUCHANAN & GIBBONS ( 197 4) bifidobacteria belong to the family Actin-
omycetaceae that contains the following genera: I. Actinomyces, II. Arachnia, 111.
Bifidobacterium, IV. Bacterionema, V. Rothia. Table 9.5 shows differentiating char-
acteristics ofthe genus Bifidobacterium (see also Table 2.5).
Tentative identification of the genus Bifidobacterium isolated from intestinal or
clinical materials can be made on the basis of colony form and microscopy although
Lactobacillus, Actinomyces, Propionibacterium and Eubacterium may closely resem-
ble each other in their microscopical appearance. Reliable differentiation between
these genera, however, can only be achieved by biochemical tests as shown in Table
9.5. Morphology and analysis as to the presence ofthe fermentation products (acetic
and lactic acids) were considered tobe the most important diagnostic tests for the
genus Bifidobacterium (SYKES & SKINNER, panel discussion, 1973). Almost all bio-
chemical tests such as the demonstration of catalase, nitrate reduction, formation of
indole, liquefaction of gelatin, gas formation from glucose and response to the rham-
nose, sorbose, glycero1, erythritol, adonito1 and dulcitol arenegative in the presence
of the genus Bifidobacterium (MITSUOKA & KANEUCHI, 1977).

Table 9.5 Key for differentiation of Bifidobacterium from related genera

Characters Bifido· Actinomyces Propioni- Eubacterium Lactobacillus

bacterium bacterium

Majorfermentation
productsb AL s p B,AF,none L
Grow aerobically - + V - V
Gasformation from
glucose - - - V V
-
Catalase production - V + - -
+-
-
Nitrate reduction - + V -
Indole production - - V - -
- +
-
Gelatin liquefaction - + V -
Acidfrom:
Rhamnose - V - + - + V
Sorbose - V V - + - +
- + +
Glycerol - - +- -
+
-
+
Erythritol - - + -
+
-
+
Adonitol - - V - -
Dulcitol - - - + - + - +

Legend: + = positive, - = negative, V = variable, +- = major positive, - + = majornegative

bA = acetic acid, B = butyric acid, F = formic acid, L = lactic acid, P = propienie acid,
S = succinic acid
Source: MITSUOKA & KANEUCHI ( 1977)

157
9.8 Species and biotypes differentiation

Species differentiation within the genus Bifidobacterium is mainly based on carbo-

hydrate fermentation which was considered tobe an important fact (SYKES & SKIN-
NER, panel discussion, 1973); MnsuoKA & KANEUCHI, 1977). Various culture media
were used for the examination of carbohydrate fermentation: a) DEHNERT (1957 b)
and MAYER and co-workers (1965 b) used the Tomarelli medium; b) MnsuoKA
( 1969 b) described a basal medium forthe examination of carbohydrate fermentation
ofbifidobacteria isolated from animals; c) Several members ofthe panel carried out
the tests as described by BARNES & IMPEY (1968) with orwithout the addition of0.5%
laked blood (SYKES & SKINNER, 1973).
The API - identification system for Lactobacillus (Vixo tab 38, La Balme-Les-
Grotte, France) has been modified by CATIEAU and co-workers (1973) forthe ident-
ification ofthe bifidobacteria species. The samples are incubated anaerobically (BBL
Gas pack) for 48 hat 37° C. Other rapid methods were also used (SYKES & SKINNER,
1973). Table 2.6 shows fermentation reactions of bifidobacteria according to BER-
GEY's MANUAL (ßUCHANAN & GIBBONS, 1974). Further schemes for differentia-
tion are given by HOLDEMAN and co-workers (1977) and MITSUOKA & KANEUCHI
(1977).

158
Appendix 1: Glossary of terms

According to JACOBS and co-workers ( 19 57); BENDER ( 197 5); HERBERT & WILKINSON
(1977); MACDONALD CRITCHLEY (1978) and B/akiston's Pocket Medica/ Dictionary
(1979).

Achlorhydria. The absence of free hydrochloric acid in the gastric juice. (See Hypo-
chlorhydria).
Aglycon. The non-sugar part of a glycoside. (See Glycosides ).
Anaemia. A shortage of red blood cells. Nutritional anaemia is the commonest form
and is due to iron deficiency. Pernicious anaemia is due to a failure to absorb
vitamin B12. (See Intrinsic factor).
Anastomosis. A surgical communication made between the blood vessels or between
two hollow organs or by two parts of the same organ.
Angstrom unit. One ten-millionth part of a millimetre, or one ten-thousandth part of a
micron. Symbol A.
Antibiosis. A phenomenon among micro-organisms in which one type of species
kills, injuries, or inhibits the growth of anotherspecies as a form of self-protection.
Antibody. Specific substances, natural or induced by exposure to an antigen, which
have the capacity to react with specific or closely related antigens. In the serum,
they occur within the immunoglobulins.
Antigen. Any substance eliciting an immunologic response, such as the formation of
antibody specific forthat substance.
Anus. The termination of the rectum.
Appendicitis. Intlammation ofthe vermiform appendix.
Bacteraemia. The presence of living bacteria in the blood.
Bile. A fluid produced by the liver and stored in the gall-bladder; it contains bile salts,
cholesterol, Iecithin and various pigments.
Blind-loop syndrome. The existence ofby-passed segments or diverticula in the small
intestine, resulting in stasis, abnormal bacterial tlora, diarrhoea, weight loss,
vitamin deficiency and megaloblastic anaemia.
Caecum. Firstpart of the Iarge intestine, separated from the small intestine by the ileo-
colic sphincter.
Cardia. The oesophageal orifice and adjacent part of the stomach.
Cell-mediated immunity. Immunity maintained by T lymphocytes.
Cellular immunity (syn. for macrophage immunity). The term originated by Metchni-
koff to refer to an increased ability of phagocytic cells to destroy or to digest parasi-
tic organisms.
Cellulose. Polysaccharide composed of long chains of glucose units, and occurring
together with hemicelluloses and Iignin in plant cell walls.
Chromosome. The rod-shaped structure which bears the genes in the nucleus of a cell
which are formed during mitosis from the chromatin.
Cirrhosis. Any diffuse fibrosis which destroys the normallobular architecture of the
liver. (See Fibrosis).
Colon. The part of the large intestine beginning at the caecum and terminating at the
end of the sigmoid tlexure.

159
 Commensalism. A relationship between species of organisms in which one receives
benefit and the other neither benefit nor harm.
Complement. An enzymatic system of serum proteins that is activated by many anti-
gen-antibody reactions, and which is essential for antibody-mediated bacteriolysis,
and immune haemolysis, and plays an important part in some biological reactions,
e. g. phagocytosis, opsonization, etc. Complement is made up of many compo-
nents. Symbol, C.
Dextrins. Mixture of soluble compounds produced by partial breakdown of starch by
acid, heat or enzymes.
Diverticulitis (syn. Diverticular disease ). Inflammation of a diverticulum.
Diverticulosis. The presence of diverticula in the intestine.
Diverticulum. An outpouching or sac arising from a hollow organ or structure. In the
acquired form, it represents a herniation of the mucous membrane through the
muscular wall of the organ.
Duodenum. The first part of the small intestine, beginning at the pylorus. It is the most
fixed part of the small intestine, and contains the openings of the pancreatic duct or
ducts and the bile duct.
Ecology, bacterial. Interrelationships of bacteria and their environments.
Enteritis. Any inflammation ofthe intestinal tract, particularly ofthe mucosa.
Entero-colic. Pertaining to the small intestine and colon.
Enterocolitis. Inflammation of the small intestine and colon.
Erythrocytes. Red blood cells carrying the red colouring matter, haemoglobin, which
is the means of transporting oxygen and carbon dioxide in the blood stream.
Fibrosis. An increment in fibrous connective tissue.
Fistula. An abnormal communication between two surfaces or between a viscus or
other hollow structure and the exterior.
Focus. The major seat of a disease.
Gall-bladder. Organ that stores the bile produced by the liver.
Gamma-A globulin. The immunoglobulin which comprises about l 0% of the anti-
hodies of human serum, where it occurs as a monomer. lt constitutes the major
immunoglobulin in such secretions as intestinal fluid, saliva, bronchial secretions,
colostrum, where it occurs as a dimer linked to secretory piece, an additional pep-
tide; it does notcrosshuman placenta. lt is not always major colostral immuno-
globulin of cow, sheep and pig. Symbol, lgA.
Gamma-D globulin. An immunoglobulin occurring in small amounts in normal
human serum. Symbol, lgD.
Gamma-E globulin. The immunoglobulin associated with reaginic antibodies. Sym-
bol, lgE.
Garnma-G globulin. The immunoglobulin comprising about 80% ofthe serum anti-
hodies of the adult; it crosses human placenta, and fixes complement. Four sub-
classes in man IgG l, IgG2, IgG3, IgG4. Symbol, IgG.
Gamma-globulin. A designation for immunoglobulins of different molecular weights
and for certain proteins related to them by chemical structure, many ofwhich have
known antibody activity.
Gamma-M globulin. The immunoglobulin comprising 5-10% ofthe serum antibod-
ies, and including Wassermann and heterophile antibodies, cold agglutinins, iso-
hemagglutinins and antiborlies to the endotoxins ofGram-negative bacteria. Sym-
bol, lgM.
160
Gastrectomy. Excision ofthe whole or apart ofthe stomach.
Gastricjuice. The secretion ofthe glands ofthe stomach, having an acid pH and con-
taining hydrochloric acid, pepsin, mucin, andin infants, rennin.
Gastritis. Inflammation of the stomach.
Gastro-colic. Pertaining to the starnach and the colon.
Gastroenteritis. Inflammation of the mucosa of the starnach and intestines.
Gene. The physical unit of hereditary traits in the chromosome.
Geriatrics. The branch of medical science that is concemed with aging and its dis-
eases.
Glucuronic acid. The acid derived from glucose by the oxidation ofthe group on car-
bon atom number six. Many toxic substances are excreted from the body combined
with glucuronic acid as glucuronides.
Glycogen."Polysaccharide composed of glucose units; the form in which carbohy-
drate is stored in the animal body, in the liver and muscles.
Glycosides. Compounds consisting of a sugar attached to another molecule. When
glucose is the sugar they are called glucosides. Many varieties occur in plants, and
some are useful medicinally, such as digitalis and rutin.
Haem. The iron-containing pigment which in combination with protein forms the
haemoglobin ofthe red blood cells. The iron is in the ferrous state.
Haemoglobin. The red colouring matter, the respiratory pigment, of erythrocytes
composed ofthe protein globin linked to four haem molecules. Haemoglobin com-
bines reversibly with oxygen which it carries from the lungs to the tissues, and with
carbon dioxide which it carries from the tissues to the lungs where it is excreted.
Oxidation ofthe ferrous ion ofhaemoglobin to the ferric state produces methaemo-
globin.
Hemicelluloses. Carbohydrates composed of polyuronic acids combined with
xylose, glucose, mannose and arabinose. They occur tagether with cellulose and
Iignin in plant cell walls.
Haemorrhoid. A varix in the lower rectal or anal canal. (See Varix).
Hepatic. Pertaining to or involving the liver.
Hepatic coma. A state of unconsciousness observed in patients severely ill with liver
disease.
Hepatic encephalopathy. Disturbance of central nervaus function occurring in ad-
vanced liver disease.
Hepatitis. Inflammation ofthe liver.
Hernia. The abnormal protrusion of an organ or apart through the containing wall of
the abdominal cavity, beyonditsnormal confines.
Humour. Any fluid or semifluid part ofthe body.
Humoral antibody. Any antibody occurring in solution in plasma, lymph or another
fluid part ofthe body.
Humoral immunity. Immunity maintained by antibody molecules secreted by B lym-
phocytes and plasma cells and circulating throughout the plasma, lymph and other
body fluids. In contrast, cell-mediated immunity is maintained by T lymphocytes.
Hypertension. Excessive tension or pressure, particularly that effected by bodily
fluidssuch as blood or aqueous humour.
Hypochlorhydria. Diminished hydrochloric acid secretion by the stomach.
Hypogammaglobulinaemia. A decrease in plasma gamma globulin.

161
Ileum. The lower part of the small intestine, extending from the jejunum tothelarge
intestine.
Indoxyl. A substance found in the urine which is a metabolic derivative of the amino
acid tryptophan. It is excreted as indoxyl potassium sulphate, known as indican.
Jejunum. The part of the small intestine extending between the duoden um and the
ileum.
Immunity. The condition of a living organism whereby it resists and overcomes infec-
tion or disease.
Immunogen. Any substance capable of stimulating an immune response.
Immunoglobulin. Any one of the proteins of animal origin having known antibody
activity, or a protein related by chemical structure; and occurring in plasma, urine
and other body tissues and fluids.
Interferon. A protein, formed by animal cells in the presence of a virus or other indu-
cing agent, that prevents viral reproduction and is capable of inducing in fresh cells
of the same animal species resistance to a variety of viruses.
Intestinal juice. Digestive juice produced by the intestinal glands lining the small
intestine. It contains the enzymes aminopeptidase and dipeptidase, amylase, mal-
tase, Iactase, sucrase, Iipase, esterase, nucleases, nucleotidase and the activator
enterokinase (activates trypsinogen and chymotrypsinogen of the pancreatic
juice).
Intestine. The part of the digestive canal extending from the pylorus to the anus, and
consisting of the small and large intestine.
lotriosie factor. Vitamin B12 from the diet requires an unidentified substance from the
gastric mucosa (known as intrinsic factor) either to aid its absorption or to form a
complex called the haemopoietic factor. In pemicious anaemia there appears tobe
a deficiency of the intrinsic factor and the vitamin B12 is not absorbed.
Leukocytes, white blood cells. The colourless cells ofthe blood having a nucleus and
cytoplasm. Those occurring in normal blood are divided into non-granular leuko-
cytes, including lymphocytes and monocytes, and granular leukocytes, including
neutrophils, eosinophils, basophils.
Lignin. A high molecular weight aromatic compound, associated with the carbohy-
drates of the cell wall of plants but not itself a carbohydrate.
Liver. The largest glandular organ in the body. Its functions include secretion ofbile,
storage of glycogen and fat, protein breakdown and detoxication.
Lymph. The fluid intermediate between the blood and the tissues; the medium in
which oxygen and nutrients are conveyed from the blood to the tissues and waste
products back to the blood.
Lymphatic tissue. Tissue consisting of networks of reticular and collagenous fibres
and lymphocytes.
Lymph nodes. Masses oflymphatic tissue, intercalated in the course oflymph vessels.
Lymph nodule. A small mass of dense lymphatic tissue in which new lymphocytes are
produced.
Lymphocyte. A leukocyte occurring in the lymphatic tissue, blood and lymph. It is a
spherical cell, with a large, round nucleus and very scanty cytoplasm. These cells
are associated with all aspects of specific immunity; B lymphocytes being asso-
ciated with humoral immunity, and T lymphocytes with cell-mediated immunity.
Lysozyme. An enzyme that digests certain high-molecular weight carbohydrates. Bac-

162
 teria that contain these carbohydrates (composed of specific polysaccharide units)
as part oftheir cell wall disintegrate or lyse under attack by Iysozyme. Widely distri-
buted (tears and other secretions), but particularly in egg-white.
Macrocyte. An erythrocyte having either a diameter or a mean corpuscular volume
exceeding by more than two standard deviations that of the mean normal.
Macrocytosis. The presence of macrocytes or abnormally large erythrocytes in the
blood.
Macrophage. A phagocytic cell betonging to the reticuloendothelial system; it is
important in resistance to infection andin immunological response. (See Phago-
cyte).
Malabsorption. Defective absorption of nutritive substances from the alimentary
canal.
Malnutrition. Disturbance of form or function arising from a deficiency or excess of
one or more nutrients.
Megaloblast. A large erythroblast with a characteristic nuclear pattem produced in
marrow in vitamin B12 or folic acid deficiency.
Megaloblastic anaemia. Any anaemia characterized by the presence of megaloblasts
in the hone marrow, usually associated with macrocytosis in the peripheral blood.
Methaemoglobin. The oxidized form ofliaemoglobin, in which the iron atom is triva-
lent, and which is not able to combine reversibly with oxygen.
Methaemoglobinaemia. An excess of methaemoglobin in the blood.
Micron, unit. One-thousandth of a millimetre; unit of measurement ofbacterial size.
Mutant. An organism or micro-organism with a changed or new gene.
Oesophagus. The musculomembranous canal, extending from the pharynx to the sto-
mach.
Pancreas. A gland in the abdomen with two functions; it secretes the pancreatic juice
and the hormone insulin.
Pancreatic juice. The secretion of the pancreas; a thick fluid, strongly alkaline, con-
taining proteolytic, lipolytic and amylolytic enzymes.
Pepsin. The proteinase of gastric juice which hydrolyzes certain of the linkages of
proteins to produce peptones. lt is secreted as the inactive precursor pepsinogen,
which is activated by acid.
Peritoneum. The serous membrane lining the interior of the abdominal cavity and
surrounding the contained viscera. (See Viscus).
Peritonitis. Inflammation of the peritoneum.
Phagocyte. A cell having the property of engulfing and digesting foreign or other par-
ticles or cells harmful to the body. Fixed phagocytes include the cells ofthe reticulo-
endothelial system and fixed macrophages. Free phagocytes include the leuko-
cytes and free macrophages.
Plasma. The fluid portion of blood or lymph, composed of a mixture of many pro-
teins.
Plasma cell. The antibody-producing leukocyte into which aB lymphocyte differen-
tiates upon Stimulation by antigen. It is present in lymphoid tissue and increased in
numbers in the draining lymph node and at the site of entry of antigen following
antigenic stimulation.
Plasmacyte. See plasma cell.
Plasmid. An extra chromosomal hereditary unit.

163
Portal cirrhosis. Progressive fibrosis centered in the portal areas ofthe liver.
Portal system. The portal circulation, usually the hepatic portal vein and its tributa-
ries.
Portosystemic. Pertaining to the portal system.
Properdin. A macroglobulin of normal plasma capable ofkilling various bacteria and
viruses in the presence of complement and magnesium ions, and involved in the al-
ternative pathway of complement activation.
Pylorus. The circular opening of the stomach into the duodenum.
Rafrmose. A trisaccharide composed of one molecule each of glucose, fructose and
galactose; it hydrolyzes to fructose and melibiose, which in turn hydrolyzes to glu-
cose and galactose. It is present in sugar-beet molasses, cotton seed, soybeans,
beans and Australian manna.
Rectum. The lower part of the large intestine, extending from the sigmoid flexure to
the anal canal.
Regionalenteritis (Crohn's disease). A chronic, non-specific, granulomatous process
frequently involving the terminal part of the ileum, but occasionally extending into
the colon or arising in the moreproximal parts ofthe small intestine. Clinical phen-
omena: recurrent crampy abdominal pain accompanied by diarrhoea, fever, anor-
exia and weight loss.
Rennin. Digestive enzyme that clots milk, and occurring in the gastric juices of young
mammals. It is secreted as the precursor prorennin, and activated by hydrochloric
acid.
Reticuloendothelial system. The macrophage system, which includes all the phago-
cytic cells ofthe body, except the granulocytic leukocytes. They include the histio-
cytes and macrophages of loose connective tissue, the reticular cells of lymphatic
and myeloid tissues, the blood monocytes, etc.
Scleroderma. An increment in collagenous connective tissue in the skin, either focal
or diffuse, the latter is associated with similar changes in the viscera.
Septicaemia. The invasion of the blood stream by micro-organisms from a focus or
foci in the tissues, and possibly with the micro-organisms multiplying in the blood.
(See Focus).
Spleen. An abdominal viscus located below the diaphragm on the left side. It is the
largest lymphatic organ of the body and also functions as a producer of red blood
cells. (See Viscus).
Stachyose. A non-reducing tetrasaccharide composed of one unit of glucose, two of
galactose and one of fructose; it is hydrolyzed to fructose and manninotriose. It is
present in many foods such as soybeans, beans, lupins, etc.
Starch. Complex polysaccharide composed of glucose units. The form in which
carbohydrate is stored in the plant.
Steatorrhoea. Excess offat in the stools. It may be due to Iack ofbile, Iack oflipase in
the digestive juices, or defective absorption of fat.
Stricture. A circumscribed narrowing of the Iumen of a canal or hollow organ.
Syndrome. A group of symptoms and signs, which when considered together, are
known or presumed to characterize a disease or lesion.
Synergism, bacterial. The ability of two or more species of organisms to bring about
chemical changesthat neither can bring about alone.
Thymus. A lymphoepithelial organ, which is the site of differentiation ofT lympho-

164
 cytes, and is a centrallymphoid organ controlling many aspects of immunologic
reactivity.
Ulcerative colitis. An inflammatory disease of unknown aetiology involving primar-
ily the mucosa and submucosa ofthe colon. It is manifested clinically by abdomi-
nal pain, diarrhoea, and rectal bleeding.
Urea. The waste nitrogen of most mammals is excreted in the urine as urea. It is
formed in the liver by the urea cycle.
Varix. A tortuous, enlarged artery or lymphatic vessel.
Viscus (pl. viscera). Any one ofthe organs enclosed within one ofthe four great cavi-
ties, the cranium, thorax, abdomen or pelvis; especially an organ within the
abdominal cavity.
Vermiform appendix. The small, blind gut projecting from the caecum.
Xylose. Pentose sugar occurring in plant tissues as complex polysaccharide; 40%
sweetness of sucrose.

165
Appendix 2: Culture media

The following culture media used for the isolation, culturing and characterization of
bifidobacteria will be discussed.

2.1 Natural (complex) culture media

2.1.1 Liver-cystine-milk sugar agar according to BLAUROCK (193 7)

BovENTER ( 1949) indicated the above agar medium as optimal for bifidobacteria. lt
may also be suitable as a liquid (without agar) medium.

2.1.1.1 Formula

Per litre
Agar 20.0g
Cystine 0.1 g
Lactose lO.Og
Liver infusion (fresh) lOOO.Oml
Peptone (Difco) lO.Og
Sodium chloride 5.0g
pH 7.4

2.1.1.2 Preparation

500 g ofbeefliver and 100 ml water boiled for 2 hours, filtered and diluted to 1000 ml.
- Sterilization at 108-109°C for 30 min. The maximum period offreshness is 7-10
days (RA YNAUD & LEVESQUE, 1959).

2.1.1.3 Modification

The addition of sorbic acid ( 1.2 %) previously neutralised to pH 6.4 and filtered sterile
improves the selectness and enables the isolation ofbifidobacteria from the stools of
children and adults (RA YNAUD, 1959; RA YNAUD & LEVESQUE, 1959).- For the isola-
tion of bifidobacteria from the stools of infants, the cystine content is reduced to
2.5-10 mg/1 resulting in better bacterial growth (RA YNAUD & LEVESQUE, 1959).

