European Journal of Clinical Nutrition (2002) 56, Suppl 4, S2–S15

ß 2002 Nature Publishing Group All rights reserved 0954–3007/02 $25.00

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Fermented milks: a historical food with modern

applications — a review
AY Tamime1*
1
Dairy Science and Technology Consultant, Ayr, Scotland, UK

Background: This paper was presented at the symposium which was organized by Instituto Danone Mexico in May 2001, and it
provides an overview of the current scientific knowledge on fermented milks concerning the historical developments,
manufacturing stages, classification of such products, and nutritional aspects. Particular attention has been paid to the
human health benefits associated with the consumption of these products, the use of probiotic starter cultures and their
industrial applications, and the significance of using a trained sensory panel for the evaluation of probiotic fermented milks made
with different commercial blends of starter cultures. The paper also highlights the future research areas for the exploitation of
starter microflora (Lactobacillus, Bifidobacterium and Enterococcus species) in fermented milk products.
Conclusion: This review indicates that the complex metabolism of the starter cultures is well established; however, more
information is still needed on specific microbial metabolites such as polymerization of milk sugars for the production of
exopolysaccharides and the modification of the milk peptides and secretion of bacteriocins. More clinical studies are still
required to establish the ‘functional’ health benefits of probiotic fermented milks to humans.
Sponsorship: The visit to Mexico City was supported by Instituto Danone Mexico.
European Journal of Clinical Nutrition (2002) 56, Suppl 4, S2 – S15. doi:10.1038/sj.ejcn.1601657

Descriptors: review; fermented milks; starter cultures; probiotic microflora; health benefits

Introduction
Fermented milks are widely produced in many countries.
This type of process is one of the oldest methods used to
extend the shelf-life of milk, and has been practised by
human beings for thousands of years. The exact origin(s) of
the manufacture of fermented milks is difficult to establish,
but it is safe to assume that it could date to more than
10 000 y ago as the way of life of humans changed from food
gathering to food producing (Pederson, 1979). This change
also included the domestication of certain mammals such as
the cow, sheep, goat, buffalo and camel; it is most likely that
the transition occurred at different dates in different
countries. However, archaeological evidence of certain
civilizations (Sumerians, Babylonians, Pharos and Indians)
suggests that they were well advanced in agriculture and in
the production of fermented milks.
*Correspondence: AY Tamime, Dairy Science and Technology Consultant,
24 Queens Terrace, Ayr KA7 1DK, Scotland, UK.
E-mail: adnan@tamime.fsnet.co.uk
It is likely that the origin of these products was the Middle
East and the Balkans, and the evolution of fermented milks
through the ages could be attributed to the culinary skills of
the inhabitants living in these regions. Today’s fermented
milk products are manufactured in many countries, and the
stages of manufacture, which is still a complex process,
combine the art of such ancient craft and science (micro-
biology and enzymology, physics and engineering, and
chemistry and biochemistry) together.
This article summarizes the research findings on yoghurt
and probiotic fermented milk products made from cow’s
milk over the past century.
Diversity of fermented milks
Fermented milks are manufactured throughout the world,
and approximately 400 generic names are applied to tradi-
tional and industrialized products (Kurmann et al, 1992), but
in actual essence the list may only include few varieties. In
the 1980s, Kurmann (1984) attempted, in part, the classifica-
tion of fermented milks into a ‘family tree’ (see Figure 1;
Bylund, 1995), which was based primarily on the optimum

Figure 1 The family tree of fermented milk types. Adapted from Kurmann (1984).
growth requirements of the starter cultures (ie mesophilic
and thermophilic microflora).
Nevertheless, taking into account the microorganisms
that dominate the product, including their principle
metabolites, Robinson and Tamime (1990) proposed a
scheme for the classification of fermented milks as follows:

lactic fermentations — (a) mesophilic type, eg cultured
buttermilk, filmjölk, tätmjölk and långofil; (b) thermo-
philic type, eg yoghurt, Bulgarian butter-milk, zabadi,
dahi; and (c) therapeutic or probiotic type, eg acidophilus
milk, Yakult, ABT, Onka, Vifit); products within this
group constitute by far the largest number known
worldwide;