2.1.1.4 The appearance of colonies

The sharp-edged, porcelain white, circular, smooth colonies with cream coloured
center (with 1-2 mm in diameter) appear on the BLAUROCK medium 24 hrs and at the
most 48 hrs after incubation; rough colonies R-type, may also appear to a lesser
extent, in addition to smooth, S-type, colonies. Microscopic pictures show a remark-
able pleomorphism with rod, club and spatulate forms. Club forms predominate,

166
whi1e branched forms se1dom appear. Gram-positive rods are 2-5 microns long and
0.5-0.7 microns wide. (DEHNERT, 1961 a).- According to RAYNAUD (1959) co1onies
ofbifidobacteria (iso1ated from the stoo1s of infants) are somewhat irregu1ar in shape
with a dark co1oured center.

2.1.1.5 References:

BLAUROCK (1937), BOVENTER (1938), BLAUROCK (1939; 1940), MAASEN & ALBERT-
SEN (1940), BOVENTER (1949), TOMARELLI et a/., (1949a), NORRIS et a/., (1950),
MAYER & MOOSER (1950), RAYNAUD (1959), RAYNAUD & LEVESQUE (1959), KALZ
(1961 a), DEHNERT (1961 a), PECH & MüLLER (1962), SCHNEEGANS et a/., (1966),
MüLLER & PEcH (1967), GEDEK .0969), KosiKOWSKA ( 1978).

2.1.2 Tornato-casein peptone-yeast autolysate agar according to ÜRLA-JENSEN

(1943).

The culture medium is based on a tomato agar originally described by WEISS & REn-
GER ( 1934 a) and subsequently modified by ÜRLA-JENSEN ( 1943).

2.1.2.1 Formula

According to FRISELL ( 1951) Per litre

Agar 15.0g
Ascorbicacid(O.I %) l.Og
(or cysteine 0.2 %)
Caseinpeptonesolution (I %) 100.0m1
Dextrose (2 %) 20.0g
Peptone (Difco) 5.0g
Tapwater 800.0g
Tornato puree (Bäncke) 50-60g
Yeast autolysate solution (I %) IOO.Oml
pH 6.4-6.5

2.1.2.2 Preparation (According to FRISELL, 1951)

2.1.2.2.1 Caseinpeptone
Casein powder 80 g, pepsin (1 :900) 2 g, tap water 700 g, and concentrated hydro-
chloric acid 10 g, are mixed in a glass flask, which is then closed with a cotton-wool
plug soaked in chloroform. The flask is kept in an incubator at 38° C for 12 days, and
shaken each day; after this, the contents are removed and filtred. Sodium phosphate 4
g per litre, and magnesium sulphate 2 g per litre are added to the filtrate. The pH is
adjusted to 6.7. The mixture is then transferred to smaller flasks and autoclaved at
115° C for 15 minutes.

2.1.2.2.2 Yeast autolysate

Three litres of distilled waterare boiled and then cooled to 60° C. Four kilogrammes

167
of dry yeast are crushed in the water to form a homogeneous mixture. This latter is
then poured into a sterile container fitted with a Iid, and heated to 49-50° C in a water
bath, after which it is kept in an incubator at that temperature for 4 days (The Con-
tainer must be sufficiently large and have a well-fitting Iid). The solution formed is
then heated to l 05° C for 30 minutes, and thereafter filtered. The filter paper and con-
tents are mixed with 0.5 litres water in a dish, then heated to 60° C and filtered. Both
filtrates are mixed, and now contain approximately I per cent nitrogen. The pH is
adjusted to 6.0 and the mixture autoclaved at 115° C for 15 minutes.

2.1.2.2.3 The peptone is dissolved in some of the water and mixed, by careful stir-
ring, with the yeast autolysate, casein solution and tomato puree. The mixture is then
brought to a boil. Approximately 15 g (preferably chopped) agar is melted in the
remainder ofthe water and added to the mixture.

2.1.2.2.4 The mixture is chilled to approximately 40° C, and the white of one egg,
suspended in approximately 50 ml of water, is then added. A check is made that the
pH is below 7.0, as some ofthe amino acids and part ofthe vitarnin B content ofthe
culture medium are destroyed on heating in an alkaline environment.

2.1.2.2.5 The mixture is autoclaved at 1l5°C for 10 minutes, and then filtered until
the medium is as clear as possible. The quantity obtained is measured. The pH is
adjusted to 6.9. Glucose, ascorbic acid and sodium azide are added. The ascorbic acid
is dissolved in a little water and adjusted to a pH of approximately 6. (Cysteine
hydrochloride can be substituted for a part of the ascorbic acid). The solution is
poured into tubes to the l 0 ml mark. It is sterilized once at 120° C for 8 minutes and
then quickly cooled. The final pH should be 6.4 to 6.5.
The quality of a culture medium is dependent upon the freshness of its constitu-
ents and the cu1ture medium itse1f. The period of storage of the medium is approxi-
mately 7-10 days.

2.1.2.3 Modification of formula and preparation

In Frisel/'sinvestigation, sodium azide (0.01% = 0.1 g) was added to the original pre-
paration in order to prevent the growth of E. coli, which at times ferments the column
of culture medium and makes the reading difficult or impossible. Tests have shown
that sodium azide in this concentration does not demonstrably affect the growth of
B.bifidum (FRISELL, 1951). HAENEL & MüLLER-ßEUTHOW (l957a) give the exact
data about how this culture medium with various modifications can be prepared. The
pH amounts to 7.0. The addition of sorbic acid (0.04% dissolved in weak alkali
increases the selectness in the isolation of bifidobacteria from the faeces of adults.-
LERCHE & REuTER ( 1961) add 1 ml ofTween 80 (Sorbitan monoleat) to the medium.

2.1.2.4 The appearance of colonies

B. bifidum forrns lens-shaped, white colonies which are, unlike those of L. acidophi-
lus, smooth-edged (ÜRLA-JENSEN, 1943).

168
2.1.2.5 References:

ÜRLA-JENSEN (1943), ÜLSEN (1949), FRISELL (1951), HAENEL & MüLLER-ßEUTHOW

(1957 c), LERCHE & REUTER (1961).

2.1.3 Alkaline blood agar with dextrose and china blue according to HACKEN-
THAL & ßiERKOWSKI (1951), modified by ßiERKOWSKI (1956)

This culture medium designed for the differentiation of streptococci was applied by
KLuDAS (l959b) for the detection ofbifidobacteria which are better differentiated
from other organisms using indicator dye.

2.1.3.1 Formula
Per litre
Agar 20.0g
Bluish (6 B extra from Gübler, Stuttgart, W. Ger-
many) l% solution 30.0ml
Dextrose lO.Og
Horse flesh infusion fresh lOOO.Oml
Peptone (powder) lO.Og
in 50% solution 40.0ml
Sodium chloride 5.0g
Wetherblood 70.0ml
pH 7.5

2.1.3.2 Preparation

2.1.3.2.1 Preparation ofthe horse flesh infusion according to HALLMANN (1953):

Horse flesh, possibly non-fat, is cut with a knife or even better, is milled. A quantity of
water double this weight is added to the horse flesh and all is stored during the night at
4-6° C, followed by heating to 40-50° C for a good many hours, then cooked for 30
min in a steam cooker or over a flame. Mterwards cooling is carried out (to stiff possi-
ble fat), followed by filtering through a double filter (cotton-wool is placed over the
filter paper). The horse flesh is pressed on the filter cloth and then the original volume
(1000 ml) is adjusted by adding distilled water. This horse flesh infusion is either
immediately used or sterilized in an autoclave.

2.1.3.2.2 Preparation of peptone solution:

50 g peptone is dissolved in 100 ml of distilled water. The peptone solution (large
amounts of l-21) is kept in stock (on ice) until sterilization for 15 min. in autoclave.
The colouring effect is better when the peptone solution is older.

2.1.3 .2.3 Preparation ofwater blue solution:

l per cent of water blue solution ( l g of powdered dye is dissolved in 99 ml of distilled
water over a flame) is filtered, sterilized in an autoclave for 15 min., cooled and stored
in thedark.

169
2.1.3 .2.4 Preparation of a basic agar mixture:
5 gofNaCI, 10 g ofpeptone and 20 gofagarare added to 1000 ml ofthe horse flesh
infusion. The pH is adjusted to 7.5 using I 0% soda solution. Sterilization in autoclave
and filterlog follow.

2.1.3.2.5 I g of dextrose, 4 ml of peptone solution and 3 ml of I % water blue solu-

tion are added to I 00 ml ofthe basic agar mixture. Sterilization follows for 20 min. in
an autoclave.

2.1.3 .2.6 After cooling to 50° C, 7 ml of sterile wether blood, warmed to 37o C (fibre
previously removed) is added to I 00 ml of agar medium and poured into plates. Solid-
ified plates have the appearance of anormal blood agar medium, excepting the gentle
blue colour.

2.1.3.3 Modification

Amodification was proposed by REuTER (1963) and applied by BAJARD (1977).

2.1.3.4 The appearance of colonies

E. coli forms grey and B. bifidum brown colonies (KLUDAS, 1959 b). - The water blue
indicator present in the culture medium causes various colours for every particular
bacterial species grown on plates. LERCHE & REUTER ( 1961) and LIEBSCHER ( 1964 b)
reported on the variations of colour as regards bacterial colonies including bifidobac-
teria.

2.1.3.5 References:

KLUDAS (1959b), LERCHE & REUTER (1961), LIEBSCHER (1964b), KALZ (1961 a),
WERNER (1964), HOFFMANN (1966), BRAUN et a/., (1967), ÜEDEK (1969), BAJARD
(1977).

2.1.4 VL-Agar (Meat-Yeast-Agar) according to BEERENSand co-workers

(1963)

This isabasicmedium for many culture media with modifications (see also 2.6.18,
dried media). This culture medium was used by RAYNAUD (1959) fortransfers ofthe
original Tissier's strain.

2.1.4.1 Formula (Pasteur Institute Paris, Production)

Per litre
Agar 18.0g
Cysteine hydrochloride 0.3g
Distilled water 1000.0ml
Glucose 2.0g
Meat extract (Liebig) 3.0g

170
 Per litre
Tryptone 10.0g
Sodium chloride 5.0g
Yeast extract 6.0g
pH 7.4-7.5

2.1.4.2 Modification

a) A basic medium, without g1ucose and agar added, may be easily modified; b) Most
often 10% ofhorse or sheep's b1ood is added; c) Modified VL- medium with glucose
and gycocholate is used for the study of deconjugation ofbile sa1ts (CATTEAU et a/.,
1971); d) VL- agar can be supp1emented with 5% b1ood or laked b1ood or with liver
and faecal extracts 5 %. VL with 0.5% of laked b1ood is an optimal liquid growth
medium (SYKES & SKINNER, 1973); e) Fluid VL- medium with 1%glucose is used for
determination of volatile products of metabolism (BULLEN & TEARLE, 1976); t)
BARNES & IMPEY ( 1972) added 0,5% oflaked blood; g) The addition ofkanamycin is
possible to suppress enterobacteria (Pasteur Institute Production); h) Modification
made by BARNES & IMPEY (1971) contains (g/1): Tryptone (Oxoid), 10 g; sodium
chloride 5 g; beef extract (Lab-Lemco, Oxoid) 3 g; yeast extract (Difco) 5 g; cysteine
hydrochloride 0.4 g; glucose 2.5; agar (New Zealand) 0.6 g; pH 7.2-7 .4. Forthe solid
medium, agar (New Zealand) 12 g/1 is added.

2.1.4.3 References:

BEERENS et a/., (1963); BEERENS et a/., (1965); CATTEAU et a/., (1971); BARNES &
IMPEY (1971), (1972); SYKES & SKINNER, panel discussion, (1973).

2.1.5 BL-Agar (G/ucose-Blood-Liver-Agar) according to ÜCHI and co-workers

(1964) modified byTERAGUCHI and co-workers (1978).

BL-agar is an optimal culture medium for the detection of bifidobacteria. It was first
described by ÜCHI and co-workers (1964), then by MnsuoKA and co-workers
(1965b) andin a somewhat modified form was written up by TERAGUCHI and co-
workers ( 1978). It is occasionally called the Mitsuoka culture medium. SHIMADA and
co-workers ( 1977) showed that VL-agar is a good medium for the detection of bif-
idobacteria and 1actic bacteria in fermented milk products.

2.1.5.1 Formula according toTERAGUCHI and co-workers (1978)

Per litre
Agar (Difco) 15.0g
L-cysteine hydrochloride 0.5g
Glucose 10.0g
Lab-Lemco (Oxoid/Liebig meat extract) 3.0g
Liver extract solution 150.0ml
Phytone(BBL) 3.0g

171
 Per litre
Proteose peptone noo 3 (Difco) lOoOg
SolutionA 1000ml
SolutionB SoOml
Starch soluble OoSg
Tween 80 (Sorbitan monoleat) l.Og
Trypticase (BBL) SoOg
Water, distilled 8l5o0ml
Y east extract (Difco) SoOg
pH 702

Solution A:
Potassium dihydrogen orthophosphate (KHzP04) 2500g
di-Potassium hydrogen orthophosphate (KzHP04) 25o0g
in 250 ml of distilled water
Solution B:
Ferrous sulphate (FeS04 ° 7Hz0) OoSg
Magnesium sulphate (MgS04 ° 7Hz0) 10o0g
Manganoussulphate(MnS04 ° 4Hz0) 0.337 g
Sodium chloride O.Sg
in 250 ml of distilled water

201.502 Preparation

2ol.So2o1 Liverextract solution

10 g ofliver powder (KYOKUTO) are extracted with 170 ml of distilled water for l hour
at 50-60° C and boiled for 10 min and filtratedo

201050202 Solution A
KH 2 P04 25 g and KzHP04 35 g in 250 ml of distilled watero

2ol.5 02.3 Solution B

FeS04 o 7H 20 0.5 g, MgS0 4 ° 7H 20 I 0 g, MnS0 4 ° 2H 20 00337 g and Na Cl 005 g in
250 ml of distilled watero
20105 0204 All constituents of the culture medium, except L-cysteine hydrochloride,
are dissolved in water, with the pH adjusted to 7 02 and sterilised at 120° C for l 0 mino
After cooling to soo C, cysteine hydrochloride in a 5 %sterile solution is addedo

201.5.3 Modification

The addition of 50 ml ofhorse blood per litre (ÜCHI et a/0, 1964) after Sterilisation and
cooling to soo Co

2ol.So4 The appearance of colonies

On BL-agar plates, bifidobacteria may be easily recognized from other organisms by

their different forms of colonies, as weil as by their colour and odour (MnsuoKA et
alo, 1965 b )o

172
2.1.5.5 References:

ÜCHI et al., (1964); MITSUOKA et al., (1965b); SYKES & SKINNER (panel discussion,
1973); SHIMADA et al., (1977); TERAGUCHI et al., (1978).

2.1.6 Bifidobacterium-medium (LAUER & KANDLER, 1976)

The bifidobacterium-medium, as it is commonly called, was composed by LAUER &

KANDLER ( 1976) on the basis of enrichment medium for clostridia.
The liquid medium is used for transfers of bifidobacteria strains and the solid
medium for plate counts of cultures used in manufacturing fermentedmilk products.
All known species ofbifidobacteria demoostrate good growth on the bifidobacteria-
medium ( German Collection of Microorganisms, 1977).

2.1.6.1 Formula

According to German Collection of Microorganisms ( 1977)

Per litre
Ascorbicacid(sodiumsalt) 1% 10.0g
Caseine peptone, tryptic digest 10.0 g
Cysteine hydrochloride 0.05% 0.5 g
Dextrose 10.0 g
Meatextract 5.0g
di-Potassium hydrogen orthophosphate (K2HP04) 3.0 g
Tap water I 000.0 ml
Tween 80 (Sorbitan monoleat) 1.0 ml
Yeastextract 5.0g
pH 6.70

2.I.6.2 Preparation

After sterilization, add a solution of sodium ascorbate and cysteine hydrochloride

being careful not to contaminate.- Medium not freshly prepared should be heated in
a steamer for I 0 min. before addition of reducing substances.

2.I.6.3 Modification

Originally only 5 g of glucosewas added by LAUER & KANDLER (1976). Later came
the addition of 15 g agar per litre for the preparation of Bifidobacteria-Agar ( Labora-
tory Wiesby Niebüll, 1980).

2.I.6.4 References:

LAUER & KANDLER (1976); NICOLAI (I976); German Collection of Microorganisms

(1977); Laboratory Wiesby Niehüll (1980).

I73
2.1. 7 Modified Rogosa 's Agaraccording to SHIMADA and co-workers ( 1977)

This is used for the viable cell counts of bifidobacteria in fermented milk products
(SHIMADA et a/., 1977).

2.1.7.1 Formula

Per litre
Agar 15.0g
Ammoniumcitrate 2 H20 2.0g
L-cysteine hydrochloride 0.3g
Ferrous sulphate FeS04 · 7 H20 35mg
Lactose lO.Og
Magnesium sulphate MgS04 · 7 H20 575mg
Manganaus sulphate MnS04 · 4 H20 120mg
Potassium dihydrogen orthophosphate KH2P04 3.0g
di-Potassium hydrogen orthophosphate K2HP04 3.0g
Trypticase (trypsine-peptone) lO.Og
Tryptose 3.0g
Tween 80 (Sorbitan monoleat) l.Og
Water, distilled lOOOml
Yeast extract 5.0g
pH 7.0-7.2

2.1.7.2 Preparation

After sterilization add aseptically a solution of cysteine hydrochloride to the agar

making sure not to contaminate it.

2.1.7.3 Modification

See Appendix Chap. 2.6.11.

2.1.7.4 References:

MITSUOKA et a/., (l965b); SHIMADA et a/., (1977); see Appendix Chap. 2.6.11.

2.1.8 Chopped Meat Medium according toATCC(J980)

This is a basic medium for the propagation of some strains of B. adolescentis and
B.breve(see ATCC, 1980).
Ground beef(free offat) 500.0g
Distilled water lOOO.Oml
NNaOH 25.0ml
Use lean beef or horse meat. Remove fat and connective tissue before grinding.
Mix meat, water, and NaOH and bringto a boil while stirring. Cool to room tempera-

174
ture, skim fat off surface, and filter, retaining both meat particles and filtrate. Add suf-
ficient distilled water to the filtrate to restore the lliter original volume. To the filtrate
add:
Per litre
Peptone 30.0g
Yeast extract 5.0g
K2HP04 5.0g
0.025% resazurin solution 4.0ml

Boil, cool and add 0.5 g of L-cysteine hydrochloride, adjust pH to 7.0. Under 0 2-
free nitrogen and 3% hydrogen, dispense 7 ml over 1 part meat particles to 4-5 parts
fluid per test tube. Cap with rubber stoppers und er N 2 and H 2 and autoclave in pres-
sure for 15 minutes under fast exhaust.

2.1.9 Bifidobacterium Medium according to ATCC {1980)

Medium used for propagating some strains of B. infantis (ATCC, 1980).

2.1.9.1 Formula
Per litre
Casitone lO.Og
Glucose 20.0g
Peptone lO.Og
Tornato juice, diluted lOOO.Oml
Tryptone lO.Og
Tween80 2.0ml
Yeast extract lO.Og
pH 6.80

2.1.9.2 Preparation

To prepare diluted tomato juice, combine one part LIBBY'S tomato juice with two
parts distilled water. Boil and filter through paper. - Autoclave for 30 minutes at
ll0°C.

2.1.10 Trypticase-Phytone-Glucose Medium according to ATCC {1980)

Used for propagating some strains ofbifidobacteria species (B. angulatum, B. anima-
lis, B. asteroides, B. catenulatum, B. dentium, B. indicum, B. infantis, B. pseudolon-
gum, B. thermophilum, B. ruminale, B. suis) (see ATCC, 1980).

2.1.1 0.1 Formula

Per litre
Agar 8.0g
Cysteine 0.5g

175
 Per litre
Ferric chloride (FeCh) trace
Glucose l5.0g
Magnesiumchloride (MgCb) 0.5g
Phytone (BBL) 5.0g
di-Potassium hydrogen orthophosphate (K2HP04) 2.0g
Trypticase (BBL) lO.Og
Yeast extract 2.5g
Water, distilled lOOO.Oml
Zinc sulphate (ZnS04) 0.25g

2.1.1 0.2 Preparation

Dissolve separately (ferric chloride and zinc sulphate) in water and add to the bulk of
themedium.

2.1.1 0.3 Modification

Tween 80 (2 ml/1) is added for propagating strains of B.magnum and B.pullorum

(See ATCC, 1980).

2.1.11 Tornatojuice agar

This formu1a according to ATCC is used for propagating a certain strain of B. bifidum
(G. Malyothstrain) (ATCC, 1980).

2.2 Selective culture media

2.2.1 Selective agar I according to HAENEL and co-workers (1970)

This se1ective culture medium is used for the determination ofboth p1ate counts and
types of B. bifidumwhich originate from the stools ofinfants and adu1ts.lt represents
a modification of the NH-agar (HAENEL et al., (1970).

2.2.1.1 Formu1a

Per litre
Agar lO.Og
Brom creso1 green so1ution 2.5ml
Carbohydrate (see preparation) l5.0g
Cysteine hydroch1oride 500mg
Cystine 500mg
(Casein?)-Peptone lO.Og
Humanblood 30ml
Meat extract 0.3g

176
 Per litre
Neutral red solution 0.1 ml
Salt solution 2.5ml
Sodium azide IOOmg
Sodium chloride 3.0g
di-sodium hydrogen orthophosphate 2.0g
Na2HP04 · 2 H20
Water, distilled IOOO.Oml
Tween 80 (Sorbitan monoleat) l.Oml
Yeast extract 4.0g
pH 7.0

2.2.1.2 Preparation

a) Brom cresol solution: 0.2 g in 40 ml water and 4 drops of 40% KOH solution
added. b) Neutral red solution: 0.05 g in 20 ml of96% alcohol. c) Salt solution: 250 ml
solution contain I 0 mg MgS04 · 7 H20, 0.5 g FeS04 · 7 H20 and 0.337 g
MnS04 · 2 H 20. d) The addition of cysteine hydrochloride after dissolving ingre-
dients, immediate before autoclaving, and the addition ofblood after autoclaving. e)
Carbohydrates: the separate addition in 8 parallellines of dextrose, Iactose, arabi-
nose, xylose, cellobiose, sorbitol, melizitose and salicin respectively, to the liquefied
culture medium, and heating in steamer at 1oooc for 15 min.

2.2.1.3 The appearance of colonies

B. bifidum from the stools of infants is counted, and taking into consideration its mor-
phology and the indicator dye reaction, typed using a simple schema of carbohydrate
fermentation. The latter is based on the works of DEHNERT and REUTER. The same
type of B. bifidum always shows the same colony form on the petri dish irrespective of
the kind of carbohydrate used. Various types of B. bifidum grown on the same petri
dish show different colony forms and indicator reductions (HAENEL et al., 1970).

2.2.2 Selective mediumfor the enumeration ofbifidobacteria in dairy products

(NPNL-agar) according toTERAGUCHI and co-workers (1978).

An improved selective medium was developed for the enumeration ofbifidobacteria

in dairy products containing bifidobacteria, lactobacilli and streptococci. NPNL-
agar was BL-agar without blood-containing neomycin sulphate, paromomycin sul-
phate, nalidixic acid and Iithium chloride.

177
2.2.2.1 Formu1a

BL-agar (see 2.1.5) without blood and with NPNL-selective solution:

Per litre
Lithiumchloride (3 mg/ ml) 3.0g
Nalidixicacid(l5jlg/ml) 15mg
N eomycin sulphate 100mg
(1 00 jlg/ ml)
Paromomycin sulphate 200mg
(200 jlg/ml)

2.2.2.2 Preparation

See BL-agar. The NPNL-selective solution is added tagether with cysteine hydro-
chloride after the culture medium and has been sterilized and cooled to 50° C.

2.2.2.3 Modification

In the case of the anaerobic jar being dispensed with a modified NPNL-agar in com-
bination with the shake culture tube method and an additional agar medium overlaid
also proved to be useful for the same purpose. Modified NPNL-agar was BL-agar
without blood-containing neomycin sulphate (30 jlg/ml), paromomycin sulphate
(50 jlg/ml), nalidixic acid ( 15 jlg/ml) and Iithium chloride (3 mg/ml).

2.2.2.4 References

MÜLLER (1976); TERAGUCHI et a/., (1978).

2.2.3 Selective modified Rogosa's-Agar

The selective agar consists of 1000 ml modified Rogosa's agar according to SHIMADA
and co-workers (1977) (see 2.1.7) with the addition of50 ml ofthe following solution
which suppresses the growth of lactic acid bacteria in fermented milk products. To
prepare 100 ml of selective solution: 30 g sodium propionate is dissolved in 90 ml dis-
tilled water. Then I 00 mg paromomycin sulphate, 400 mg neomycin sulphate, and 6 g
Iithium chloride are added, and after dissolving these substances the solution is
diluted with distilled water to I 00 ml.

2.2.4 Selective BL-agar

The selective BL-agar according to SHIMADA and co-workers (1977) has the same
composition, including the added selective solution as the selective modified
Rogosa's medium (see 2.2.3).

178
2.2.5 Differentmedia with selective solution

The addition of selective solutions to various culture media may show the selective
effect (see Table 9.4).

2.3 Semi-synthetic and related culture media

Various semi-synthetic or related culture media have been used for culturing bifido-
bacteria:
- Culture medium according to TEPLY & ELVEHJEM ( 1945);
- Ascorbic-amino-lactose agar according to PLOTHO ( 1947);
- Culture medium according to MALYOTH & BAUER (1953);
- Culture medium called GARCHES, Pasteur Institute Garches, (RA YNAUD, 1959).
However the culture medium proposed by ToMARELLI and co-workers
( 1949 a) and its modifications by NoRRIS and co-workers (1950) and GYöRGY and co-
workers (1954 a) have been the ones most often mentioned in the literature.

2.3.1 Culture medium according toTOMARELLI, NoRRIS, GYÖRGY, HASSINEN

andBERNHART (1949a), called Tomare/li medium

2.3.l.l Formula

Assay Medium with double strength: Per litre

Adenin 0.020gr
Alanine 0.400gr
Ascorbic acid 2.000gr
Asparagine 0.200gr
Casein, enzyme hydrolyzed
(N-Z-Case, Sheffield) lO.OOOgr
Cystine 0.400gr
Glucose 40.000gr
Guanine 0.020gr
di-Potassium hydrogen
orthophosphate (K2HP04) 5.000gr
Salts B *) lO.Oml
Sodium acetate (anhydr.) 50.000gr
Tryptophane 0.400gr
Uracil 0.020gr
Xanthine 0.020gr

p-Amino-benzoic acid 0.020mg

Biotin 0.008mg
Calcium pantothenate 0.800mg
Folieacid 0.020mg
Pyridoxine hydrochloride 2.400mg

179
 Per litre
Riboflavin 0.400mg
Thiaminehydrochloride 0.400mg
pH 6,8

*) Salts B:
Ferrous sulphate (FeS04 · 7 H20) 0.500gr
Magnesiumsulphate(MgS04 · 7 H20) IO.OOOgr
Manganous sulphate (MnS04 · 2 H20) 0.337 gr
Sodium chloride in 250 ml distilled water 0.500gr

I 0 mg sterile ascorbic acid added to each I 0 ml of single strength medium

after sterilization.

2.3.1.2 Modifications

BRAUN (1959) added beta-aethyl-N-acetyl-d-glucosaminide to a similarly composed

medium in place of human milk. - Cystine is substituted by cysteine, which is more
easily soluble (MAYER et al., 1964a). - The constituents of Tomarelli's medium,
which is commonly used for the incubation of the organisms, was slightly modified
by decreasing sodium acetate and increasing enzymatic casein hydrolyzate (poly-
peptone) because of growth suppression by the original medium of some strains
(YOSHIOKA, 1971).

2.3.1.3 App1ications

MA YER and co-workers ( 1965 a) carried out numerous investigations on the metabo-
lism of B. bifidum using Tomarelli's solution.

2.3.2 Cu/ture medium according to NORRIS, FLANDERS, TOMARELLI and

GvöRGY (1950), called Norris medium

This medium based on the modification of the medium from ToMARELLI and co-
workers (1949a).

2.3.2.1 Formula

Basic formula from the TOMARELLI and co-workers (1949 a) medium with the follow-
ing modifications:
double strength
Per litre
Ascorbic acid per litre l.OOOg
Cyanocobalamin (B12 new) 0.010mg
Folieacid (higher quantity) 0.200mg
Pancreatin (new) 0.200g
Sorbitan monoleate (Tween 80 new) 2ml
pH 6.8

180
2.3.2.2 Modification, called Györgymedium

Addition of defatted breast milk to a similar medium formula (GYöRGY et al.,

1954a).
A modification of medium from TOMARELLI and co-workers ( 1949 a) with addi-
tion of 10/100 m1 human milk, skimmed or 2% in single strength medium, is used for
propagating of B. bifidum var. pennsylvanicus (called Lactobacillus bifidus medium,
see ATCC, 1980).

2.4 Full-synthetic culture media

The following full-synthetic culture media have been developed:

- Minimal culture medium according to HASSINEN and co-workers ( 1951)
- Full-syntheticculturemediumaccordingto PETUELY & LYNAU (1954)withitsmod-
ifications from
- PETUELY(l956c)and
- GYLLENBERG & NIEMELÄ (1959)

2.4.1 Minima/cu/turemedium according to HASSINEN, 0URBIN, TOMARELLI

andßERNHART (1951)

This is the first full-synthetic culture medium for bifidobacteria strains with minimal
nutritional requirements.

2.4.1.1 Formula

double strenth
Per litre
Ammoniumacetate 4.000gr
Biotin 0.008mg
Calcium pantothenate 0.800mg
Cystine 0.400gr
Lactose 70.000gr
di-Potassium dihydrogen orthophosphate 5.000gr
Sodium acetate 50.000gr
Salts B*) 10ml
Distilled Water(diluted to) lOOOml
pH 6.8

*) SaltsB:
Ferrous sulphate (FeS04 · 7 H20) 0.500gr
Magnesium sulphate (MgS04 · 7 H20) lO.OOOgr
Manganaus sulphate (MnS04 · 2 H20) 0.337 gr
Sodium chloride in 250 ml distilled water 0.500gr

181
2.4.1.2 Preparation

l 0 mg sterile ascorbic acid added to each l 0 ml of single strength medium after sterili-
zation.

2.4.2.3 Modification according to GYLLENBERG & NIEMELÄ ( 1959), enurlleration

Dehnertsubtype B1

Mediumfora selective enumeration modified so as to contain riboflavin and pante-

thine, arabinose (not Iactose) in order to satisfy the requirements of Bifidobacteria
subtype B1 according to DEHNERT (l957b).

2.3.2.3.1 Formula
Per litre
Agarwashed 20.000gr
Ammonium sulphate (NH4)2 S04 4.000gr
L( + )-arabinose lO.OOOgr
Biotin 0.005gr
Cystein hydrochloride 0.400gr
Ferrous sulphate (FeS04 · 7 H20) O.OlOgr
Magnesium sulphate (MgS04 · 7 H20) 0.200gr
Manganous sulphate (MnS04 · 4 H20) 0.007 gr
Pantethine 0.500mg
di-Potassium hydrogen orthophosphate (K2HP04) 5.000gr
Riboflavin 0.250mg
Sodium ascorbate lO.OOOgr
Sodium chloride (NaCl) O.OlOmg
Tween80 l.OOOgr
Water, distilled lOOOml
pH 6.8-7.0

2.4.2.3.2 Preparation
This medium is prepared in two lots, one ofwhich contains all the components except
arabinose and ascorbic acid at double strength. This Iot can be sterilized by ordinary
autoclaving. The other Iot is prepared by dissolving l g ascorbic acid in distilled
water; 1-N NaOH is added tobring the pH into the range of6.8-7.0. 1 g arabinose is
then dissolved in this Na-ascorbate solution, and distilled water is added tobring the
volume to 50 ml. This solution is sterilized by filtration. Equal volumes of the ascor-
bate-arabinose solution (heated to 55-60°C) and the melted agar Iot (cooled to
55-60°C) are then mixed, the mixture is poured in plates and allowed to solidify.
Overheating of the ascorbate-arabinose solution must be avoided since it gives rise to
oxidation of the ascorbic acid.

182
2.5 List of culture media non-available in commercial trade for culturing of
bifidobacteria

Cu1ture media used for cu1turing of bifidobacteria as weil as those non-avai1ab1e in

commercia1 trade are presented be1ow. Some important media are indicated with (*)

Reference Designation of the culture medium

ADAM(1922b) Haematin broth (cit. by FELDHEIM, 1958)

BARNES & IMPEY ( 1972)
BACHMANN & VIEHMANN
(1953)
BECK(1967)
BESSAU(1943)
(*) BLAUROCK(1937)
BRAUN & MARGET ( 1952 b)
DEHNERT(1975b; 1961 a)
EGGERT & GAGNON (1933)
Evoo(1966)
FRISELL ( 1951)
GYLLENBERG & NIEMELÄ
(1959)
(*) GYöRGY et al. (1954a)
(*) HACKENTHAL&BIER-
KOWSKI(1951)
HAENEL & MüLLER-BEU-
THOW(1956a)
HAENEL& MüLLER-BEU-
THOW(1956a)
HAENEL & MüLLER-BEU-
THOW(1957 C)
(*) HAENEL et a/. ( 1970)
(*) HASSINEN eta/.(1951)
HENNEBERG(1934)
HOFFMANN(1966)
HOLZAPFELet a/. (1969)
KALz(1961 a, b)
Modification ofthe medium MnsuoKA et al.,
(1965b)
Bifidobacteria cu1ture medium with Alete-early
diet
Medium for cu1ture, iso1ation and maintenance of
bifidobacteria
Whey-cystine-p-aminobenzoic acid-liver medium
Liver-cystine-1actose-agar
Human milk agar modif. by MITSUOKA et al.,
(1965a)
Human milk agar
AlkaHne b1ood agar, called EG-agar, introduced
and modified by ÜCHI et al., ( 1964)
Liquid cu1ture medium fortransfers ofbifidobac-
teria cu1tures
See0RLA-JENSEN(1943)
Agar for se1ective enumeration. Modification of
the full-synthetic medium from PETUELY& LYNAU
(1954)
Modification ofthe medium from TOMARELLI et
al. (1949 a) and NORRIS et al. (1950), modif.
TAMAKI(1959)andKUNIYA(1959)
SeeKLuoAs(l959b)
Beerwort agar
Tornato juice-peptone-milk-agar cit. by FELDHEIM
(1958)
Sorbicacidagar 1,2,3withpH5.0
Se1ective agar medium I
Full-synthetic cu1ture medium
China b1ue-1actose-agar
Yeast extract-caseine hydro1yzate-dextrose-blood
agar with added Penicillin-G
Medium formass-culture
Penicillin-agar (for developing ofthe typical mor-
pho1ogy)

183
 KENDALL ( 191 0) Acetic acid-glucose broth
KlSZAet a/. (1974) Milk and whey digested media
KLUDAS(l959b) Culturing on simple culture medium as flour
extract together with E. coli (Symbiotic culture)
(*) KLUDAS(l959b) Introduction ofthe blood agar from HACKENTHAL
& BIERKOWSKI modif. by LERCHE & REUTER
(1961)
(*) LAUER & KANDLER (1976) Bifidobacteria-medium
LERCHE & REUTER ( 1961) Combined agar (on the basis ofwork by HAENEL &
MüLLER-BEUTHow(l957 c)
MALYOTH&BAUER(l953) Semi-synthetic culture medium
MAYER(1948) Peptone-pepsin-yeast autolysate medium
MAYER(l948) Milk-pepsin-yeast autolysate medium
MA YER ( 1948) Li ver extract medium
MA YER ( 1948) Liper-pepsin-peptone-yeast agar (called bifido-
bacteria-plate)
MAYER et a/. (1964b) Potato extract (used for research of metabolism
MITSUOKA et a/. ( 1965 b) BS-agar 1 with addition of selective substances
MITSUOKA et a/. ( 1965 b) BS-agar 2 with addition of selective substances
MITSUOKA et a/. (1965 a) BS-agar 1 and 2
(*) MITSUOKA et a/. ( 1965 b) BL-agar with addition of selective substances
MUTAI eta/.(1976) VL-G-broth medium for culturing ofbifidobacteria
NEGISHI Negishi's selective agar medium with add sodium
azide forthe enumeration offaeces bacteria (cited
byYOSHIOKA, 1971)
(*) NORRIS et a/. ( 1950) Semi-synthetic culture medium (called sometimes
Norris-medium)
(*) ÜCHI et a/. (1964) Glucose-blood-liver-agar(BL-agar)
ÜCHI et a/. ( 1964) Modified alkaline blood agar according to
EGGERT & GAGNON ( 1933) (EG-agar)
(*) ÜRLA-JENSEN ( 1943) Tornato-casein peptone-yeast autolysate agar (see
modification by FRISELL, 1951)
Pasteur Institute Garches See RA YNAUD (1959)
PETUEL Y( 19 56 C) Full-synthetic ascorbic acid culture medium. A
modification by PETUELY & LYNAU (1954). See
also modification by ÜYLLENBERG & NIEMELÄ
(1959)
PLOTHO(l947) Ascorbic-amino-lactose agar (semi-synthetic cul-
turemedium)
RAYNAUD(l959) Application of the semi-synthetic culture medium
from TEPLY & ELVEHJEM ( 1945)
RAYNAUD(l959) Semi-synthetic culture medium, called "Garches"
(Pasteur Institute Garches, France)
RüHLE(l924b) Loeffler-serum plate introduced in culturing of
bifidobacteria (FELD HEIM, 19 58). See also und er
Commercial culture media
(*) SHIMADA et a/. ( 1977) Modified Rogosa's agar

184
 TEPLY & ELVEHJEM ( 1945)
(*) TERAGUCHI et al. (1978)
TisSIER ( 1900)
(*) ToMARELLI et al. ( 1949 a) Semi-synthetic culture medium (called sometimes
ToRREY ( 1917)
Semi-synthetic medium that is used for assay of
folic acid with L. case~ modified by Norris et al.,
( 1950) for culturing ofbifidobacteria
Neomycin-paromomycin-nalidixicacid-lithium
chloride agar (NPNL-agar). Selective medium for
enumeration in dairy products.
Dextrose-deep agar (Veillon-agar). See ßASTN
{1914), RAYNAUD(1959)
Tomarelli-medium)
Liver-glucose-blood agar

2.6 Commerclal culture media

The manufacturers of culture media are presented below in alphabetical order:

BBL BBL Manual of products and laboratory procedures. Sixth reprinting,
1973.- Division Becton, Dickinson and Company, BD Cockeysville,
Maryland21030, USA.
DIFCO Difco Manual of dehydrated culture media and reagents for microbio-
logical and clinicallaboratory procedures, 9th edition, reprint 1963.-
Difco supplementary Literature, Octobre, 1968. Difco Laboratories,
Detroit, Michigan 48201, USA.
MERCK Microbiological Manual. Dehydrated culture media, basis of culture
media and special preparations for microbiology. E. Merck, Darmstadt,
W.Germany.
OXOID The Oxoid Manual of culture media ingredients and other laboratory
services. Fourth edition 1979. Published by Oxoid Limited, Wade Road,
Basingstocke, Hampshire RG240PM, England
PASTE UR Institute Production, 36 rue du Docteur Roux, Paris 15 e, France.
PRODUCTION

2.6.0 Actinomyces Broth, BBL no. 10919/pH 6.9

Basicmedium for cultivation and/or maintenance of Actinomyces species. Applica-

tions: Used for propagating certain strains of Bifidobacterium adolescentis (No.
15423 and 15424, pathogen strains; Actinomyces eriksonü) (see ATCC, 1980).

2.6.1 Anaerobic Agar, BBL no. 10925/pH 7.2

Bacto Brewer Anaerobic Agar, Difco no. B279/pH 7.2

Anaerobic Agar was designed for cultivation of members of the genus Clostridium
and other anaerobic organisms. Applications: Brewer Anaerobic- Agar Difco for bi-
fidobacteria cultivation (WERNER & SEELIGER, 1963 b; WERNER, 1964).

185
2.6.2 Bacto Elliker Broth, Difco no. 0974/pH 6.8

For cu1turing Streptococci and Lactobacilli in dairy products. Applications: It was

used with 1.5% agar for p1ate counts and propagation of B. bifidum var. pennsylvani-
cus. Liquid medium was used for cell preparations and growth assay (JAO et al.,
1978).

2.6.3 Brain Heart Infusion Agar, BBL no. 11064/pH 7.4

Bacto Brain Heart Infusion Agar, Difco B 418 (Manual9th ed.)/pH 7.2
Brain Heart Infusion Agar, Oxoid no. CM 375/pH 7.4

It is recommended as asolidmedium for the cu1tivation of fastidious pathogenic bac-

teria and fungi (possib1e to add b1ood). Applications: Use for detection ofbifidobacte-
ria (SYKES & SKINNER, pane1 discussion, 1973).

2.6.4 Columbia Agar Base, BBL no. 11123/pH 7.3

Bacto Columbia Blood Agar Base, Difco no. 0792/pH 7.3
Columbia Agar Base, Oxoid no. CM 331/pH 7.3

Co1umbia Agar Base containes peptones derived from both casein and meat, and pro-
vides an efficient base forpreparation ofboth b1ood and chocolate agar media. It may
also be used as a base forvarious selective and identification media. Applications: Use
of Columbia-Agar (Oxoid) containing 7% of horse b1ood and neomycin su1phate
( 100 j.lg/ml) for iso1ation (SYKES & SKINNER, pane1 discussion, 1973).

2.6.5 Eugon Brothand Agar, BBL no. 11234 and 11229/pH 7.0

Eugonagar is a medium designed to obtain eugonic (luxuriant) cu1tures of bacteria,

including many organisms ordinari1y regarded as difficult to cu1tivate. With b1ood
added, Eugonagar enables rapid growth of such fungi and bacteria. Applications: a)
With sheep's b1ood added Eugon-Agar BBL is used for cu1tivation (WERNER & SEELI-
GER, 1963 b). b) WERNER (1964) used this medium for detection of bifidobacteria
from the mouth of adu1ts. c) Eugon agar was supplemented with 0.2% yeast extracts,
as well as with 5 mg/1 of haemin for antibiotica sensitivity testing of bifidobacteria
(FINEGOLD et al., 1967). d) It is used for alternate transfers of B. bifidum TISSIER and
its variant pennsylvanicus (BECK, 1967). e) Used for propagating some strains of bif-
idobacteria species (B.longum subsp. animalis, B.pseudolongum, B. thermophilum)
(See A TCC, 1980).

186
2.6.6 LAgar, BBL no. 11324/pH 7.0
Bacto L Agar, Difco no. 0594

L agar, or Lactalysat agar, was devised by VERA (1947) for cultivation oflactobacilli
species. Applications: WERNER (1964).

2.6. 7 Loeßler's Medium, BBL no. 11353/pH 7.6

Loeßler's Serum Medium, Oxoid no. PM 200

Loeffier's medium is used for Iabaratory studies of nose and throat specimens, parti-
cularly for detection of corynebacteria. Applications :a) To promote the growth ofbif-
idobacteria {ROHLE, 1924b). b) For detection of bifidobacteria (CRUICKSHANK,
1925). c) Loeffier's diphtheria culture medium is used for cultivation ofbifidobacte-
ria {BLAUROCK, 1937).

2.6.8 M.R.S. Broth (Medium de Man, Rogosa, Sharpe), Oxoid no.

CM 361/pH 6.2

This medium is used for the isolation and counting of lactobacilli. Applications: a)
M.R.S. medium is used with an added I % ascorbic acid and 0.02% cysteine hydro-
chloride for mass-cultures. For identification, a basal medium without added glucose
and ascorbic acid has been used (KocHet al., 1970a). b) M.R.S. containing cysteine
hydrochloride is considered as an optimalliquid growth medium (SYKES & SKINNER,
1973). c) lt is used for transfers of bifidobacteria strains of various origin on the
M.R.S. medium containing added cysteine (0.3 g/pH 6.5) (CATIEAU et al., 1973). d)
Difco Lactobacillus MRS broth has been used for propagating B. coryneforme, strain
no. 25111 (see ATCC, 1980).

2.6.9 Peptonized Milk, BBLno.11903/pH?

Peptonized Milk, Oxoid no. L32

Peptonized milk can be used (as a simple solution, solidified with agar only, or as a
constituent of more complex media) for the investigation of lactic acid bacteria such
as L. acidophilus and bifidobacteria.