yeast – lactic fermentations (kefir, koumiss, acidophilus
yeast milk); and

mould – lactic fermentations (villi).
Tamime and Marshall (1997) have detailed the manufac-
turing stages of these types of fermentations. However, the
so-called fermented milk ‘drinks’ and=or beverages including
the carbonated products should be classified separately
rather than being known as fermented milks; such an
approach will minimize confusion among consumers. Never-
theless, some closely related products are manufactured from
fermented milks by de-wheying, and some examples are
labneh, skyr and ymer. The different methods available to
manufacture concentrated fermented milks are as follows:
Fermented milks — a review
AY Tamime
S3
(a) cloth bag or Berge system; (b) mechanical or nozzle
separators; (c) ultrafiltration (UF); and (d) product formula-
tion (Tamime & Robinson, 1999).
Patterns of consumption
Until the 1950s, production and=or consumption of yoghurt
(ie natural type) was confined to communities in the Middle
East, the Balkans, India, Eastern Europe, to ethnic groups
living in different parts of the world, and to those who
perceived that the product was beneficial to health. How-
ever, consumers’ attitudes towards yoghurt have changed,
possibly for the following reasons: (a) as refrigeration became
widespread worldwide, the product became widely distribu-
ted and readily available on the market; (b) the introduction
of a ‘new’ generation of yoghurts (eg addition of fruits and
sugar) gave the product an entirely fresh image and it
became an inexpensive snack or dessert; and (c) the advent
of incorporation of probiotic bacteria into the product have
enhanced the health benefits of fermented milks.
Consumption figures reflect the expanding markets in
some selected countries between 1970 and 1999 (Table 1).
Until the early 1990s, fermented milks were classified as
buttermilk, yoghurt and others according to the statistical
data published by the International Dairy Federation, and
such information provided consumption preferences of con-
sumers in different countries; the current approach of
European Journal of Clinical Nutrition
 Fermented milks — a review
AY Tamime
S4
Table 1 Per capita annual consumption (kg=head) of milk drinks increased from 1500 to > 91 000 tonnes between 1986 and
and fermented products including yoghurt 1990 (Renard, 1998).
Country 1975 1980 1990 1999

Australia 1.0 1.8 3.5 5.8 Definition and terminology of fermented milks
Austria 7.9 9.8 10.4 15.1
including yoghurt
Belgium 11.8 7.7 8.4 20.3
Canada 5.1 2.3 3.7 4.2 The existing legal standards or the provisional regulations of
a
Chile NR 1.4 3.9 6.7 yoghurt in many countries classify the product on the basis
Czech=Slovakia 3.9 7.3 NR 20.0 of chemical composition or fat content (full, semi-
Denmark 36.1 26.7 21.6 29.8
skimmed=medium or skimmed=low); the reviews by Robin-
Federal Germany 16.5 10.1 14.2b 25.5b
Finland 32.6 41.0 38.3 NR son and Tamime (1976), Pappas (1988), Anonymous (1989,
France 9.6 9.3 16.4 27.4 1996), FAO=WHO (1990), and Tamime and Robinson (1999)
Iceland 1.7 5.7 24.6 NR are recommended for further reading.
Israel 14.1 14.3 NR 29.3
The International Dairy Federation (IDF, 1992a,b) pub-
Italy NR 1.3 4.0 NR
Japan 2.5 2.4 7.8 NR lished general standards of identity of fermented milks that
Netherlands 24.7 27.3 32.5 NR could be briefly defined as follows: ‘Fermented milks are
Norway 9.8 10.1 14.9 19.9 prepared from milk and=or milk products (eg any one or
Poland 3.2 2.0 NR 7.9
combinations of whole, partially or fully skimmed, concen-
Spain 3.4 6.0 8.0 15.4
Sweden 24.1 23.5 29.1 30.2 trated or powdered milk, buttermilk powder, concentrated or
Switzerland 16.4 14.8 19.0 NR powdered whey, milk protein (such as whey proteins, whey
UK 1.7 2.8 4.4 NR protein concentrates, soluble milk proteins, edible casein
USA 9.9 3.1 3.5c NR
and caseinates), cream, butter or milkfat — all of which
a
NR ¼ not reported. have been manufactured from raw materials that have
b
Data includes German Democratic Republic. been at least pasteurized) by the action of specific
c
Data for 1993.
microorganisms, which results in a reduction of the pH
Data compiled from Tamime and Robinson (1999) and IDF (2000).
and coagulation. It is of interest that in some countries the
fortification of milk components with dried dairy ingredi-
ents during the manufacture, of yoghurt for example, is
per capita consumption figures covers milk drinks and fer- against the existing legal standards.
mented milks including yoghurt. It could be argued, how- Other IDF specifications include the following aspects: (a)
ever, that this classification of these products may not the starter cultures shall be viable, active and abundant
provide a clear picture or patterns of consumption. Further- ( 107 cfu=g) in the finished products at all times of sale for
more, in some countries, the consumption of buttermilk is consumption; (b) these products may or may not be homo-
not properly defined because: genized, must at least be pasteurized, contain certain per-
mitted additives (optional), and have a shelf-life up to 30