2.6.10 Reinforced Clostridial Agar, BBL no. 11563/pH 6.8

Reinforced Clostridial Agar (R.C.M. Agar), Oxoid no. CM 151/pH 6.8

This agar is suitable for the cultivation and enumeration of clostridia and other anaer-
obes, lactobacilli, and many other species of bacteria. Applications: a) RCM-Oxoid
medium with pyrogallol-seal may be used for the detection of bifidobacteria in yog-
hurt (milk products) (SCHULER-MALYOTH, 1968). It may also be used to detect of bif-
idobacteria from fermented milk (BROWN & TowNSLEY, 1970). b) With a 2% agar

187
addition it can be used for detection (RoBERT, 1971 ). c) For anaerobic cu1tivation of
freeze-dried cultures of B. bifidum (KlszA & ZIAJKA, 1973). d) Used as a modified
Reinforced Clostridial Medium (RCM) Oxoid CM 149 formulation from which sol-
uble starch, sodium acetate and agar were omitted. It formed the nutrient basis of ace-
tate-buffered RCM and unbuffered RCM for a study ofthe growth ofbifidobacteria
(BULLEN & TEARLE, 1976). e) A number of the panel discussed using RCM modified
in various ways (SYKES & SKINNER, 1973) for selective isolation: aa) At pH 5.0, with
an agar concentration of 0.75%: (New Zealand agar) to encourage larger colonies.
Cotton blue, 0.1 % incorporated to help differentiate bifidobacteria, the colanies
appearing pale blue on first isolation (BULLEN et al., 1973). bb) PEACH & DRASAR
(1972) listed from WIEL-KORSTANJE & WINKLER (1970) a modification containing
China blue 0.03 %. It was emphasized that the China blue must be the ferri-ferrocya-
nide complex (no. l 0365, Chroma-Association, Stuttgart, W. Germany). Bifidobacte-
ria have brown colonies, the colour differentiation is less marked in a subculture. This
was named by BARNES and co-workers ( 1978) as China blue agar. cc) A modification
of the medium described by MITSUOKA and co-workers ( 1965 b) was used by BARNES
and co-workers (1978) but filamentous, nonsporing, Gram negative anaerobes and
clostridia also grew. This was named by BARNES and co-workers ( 1978) as Bifid agar.
dd) RCM with 1 % of liver extract, pH 6.8 considered as optimal liquid growth
medium.

2.6.11 LBS Agar, BBL no. 11326/pH 5.5

Bacto Rogosa SLAgar, Difco no. 0480/pH?
RogosaAgar, Oxoid no. PM221/pH 5.8

Se1ective medium for the cultivation of oral and faecallactobacilli. It is prepared

according to a modification of the formula of Rogosa, Mitche/1 and Wiesmann. The
above mentioned three culture media have the pH value 5.4, which is too low for bif-
idobacteria. Applications:a) WERNER (1964) used the LBS medium (BBL) forthe iso-
lation ofbifidobacteria from the mouth of adults. b) LBS agar (BBL) with addition of
1% meat extract (Oxoid) and 1.5% sodium acetate. Aftercooling to 50° C 0.37 V/V%
acetic acid is added and poured into petri dishes (MITSUOKA et al., l965b). c) See
modified Rogosa's agar, Appendix Chap. 2.1.7 with a pH of7.0-7.2. d) Detection of
B. bifidum with the addition of 100 ~g/ml neomycin (BRANCA et al., 1979).

2.6.12 Culture Medium Rosenow with cysteine, Pasteur Production no. P-

5.568/pH 7.2

This liquid medium is mainly used for the cultivation of anaerobic bacteria (sealing
with paraffin). Applications: a) Addition of 80 ~g/ml of nalidixic acid (BEERENS &
TAHON-CASTEL, 1965). b) Transfers and cultivation of bifidobacteria strains of var-
ious origin (CATTEAU et al., 1973).

188
2.6.13 Thioglycollate Medium Brewer Modified, BBL no. 11715/pH 7.2
Bacto Fluid Thioglycollate Medium, Difco no. 0256/pH?
Thioglycollate Medium (Brewer), Oxoid no. CM 23/pH 7.2

Medium permitting growth of strictly anaerobic and microaerophilic as weil as aer-

obic organisms. Applications: a) For isolation of strains from the stools of infants and
from vaginal secretion using Thioglycollate medium with modified recipe, which is
no Ionger manufactured by BBL and Difco. (PEcH & MüLLER, 1962). b) Cultivation
ofbifidobacteria from vaginal secretions (MüLLER & PEcH, 1967). c) Subculturing on
Cysteine-Thioglycollate medium (modified formula) (MITSUOKA, 1969 a).

2.6.14 Bacto Thiol Broth, Difco no. 0307

Recommended for culturing of organisms from body fluids and other materials con-
taining penicillin, streptomycin and sulpha drugs. Applications: a) B. breve was
grown anaerobically on Thiol Medium Difco (ROMANO et al., 1979). b) Used for pro-
pagating strains from different bifidobacteria species (B. adolescentis, B. bifidum,
B. breve, B. infantis, B.longum) (See A TCC, 1980).

2.6.15 Tornato Juice Agar, BBL no. 11738/pH 6.1

Bacto Tornato Juice Agar Special, Difco no. 0389/pH 5.0
Tornato Juice Agar, Oxoid no. CM 113/pH 6.1

Tornatojuice agar is recommended for the plate count and cultivation oflactobacilli
(especially L. acidophilus). Applications: a) lt is suitable for the isolation of bifido-
bacteria from the faeces of adults (Bacto-Tomato Juice Agar, Difco) (HAENEL &
MüLLER-BEUTHOW, 1957 c). b) lt is used for the cultivation of B. bifidum (HosATTE,
1964). c) Tornato Juice Agar, Difco is used for identification (SCHNEEGANS et al.,
1966). d) B. bifidum was maintained as stabculture in Tornato Agar (Oxoid) contain-
ing 2% glucose and 0.2% cysteine (DE VRIES & STOUTHAMMER, 1967; DE VRIES et al.,
1967).

2.6.16 Trypticase Soy Broth, BBL no. 11767/pH 7.3

Bacto-Tryptic Soy Broth, Difco no. 0370/pH 7.3
Tryptone Soya Broth (Soybean Casein Digest Medium USP XV/li)
Oxoid no. CM 129/pH 7.3

Soy Brothis a stable, efficient and reliable generat medium for culturing organisms
from blood or special fluid (with blood added). Applications: The use of BBL-Trypti-
case Soy Broth constitutes an optimal liquid growth medium (SYKES & SKINNER,
panel discussion, 1973).

189
2.6.17 VF (Agar Meat Liver 6 %), Pasteur Production/pH 7.4-7.6

This is a semi -liquid medium used f or detection of anaerobic bacteria. App/ications: a)

Application for the isolation ofbifidobacteria strains of faecal origin (BEERENS et a/.,
1957). b) VF is used in a slightly modified form. Without glucose it is used as a basal
medium for ferrnentation and other tests (BARNES & IMPEY, 1971). c) For the plate
counts of bifidobacteria (B.bifidum) in applied veterinary preparations (RoBERT,
1971 ). d) Used for propagating some strains of B. bifidum, including the strain from
TISSIER (Collection of the Institute Paste ur, 197 5).

2.6.18 VL (Agar Meat Yeast), Pasteur Production/pH 7.4

This is a medium perrnitting isolation of anaerobic bacteria. It is agar with blood

added. Also see Chap. 2.1.4 Appendix.

2. 7 Dilution solutions for samples and enumeration

2. 7.1 Buffer solution according to HAENEL (1 960)

Per Iiter
Agar l.Og
Potassium dihydrogen orthophosphate KH2P04 4.5g
di-sodium hydrogen orthophosphate Na2HP04 6.0g
Thioglycollic acid 97% 0.3 ml
Water, distilled 1000 ml
pH 6.6

2.7.2 Buffer so/ution according to ÜCHI and co-workers (1964)

Per Iiter
Agar(Difco) l.Og
Cysteine hydrochloride 0.5g
Potassium dihydrogen orthophosphate KH2P04 4.5g
di-sodium hydrogen orthophosphate Na2HP04 6.0g
Tween 80 (Sorbitan monoleat) l.Og
Water, distilled 1000 ml
pH 6.8

2. 7.3 Various diluants

0.1% Corn steap Iiquor and 0.1% yeast extract solution (SHIMADA et al., 1977). RCM,
LV and saline water with 0.05% Cysteine hydrochloride (SYKES and SKINNER, panel
discussion, 1973).

190
Appendix3

Catalogue of the Supply Sources for Diverse Strains and Coltures of Bifidobacteria

1. Starter cultures for technical use

1.1 Pure cultures
B. bifidum K. K. Yakult Honsha, 1-1-19, Higshi,
YIT-4002 Shinbshi, Tokyo, 105,Japan
(Ferm-P-No. 3371)
YIT-4005
(Ferm-P-No. 3372)
B. breve K. K. Yakult Honsha
YIT-4006
(Ferm-P-No. 3906)
B.longum Labaratory Wiesby, D-226 Niebüll,
Type2 W. Germany (see advertisement)
1.2 Mixed cu1tures
1.2.1 Type BAT
MSK/1 (Bifidobacteria, L. acidophi- Labaratory Wiesby
lus, S. thermophilus)
B. bifidum, L. acidophilusand S. Biogarde, evog, Rheinstrasse, 640,
thermophilus FL-9496 Baizers (Liechtenstein). See
advertisement.
1.2.2 Type BAP
Bifidobacteria, L. acidophilus and Dairy Research Institute, 11 000
Pediococcus acidilactici Praha 1,Jindrisska5,CSSR
1.2.3 Differenttypes
MSK/R/ 1 (Bifidobacteria, L. acido- Labaratory Wiesby
philus, L. bulgaricus, S. thermophi-
lus)
MSK/VI 1 (MSK/R/ 1 with mucous Labaratory Wiesby
strains of L. bulgaricusand S.
thermophilus)
MSK/Q/1 (Bifidobacteriaand L. Labaratory Wiesby
acidophilus)

2. Type Culture Collection strains

Bifidobacterium adolescentis (REUTER)

ATCC: 1523, 1524, 15703*, 15704, 15705, 15706
DSM: 20083* (ATCC 15703), 20084 (ATCC 15423), 20087, V. Scardovi RU 424

Bifidobacterium angulatum (SCARDOVI & CROCIANI)

ATCC: 27535*, 27669,27670,27671
DSM: 20098* (ATCC 27535), 200225 V. Scardovi F 158

191
Bifidobacterium animalis(MITSVOKA; SCARDOVI & TROVATELLI)
ATCC: 25527*, 27536,27672,27673,27674
DSM: 20104* (ATCC 25527), 20105 (ATCC 27536)

Bifidobacterium asteroides (SCARDOVI & TROVATELLI)

ATCC: 25909, 25910*
DSM: 20089* (ATCC 2591 0), 20431 (ATCC 25909)

Bifidobacterium bifidum (TISSIER; ÜRLA-JENSEN)

ATCC: 11146, 11147, 11863 (subsp. pennsylvanicus), 15696, 17930
DSM: 20082,20215, 20456* (Tzssierstrain), 20239 (subsp. penn. ATCC 11863)
PASTEUR: 56.7* (Tissierstrain); 63.78; 64.58; 64.59; 64.60; 64.61; 64.62; 64.63;
64.64; 64.65; 64.66; 64.67; 64.68; 64.69; 64.70; 64.71 ; 64. 72.
Bifidobacterium breve (REUTER)
ATCC: 15698, 15699, 15700*, 15701
DSM: 20213* (ATCC 15700), 20091 (ATCC 15698)

Bifidobacterium catenulatum (SCARDOVI & CROCIANI)

ATCC: 27539*, 27675,27676,27677
DSM: 20103* (ATCC 27539), 20224,20437

Bifidobacterium coryneforme (SCARDOVI & TROVATELLI)

ATCC: 25911 *
DSM: 20216* (ATCC 25911)

Bifidobacterium dentium (SCARDOVI & CROCIANI)

ATCC: 27534*, 27678,27679,27680
DSM: 20221, 20436* (ATCC 27534)

Bifidobacterium globosum (SCARDOVI et al.)

Synonym Bifidobacterium pseudolongum
ATCC: 25865*
DSM: 20092* (ATCC 25865)

Bifidobacterium indicum (SCARDOVI & TROVATELLI)

ATCC: 25912*, 25913
DSM: 20214* (ATCC 25912), 20216 (ATCC 25911)

Bifidobacterium infantis (REUTER)

ATCC: 15697*, 15702,25962,27920
DSM: 20088* (ATCC 15697), 20090 (ATCC 15702), 20223 (ATCC 25962), 20218
(ATCC 17930)

Bifidobacterium lactentis (REUTER)

Subjective synonym of Bifidobacterium infantis
ATCC: 25962*

192
Bifidobacterium liberorum (REUTER)
Subjective synonym of Bifidobacterium infantis
ATCC: 15702*
DSM: 20090* (ATCC 15702)

Bifidobacterium longum (REUTER)

ATCC: 15707*, 15708
DSM: 20219* (ATCC 15707)

Bifidobacterium longum subsp. animalis (MnsuoKA)

Synonym of Bifidobacterium animalis
DSM:20097

Bifidobacterium magnum (SCARDOVI & ZANI)

ATCC: 27540*, 27681,27682
DSM: 2022* (ATCC 27540), 20220

Bifidobacterium parvulorum (REUTER)

Subjective synonym of Bifidobacterium breve
ATCC: 15698*
DSM: 20091 * (ATCC 15698)

Bifidobacterium pseudolongum (MnsuoKA)

ATCC: 25526*, 25864, 25865
DSM: 20099* (ATCC 25526), 20085,20092 (ATCC 25865), 20094,20095

Bifidobacterium pullorum (TROVATELLI et al.)

ATCC: 27685*
DSM: 20433* (ATCC 27685)

Bifidobacterium ruminale (SCARDOVI et al.)

Subjective synonym of Bifidobacterium thermophilum
ATCC: 25866*, 27537,27538,27683,27684,27686,27916,27917,27918,2791 9
DSM: 20212* (ATCC 25866)

Bifidobacterium suis (MATTEUZZI et al.)

ATCC: 27531,27532, 27533*
DSM: 20211 * (ATCC 27533)

Bifidobacterium thermophilum (MITSUOKA)

ATCC: 25525*, 25866,25867
DSM: 20210* (ATCC 25525), 20212 (ATCC 25866), 20440

Legend:
ATCC = The American Type Cu1ture Collection, Cata1ogue of Strains I, Fourteenth Edition, 1980.
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Mary1and 20852, USA.

DSM = German Collection of Microorganisms. Cata1ogue of Strains, Second Edition ( 1977). Asso-

193
ciation for the Investigation of Radiation and the Environment GmbH, München D-8042 Neuher-
berg, Post Oberschleissheim, Ingoldstädter Landstr. I, W. Germany

PASTE UR = Collection ofthe Pasteur Institute. Catalogue ofBacterial Strains (1975). Pasteur Insti-
tute, Paris, France.
* = Type strain

The choice of strains for technical use (e. g. incorporation in cultured dairy
foods) should be made considering the following points: (a) the description of a parti-
cular strain should be looked up in the catalogue; (b) strains isolated from pathogenic
material should not be used; (c) preference is tobe given to the use ofstrains isolated
from human material for the manufacture of cultured foods for human beings.

194
Bifidobacteria
Bibliography 1899-1980/81

1. lntroduction
The following bibliography mentions publications dealing either partially or com-
pletely with bifidobacteria.

l.l Statistics of references cited, given in chronological order

The following Table l gives a survey of the number of publications appearing in the
period from 1899 to 1981, which deal with bifidobacteria. Table 2 gives a summary of
publications which have appeared since 1900 in periods of l 0 years. lt can be
observed that the number of publications has increased since 1949 I 19 50-19 59 from
4-7 per year (on the average) to 25-40.

Table 1 Chronologieallist of the number of publications which appeared per year in the period
from 1899to 1981
Year Number of publi- Year Numberofpubli- Year Number of publica·
cations cations tions
1899 I 1928 2 1956 26
1900 4 1929 5 1957 40
1901 I 1930 5 1958 20
1902 2 1931 5 1959 33
1903 4 1932 5 1960 30
1904 I 1933 7 1961 28
1905 5 1934 6 1962 23
1906 5 1935 6 1963 24
1907 6 1936 5 1964 32
1908 5 1937 7 1965 45
1909 7 1938 6 1966 30
1910 5 1939 6 1967 41
1911 3 1940 7 1968 38
1912 3 1941 8 1969 43
1913 3 1942 2 1970 47
1914 10 1943 6 1971 48
1915 7 1944 6 1972 34
1916 0 1945 I 1973 22
1917 3 1946 I 1974 43
1918 0 1947 I 1975 26
1919 5 1948 7 1976 33
1920 3 1949 12 1977 38
1921 9 1950 19 1978 49
1922 6 1951 10 1979 39
1923 5 1952 II 1980 20*)
1924 6 1953 31 1981 7*)
1925 7 1954 28
1926 2 1955 31
1927 3
*) survey incomplete

195
Table 2 Number of publications per decade and average per year (year's average per decade)

Decade Number ofpublications Average number per year

1900-1909 40 4
1910-1919 39 3,9
1920-1929 48 4,8
1930-1939 58 5,8
1940-1949 51 5,1
1950-1959 249 24,9
1960-1969 334 33,4
1970-1979 379 37,9

1.2 Compilation of theses and other extensive publications in chronological

order and with alphabeticallist of patents

Extensive publications are enumerated chronologically, and patents are listed alpha-
betically. The firstpatent concerning bifidobacteria appeared in 1954.

1.2.1 Theses

Tissier ( 1900) Torzewski ( 1966)

Maurer(l929) Seidel (1966)
Delbove ( 1932) Salahi-Arab ( 1968)
Weiss(l933) Wunderwald ( 1968)
Breit ( 1935) Youssef(l968)
Moser (1948) Cruz, de Ia ( 1968)
Vogel (1952) Bounjoua (1970)
Reimers(l955) Nicolai, von ( 1971)
Birkenbach ( 1957) Robert (1971)
Hämmerling (19 57) Wie!, vander(l973)
Manciaux(l958) Jao(l975)
Brand (1959) Mate(I975)
Romahn(l961) Benner ( 197 5)
Kalz( 1961 b) Süss(l977)
Hosatte ( 1964) Kosikowska ( 1979)
Weis(l965)
Schmittbühl ( 1965)

1.2.2 Post-doctoral publications (Germanpapers called "Habilitationsschrift'')

Wal eh ( 1956a)
Haenel ( 1960)
Dehnert (196la)
Nicolai, von (197 6)

196
1.2.3 Extensiveresearchpapers

Weinberg, Nativelle & Pn!vot (1937)

Orla-Jensen, Olsen & Geill ( 1945)
Boventer ( 1949)
Olsen ( 1949)
Frisell (1951)
Petuely ( 1957a)
Stenger & Wolf (1962)
Yoshioka (1971)

1.2 .4 Collections of various papers

About Lactobacillus bifidus: Levesque (1959a), Raynaud (1959), Raynaud & Guin-
tini (1959), Raynaud & Levesque (1959), Levesque etal., (1959), Levesque (l959b).
Sem. Höp. Paris 35 (4/1), 237-270 (Pasteur Institute Garches, Paris, France).

1.2.5 Alphabetic Iist ofpatents

Daiichi Seiyaku Co. Ltd. ( 1972)

Eurorga SA ( 1969)
Evog ( 1965a, b ), ( 1966)
György etal., (1954d), (1955a,b,c,d)
Hinterwaldner ( 1971)
Kawashima ( 1964)
Kitabara etal., (1966)
Kyowa Hakko Kubushikoisha Co. Ltd. ( 1970)
Legrand ( 1977)
Morinaga Milk Ind. Co. Ltd. (1970), (1973), (1974)
Mutai etal., (1976), (1978a,b,c), (1980)
Nisshin Flour Milling Co. Ltd. (1969a, b ), ( 1976)
Nouvel (1964)
Ogasa ( 1979), ( 1980)
Petue1y (1957e), (1959), (1966)
Rehm (1970)
Roberts ( 1977a, b)
Samezima etal., (1970)
Sawada & Misaki ( 1967)
Schul er (1969a, b, c), (1971 ), (1972)
Schul er & Hinterwaldner ( 1971)
Snowbrand (1970a,b)
Stenval (1976), (1977)
Taisho Pharmaceutical Co. Ltd. (1960), (1964), (1965)
Tomarelli etal., (1954b), (1957)
Watson ( 1957)
Yakult Honsha Co. Ltd. ( 1970)
Zilliken ( 1966)

197
1.3 How to use the bibliography

The following helpful suggestions may be given as to how to use the bibliography.

lndication of year:
1921 a; 1921 b, etc. indicate serial publications by the same author, or the same author
with various coauthors.

Author (s):
The authors' names are alphabetically listed. Single authors or a group of authors
with the same sumames but different Christian names are alphabetically listed
according to their Christian names. In some cases the Christian name is not men-
tioned due to the lack of data.

Titles:
If the titles of papers have been translated from the originallanguage, such translated
titles are enclosed in square brackets. The title-translations are taken either from
abstracts cited orfrom the author's summary. In orderto save space, the original title
is not mentioned if there is an English translation.

Titles of periodicals:
Abbreviations of the title of journals have been made according to the system pro-
posed by KARGER (1958):
(a) all nouns are written with capitalletters, even ifthey were written insmall letters in
the original title, e. g., in French (Archives d'anatomie microscopiques = Arch. Anat.
micr.).
(b) all adjectives are written with smallletters, even ifthey were written in capitallet-
ters in the original title, e. g., in English (Journal ofthe American Medical Association
= J. amer. med. Ass.).
(c) articles and conjunctions are omitted (e. g. The J oumal of Labaratory and Clinical
Merleeine = J. Lab. clin. Med.).

List under 1.4 gives a comprehensive list of the abbreviations generally used. In one
list, all abbreviated titles of joumals are omitted so as to save space. Lesser known
titles of joumals (in languages less frequently used) are written in full.

Patents:
The number ofthe patent is indicated and the date of priority is given, when known, in
parenthesis.

Abbreviations of langnage:
References of the originallanguage in which a paperwas published is given using the
following abbreviations (according to Dairy Sciency Abstracts, 1981):

198
lAnguage ofpaper Abbreviations !Anguage ofpaper Abbreviations
Arabic Ar Japanese Ja
Bulgarian Bg Norwegian No
Czech Cs Persian Pe
Danish Da Polish PI
Dutch NI Portugese Pt
English En Rumanian Ro
Estonian Ee Russian Ru
Finnish Fi Serbo-croat Sh
French Fr Slovak Sk
German De Slovene Sn
Greek Gr Spanish Es
Hebrew He Swedish Sv
Hungarian Hu Turkish Tr
Italian It Ukrainian Uk
Ifthe originallanguage is unknown there is no reference.

Cited in or by
(a) References to Abstracts. The following reference abbreviations are used to referto summaries of
publications in Abstracts:

Abbreviations Abstracts
Bioi.Abstr. Biological Abstracts (with numberofvolume and numberof
abstract)
DSA Dairy Science Abstracts (with numberofvolume and number
ofpage)
Zbi.Bakt. Zentralblatt für Bakteriologie, Parasitenkunde und Hygiene
(Abteilung Referate)
Milchwissenschaft Milchwissenschaft (Abteilung Referate)

(b) Reference to authors. In order to avoid repetitions, save space and reduce costs, reference to
authors is given thus: cited by Boventer ( 1949). This reference refers us to papers mentioned in other
entries in the Bibliography.

1.4 List of generally used abbreviations for various periodicals

A Iist has been compiled of the generally used abbreviations for various periodicals. It
is the system developed and elaborated by Karger ( 1958). Most of the titles of jour-
nals given can be seen in" World List ofScientific Periodicals" and "List of Journals
Scanned" in Dairy Science Abstracts (see Reference List for both data sources).

199
Alphabeticallist of general abbreviations

This Iist of abbreviations should help to find easier the title of periodicals.

A Brit. British
Abh. Abhandlungen brit. british
Abstr. Abstracts Brit.Emp. British Empire
Abt. Abteilung Bull. Bulletin(s)
Acad. Academy, academia
Advanc. Advances
Aerztl. Aerztliches
c
Calif. California
Agric. Agricultural
Camb. Cambridge
Aliment. alimentaire(s)
Canad. Canadienne
Am er. America
canad. canadian
amer. American
Chem. Chemistry, Chemie, Che-
An. Anais, Anales
misch(e)
Ann. Annalen, Annales, Annali,
Chem. Chemical
Annals
ehern. chemical
Annu. Annual Chirurgie
Chir.
annu. annual clinic(al), clinique
clin.
Anon. anonymus, anonym, anony-
Coli. College
mous Comparative
Comp.
Anz. Anzeiger comparative
comp.
Appl. Applied
Conf. Conferance, Conference
appl. applied
conf. conference, conference
Arb. Arbeiten
Confin. Confinia
Arch(s). Archeion, Archiv, Archiva,
Congr. Congres(s), Congres, Con-
Archives, gresso
Archivii, Archivio, Archivo,
Contrib. Contributions
Archivos,
C.R. Comptes rendus
Archivom
es! eeskoslovenskä
argent. argentino(s)
Arq. Arquivo(s)
Ass. Association D
Asoc. Asociatione diet. dietetique
Austr. Australia Dietol. Dietologia, Dietology
austr. australian Dis. Disease(s)
Diss. Dissertation
B diss. dissertation
Bact. Bacteriology DSA Dairy Science Abstracts
bact. bacteriology Dtsch. Deutsche
B.Aires Buenos Aires dtsch. deutsch
Bakt. Bakteriologie
Beitr. Beiträge
Belg. Belgique E
belg. belgica, beige Edn. Edition
Ber. Berichte edn. edition
Bibi. Bibliothek, Bibliotheca eng!. English
Biochem. Biochemical, Biochemische Envr. Environnement
Biochim. Biochemistry Enzimol. Enzymologia
Bio!. Biologie, Biology Enzymol. Enzymologie
bio!. biologic, biological, biologisch Ergebn. Ergebnisse
Biophys. Biophysical Ernähr. Ernährung
biophys. biophysical Esp. Espana
Bol. Boletim, Boletin, Boletines esp. espanol
Boll. Bolletine Eur. European
brasil. brasileira, brasiliana exp. experimentelle, experimental

200
F Kinderärztl. Kinderärztliche
Fed. Federation Kinderheilk. Kinderheilkunde
Fenn. Fennia Klin. Klinik
Forschungsber. Forschungsberichte klin. klinische
Fortbild. Fortbildung Kongr. Kongress
Fortschr. Fortschritte
Pound. Foundation L
fran-r. fran-rais(es) Lab. Labaratory
lat. latina
G Lebensmittelhyg. Lebensmittelhygiene
G. Giornale Lond. London
Gac. Gaceta Lpz. Leipzig
Gaz. Gazeta, Gazette, Gazzetta
Geburtsh. Geburtshilfe M
Gegenw. Gegenwart Maandschr. Maandschrift
gen. general Mbl. Monatsblätter
Germ. Germany Med. Medizinische, Medicin,
Ges. Gesellschaft Medici,
Gesundh. Amt Gesundheitsamt Medical
Gesundh. Ws. Gesundheitswesen med. medical
Gynec. Gynaecology Med. Medical
Gynäk. Gynäkologie med. medical
Mem. Memoires
H Mex. Mexiko
H. Hefte mex. mexicano
Hb. Handbuch Mh. Monatshefte
hisp. hispanica Microbiol. Microbiology, Microbiologia,
Höp. Höpital Microbiologie, Mikrobiologie
hosp. hospitalier(s), hospitaliere(s) microbiol. microbiological
Hung. Hungaria Milchwiss. Milchwissenschaft
Hyg. Hygiene, hygiene Milchztg. Milchzeitung
Mitt. Mitteilungen
I
Mod. Moderne
lg. !giene
Molk.Ztg. Molkerei-Zeitung
Immfrschg. Immunitätsforschung
moloch. Molochnaya, Molochnoe
Ind. Industria, Industry
Mschr. Monatsschrift
Inf. Informations
mthl. monthly
infect. infections, infectious
Münch. Münchner
InfektKr. Infektionskrankheiten
inn. innere
N
lnst. Institut(e)
N. North
Int. Internationales
Natl. National
int. international, internationalis,
Naturforsch. Naturforschung
internazionale
Naturw. Naturwissenschaft
intern. internal
Nederl. Nederland
Ist. Istituto
nederl. nederlandsch
Ital. Italia
neerl. neerlandica
ital. italiano, italiani
N.F. NeueFolge
Nomencl. Nomenclature
J
Nutrit. Nutrition, nutrition
J. Journal,Jornal
N.Y. NewYork
Jap. Japonese,Japonica
jap. japonese,japonica
Jb. Jahrbuch 0
Jber. Jahresbericht( e) Obstet. Obstetrics
Oeff. Oeffentliches
K Oesterr. Oesterreichische
Kbh. Kebenhavn Org. Organisation, Organization

201
orient. oriental, orientalica Sch. Schola
Orig. Originale Schweiz. Schweizerisch
schweiz. schweizerisch
p Sei. Science
Pädiat. Pädiatrie sei. science
Pädiol. Pädiologie Scot. Scotish
Paediat. Paediatrische scot. scotish
Pediat. Pediatric(s), Pediatrya Sect. Section
Pediat. Pediatrie Sem. Semana, Semaine
Pat. Patent Settim. Settimana
Path. Pathology, Pathologie sieroter. sieroterapia
Pediat. Pediatrics, Pediatrie, Peditria Soc. Societas, Society, Sociologie
pediat. pediatrics St. State
pediat. pediatrique Sth. South
Penn. Pennsylvania sth. southern
Pharm. Pharmaceutical, Pharmaceu- Stockh. Stockholm
tique Strahlenther. Strahlentherapie
Physik. Physikalische Sper. Sperimentale
Physiol. Physiology sper. sperimentale
physiol. physiologische Summ. Summary
Pneumon. Pneumonia sup. superiore
Poult. Poultry Suppl. Supplement
Prax. Praxis Symp. Symposium
ProbI. Probleme Syst. Systematic
Proc. Proceedings
Proctol. Proctology
T
Progr. Progres, Progresos, Progress,
T. Tidskrift, Tijschrift
Progresso
Taxon. Taxonomy
PubI. Publicaciones, Publica~es, TechnoI. Technology
Publications
ter. terapia
Ther. Therapie, Therapia
Q ther. therapeutiques
Quad. Quaderni Therap. Therapeutiques
Quart. Quarterly therap. therapeutiques
quart. quarterly Trab. Trabajos, Travalhos
Trans. Transactions
R Trav. Travaux
Rass. Rassegna
R.C. Rendieanti u
Rec. Record Ugeskr. Ugeskrift
Rech. Recherehes Umsch. Umschau
Rdsch. Rundschau Un. Unio,Union
Ref. Referate Univ. Universität, Universite,
Rep. Report University
Res. Research Urol. Urologique
Rev. Revies, Revista, Revue Urug. Uruguay
Rif. Riforma u.s. United State
RiodeJ. Rio de Janeiro
Riv. Rivista
V
rozhl. Rozhledy
Verh. Verhandlungen
Vestn. Vestnik
s Vet. Veterinary
Sammelbl. Sammelblatt vet. veterinary
S.Ber. Sitzungsbericht(e) Vitaminol. Vitaminology
scand. scandinavic, scandinavian, vol. volume
scandinave Vjschr. Vierteljahresschrift

202
w z
West West z. Zeitschrift
west western Zbl. Zentralblatt
Wien Wien er Ztg. Zeitung
Wschr. Wochenschrift Zootech. Zootechnic

y
Yb. Yearbook

203
2. Alphabeticallist of papers about Bifidobacteria
from 1899-1981
1921a ADAM, A., Über Darmbakterien. II. Züchtung des B. bifidus auf Hämatinnährböden.
Z. Kinderheilk. 29,65-67 [De]. Cited in Zbl. Bakt. (I. Abt. Ref.) 73, 134 (1922).
1921 b ADAM, A., Über Darmbakterien. III. Über den Einfluß der H-Ionenkonzentration des
Nährbodens auf die Entwicklung des Bacillus bifidus. Z. Kinderheilk. 29,306-320 [De]. Cited
inZbl. Bakt. (I. Abt. Ref.)73, 136(1922).
1921c ADAM, A., Über Darmbakterien. IV. ÜberdasH-Ionenoptimum der Köpfchenbakterien des
Mekoniums. Z. Kinderheilk. 30, 265-272 [De]. Cited in Zbl. Bakt. (I. Abt. Ref.) 74, 335
(1923).
1922a ADAM, A., Über Darmbakterien. V. Grundlagen der Ernährungsphysiologie des B. bifidus.
Z. Kinderheilk. 31, 331-366[De]. Cited inZbl. Bakt. (1. Abt. Ref.) 74,335-336 (1923).
1922b ADAM, A., Über die Bedeutung der Eigenwasserstoffzahl (des H-Ionenoptimum) der Bakte-
rien. Zbl. Bakt.(I.Abt. Orig.)87,481-486[De].
1925 ADAM, A., Die Entstehung der Bifidusvegetation. Jb. Kinderheilk. 110, 186-196 [De]. Cited in
Zbl. Bakt. (!.Abt. Ref.)82, 528(1926).
1927 ADAM, A., Die Entstehung der Bifidusvegetation. Jb. Kinderheilk. 117, 15 [De]. Cited in Zbl.
Bakt.(l. Abt. Ref.)89, 378 (1928).
1949 ADAM, A., (Substitute for human milk in infant feeding. A contribution to the establishment of
an artificial bifidus flora). Mschr. Kinderheilk. 97, 500-507 [De]. Cited in DSA 14, 339
(1952).
195 5 ADAM, A., Praxis der modernen künstlichen Ernährung des gesunden und kranken Säuglings.
Öff. GesundhAmt.IO, 345 [De]. Cited by Haenel (1960).
1956 ADAM, A., (Pathogenese, clinic and therapy, pp. 38-490). In: "Säuglings-Enteritis"(ADAM,
A.,ed.) [De]. Georg Thieme, Stuttgart, German Federal Republic.
1925 ADAM, A. & FROBOESE, C., Anatomie und Bakteriologie des Darmes bei Durchfallerkran-
kungen des Säuglings. Mschr. Kinderheilk. 29,562 [De]. Cited in Zbl. Bakt. (I. Abt. Ref.) 79,
233 (1925).
1922a ADAM, A. & KlssoFF, P., Über Darmbakterien. VII. Zur Biologie der Darmflora des
Säuglings. Ernährungsphysiologie des B. acidophilus im Verhältnis zu der des B. bifidus.
Z. Kinderheilk. 34,207-212 [De]. Cited in Zbl. Bakt. (I. Abt. Ref.) 75,558 (1923).
1922b ADAM, A. & K!SSOFF, P., Über Darmbakterien. VIII. Zur Biologie des Säuglings. Das quanti-
tative Verhältnis von B. bifiduszu B. acidophilus. z. Kinderheilk. 34, 213-215 [De]. Cited in
Zbl. Bakt. (I. Abt. Ref.)75, 559(1923).
1973 ADRIAN, J., "Valeura/imentaire du lait "pp. 152-155. Maison Rustique, Paris, France, [Fr].
1971 AKIMOTO, H., (Studies on the survival of Lactobacillus bifidus). Acta paediat. jap. (Domestic
Edn) 75,945-955 (Ja). En summ. Acta paediat.jap. (Overseas Edn) 13 (27), 49. Cited in DSA
34,868 ( 1972).
1975 AKoPOVA, A. 1., (Frequency of Bacterium bifidum excretion in adults with different gastroin-
testinal diseases) Mikrobiol. Zh (Kiev) 37 (3), 337-340 [Ru]. Cited in Bio!. Abstr. 62 [49299]
4859(1976).
1903 ALBARRAN, J. & COTTET, J ., "Le ro/e des microbes anaerobies dans /'infection urinaire "p. 8 5.
Presse Medicale France [Fr]. Cited by Rach & Reuss ( 1909).
1963 ALEXANDER& DAVIES,J. comp. Bact. 73, 17. Cited by Robert(I97J).
1962 AMANO, A., On the blood group substances of Lactobacillus bifidus. Proc. jap. Acad. 38 (7),
368-370 [En]. Cited in DSA 25,425 (1963).
1969 ANON, (Yoghurt with Bifidus bacteria). Molkereizeitung (Hildesheim) 23 (34), 1115 [De].
Cited in DSA31, 631 (1969)
1970 ANON, Tokyo Univ. Pharmacistics Studying. Bitidus factor and carrot extract. Jap. med. Gaz.
7 (12), 5-11.
1974 ANON, More European ideas forthe development of cultured milk products. Milk Industry74
(5), 19-20[En]. Cited in DSA36,448 (1974).
1978 ANON, Soups and a health drinkinTetra Briks in Japan. Packaging49 (584), 37 [En]. Cited in
DSA41, 406 (1979).
1970 AR IES, V. & HILL, M. J., Degradation of steroids by intestinal bacteria: II. Enzymes catalysing

204
 the oxidoreduction of the 3 alpha-, 7 alpha- and 12 alpha-hydroxyl groups in cholic acid and
the dehydroxylation ofthe 7-hydroxyl group. Biochem. Biophys. 202 (3), 535-543. Cited in
Bio!. Abstr. 51 [I 00129]9842 (1970).
1967 ATABEKOVA, K. S., (A study ofthe normal intestinal microflora present in nursing children).
Med. Zh. Usb. 2, 34-37. Cited in Bio!. Abstr. 50 [60 I 03]5739 ( 1969)
1980 ATCC, "Catalogue ofStrains "I. [En]. 14th ed. Arnerican Type Cuttute Collection, 1230 I Par-
klawn Drive, Rockeville, Maryland 20852, USA.
1953 BACHMANN, H. & VIEHMANN, P., Ein neuer Bitidusnährboden mit Alete-Frühnahrung.
Z. Kinderheilk. 73,610 [De].
1910 BAHRDT & BEIFELD, Über die Wirkung der Nährungskomponenten der Frauenmilch auf die
Darmflora des Säuglings. Jber. Kinderheilk. 72, 71 [De]. Cited by Rach & Reuss (1909).
1959 BAILEY, R. W. & CLARKE, R. T. J., Biochem. J. 72,49-54 [En]. Cited by C1arke & Bauchop
(1977).
1962 BAILEY, R. W. & RoBERTON, A. M., Carbohydrases of a rumen strain ofLactobacillus bifidus.
The intracellular alpha-1-+6-glucosidase (isomaltodextrinase). Biochem. J. 82, 272-277 [En].
1977 BAJARD, A., (Microbiological control of preparations containing lactic acid bacteria). Rev.
Inst. Pasteur, Lyon 10 (4), 313-332 [Fr].
1974/75 BALLABRIGA, A., HILPERT, H. & ISLIKER, H., lmmunity of the infantile gastrointestinal
tract and implications on modern infant feeding. Nestle Research News 1974/75 pp. 24-25
[En]. Cited in DSA 38,843 (1976)
1966 BAMBERG, H., Über die Sanierung von Typhusdauerausscheidern mit Arnpicillin. Med. Welt
39,2086 [De]. Cited by Süss ( 1977).
1957 BAMBERGER, P., Zur Morphologie und Biochemie des Bacterium bifidum. Mod. Prob!. Pädiat.
(Basel-NewYork)2, 13-23[De].
1956 BAMBERGER, PH. & DEHNERT, J., Über die Invarlabilität der biochemischen Leistungen des
"Bakterium bifidum "als Grundlage für eine Klassifikation. VIII. Int. Kongr. Kinderheilk.,
Kopenhagen, Denmark [De].
1956 BARABOI, T. A., (Oxidation-reduction processes in the digestive tract of infants on different
diets) Voprosy Pitanija 15 (6), 16-22 [Ru].
1952 BARBERO, G. J., RUNGE, G., FISCHER, D., CRAWFORD, M. N., TORRES, F. E. & ÜYÖRGY, P.,
Investigations on the bacterial flora, pH and sugar content in the intestinal tract ofinfants. J.
Pediat. 40, 152-163 [En].
1930 BARENBERG, L. H. & ABRAMSON, The effect of Iarge amounts of milk sugar on the stools and
nutrition ofinfants. Arch. pediat. 47, 1-7 [En].
1914 BARKER, The diet in typhoid fever. J. amer. med. Ass. 63,929 [En]. Cited by Weinberg etal.
(1937).
1979 BARNES, E. M., The intestinal microflora of poultry and game birds during life and after sto-
rage. J. appl. Bact. 46 (3), 407-419 [En]. Cited in DSA41, 776 (1979).
1970 BARNES, E. M. & IMPEY, C. S., The isolation and properties ofthe predominantanaerobic bac-
teria in the caecaof chickens and turkeys. Brit. Poult. Sei. 11 (4), 467-481. Cited in Bio!. Abstr.
52 [49792]5039 ( 1971 ).
1971 BARNES, E. M. & IMPEY, C. S., The isolation ofthe anaerobic bacteria from chicken caeca with
particular reference to members of the family Bacteroidaceae. In: "Isolation ofAnaerobes"
(D. A. Shapton and R. G. Board, eds.). pp. 115-123, Academic Press, London [En].
1972 BARNES, E. M. & IMPEY, C. S., Some properties of the nonsporing anaerobes from poultry
caeca. J. appl. Bact. 35, 241 [En].
1979 BARNES, E. M., IMPEY, C. S. & STEVENS, B. J. H., Factors affecting the incidence and anti-Sa1-
monella activity ofthe anaerobic caeca flora ofthe young chick. J. Hyg. 82 (2), 263-284 [En].
Cited in Bio!. Abstr. 68, 547 4 ( 1979).
1972 BARNES, E. M., MEAD, G. C., BARNUM, D. A. & HARRY, E. G., The intestinal flora of the
chicken in the period 2 to 6 weeks of age, with particular reference to the anaerobic bacteria.
Brit. Poult. Sei. 13,311 [En].
1978 BARNES, E. M., MEAD, G. C., IMPEY, C. S. & ADAMS, B. W., Analysis ofthe avian intestinal
flora. In: Lovelock & Davies ( 1978), pp. 89-105 [En].
1957 BARNESS, L. A., BAKER, D., GUILBERT, P., TORRES, F. E. & GYöRGY, P., Nitrogen metabolism
of infants fed human and cow's milk. J. Pediat. 51 (1), 29-39 [En]. Cited in DSA 19, 928
(1957).