traditional=natural buttermilk is the by-product of butter
days at 4 – 7 C; (c) post-fermentation heat treatment shall
making from ripened or cultured butter;
not be allowed; and (d) de-wheying after fermentation is

cultured buttermilk is the fermentate of skimmed milk
only permitted during the manufacture of concentrated
followed by the addition of butter flakes; and
fermented milk products such as labneh, ymer, skyr and

sweet buttermilk is not fermented; hence the data for
stragisto. Furthermore, the identity of the starter cultures is
buttermilk in such statistical tables have to be assessed in
also detailed and, in the case of yoghurt, Streptococcus thermo-
a cautious manner.
philus (formerly known as S. salivarius subsp. thermophilus)
It is safe to conclude that fermented milk products that and Lactobacillus delbrueckii subsp. bulgaricus are normally
fall within the category of lactic fermentations (mesophilic used because of the associative growth that exists between
type) are widely consumed in northern European countries, these two microorganisms, whilst in some countries, Lacto-
whilst the yeast – lactic fermented milks are popular in the bacillus helveticus and Lactobacillus delbrueckii subsp. lactis (ie
Russian Federation, eastern European countries and Mongo- non-traditional organisms) are sometimes mixed with the
lia. Conversely, natural yoghurt is popular in countries starter culture. However, the name(s) of the so-called ‘bio’,
between the Balkans and the Indian sub-continent, while therapeutic and=or probiotic fermented milks is not well
the fruit flavoured and=or flavoured products are normally established and should be addressed because consumers
consumed in the rest of the world. and=or some manufacturers in different countries consider
Little data is available on the world production figures of all these products to be yoghurt (for more details refer to
probiotic-fermented milks but during the last decade produc- subsequent section).
tion has increased dramatically in some selected countries. The milk fermentates by mesophilic microorganism
For example, the French market of fermented milk include the products that are grouped under lactic and
containing Bifidobacterium spp. and Lactobacillus acidophilus yeast – lactic fermentations. The starter microorganisms of

European Journal of Clinical Nutrition


the former group belong to the genera of Lactococcus(Lac. lactis
subsp. lactis, Lac. lactis subsp. lactis biovar diacetylactis and Lac.
lactis subsp. cremoris) and Leuconostoc (Leu. mesenteroides
subsp. cremoris and Leu. mesenteroides subsp. dextranicum),
and they are normally used as mixed cultures. However, the
microflora of the latter products is complex and not always
constant; numerous species of lactic acid bacteria, yeasts and
moulds have been found, for example, in the kefir grains.
Tamime and Marshall (1997) have detailed the organisms that
have been identified by many researchers in many countries.
Traditionally, many products are made from ‘natural’
yoghurt in the Balkans and the Middle Eastern countries
and these can be briefly described as follows: (a) dilution of
the fermentate with water for the production of drinking
yoghurt; (b) concentration of the yoghurt by de-wheying for
the manufacture of strained yoghurt and yoghurt cheese; the
latter product is formed into small balls and preserved in
olive oil; (c) cultured butter and=or ghee can be made by
churning full-fat yoghurt, and the aqueous phase is either
utilized as drinking yoghurt or mixed with parboiled cracked
wheat which is later sun-dried and milled into powder (such
product is known as Kishk); and (d) surplus of yoghurt is
preserved by drying or gently heating yoghurt for few hours
over low and smokey fires (the end product is known as
smoked yoghurt). Nevertheless, at present the classical
approach to categorize yoghurt products is based on its
physical characteristics as shown in Table 2 and, in particu-
lar, yoghurt is further sub-divided into different groupings
based on the following aspects:

legal and=or proposed standards classify the product, for
example, on the fat content (full, medium or low);

physical nature of the product (ie set or stirred; drinking
yoghurt is normally considered as stirred-type of low
viscosity);

flavours of yoghurt are divided into plain=natural,
fruit or flavoured; the latter two products are normally
sweetened;

probiotic, bio and=or therapeutic yoghurts contain
non-traditional organisms beside Lb. delbrueckii subsp.
bulgaricus and S. thermophilus (see Table 5);

miscellaneous yoghurts include pre-fermentation proces-
sing (lactose hydrolysis or replacement of the animal fat
with vegetable oil) or post-fermentation processing (vita-
min fortification or heat treatment).
Table 2 Proposed scheme for the classification of
yoghurt products
Category Physical state Yoghurt products
I Liquid=viscous Yoghurt
II Semi-solid Concentrated=strained
III Solid Frozen
IV Powder Dried
After Tamime and Robinson (1999).
Reproduced by courtesy of Woodhead Publishers, UK.
Fermented milks — a review
AY Tamime
S5
Technology of manufacture
Currently, the technological aspects of fermented milks are
well established and extensively reviewed (Rasic & Kur-
mann, 1978; IDF, 1984, 1986, 1988, 1992c; Chandan,
1989; Tamime & Marshall, 1997; Tamime & Robinson,
1999). The principal manufacturing stages and=or processes
for all fermented milk products (set- or stirred-type) have
many features in common, which can be briefly described
as follows:
Preparation of the milk base
The fat and the non-fat solid (SNF) contents of the milk are
standardized and fortified to ensure compliance with exist-
ing legal standards. The fat level may range between < 0.5
and  5.0 g=100 g, whilst the SNF is increased to  14 g=
100 g (of which 5 g=100 g is protein). The fortification of the
latter fraction(s) enhances the viscosity of the product, and
the methods available include the addition of powders or
concentration by evaporation or membrane filtration. It is
recommended that recombination of powders is carried out
at  40 C, and followed by filtration (to remove any
undissolved or scorched particles) and de-aeration (to
remove the air which is incorporated during recombination
stage). The presence of air in the milk base does not provide
the appropriate growth conditions of Lactobacillus acidophi-
lus and bifidobacteria. It also increases heat-exchanger
fouling, and affects the characteristics of the gel, especially
the products fermented with mesophilic lactic starter
cultures.
Homogenization
Advantageous physical – chemical changes caused by homo-
genization of the milk base occur during the manufacture of
fermented milk, which may include: (a) a reduction in the
diameter of fat to < 2 mm, these small globules being less
inclined to coalesce into larger units and rise to the surface;
(b) the milk becomes whiter in colour due to enhanced light
reflectance and scattering; (c) an increase in viscosity of the
product due to interaction and=or adsorption of the fat
globules onto the casein micelles; and (d) a decrease in
syneresis of the gel due to increase in its hydrophilicity
and water-holding capacity as a result of the interaction(s)
of the proteins.
A typical operating pressure used during the processing
of the milk base ranges between 15 and 20 MPa at 60 – 90 C,
and the homogenizer (single-stage type) is placed upstream
during the production of fermented milk. However, during
the manufacture of buttermilk, homogenization of the
milk base takes place on the downstream after the heat
treatment stage and, after acidification, the gel is homo-
genized at 5 – 10 MPa and 20 C; alternatively, two-stage
homogenization of the milk base may be used (Tamime
& Marshall, 1997; Tamime & Robinson, 1999; Tamime
et al, 2001).
European Journal of Clinical Nutrition
 Fermented milks — a review
AY Tamime
S6
Heat treatment
Different temperatures are used for the heat treatment of the
milk base during the manufacture of fermented milk, and
can vary depending on the time and temperature combina-
tions. Some examples are: (a) 85 C for 30 min; (b) 90 – 95 C
for 5 min; and (c) 105 C for 10 s. Thermal treatment of the
milk base induces numerous physical and chemical changes
that are complex and multi-functional; the topic has been
extensively studied (see the reviews by IDF, 1995; Tamime &
Marshall, 1997; Tamime & Robinson 1999), and in the
context of fermented milks the effects of heat treatment are:

destruction and=or elimination of pathogens and other
microorganisms present in the milk base and, hence, the
processed milk provides a good growth medium for the
starter cultures;

changes in the physical – chemical properties of the
milk constituents, mainly the denaturation of the b-
lactoglobulin, which forms a complex with k-casein;
such interaction minimizes syneresis and improves the
firmness of the gel; the protein=fat interaction in milk
should not be overlooked;

production of components that are stimulatory to the
starter cultures.
Starter culture
The heated milk base is cooled to the desired incubation
temperature of the product; some examples are: (a) yoghurt
at 40 – 45 C for up to 3 h, at 30 C for 18 h or  5 h when
using direct-to-vat inoculation (DVI) starter culture; (b) pro-
biotic products at 37 C for a few hours or up to 5 – 7 days
depending on the organism(s) used; (c) buttermilk at 20 –
30 C for up to 10 – 20 h; and (d) kefir at 22 C for 16 – 24 h
depending on the type of kefir grains used (Tamime &
Marshall, 1977; Wszolek et al, 2001).
Gel formation in milk is mediated by acid coagulation
due to the activity of the starter cultures and their enzymes
to hydrolyse the lactose and to a lesser degree the proteins.
The mechanisms involved of casein dissociation and aggre-
gation during acid-induced gelation of milk are pH-, ion
concentration- and temperature-dependant, and these
aspects of gelation are similar for set- or stirred-type fer-
mented milks; in the former type, the milk is incubated in
the retail container, whilst for the stirred product the milk
is incubated in large fermentation tanks.
Cooling and miscellaneous handling
Cooling is only method used to control the metabolic
activity of the starter cultures and their enzymes in order
to retain an abundant count of these organisms in fermen-
ted milks. The primary objective is to cool the product to
 5 C, and the process of cooling commences at  4.6 pH,
which is carried out using a one- or two-phase cooling. The
latter method is widely applied during the manufacture of
European Journal of Clinical Nutrition
fruit-flavoured fermented milk products; the first stage of
cooling, for example for yoghurt, reduces the temperature
from 45 to 20 – 25 C prior to the addition of fruit and packa-
ging, and the second phase of cooling takes place in the cold
store (chill tunnel is optional), where the product is cooled to
< 10 C (Tamime & Robinson, 1999; Tamime et al, 2001).
Historical background on probiotic fermented milks
The gross chemical composition of fermented milks provides
a useful indication of the potential nutritional value of these
products. The main components are protein, fat,
carbohydrate, minerals and vitamins. However, the bioactive
peptides (eg casomorphins, a- and b-lactorphin, immuno-
peptides, lactoferricin or phosphopeptides), which are
claimed to be health-enhancing components, should not
be overlooked (Meisel & Bockelmann, 1999). The beneficial
health properties of these products and their use in the
treatment of body ailments dates back to few thousands
years ago; they have been also mentioned in Biblical scrip-
tures, and some ancient scientists have prescribed them as
medicine for curing metabolic disorders of the stomach and
the intestine (Oberman & Libudzisz, 1998). However, the
health aspects of fermented milks including yoghurt became
apparent in the late 1800s and the early part of the 1900s; in
part, this could be attributed to the scientific observations
and=or explanations by Tissier and Moro (Rasic & Kurmann,
1983) and Metchnikoff (1910). Tisser had isolated Bacillus
bifidus communis (currently known as Bifidobacterium bifidum)
and this microorganism was predominant in the faeces of
breast-fed infants, whilst Moro around the same period
isolated Bacillus acidophilus (currently known as Lactobacillus
acidophilus) from the stools of breast-fed infants. Metchnikoff
(1910) put forward the beneficial effects of lactic acid bac-
teria (Bulgarian bacillus, which was later designated Lactoba-
cillus bulgaricus and currently known as Lb. delbrueckii subsp.
bulgaricus), that were present in ‘yahourth’, and postulated
the ‘longevity-without-aging’ of the Caucasians with high
consumption of such product.
As a consequence, the views and=or observations of Tisser,
Moro and Metchnikoff caused a considerable flurry of inter-
est among scientists and=or doctors regarding the beneficial
role of certain microorganisms (eg Bifidobacterium spp. and
the yoghurt starter cultures) that can colonize and possibly
displace toxin-producing organisms that are present in the
intestinal tract of humans. These aspects have initiated
scientific studies on the human health benefits associated
with lactic acid bacteria in fermented milks, and it was not
until the early part of the last century that Lb. acidophilus was
identified as being able to colonize and proliferate in
humans rather than Lb. delbrueckii subsp. bulgaricus and
S. thermophilus (Rettger & Cheplin, 1921; Rettger et al,
1935). Research studies in this field have been enormous
over the past years, and the knowledge acquired in terms of
human health benefits of fermented milks has helped, in
part, to increase the consumption of these products in many