205
1914 BASTN, J., Beiträge zur Methodik der Untersuchungen der Bakterienflora des Säuglings-
stuhles und zur Kenntnis seinerwichtigsten Bakteriengruppen. Z. Hyg. InfektKr. 77,282-288
[De].
1975 BAUER, H., SIGARLAKIE, E. & FA URE, J. C., Scanning and transmission electron microscopy of
three strains of Bifidobacterium. Canad. J. Microbiol. 21, 1305-1316 [En].
1956 BAUMANN, H. E. & FoRSTER, E. M., Characteristics of organisms isolated from the rumen of
cows fed highand lowroughage rations. J. Bact. 71,333-338 [En].
1967 BECK, J., (New medium for the culture, isolation and maintenance of Bifidobacterium bifidum
(L. bifidus) ). Ann. Bio!. clin. 25 (10/12), 1255-1260[Fr]. Cited in DSA30, 271 (1968).
1965 BEERENS, H., Aore intestinale normale et dietetique. In: "Microbiologie et Dietetique "[Jour-
nee Nationale de Dietetique]. Doc. Techn. Pharm., S.U.T.I., 175 Rue du Faubourg, Poisson-
niere, Paris, France [Fr].
1957 BEERENS, H., GERARD, A. & GuiLLAUME, J., Etude de 30 souches de Bifidobacterium bifidum
(Lactobacillus bifidus). Caracterisation d'une variete buccale. Comparaison avec !es souches
d' origine fecale. Ann. Inst. Pasteur, Lilie 9, 77-85 [Fr].
1980 BEERENS, H., RoMOND, C. & NEUT, C. Influence ofbreast-feeding on the bifid flora ofthe
newbom intestine. Amer. J. clin. Nutrit. 33, 2434-2439 ( 1980) (En).
1965 BEERENS, H. & TAHON-CASTEL, M. M., "Infections humaines a bacteries anaerobies non toxi-
genes". Presses Academiques Europeennes, Bruxelles [Fr].
1956 BELL, D. J., CROMBIE, L. & GRANT, J. K., Oligosaccharides of milk: their relation to the "bifi-
dusfactor"and to blood group substances. Chemical Society, London: Annual Reports on
the Progress ofChemistry 6, 1955 [En].
1968 BENDIG, J., HAENEL, H. & BRAUN, 1., (The fecal microecology in school children.l. Communi-
cation. The quantitative composition ofthe fecal flora). Zbl. Bakt. (I. Abt. Orig.) 209,81-89
[De].
1975 BENNER, J., ("Production of D- and L-/actate in yoghurt, cultured milk and kefir") Thesis,
Tierärztliche Hochschule, Hannover, German Federal Republic[De].
1957 BERG ER, H., Zum Problem "humanisierte" Milchen. Mod. Prob!. Pädiat. 2, 70--84 [De].
1956 BERG ER, H., WAVRE, D. & GILARDI, A., L'alimentation du nourrisson ä terme premature au
lait H umana. Etude clinique. Ann. Paediat. (Base) 186, 383-406 [Fr].
1934 BERGEY, D. H., BREED, R. s., HAMMER, B. w., HUNTOON, F. M., MURRAY, E. G. D. & HAR-
RISON, F. C., "Bergey's Manual of Determinative Bacteriology ".4th ed. Williams & Wilkens
Co., Baltimore, USA[En].
1939 BERGEY, D. H., BREED, R. S., MURRAY, E. G. D. & HITCHENS, A. P., "Bergey's Manual of
Determinative Bacterio/ogy ". 5th ed. Williams & Wilkens Co., Baltimore, USA [En ].
1923 BERGEY, D. H., HARRISON, F. C., BREED, R. S., HAMMER, B. W. & HUNTOON, F. M., "Bergey's
Manual of Determinative Bacterio/ogy". Ist ed. Williams & Wilkens Co., Baltimore, USA
[En].
1925 BERGEY, D. H., HARRISON, F. C., BREED, R. S., HAMMER, B. W. & HUNTOON, F. M., "ßergey's
Manual of Determinative Bacteriology" 2nd ed. Williams & Wilkens Co., Baltimore, USA
[En].
1930 BERGEY, D. H., HARRISON, F. C., BREED, R. S. HAMMER, B., W. & HUNTOON, F. M., "ßergey's
Manual of Determinative Bacteriology" 3rd ed. Williams & Wilkens Co., Baltimore, USA
[En].
Bergey's Manual of Determinative Bacteriology, see the following authors (for different edi-
tions):
1923-Bergeyetal.,lsted.
1925- Bergey et al., 2nd ed.
1930- Bergey et al., 3rd ed.
1934- Bergey et al., 4th ed.
1939- Bergey etal., 5thed.
1948- Breed et al., 6th ed.
1957- Breed et al., 7th ed.
1974- Buchanan & Gibbons, 8th ed.
1902 BERG HOLM, H., Über Mikroorganismen des Vaginalsekretes Schwangerer. Arch. Gynäk. 66,
497 [De]. Cited by Feldheim ( 1958).
1965 BERNARD, 1., Indications de B. bifiduslyophilise en gynecologie. Inf. Ther. 3 (5/mai)[Fr].

206
1967 BERNDT, H., Gastroenterologia, Suppl. vol. I07. Cited in Prospectus Omniflora, Med. J. Carl
Pflüger 1, Berlin 44, German Federal Republic.
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259
Index of Organisms

A Bacteroides fragi/is
ammonia 69
Acidaminococcus 54 infaecalflora(man) 53,62
A.fermentans 54 indole 69
inman 54 metabolism 53
inpigs 54 polysaccharides 66
Actinobacterium bifidum pyruvate 69
taxonomy 16 soft-tissue infections 65
Actinomyces 157 urea 53
Actinomyces bifidus B. melaninogenicus
taxonomy 16 soft-tissue infections 65
Actinomyces eriksonii B. oralis
taxonomy 19 inmouth(man) 53
Actinomycetaceae 19,51, !57 Bifidobacterium 51
Arachnia 157 antibiotics 31,75
antigenicity 15
babyfood 4,5, 7, 134,136-138
8 bileacids 30, 32, 70, 110
catalase 12
Bacillaceae 55 cell wall 13
Bacillus 55 cultural properties I 0, II, 20, 115
inanimals 55 culturemedia 145-153, 166-190
inman 55 cultured milk products 7, 90, 102-134
Bacillus acidophilus 3 cultures 108-117,133,191-194
taxonomy 52 dietandnumbers 72-74
Bacillus bifidus 3 differentiation 18, 157
taxonomy 16 DNA 15
Bacillus bifidus communis 3 fermentation 19,35-41
taxonomy 16 158, Fig. 3.1
Bacillus cereus 55 flavour 37,92
food poisoning 55 growthsubstances 42-50,114-116
Bacterionema 157 identification 21, 157-158
Bacterium bifidum immune response 94-95
taxonomy 16 in animals 26, 52
Bacteroidaceae 53 in man 22-25, 52, 58, 60, 61,64
Bacteroides 53 incubation and numbers 121, 122, Fig. 7.15
antibiodes 31,7 5 intracellular enzymes 12
antibodies 78 isolation 144
bile acids 30, 32,70 lactulose 43-45, 67, 72,96-97, 143
cholesterol 70 Iipids and phospholipids 14-15
dietandnumbers 72 morphology 8-1 0
glucuronides 68 nitrate 12, 85-86
hydrogen 53 nitrosamines 69
in animals 53 numbers and consumption 129
in man 24, 53, 58,60 organicacids 12,30,35, 79,81
lactulose 44 physiology 11-12
nitrosamines 69 polysaccharides 67
organicacids 53 prophylaxy(animals) 101, 134,142
polysaccharides 66 prophylaxyandtherapy(man) 95-100,134,
soft-tissue infections 53,65 138-142
taxonomy 53 survival 125-129
Bacteroides bijidus 3 taxonomy 3-5, 6, 16,21, 51
taxonomy 16 urea 68
vitamins 71, 85

260
B. adolescentis B. bifidum var. pennsylvanicus (Continued)
bacteriophage 32-34 neuraminidase 46
carbohydrate fermentation 12, 19 nutrition II
cell wall 13 pleomorphism 9
cultural properties 20 B. breve
cultures 122 cell wall 14
enzymes 39,40 cultural properties 20
fermentation 19, 40 cultured milk products 133
in animals 26 cultures 122
in man 25,52,62 enzymes 40
morphology and physiology 20 fermentation 19,20
soft-tissue infections 65 in man 24, 25, 52
taxonomy 17,19,21 morphology and physiology 20
B. angulatum taxonomy 17-20
taxonomy 17,18,21 B. catenulatum
B. asteroides taxonomy 17,18,21
cell wall 14 B. comutum
enzymes 40 taxonomy 17, 19
fermentation 19 B. coryneforme
in bees 26,52 fermentation 19
taxonomy 17,18,21 in bees 26, 52
B. bifidum taxonomy 17,19,21
acidproduction 39,112,118 B. dentium
antagonistic effect 27, 28, II 0 inman 18
antibiotics 31 taxonomy 17,18,21
antigenicity 15 B. eriksonii
babyandchildrenfood 5,6,81,86, 134,136, soft-tissue infections 65
156 taxonomy 17, 19
cell wall 13-14 B. g/obosum
cultural properties 20 in animals 18
cultured milk products 5, 90,91, 132-133 taxonomy 17,18,21
cultures I 08, 117, 118, 122, 130 B. indicum
enzymes 40-41 catalase 12
fermentation 19,37,41 cell wall 14
flavour 92 enzymes 40
folicacid 92 fermentation 19
growth factors 48, 49,50 in bees 26, 52
in man 24, 25,94 taxonomy 17,18,21
isolation 144 B. infantis
liverdisease 81, 98, Fig. 6.7, Fig. 6.8 cell wall 14
morphology and physiology 20, Fig. 2.3 cultural properties 20
nitrogen 83 cultures 122
nutrition II enzymes 40, 41
pH 11,111 fermentation 19
prophylaxy and therapy (man) 5, 7, 87-88, in man 24, 25, 52, 58
95,96,138-141 morphology and physiology 20
proteolytic activity 12,90 prophylaxy and therapy (man) 138
taxonomy 16, 52 taxonomy 17-20
urease 111 urease 12
veterinary use 142 B. /actentis
vitamins 85,92 fermentation 19
B. bifidum var. pennsylvanicus neuraminidase 46
antagonistic effects 27 taxonomy 17-20
growthfactors 6,45,4~9 B. liberorum
human milk 6, 45 cell wall 14
human secretions 45,46 enzymes 40
Iipids and phospholipids 15 fermentation 19

261
B. Iiberorom (Continued) B. thermophilum (Continued)
taxonomy 17-20 cell wall 14
B.longum cultural properties 20
bacteriophage 34 enzymes 40
bifidus sweet milk 131 fennentation 19
cell wall 14 in animals 21, 26, 52
cultural properties 20 morphology and physiology 20
cultured milk products 90,91, 132 taxonomy 17, 18,19,21
cultures 108, 122 veterinary use (pigs) 142
enzymes 40 Butyribacterium 52
fennentation 19
in man 24, 25, 52, 62,94
morphology and physiology 20 c
prophylaxy and therapy(man) 5, 95, 140
taxonomy 17-21 Candida 31
B. longum subsp. animalis C. albicans
fennentation 19 intestinal tract (man) 56
in animals 26 moniliasis 28, 56, 75,87
taxonomy 17, 18, 26 C. krnsei, in man 56
B. longum subsp. longum C. paropsilosis, in man 56
fennentation 19 C. tropicalis, in man 56
inman 24,25 Catenabacterium 52
B. magnum Cil/obacterium 52
in faeces (rabbit) 18 Clostridium 55
taxonomy 18 amines 27,69
B. parvulornm ammonia 68
cell wall 14 bile acids 30,70
enzymes 40 bilirubin 71
fennentation 19 diet and numbers 73
taxonomy 17-21 faeces(animals) 55
B. pseudolongum faeces (man) 23, 24, 55, 58,62
cell wall 14 glucuronides 68
cultural properties 20 lactulose 97
enzymes 40 nitrosamines 69
fennentation 19 phenols 69
in animals 26, 52 pyruvate 69
morphology and physiology 20 soft-tissue infections 65
taxonomy 17-21 urea 68
veterinary use 142 Cl. bifermentans
B.pullornm faeces (man) 55
enzymes 40 Cl. peifringens (Cl. welchii)
infaeces(chicken) 18 faeces (man) 55
taxonomy 18 food poisoning 55
B. rnminale gastroenteritis 64
amino acids 12 Cl. sporogenes
bacteriophage 33, Fig. 2.9, Fig. 2.10 faeces (man) 55
in animals 21 Cohnistreptothrix bifidus
taxonomy 17, 18,19,21 taxonomy 16
B. suis Corynebacterium 58
cell wall 14 C. bifidum
enzymes 40 taxonomy 16
fennentation 19,21 C. pseudodiphtheriticum 56
inpig 26,52 C. xerosis 56
taxonomy 17,18,21
B. thermophilum
aminoacids 12
bacteriophage 34, Fig. 2.9, Fig. 2.10

262
 E L

Enterobacter 31 Lactobaci/laceae 52
Enterobacteriaceae 54 Lactobacillus 6, 52
Escherichia 54 antibiotics 3 I, 7 5
E. coli antibodies 78
amines 69 bile acids 30,70
ammonia 68 cell wall 14
antigens 78 differentiation 157
antitoxins 5, 82 DNA 15
bilirubin 70 glucuronides 68
diet and numbers 72, 75 identification 158
faeces (animals) 54 in animals 52
faeces (man) 54,75 in man 22, 24, 32, 52, 58, 60, 62
freeze-dried preparations 5, 95 lactulose 72,97
glucuronides 68 Iipids and phospholipids 14
indole 69 microscopy 157
inhibitory substances 54,79 nitrate 85
lactulose 45 nitrosamines 69
metabolism 54, 68, 69 L. acidophilus 6
nitrosamines 69 amines 27
pathogenicity 27-30,55,64,69, 82,87 baby food 86,87
phenols 69 bifidus sweet milk 131
R-factor 71 culturedmilkproducts 90,91, 102,131-133
Eubacterium 52 cultures 102,112,117-120,130
antibiotics 31 dietary adjuncts 27,31, 64, 94,95
bile acids 30,70 folicacid 92
differentiation 157 freeze-dried preparations 5, 7, 81, 95, 138,
inman 58,62 140,141
microscopy 157 in animals 94
soft-tissue infections 65 inman 52,94
urease 68 inhibitorysubstances 30, 79,110
E. aerofaciens lactulose 44
in animals 52 prophylaxy and therapy (man) 87, 88, 138,
inman 52,62 140,141
E. rectale prophylaxy and therapy (pigs) I 0 I
inman 52 vitamin B,z 92
L. bifidus
pH sensitivity !II
F prophylaxy and therpay (man) 95
taxonomy 3,8, 16
Fusobacterium 53 L. bifidusvar. pennsylvanicus
ammonia 69 taxonomy 16
in man 23, 53, 58 L. brevis
indole 69 cu1tured milk products 90
pyruvate 69 L. bu/garicus
cultured milk products 90,91, 92
folicacid 92
K prophylaxyandtherapy(man) 95,141
survivalin the gut 91
Klebsiella 55 L. casei
large intestine (man) 55 cultured milk products 90
potential pathogen 31, 7 5 inman 52
K. pneumoniae Iactate dehydrogenase 41
fixing gaseous nitrogen 55 survival in the gut 91
L. cellobiosus
inman 52

263
L. fermenturn P. asaccharolyticus
inman 52 urinry tract (children-girls) 65
survivalin the gut 91 Peptostreptococcus
L. lactis adults (faecal flora) 58,62
cu1tured milk products 90 genus 53
prophylaxy(animals) 101 organicacid(man) 79
L. parabifidus soft-tissue infections (man) 65
taxonomy 16 urease 96
L. plantarum weanling 23,58
cultured milk products 90 Propionibacteriaceae 52
inman 52 Propionibacterium 52, 157
L. salivarius appearance 157
inman 52 soft-tissue infections (man) 65
prophylaxy (animals) I 0 I P. acnes
Leptotrichia, in man 53 animals 52
Leuconostoc cremoris (L. citrovorum) man 52
cultured milk products 90, 133 P. freudenreichii subsp. shermanii
cultures 113 folicacid 92
survival in the gut 91 Kefir 92
L. dextranicum Vit. B12 92
cultured milk products 90 Proleus
cellulose (diet) 73
inhibition 27
M intestinal flora (man) 55,64
lactulose (calves) 143
Megasphaera elsdenii 54 lactulose (man) 45
inman 54 tetracycline 31, 75
inpigs 54 P. vulgaris
Micrococaceae 56 ammonia(man) 69
indole(man) 69
pyruvate(man) 69
N Pseudomonas
genus 56
Neisseria tetracycline 31
in faeces (man) 54 P. aeruginosa
genus 54 Iactulose(cattle) 143
inmouth 54 urinary tract infections 56
intestinal flora (man) 54
Nocardia bifida
taxonomy 16 R

Ramibacterium
p genus 52
Ruminococcus
Pediococcus adults (faecal flora) 58
taxonomy 222 genus 53
P. acidilactici intestinal peristalsis 79
in cultured milk 90,91, 112, 132, 191 organicacid(man) 79
in infant food 86,211 R. albus
Penicillium roquefortii cellulose(man) 66
culturing in milk 208 cellulose(ruminants) 53
Peptococcaceae 53 xylan(man) 66
Peptococcus 53 R. bromii
dexycline 252 man(faecalflora) 53,62
faeces(man) 54 pigs (faecal flora) 53
mouth(man) 54 rawstearch(man) 66
soft-tissue infections (man) 65

264
R. jlavefaciens Streptococcus (Continued)
cellulose (ruminants) 53 old people 24
R. ramosum S. agalactiae
antagonistic effect 27 phosphotransferase system 240
Rothia S. bovis
genus 157 man(faeces) 54
ruminants (faeces) 54
S. cremoris
s cultured milk products 90, 113, 132
progurt 132
Salmonella S.faecalis
young chick 205 carbohydrate diet 72
s. typhi glucoronides (hydrolyze) 68
ampicilline 205 man (intestinal flora) 54
inhibition 27 mixed culture 122
tetracycline 217 phosphotransferase system 240
S. typhimurium secondaryamines(man) 69
bacteriotstatic effect 30 S.faecium
Sarcina carbohydratediet 122
genus 53 intestinal flora (man) 54
S. ventriculi S. lactis
vegetarian humans 53 buttermilk 90
Se"atia culturedmilk 133
tetracyclines 31 gastric transit 91
S. marcescens Kefir 90
intragastric survival (Fig. 5.3) pharmaceutical preparation 95
Shigella S. lactis subsp. diacetylactis
breast-fed infant 27,82 cultured milk 132
guinea pigs (antibiotic-treatment 79 S. mitis
guinea pigs (germe-free) 79 inmouth(man) 54
mice (antibiotictreatment) 79 S. salivarius
S. dysenteriae in mouth (man) 54
inhibition 27 S. sanguis
S.jlexneri inmouth(man) 54
antagonistic effect 211 S. thermophilus
Staphylococcus after-acidification 127
adult (intestinal flora) 51, 64 Biogarde(sourmilk) 90,Fig.7.12
antagonisticeffect 27 cultured milk products 90,91, I 02, 123, 128
cellulose(diet) 73 fructose 1,6-diphosphate 41
genus 56 gastric transit 91
infant (intestinal flora) 61, 63 mixedculture 112,117,118,119,133,191
insaliva 58 pharmazeutical preparation 95
lactulose (adult) 97 photomicrograph Fig. 113
S. aureus yoghurt 90,92
antibiotic resistance 71
enterocolitis 56
enterotoxin 56 T
gastrointestinaltract (man) 56
infant(intestinal flora) 61,63 Thermobacterium
tetracyclines 31, 75 cell wall 14
Streptobacillus faecalis T. bifidum
(Syn. Bacillus acidophilus) 207 biotypes 229,231
Streptococcus Tisseria bifida
genus 81 taxonomy 16
infant (intestinal flora) 61,63 Torulopsis glabra
man (intestinal flora) 60 gastrointestinal tract(man) 56
man (saliva) 58

265
 V Vibrio cholerae (Continued)
mice 79
Vei/lonel/aceae 54 pigs 79
Veillonella small intestine 64
bile acids 70
fermentation 54
genus 54 y
man(faeces) 24
man(intestine) 24 Yeasts
man(saliva) 58 antibiotictherapy 56
man(stomach) 24 cultured milk 130
V. a/ca/escens 54 gastricjuice(man) 32
V. parvu/a 54 man (intestinal flora) 24, 32, 60,64
Vibrio cholerae salivarysamples(man) 56
antibiotictreatment 79 stomach (man) 24
antitoxine 5, 82

266
Subject Index
Pagenumbers in fat indicate figures; those followed by a T signify a table.

A Antibiotic (Continued)
sensitivity 31, 71,110
Abscesses, various 65 Antibiotics 31
Aceticacid 35,51 ampicillin 31
N-acetylglucosamine-containing saccharides 4, cepha1exin 31
11,12,45-47,67,82,45T ch1oramphenicol 31
Achtorhydria 64,69, 75 ch1ortetracyclin 31
Acid clindamycin 31,7 5
degree (bifidobacteria) 38-39 cyclacillin 31
organisms,ingested 75 erythromycin 31
production (bifidobacteria) 12, 38, 48, I 08, hetacillin 31
109,125 kanamycin 151
tolerance (bifidobacteria) 32 lincomycin 31,75
Acidophilus products 87,92 neomycin 31,97, 100,151
Acidosis 90 novobiocin 31
AD P (adenosine diphosphate) 35, 36, 38-40 oxytetracycline 31
Advertising paromomycin 31, 151
preparations, bifidobacteria 126, 139 penicillin 28,31
Alimentary tract (see Gastrointestinal tract and spiramycin 31
Gastrointestinal flora) Streptomycin 31,79
Allochthonous microorganisms 62, 78, 79,91, tetracycline 31, 75
94 Antibiotictherapy
7-alpha-dehydroxylation, bile acids 74 infants 28, 29
Aunines 27,69,74,80,83,94,96 microbial balance 80
Auninoacids 51,68, 69,74 overgrowth 28, 87
cell wall 13 resistant strains 87
cultured milk 88 side-effects 81
metabolism 83 Antibodies 76-78
nutrition II, III gutflora 76
production 12 immunofluorescent 15
Aunino oxidases 69 response 77
Aunmonia 68,69, 74,80,96-97, 100 starter bacteria 79, 82, 90,94-95
Anaerobe technique Antigen 76-78
Hungatetechnique 144 microorganisms, source, anima1s 78
simplified 153-154 starter bacteria, source 91
traditional 158 Antigenie
Anaerobicjar 155 composition 15
Anaerobicrequirements II determinants 30
oxydation-reductionpotential II, 146, 147T properties 15
oxygen sensitivity II, I 09 Antigenicity 15
Animals ( seealso Faeces) Antistaphylococcus factor 4, 82
alimentary tract 7, 8, 26, 52, 53 Antitoxins 82
Antagonistic effect 27-30, 87, II 0 (seealso In- API, identification system 158
hibitory substances) Artificially fed infants 42, 43, 83, 86 (see Bott1e-
acetic acid 81 fed)
bacteria, differents 27-28 ATP(adenosinetriphosphate) 35, 37, 38,40
buffer,presence 30 Autochthonous microorganisms 62,78
enteric infection 27 Azo-dyes 69-70
organic acids 30, 81 Azoreductase 94
Antibiotic
resistance factor 71

267
 B Breast-fed ( seealso Milk, human) (Continued)
faeces
Baby foods (see Preparations) acid 81
Bacteriocins 79 enzymes 41
Bacteriophages 32-34,34 saccharides 46
calfrumen 33 feeding 81,86-88
schematic drawing 33 growth promotion-factors 50, 83
Balance, analytical 154 history 3-4T
Bibliography, bitidobacteria 195-254 intestinal tract 22-24, 23T, 25T
Bitidogenic 134 ( seealso Growth-promoting intestine, !arge 56
factors) lactulose 45,83
compounds 42-43 protective factors 82
diet 83 Buffering capacity, milk I 04
factors 43-50 Buttermilk, cultured 7, 88, 92
Bitidus factor I 4, 45-48, 45T
Bitidus factor 2 2, 4, 7, 48, 48T
Bitidus milk (seealso Fermented milk and Pre- c
parations) 97,98
Bile acids 31-32,70,74,80 Calcium
7-alpha-dehydroxylation 74 food groups 93
enterohepatic cycle 70 infants 88
inhibitoryeffect 32 Cancer,colon 74
Bilesalts 30,32,64, 70, 73,74 clostridia 54
Bilirubin 70 colonic,pH,high 74
Bladderinfection 69 dietarytibre 74
Blind-loop syndrome 64 diets,highfat 74
Biotypes 7, 12,16,22-25,41,94 faecal enzymes 94
distribution 24, 25T toxic metabolites 74, 80
identitication 158 Cancerous 80
selective growth media 152 Carbohydrate
taxonomy 16 fermentation 12,35
Bottled-fed (see also Preparations, humanized bitidum pathway 36
and cow's milk) heterofermentative pathway 38
culture B. bitidum 86 homofermentative pathway 37
dextrin-cystin, supplement 43 Meyerhof-Emden pathway 39T
dextrin-maltose, supplement 42 species 19T
faecalflora 23,24,48,75,23,25 Carbon dioxyde 12,21, 35, 66, 68,96
faeces Carcinogens 69, 74,94
buffer 30 Caries, dental 25
enzymes 41 Catalase II, 12
saccharides 46 Cellwall
feeding 88, 86-87 N-acetylglucosamine containing sacchari-
growth-promoting factors 83, 86 des 45
history 3-5T amino acids 13
intestinal tract 23, 24, 23T, 25T composition 14T
lactulose 43,45,83,86,87 fattyacids 14,15
muramidase 50 peptidoglycan 8, 13, 14
pantethine, diet 49 phospholipid 14, 15
Breast-fed ( seealso Milk, human) species 13, 14
antagonisticeffect 27, 28,30 Cellulolitic bacteria 53
bacterioides 53 Cellulose 66,67, 72
bitidobacteria 3, 52, 81,86 diet 73,74
birth, after 22 Characterization
buffer 30 techniques 144-158
digestion, casein 49 Chenodeoxycholic acid 70
enteropathogenic E. coli 82 Cholesterol 31, 70, 74,80
faecalflora 22-24,58,75,22,23,25 enterohepatic cycle 70

268
Cholicacid 70 Diarrhoea 66, 87, 94, 141, 142
Choline 69 carrot extract 49
Cirrhosis (see Li ver cirrhosis) Diazo compounds 69
Classification, bifidobacteria Diazo linkage 70
(seealso Taxonomy and Identification) Diet
schemes 16, 17 faecalflora 72-74
Colon 32,45, 74 intestinal flora 32,72-7 4
Colonic bacteria 68, 70 Diet, composition
Colonicflora 66, 73,74 bifidogenic 83
Colonies carbohydrate, high 72
agar plate I 0 cellulose, addition 73
reading 155-156 dietary fibre, high 74
Colorectal cancer 74 egg,indican 73
Colostrum, human 45 fat, high 32, 72, 73,96
Coprostanol 70, 80 japonese type 72
Cow's milk (see Milk) meat 72, 73,74
Crohn's disease 64 mixed "westem type" 72
Culture media 166-190 lactulose 72, 73, 83, 85, 86
colonies,reading 149-150, 149T protein, high 72
commercial, dehydrated 153, 185-190 vegetarian 72
complex media II, 166-178 vetable 73,74
differents media 183-185 yoghourt 94
full synthetic media II, 152, 179-182 Dietary adjuncts 86
minimal medium 152 disaccharides 65
Optimalmedia 148,149, 148T gastric acidity 32, 81, 93,94
selectivemedia 150-151,176-179 Iactose 66
selective substances 151 T polysaccharides 65
semi-synthetic media II, 152, 179-181 Dietaryfibre 73,74
use 153-155 Dietary supplement
Cultured milk products (see Fermented milk elderly 88,93
products) Digestion
Cultures, bifidobacteria (seealso Manufacture) casein 49
B. bifidum, diet 86 gutflora 73,79-80
freeze dried 28, 86, I 0 I metabolic products 80
supply sources 191-194 · nutrition 79-80
Culturing techniques 144-158 proteins 68
Cysteine II, 146, 147T, 148T transit, fermentedmilk 32
Cystine II, 43 Dilution solutions 190
Dirnethylamine 69
Diseases, degenerative 74,80
D Diverticulosis
colon 74
Dairy preparations (see Preparations) small intestine 64
Deamination 68, 96 Duodenal secretion 70, 83
Decarboxylases 68, 69 Duodenum 32, 60,70
Deconjugate bitte salts 70, 79
Dehydratedculturemedia 153,185-190
Dehydroxylation, colon 70 E
Dental caries 25
pyrrhoae 65 Echoviruses 62
Deoxycholic acid 32, 70, 80 Ecological relationships, bifidobacteria 22-32
Deoxyribonucleic acid (DNA) 71,94 animals, occurrence 26, 53, 26, 4T
basecomposition 15,16 biotypes 16, 25,94
extrachromosal 71 carbohydratefermentation 12
DNA-DNAhomology 15,18 dental caries 25
homology 15, 18-20 distribution 25
taxonomy 15 human 24, 25, 25T

269
Ecological relationships, bifidobacteria Faecal flora (Continued)
(Continued) rats 26
human, occurrence 22-25, 22, 23, 24T, 25T swine 26
intestinal tract 22-25 human
mouth 25,52 adult 24,51-58,62,72-74,9
vagina 25, 52 bacteriophage 32
Elderlypeople 95 bottle-fed 23, 24, 82, 83, 86, 95, 23T, 25T
Encephalopathy, hepatic, Iactulose 45 breast-fed 22-25, 52, 53, 83, 8, 22, 58,
Enpac 95 23T,25T
Enteric children 57
infections 28, 86,88 old people 24
viruses 27 weanling 23,58
Enteritis, regional 64 Faeces
Enterococine 79 biles acids 32
Enterocolitis 56, 87 buffer 30
Enterohepatic cycle 70 enzymes 94
Enteropathogenic E. coli 27, 82, 87 Iysozyme
Enterotoxine 64 breast-fed 83,84
Enteroviruses 62 excretion 85
Enzymes 39-41 Fat, diet 32, 43, 72, 73,96
alpha-galactosidase 41 Fattyacids 14, 15,43,54, 73,79,82
beta-galactosidase 41, 66, 97 Feeding (see Preparations)
dextranase 12 Fermentation (seeCarbohydrate fermentation)
fructose I ,6 diphosphate aldolase 39 Fermented milk products (seealso Manufactu-
fructose 6-phosphate phosphoketolase 40 re)
glucose 6-phosphate dehydrogenase 40 acidophilus products 87, 92
isomaltodextrinase 12 advertising 126
Iactate dehydrogenase 41 babies 88
muramidase (see Lysozyme) bifidobacteria, used 90T
neuramidase 45 buttermilk 7, 88,92
6-phosphogluconate dehydrogenase 40 children 88
phosphofructokinase 39 countries,various 131-133
urease 12, II! cultures, S.lactis 86-87,92
Enzymes, activities, intestinal bacteria dietaryadjuncts 81,93,94
antibiotics, break-down 71 flavour 37,92
carbohydrates, glucosides, glucoronides 65- yoghourt 88, 89, 90, 92, 94, I 03
68 healthvalue 81-101
endegenouscompounds 70-71,70 amines, formation 94
nitrogencompounds 68-70 antibodies, starter bacteria 79, 82, 90,
vitamin, synthesis 71 94-95
Euga-Lein 139 antigens, starter bacteria 91
Eugalan Töpfer, forte 139 calcium 90,93
consumption 129,129T
F Iarge amounts 72
regular 94
Faecal flora digestibility 88, I 00
animals elderly persons 93
bovine rumens 26 gastric
calves 26 acidity 32
chickens 26 transit, time 91
dogs 26 gut flora, regularconsumption 93,94
horses 26 heating 42
mice 26 immunogenic 91
monkeys 26 immunological activity 79, 91, 94
ovine rumens 26 L( + )lactic acid 90
pigs 26,53 livercirrhosis, bifidus milk 100
rabbits 26 nitrogen compounds 90

270
Fermented milk products (seealso Manufactu- Gastrointestinal flora, animals 26, 26T (seealso
re)(Continued) Faecal flora, animals)
nutritional significance 91-93 Gastrointestinal flora, human 51-80
starterorganisms 79, 82, 89, 90, 91,94-95, abnormal 63-64
125-129 age groups, differents 56-68
vitamins 92 allochthonaus 62, 78, 79, 91,94
lactic acid bacteria 90T autochthonaus 62,78
manufacture and technology (see Manufactu- biodynamicbalance 63, 80
re) characteristics, gut microorganisms 51-56
milk powders 86 disribution species, biotypes 25T
preparations 131-133, 138 colon 22
Biogarde 91, 132 faeces (see Faecal flora)
Biokys 132 ileum
dried 131 distal 60
Progurt 132 lower 60
set 131 intestinal, motility 65
stirred 102 intestine 22-26, 51-56
sweet 131 !arge 25, 28, 30, 42, 44, 50-56, 58-64,
various 133 71-73,80,81, 24T, 25T
supplements, calcium, iron 93 small 44, 50, 51, 52, 54,58-64,71-73,75,
Filant,culture 130(seealso Mucoid strains) 87,97,24T,25T
Flatulence 66 metabolic activities 65-71
Flavour antibiotics, inactivated 71
bifidobacteria, specific 92 carbohydrates 65-68
Iabaratory 107 nitrogen compounds 68-70
Food vitamin, synthesis 71
baby 88,102,131 (seealsoPreparations) microbial balance 58
carbohydrates 66T, 67T microecology
colours 69-70 adults 56-65
fermented 133, 138(seealsoFermentedmilk infants 57, 61,63
products) mouth 8, 25, 26,52-54
groups 93, 93 overgrowth 28, 63, 64, 28T, 29T
poisoning 55,56 peristalsis 61,63, 79,81,97
Formula feeds 28, 86,88 (seealso Preparations, regions, different 58-62
cow's milk modified) saliva 58
Freeze dried culture 28, 86, I 0 I, 115 spores, transit 55
stomach 32, 50, 52, 64, 75, 80, 24T
G vitamin, synthesis 71
Gastrointestinal tract 59
beta-Galactosidase 65,66 Genus, Bifidobacterium 51-52, !57, 18T, 157T
Gastric Germ-free animals (seeGnotobiotics)
achlorhydria 63, 69,75 Glucose 6-phosphate dehydrogenase 40
acid, barrier 64,75 Glucosidases 65
acidity 24, 50, 58,65 beta-Glucosidase 65
sensitivity 32 beta-Glucuronidase 65
function Glucuronides 65,68
disorders 58 Glycine 70
disturbances 63 Glycopeptides 49
hypochlorhydria 75 Glycopolypeptide 49
juice 24 Glycoproteins 49
germicidal effect 32,59 Glycosides 65
survival, Serratia marcescens 60 Gnotobiotics
mucin 46, 47, 83 chicks, nitrogen 73
transit 91 defencesystem 76,79
gut flora 62,76
Gastro-enterocolic fistulae 64 Gnotophoric, associated 76
Gastro-colic fistulae 64

271
Gram stain 10, 30, 156 Human regions, bifidobacteria(Continued)
Gram negative bacteria 81, 82 skin 25
Gram positive bacteria 42, 72, 82, 97 softtissue 26, 52,65
Growth 11 teeth 25
human milk, absence 15 vagina 22, 25,26
inhibition, cow's milk 43 Humanized milk (see Preparations)
promoting, milk cu1ture 106, 115 Hungatetechnique 144
organicacids 81,85 Hydrochloricacid 58
requirements 11-12 Hypochlorhydria 75
temperature 11 Hypogammaglobulinaemia 64
Growth-promoting factors 43-50,82-83
cow's milk composition, modification 42-43
human-milk, intrinsic properties 50 I
specific growth-promoting 43-50
N-acety1g1ucosamine-containing sacchari- Identification 21, 157-158 (seealso Taxono-
des (bifidus factor 1) 4, 45-47, 45T my)
carrot-rot 49 biotypes 158
glycopeptides 49 definitive 21
glycoproteins 49 fermentation products 21
maize extract 50 genus 157,157T
muramidase 50(seealso Lysozyme) species 19,21, 158,20T
Iactite 44 tentative 21
Iactulite 44 Immunofluorescent antiborlies 15
lactulose (see also Lactulose) 43-45,44 Immunogenic 91
pantetheine 49 Immunoglobulins 77-78,95
peptides, enzymatic proteinshydro Iysis IgA 77,79,94
(bifidus factor 2) 4, 48, 48T IgD 77,78
potato extrat 49 IgE 77,78
Guinea pigs 26, 53 IgG 77,95
Gutflora 64,79-80,98 (seealso Gastrointesti- IgM 77,78
nal flora, human) Immunological activity
family, genus, species, biotypes 51-56 activity 62, 72
system(s) 64,79
Immunosystem 94
H intestinal flora 65
resistance factors 50, 51
Haemorrhoids 74 Incubation,fermentation 118,124
Health value 81-101 (seealso Fermented milk, Indican 73,74
health value) Indicanuria 74
Hepatic Indole 12,69, 74,80
coma 96 Infant food 84 (seealso Preparations)
disease 96 Inhibitory substances (see also Antagonistic ef-
encephalopathy 45,96 fect)
Heterofermentative pathway 38 acidolin 79
Historicalsurvey 3-7,3T-5T acidophilin 79
first period 5-6 antistaphylococcus factor 4, 82
second period 6-7 bacteriocins 79
taxonomy 16T bile salts, deconjugated 30
Holoxenic, classic 76 colicins 79
Homofermentativepathway 37 enterocines 79
Homogenization,milk 106,105 lactocidin 79
Host 24,51,62,91 microcins 79
Human milk (see Milk) organicacids 30
Human regions, bifidobacteria Inoculation, fermentation 120, 120T
gastrointestinal tract (see Gastrointestinal Intestinal
tract) bacteria ( see Faecal flora and Gastrointesti-
mouth 25,26 nal flora, human)

272
Intestinal (Continued) Livercirrhosis SO
biliary excretions 73 bifidus milk 99
motility 5S,61,64,65 detoxification function SO
mucus 73 gutflora 64
mucusal cells 73 treatment, lactulose 44, 45,97
perforation 65 Li ver diseases 96-1 00
peristalsis 59,63, 79,SI,97 Lymphocytes 76, S2
structure, effect 51 B-lymphocytes 76
Intestine (seeGastrointestinal tract) T-lymphocytes 76
Iron 93 Lysozyme(muramidase)milk S2
Irradiation, X-rays 75 cow's milk 50
Isolation techniques 144-15S faeces S3,84
technique Tissier 144 formulafeeds SS
Isomaltodextrinase 12 growth-promoting 50
mechanism, excretion 85
peros S3
K preparations 50, SS
production 30
Kefir 92, 133
Key, differentiation
Actynomycetaceae 157T M
genus Bifidobacterium IST, 157T
species 19T, 20T Maize extract 50
Kinetics, product formation 122-123 Malnutrition, small intestine 75
Koumiss 92 Manufacture, bifidobacteria containing
milkproducts 102-133
cooling, after incubation 123
L cow'smilk,raw 102-103
fatty acid, content 43
Lactate dehydrogenase 41 recombined 104
L( + )-lacticacid 21,35,41, 90,95, 90T cultures I OS-117
Lactic acid bacteria 90T asepticpreparations 116
Lactite 44 bifidobacteria used 91, 90T
Lactocidin 79 biotechnogram 109-110
Lactoferrin S2, SS bulk starter 114-116, IlST
Lactoperoxidase S2 concentrated deep frozen 120, 119
Lactose 42, S6 dietetic-therapeutic criteria II 0-111
Lactose intolerant 74, 92 filant 109
Lactulite 44 photomicrographes 112, 113, 114, 117
Lactulose survival, different pH 111 T
acidification,colon 74 technological criteria I08-109
adults 44 acid production lOS, 109, 108, 115, 123
bifidogenic 43 after-acidification 109, 125,127,128
diet 72, 73, S3, S5, S6 dairy treatment, milk I03-107
faeces,pH 44 bufferingcapacity 104
freeze-dried culture S7 flavours, sterile I 07
growthfactor 43-45 growth-promotingsubstances 49,106,115
gut bacteria 9S, 98 heating 42
hydrogenation products 44 homogenization I 06
hyperammonaemia 44 milkprocessingplant 106
liverdiseases 96-100 defects, bifidus milk products 129-130
toxic nitrogenaus substances 98 equipments 130
Large intestine (see Gastrointestinal tract) fermentation
Lecithin 69 acidification, fast and slow 121
L-isoleucine, production 12 incubation 92, 121,124
Lithocholic acid 32, 70, SO inoculation 92, 120, 120T
Liver 6S,69, 74 productformation 122-123, 122T

273
Manufacture, bifidobacteria containing Milk (Continued)
(Continued) oligosaccharides 4, 42,46
flavours 92 protective
foodcolours 69-70 effect 27
hygienic production 125 factors 82
mechanical treatment, gel 123-124 protein composition 89
packaging 124 sialic acid-containing oligosaccharides 4,
pump, metering 125 82
steps, model processing 103, 118, IlST, 130T whey 43
storage humanized milk (see Preparations)
ability 124-125 sugarTöpfer 140
afteracidification 125-129,127,128 Morphology
survivalrate 125,126, 128T N-acetylamino-sugar 9
decimal reduction 127, 127T amino-acids I 0
technological advantages I 02 Ca-ions 9
vitamin, manufacture 92 colonies (seeColonies)
Media (seeCulture media) culture media 9
Medium, fatty acid 14 human milk, absence 15
Megaloblasticanaemia 64 peptidoglycan 9
Metabolie activities pleomorphisme 9
intestinal flora 65-71 Mouth 25, 52-54
Metabolites, toxic 80 Mucin, gastric
Methaemoglobinaemia 85, 86 bifidus factor, isolation 46,47
Microecology (seeGastrointestinal flora) infants, low-birth-weight 83
Micrococins 79 Mucoidstrains 13, 109, 13T
Microscopic examination 156-157 Muramidase (see Lysozyme)
appearance 21 Murein 8, 9, 13, 14
infantstool 156,134
Milk
cow's N
chemical composition 136T
fatty acid 43 Neuramidase 45,46
muramidase 50 Nitrate reduction 12, 69, 85, 86
protein composition 89 Nitrite 69, 85,86
recombined 104 Nitrogencompounds 68-70
human II Nitroreductase 94
absencehuman milk 15 Nitrosamines, degradation, synthesis 69
N -acetylglucosamine-containing saccha- Nitrosate, sec. amines 69
rides 45 Nitrosating agent 69
antibodies 4, 82 Nutrition
antistaphylococcus factor 4 animals 101
antitoxins 4 bacteria 11-12
chemical composition 136T human
colostrum 45 adu1ts 91-95
cystine, growth-promoting 43 digestion 79-80
faeces,pH 43 infant 81-90 ( seealso Preparations)
fatty acids, essential 43 malnutrition 75
dog,fed 26 nutritional status, tropical residents 61
glycopeptides 49
glycopolypeptides 49
growth-factors 15, 43, 49,50 0
lactoferrin 4
Iactose 42 Occurrence, bifidobacteria
Iactose-N -tetraose, split 41 animals 26
!arge intestine, pH 4T humans
Iysozyme (muramidase) 4, 50 intestine 22-25, 28, 63, 64, 87, 22, 23, 28T,
mucin 46 29T

274
Occurrence, bifidobacteria (Continued) cow's milk modified 27, 28, 43, 86-87
mouth 25 beta-ethyl-N-acetyl-0-glucosamine 47
newborn 25 calcium 88
starnach 63, 64, 24T casein, hydrolysed 48
vagina 25 chemical composition 43
Oldpeople faecal flora 82
faecal flora 24 feeding 88
gastric acidity 58 formula, adaptation 88
Oligosaccharides 42, 46,47, 66, 81, 82 growth-promoting factors 82
Omniflora 140 iron, content 88
Organicacids 30 L. acidophilus 87
Osteoporosis 93 lactulose 83,85
Overgrowth, bacterial Iysozyme (muramidase) 50, 88
intestine 28, 62, 63, 64, 80,87, 28T, 29T mucin 47
starnach 63,64 nitrogen retention 83
Oxidase II vitamin B, excretion 85
Oxidation-reduction potential II, 146, 147T water, reconstitution 86
Oxido-reduction 70 humanized milk preparations 134-13 5
Oxygensensitivity,strains II, 109 bifidogeniceffect 137T
chemical composition 136T
commercial products
Eiedon 86
p Eugalan forte 98
Lactana-8. 86, 137
Pantethine 49 Petargon 86
Pathogenicity dietary adjuncts
bifidobacteria 26-27, 65, 7 5 bifidobacteria with 81, 94, 136-138
enteropathogenic E. coli 27 fresh preparations
enterotoxin, E. coli 27 manufacture, scheme 135
human soft tissue 26, 52, 65 partly humanized 135-136
pathogens, intestinal 56, 64, 79 protein composition 89
Peptidoglycan 9, 13,14 pharmaceutical 138-143
Peristalsis, intestinal 59,61, 63, 79,81, 97 animaluse
Peritonitis 65 KorolacB 142
Pernicious anaemia 63-64 KorolacD 142
Pharmaceutical preparations (see Preparations, human use 138-143
pharmaceutical) control 88
Phenol(s) 69, 74, 80,97,98 Euga-Lein 138-143
Phospholipids 14, 15 Eugalan, Töpferforte 139
Piperidine 69 lnfloran Berna 138, 139
Pleomorphism 9 Lactopriv 140
Pleural fluid, human 65 Milksugar"Töpfer" 140
Pleuropulmonary spaces 65 Omniflora 140
Poisoning, food 55,56 therapeutic (see Preparations, pharmaceuti-
Polysaccharide, production cal)
capsular 13T Processing (see Manufacture)
non-encapsulated 13T Prophylactic value (seealso Protective effect,
Preparations factors, role)
dairy (see Fermented milk products) acidosis,metabolic 90
acidophilus product 87 B. bifidum culture 87
buttermilk 27, 28 B. bifidum, piglets 143
fresh preparations 138 intestinal disturbances, animals I 0 I
formula feeds 86,87 ( seealso Preparations, Korolac, B, D, piglets 142
cow's milk modified) Protective
infantfood effect,gutmicroflora 27
adapted milk (see Preparations, humani- factors, newborn 82
zedmilk) mechanisms

275
Protective mechanisms (Continued) Starter (see Manufacture, cultures and
hast 64 Fermented milk products)
newborn 82 Steatorrhea 64
roJe Steroids, neutral 73,74
enteric infections 86 Starnach 63, 64, 24T
dyspepticconditions 87 acidity 59, 69,82
fatty acids 82 Stoa!
gut flora, resistance 27 adults 58
lactoperoxidase 82 faecal flora (see Faecal flora)
Proteins, hydrolysis pH 23,42
papain 48 sample collection 144-145
pepsin 48 Strains
trpysin 48 antibiotic resistant 87
Proteolytic activity 12, 87,90 characteristics 26
Putrefactive organisms 6, 23, 24,27 defined 32
acidity increased 83 differentiation
Pyrrolidine 69 animal 26, 26T
Pyruvate 69 human 18, 20,21, 26,67
electrophoretic mobilities 40T
mucoid 13, 109
Q pathogenic 26
rumen 12
Quark, bifidobacteria 133 serologically different 15
supply sources 191-194
Sulphonamides 69
R Survival rate, fermentedmilk 125-127, 127T,
128T
Reducing substances, culture media 146, Synthesize, B-groups vitamins 71
146T
Resistance-factors (R-factors) 71
Rumens T
calf, bacteriophage 33
bovine, bifidobacteria 26 Taurin 70
ovine, bifidobacteria 26 Taxonomy 16-21 (seea!so Identification)
Ruminants biotypes, groups 16, 17T
faeces 53,54 classification
rumen 79 genus IST, 157T
schemes 16
species 19T, 20T
s fermented reactions 19T
historical survey 16T
Saccharides, intestinal 46 Technology, dairy (see Manufacture)
Saliva 58 Therapeutic applications, animal
Samplecollection 144-145 intestinal disturbances I 0 I, 142-143
anaerobe technique 145 anti-scouring
Septicabortion 65 calves 101
Serologycally, different, strains 15 dogs 101,142
Sialic acid 46, 49 pigs 101
Sialic acid containing saccharides 4, 82 swine 101, 142
Skin, human 25 diarrhoea
Sodium deoxycholate 71, II 0 calves 142
Softtissue, infections 26, 27, 52,65 canine 142
Spezies, bifidobacteria 19,21, 158, 19T, 20T dogs 142
Spores, gastrointestinal tract 55 piglets 142
Staining procedures 156-157 enteric disturbances
Gram 156 calves 101
Loeffier's methylene blue !56 digs 101

276
Therapeutic applications, animal
intestinal disturbances (Continued)
pigs 101
gastrointestinal disturbances I 0 I
calves 143
dogs 143
pigs 101
swine 143
Therapeutic applications, human
gastrointestinal disturbances 95-96
colibacillose 141
constipation, chronic
Euga-Lein 139
Eugalan Töpferforte 139
supporting therapy, Milksugar
Töpfer 140
diarrhoea 141
dieteticregimen 95
digestive disturbances
elderlypeople 95
Infloran Berna 138
disordered flora 5T
MilksugarTöpfer 140
disturbed balance, flora
antibiotictherapy 5T, 142
antibiotic-resistant strains 87
dyseptic disturbances 87
enterocolitis, lnfloran Berna 141
EugalanTöpferforte 139
intestinal disturbances 95
lactobacilli preparation 65
Lactopriv 140
side-effects, lactobacilli 65
dyspepsia, bifidus milk 95
irradiation therapy 5T, 95
Eugalan Töpferforte 139
Lactopriv 140
regeneration,gutflora 31,64,95,134
enteric infections
infants 27,141
small intestine 88
enterocolitis
acute, Infloran Berna 138
chronic, Infloran Berna 138
milk (Iactose) intolerance
cultured milk 92
Lactopriv 140
peristalsis, intestinal
MilksugarTöpfer 140
premature inants, septicaemia 87
putrefactive organisms
MilksugarTöpfer 140
supplementary treatment
diarrhoea, acute 87
entericinfections 87
enterocolitis, infants 87
re-establishment, flora 64
Therapeutic applications, human
supplementary treatment (Continued)
hepatic diseases ( seealso Lactulose)
chronic Iiver disease 5T, 96-1 00
cirrhosis 97-100
Euga1anTöpferforte 139
lactulose
effect 44
management 45
portal encephalopathy 45, 96-100
Therapeuticconcentrations 95
B. bifidum, minimum 128
culturedmilk 127
dietetic use 125
Lb. acidophilus, preparations 95
Therapeutic criteria, strain selection 11 0
Therapeutic preparations (seeTherapeutic app-
lications and Preparations, pharmaceutical)
Tissier, dissertation 3
u
UHTtreatment, milk I 07
Ulcerative colitis 74
Urea 68,74
Urease 12,68,111
Urealysis 68
Ureolytic organisms 53,97
Urinary excretion
excretion Vit. 86 85
tract, infection 65
Urobilinogen(s) 71,80
V
Vagina 25
Virus
enteric 27
enterovirus (echoviruses) 62
rotavirus, colostrum 82
serotypes 62
Vitamins
biologic enrichment 92
manufacture, cultured milk 92
ruminants 80
synthesis 50,71, 80, 85,92
w
Water, high nitrate 86
Weaning, infant 23, 58, 81
Weanling
infant 58
piglet 94
277
Weighing, analytical balance 154 y
Whey
humanizedmilk 43,135 Yakult, cultured milk 90
whey protein, addition I 05 Yeastautolysate 38,48,167
Women Yoghourt 88, 90, 92, I 03
lacting 88 growth stimulant 89
pregnant 88 heat-treated 94

X z
X-rays, therapy 64,7 5 Zyma, Lb. acidophilus preparation 95
Xylan, utilisation 66

278
 This book teils you
how to apply
biological knowledge
in milk processing
 JUNE

1
MONDAY

JUNE

~~--------~~--------r1
JUNE

CHR. HANSEN'S LABORATORIUM A/S

3, SanktAnnae Plads · DK-1250 Copenhagen K · Denmark · Phone: (01) 142888 ·Telex: 19184 hansendk
Cultures
with more technical facilities for you
and with more dietetic benefits
for your customer. Lactic acid L (+)

Our diverse MSK cultures will satisfy your ideas

of a mild lactic product.
MSK or MSK/R cultures for yoghurt and
cultured milk.
MSKN culture for yoghurt with more viscosity.
MSK/Q culture for quark or curdled milk in
thermo technique.

Make use of our experience, please call.

Laboratorium Wiesby GmbH & Co.

D-2260 Niebüll, P. 0. Box 1366, Phone (04661) 715, Telex 221411
• Place sample on pan -
get results within 3 seconds
with the sing/e contro/ bar
• Reach target weight
quickly and dependably
with the high speed readout
• Transfer results reliably
with the stability detector
• Assure reliable results
w;th the self-calibrator
• Meet present and future needs
with the serial data output

~q,
0~
/ //
/

#'cf0-
"0 ~ /.' /

a
~0
~ ~*'"
.-s" /
//
Elec1roNC balances and wetghong systems Thermoanatytocalonstruments ~
o<~,.~,§:''~ /
~
/
/
Autornahe trtratoon systoms Laboratorv automatoon ß~ J>.., /
Mettier Instrumente AG, CH-8606 Grelfensee, Switzer1and, Telex 54592
Mettier-waagen GmbH, Postfach 110840. 0-6300 Glossen 2 ,p~
~~l~ /
Mettier Instrumenten B V, Postbus 68, Amhem, Holland ,jl ,s.'\ ~ / rfb
Mettier Instrument Cofl)Ot8toon. Hoghtstown. NJ 08520, USA .1' .§'"' ..,<r .~><" if
Solranae SA. F-92300 LevaiiOos-PerreL France <tY ~ vo- ~ .._qJ. 6338.n
~ßuH!NFORMA770N ~dmfo

~
'
''

BIOgarde culture BIOgarde product

(Sc. thermophilus, (Sc. thermophilus,
Lb. acidophilus, Lb. acidophilus,
Lb. bifidus) Lb. bifidus)

At what culturallevel
is your manufacture of dairy
products?
Benefit from the advantages of BIOgarde special cultures. They areideal for the
manufacture of sour-milk products and various cheeses .
BIOghurt/BIOgarde dairy products are characterized by good flavor. By negli-
gible after-acidulation. By prolonged durability.
By rising sales due to optimal quality.
Adapt your production to the latest state of dairy research.
Our service: free advice on application procedures and technology
for our extensive program

BIOghurt/BIOgarde companies in various countries

Germany
Deutsche BIOghurt GmbH, Kaiserplatz 2, 8000 München 40 (West-Germany)
France, Belgique, Luxembourg
STENVAL-BELGIQUE, Avenue Lloyd George 6, Bte 7, B-1050 Bruxelles
Southamerica-Panama
BIOgarde AMERICA LATINA, Casilla de Correo 2846, 1000 Buenos Aires/Argentina
Other European countries and overseas
- evog- Rheinstrasse 640, FL-9496 Baizers (Liechtenstein)

~ßuH!NFORMA770N ~dmfo
lncubators for Yoghurt and other Fermented Milk Produkts
excellently designed with a high performance

For details please contact

Joeler A.G. Apparatebau, CH-9213 Hauptwil
Switzerland, Telex 719 261 Jorg CH

TheB&L
Metering
Plant
Pumps. Blends. Meters. Controls.
Continuously.
With high accuracy.

BRAN&LDBB
P. 0. Box 13 60
2000 Norderstedt 1
Western Germany
~ (0 40) 52 20 21
 HAMBA-BK-6005-UV-C

HAMBA-BK-6005-UV-C with piston valve

HAMBA Cupfillers in UV-C-execution fillers for connection to a C.I.P. cleaning
give a Ionger shelf-life for sour and system for filling 3 products in layers.
sweet products. Cupsand Iids are Many variations are possible with the

dffl MBA
degerminated by UV-C high intensity BK-system. BK-machines in UV-C-exe-
lamps. Re-infection is prevented by cution can be delivered with capacities
pressurised sterile zone. from 4.000 up to 36.000 cups/hour.

HAMBA-Maschinenfabrik
Buchenhofener Straße 49 ·Postfach 144180
D-5600 Wuppertai11-Vohwinkel· Telefon 0202fi40030
8 591243 . West-Germany
A
Ready for use
Partly or fully automated plants for
reception station, production hall, milk
storage (raw and prepared milk),
fermented milk production or cleaning
stations - for this range of application
H&K solve your problems in the best
manner possible - and ready for use.
We offer all components:
- equipment and tanks (e.g. filter plants,
heat exchangers)
- as weil as the whole range of
tube joints.
We give solution from planning
and design to installation including
after-sales service.

- control valves (of the proven

ROSISTA-DELTA-programme) There are only a very Iew affering such
comprehensive service.
- process automation 6PPlY for further information.

Holstein und Kapperl GmbH ® Unternehmensbereich Unna <it

Zechenstr. 49 · 4 750 Unna-Königsborn (0 23 03) 1 08-1 Tx.: 08 229 24 7 ras d
You can benefit from the experience of the
authors
and
from that of the other members of the study
group as concerns practical application
procedures, consulation, etc. in cultured fresh
milk products manufacturing and development
of new milk-based products.

Consultation by correspondence
Consultation by contract
Consultation on location in
your company

Office:
International Study Group of
Fermented Fresh Milk Product Research
P.O. Box 90,
CH-1700 Fribourg 7, Switzerland
Telephone (0 37) 2813 62
and
YU-11000 Belgrade,
Vucedolska 14/1,. Yugoslavia.
Who delivers yoghurt plants
to the Balkans?
Alfa-Laval, of course, owing to our

0 Process know-how
0 Process-oriented plant engineering
0 Process equipment matched to yoghurt
production
0 Aseptic starter production
0 World-wide experience

(X ALFA•LAVAL
DAIRV & FOOD ENGINEERING

Box 1008, S-221 03 Lund, Sweden

Tel. int + 4646105000
TÖPFER-Bifidus-Preparations
EUGALAN Töpfer forte
This is used in the treatment of cirrhosis of the liver and for
portal encephalopathy.
EUGALAN Töpfer
This is used to establish an eubiotic intestinal flora.
Treatment of gastrointestinal-, bile-, and liver disturbances, as
weil as prolonged constipation.
EUGA-LEIN with Linseed
This is used to normalize the intestinal flora and to eliminate
chronic constipation. - Particularly low in calories.
EU810FLOR Töpfer
This combination of wheat bran, rich in dietary fibre,
with dried yoghurt and live bifidus bacteria, favourably
influences the function of the intestines.
Milk sugar Töpfer
Mild Iaxative with bifidus bacteria.
LACTOPRIV/8
This is used for regeneration of the intestinal flora in persons of
any age-groups who demonstrate milk intolerance. lndicated in
cases of darnage to the intestinal flora, as weil as after
radiation therapy or the administration of sulphonamides and
antibiotics.
LACTANA/8
Partially adapted baby milk food containing bifidus bacteria.
The Töpfer-bifidus-preparations contain viable bifidus bacteria
and their metabolites in a carefully dried milk base.
The Töpfer-bifidus-preparations serve the intestinal bifido-
bacteria, leading to the establishment of the natural gut flora,
and having positive effects in combating pathogenic gut
bacteria.
ln this way the Töpfer-bifidus-preparations give:
- Greater protection against enteral bacterial infections;
- A boost to the detoxification function of the liver by
lowering the contents of the toxic protein metabolites;
- A beneficial aid to the functioning of the intestines by
increased production of lactic acid in the intestines;
and normalization of the intestinal flora.
B
1. List of names of the advertisers

Company Address Communications

ALFA-LAVALAB P.O.Boxl008 Tf. 046-1 0 50 00

(Dairy & Food Engineering) S-221 03 Lund I Tgm. Alfadairy Lund
Sweden Telex32145
ALUMINIUMWERKE AG Industriestrasse 45 Tf.0711415222
RORSCHACH CH-9400 Rorschach Tgm. Aluminium Rorschach
Switzerland Telex 77 203 awrg eh
BIOGhurt/BIOgarde Company -evog- Tf.075/22129
Rheinstrasse 640 Tgm. evog, Baizers
FL-9496 Baizers
Principality of Liechtenstein
BRAN & LUEBBE P. 0. Box 1360 Tf.040/522021
(Dosier- und Mischtechnik) 2000 Norderstedt I Tgm. dosierpumpe hamburg
Western Germany Telex02174691
GIVAUDAN DÜSENDORF CH-8600 Dübendorf Tf.Ol/8204222
LTD Switzerland Tgm. Givarome Dubendorf
(Flavours forthe food industrie) Telex54356
HAMBA-Maschinenfabrik Buchenhofener Straße 49 Tf. 02 0217 4 00 30
P. 0. Box 1441 80 Tgm. Hamba-Wuppertal
D-5600 Wuppertalll- Telex 8 591 243
Vohwinkel
Western Germany
Chr. HANSEN'S 3, Sankt Annae Plads Tf. 01/1428 88
LABORATORIUM AIS D K -1250 Copenhagen K Tgm. Hansenslab, Kopen-
Denmark hagen
Telex 19184
HOLSTEIN and KAPPERT P.O.Boxl840 Tf.02303/I 08-1
GmbH Zeichenstr. 49 Telex 08 229 247 ros d
Unternehmensbereich Unna D-4750 Unna-Königsborn
Western Germany
JÖLERAG CH-9213 Hauptwil Tf. 0711813109
Apparatebau Switzerland Tgm. Jöler Hauptwil
Telex719261
LABORATORIUM WIESBY P. 0. Box 1366 Tf.046611715/6
GmbH&Co. Gotteskogstr. 40 Tgm. Laboratorium Niehüll
D-226 Niehüll Telex221411
Western Germany
METTLER INSTRUMENTE CH-8606 Greifensee Tf.9412241
AG Switzerland Tgm. mettlerbalance
Telex 54 592 mebal eh
SWISS SERUM and P. 0. Box 2707 Tf.0311344111
VACCINE INSTITUTE Rehhagstr. 79 Tgm. Serum Bern
BERNE CH-3001 Bern Telex32220
Switzerland
TOEPFERGmbH P.O. Box 1180 Tf.08374/8041
(Hersteller von pharmazeutisch- D-8961 Dietmannsried I Tgm. Lactana Dietmannsried
diätetischen Präparaten) Western Germany Telex 054 738

Legend: Tf =Telephone, Tgm = Telegram

292
2. List of equipment and machinery of the advertisers for the manufacture of
fermentedmilk products (with or without bifidobacteria)

2.1 Rawmilk Company

2.1.1 Dairy seperator Alfa-Laval AB
2.1.2 Milk standardization Alfa-Laval AB
Holstein & Kappert GmbH
2.2 Treatment of milk in the dairy
2.2.1 Heater: Plate, tubular and ultra-high-heater Holstein & Kappert GmbH
Alfa-Laval AB
2.2.2 Homogenizer Alfa-LavalAB
Holstein & Kappert GmbH
2.2.3 Evaporater Holstein & Kappert GmbH
Alfa-Laval AB
2.3 Cultures starters and equipment
2.3.1 Singlestrain cultures:
Hansen's Laboratorium A/S
Str. thermophilus, Lb. acidophilus
Laboratorium WiesbyGmbH &Co.

Str. thermophilus, Lb. acidophilus, Laboratorium Wiesby GmbH & Co.

Bifidobacterium /ongum I
2.3.2 Mixedcultures:
Yoghurt cultures Hansen's Laboratorium AIS
Laboratorium Wiesby GmbH & Co.
Str. thermophilus and Lb. acidophilus Bioghurt/Biogarde Company
Laboratorium Wiesby GmbH & Co.
Str. thermophilus, Lb. acidophilus, B.longum Laboratorium Wiesby GmbH & Co.
Str. thermophilus, Lb. acidophilus, B. bifidum Bioghurt/Biogarde Company
2.3.3 Equipment for cultivation of cultures Alfa-LavalAB
Laboratorium Wiesby GmbH & Co
Holstein & Kappert GmbH
2.4 Fermentation in incubation tanks or in retail containers
2.4.1 Process tanks for aseptic and non aseptic Alfa-Laval AB
manufacture Holstein & Kappert GmbH
2.4.2 lncubation chambers JölerAG
2.5 Cooling after incubation
2.5.1 Gel-cooler(differenttypes) Holstein & Kappert GmbH
Alfa-Laval AB
2.5.2 Cooling chambers and tunnel JölerAG
2.6 Metering pumps
Piston (plunger) pumps Bran&Lübbe
2.7 Packaging and packaging materials
2. 7 .I Filling, bottling and closing equipment Hamba Maschinenfabrik
Holstein & Kappert GmbH
2.7.2 Aluminium caps forclosing cups Aluminiumwerke AG
2.7.3 Alcan Clean Air System forpackaging machinery Aluminiumwerke AG
2.8 Process lines
Aseptic and non aseptic processing !ines Alfa-Laval AB
Holstein & Kappert GmbH
2.9 Flavours Givaudan Dübendorf AG
2.10 Laboratory equipment
Balances: analytical, micro and ultra-micro, top loading Mettier Instrumente AG

293
 Weighing systems for filling process controls Mettier Instrumente AG
Thermoanalytical instruments: Mettier Instrumente AG
thermonalyzers; automatic determination of the
melting; boiling or the dropping point
2.11 Complementary dairy equipment
Different equipment for cheese, buttermaking, Alfa-LavalAB
Ice-cream Holstein & Kappert GmbH
Packaging materials Aluminiumwerke AG
Flavours Givaudan Dübendorf AG

3. List of pharmaceutical preparations with viable bifidobacteria

3.1 Different preparations forinfantnutrition, foradults TöpferGmbH

and for different dietetic use
3.2 Lyophylized preparations forintestinal disorders for Serum and Vaccine Institut Beme
infants and adults, antibiotic treatment, chronic TöpferGmbH
constipation

294
Acknowledgments

The authors emphasize many thanks to the following companies and organizations and persons for
supporting the publication ofthis book: Alfa-Laval AB-Lund, Sweden; Aluminiumwerke AG, Ror-
schach, Switzerland; The American Type Culture Collection, Praklawn Drive, Rockville, Maryland,
USA; Bioghurt/Biogarde Company, Balzers, Principality of Liechtenstein; Bran & Luebbe, Nor-
derstedt, Western Germany; Mr. Espina, F., Valdivia, Chile; Federal Dairy Research Station, Liebe-
feld, Switzerland (Prof. Dr. H. B. Blanc, Director); Galactina AG, Belp, Switzerland; Givaudan
Dübendorf Ltd., Dübendorf, Switzerland; Hamba-Maschinenfabrik, Wuppertal-Vohwinkel, West-
ern Germany; Chr. Hansen's Laboratorium AIS, Copenhagen K, Denmark; Mr. R. Hansen, Director,
Nordeuropaeisk Mejeritidskrift, Vanlose-Copenhagen K, Denmark; Holstein & Kappert GmbH,
Unna-Königsborn, Western Germany; Dr. B. Hylmar, Dairy Research Institute, Prag, CSSR; Jöler
AG, Hauptwil, Switzerland; Prof. Dr. H. Kay, Dairy Research Institute, Kiel, Western Germany; Dr.
Martin Kludas, Berlin, Western Germany; Dr. Kobayashi, A., Nisshin Flour Milling Co., Ltd., Sai-
tama, Japon; Laboratories Gallier S. A., Paris, France; Laboratory Wiesby GmbH & Co., Niebüll,
Western Germany; Librairy of University of Fribourg, Fribourg, Switzerland; Prof. Dr. E. Lipinska,
Instytut Prezmyslu Mleczarskiego, Warszawa, Poland; Mr. E. J. Mann, Director, Commonwealth
Bureau ofDairy Science and Technology, Shinfield, Reading, England; Med. J. Carl Pflüger, Berlin,
Western Germany; Mettier Instrumente AG, Greifensee, Switzerland; Prof. Dr. J. B. Meyer,
Saarbrücken, Western Germany; Molkerei Lüneburg Hans Stamer KG, Lüneburg (Dr. A.
Röckeisen); PD Dr. H. v. Nicolai, Universität Bonn, Western Germany; Dr. T. Sozzi, Nestle S.A.,
Linor, Orbe, Switzerland; Swiss Serum and Vaccine Institute Berne, Berne, Switzerland; Töpfer
GmbH, Dietmannsried, Western Germany; Dr. N. Weiss, German Collection ofMicroorganismes,
München, W.Germany; Yakult Honsha Co. Ltd., Tokyo, Japon (Dr. Hori, Director); Dr. Y. Yosh-
ioka, Snow Brand Milk Prod. Co. Ltd., Sapporo, Japon.

Special thanks for important support to:

Centrat Association of Swiss Milk Producers (Mr. Dr. F. Hofmann, ing. agr., director and Dr. D. B.
Stüssi, vice-director), Berne, Switzerland;
Conserves S. A. Estavayer (Mr. R. Scheidegger, director, Estavayer-le-Lac, Switzerland);
Cremo S. A. (Mr. P. Reynaud, ing. agr., director), Fribourg, Switzerland;
Food Research Institute, Novi Sad, Yugoslavia;
Prof. H. Bernard Hawley, Zug, Switzerland;
Institut Agricole de !'Etat de Fribourg (Mr. P. Bourqui, ing. agr., director), Grangeneuve-Fribourg,
Switzerland;
Nestle, S. A. (Prof. Dr. J. Mauron, director), Vevey, Switzerland.

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