countries. Nevertheless, who could have imagined in the
1950s that these products would be used successfully for the
treatment of certain human diseases in the 1980s or 1990s?
(See also Shortt, 1999.)
Probiotic microorganisms
The current definition of probiotics, as agreed by European
scientists, is ‘a live microbial feed supplement that is
beneficial to health’ (Salminen et al, 1998a). However, the
microflora of the human intestinal tract in both infants and
adults is highly complex and many bacterial species have been
identified; the topic has been extensively researched, and the
following selected reviews provide relevant information with
specific emphasis on probiotic microorganisms and their
health applications to humans (Robinson, 1991; Fuller,
1992, 1997; Sanders, 1994; Salminen et al, 1996a, 1998b, c;
Marshall & Tamime, 1997a; Holzapfel et al, 1998; Salminen &
von Wright, 1998; Dunne et al, 1999; Hirayama & Rafter, 1999;
Lee et al, 1999; Tannock, 1999; Fuller & Perdigon, 2000;
Salminen, 2001). Periodically, Danone Vitapole Recherché
publishes monographs (Capron et al, 2000; Malagelada et al,
1999; Denariaz et al, 1999; Gibson et al, 2000; Hartley et al,
2001) and World Newsletter (23 issues have been published)
updating the health aspects associated with fermented milks.
As mentioned earlier, lactic acid bacteria, yeast, moulds or
combinations of these are widely used in the production of
fermented milks and cheeses. The characteristics of these
Table 3 Examples of probiotic micro-organisms that
are used during the manufacture of fermented milks
Genera Microbial species
Lactobacillus Lb. acidophilus
Lb. acidophilus strains LC1,
La5, La1, La7, Gilliland
Lb. casei strains Shirota, GGa
or LGG,a Imunitass, NCC208
Lb. rhamnosus strain GG
Lb. johnsonii
Lb. helveticus
Lb. delbrueckii subsp. bulgaricus
Lb. gasseri
Lb. plantarum
Lb. paracasei subsp. paracasei
and subsp. tolerans
Lb. reuteri
Pediococcus P. acidilactici
Bifidobacterium B. bifidum, breve, longum,
adolescentis, infantis,
lactis, animalis
Lactococcus Lc. lactis subsp. lactis
Enterococcus E. faecium, faecalis
Saccharomyces S. boulardii
b Treatment of relapsing Clostridium difficile
a
This strain could be confused with Lb. rhamnosus GG due
to latest nomenclature classification.
b
Most likely application is during the manufacture of kefir.
Data compiled from Tamime et al (1995), Tamime and
Marshall (1997), Shortt (1997), Lee et al (1999) and
Sanders and Huis in’t Veld (1999).
Fermented milks — a review
AY Tamime
S7
microorganisms have been recently reviewed by Tamime
(2002), and only the relevance of probiotic microflora in
fermented milks will be discussed. It is evident that these
bacterial species have to withstand the acidic and bile salts
conditions in the gastro-intestinal tract of humans when
compared with ‘traditional’ dairy starter cultures, and reach
and=or colonize the large intestine. Researchers in different
countries have identified many probiotic microorganisms,
and Table 3 illustrates some examples.
The recent taxonomy and physiology of probiotic lacto-
bacilli species have been reported by Klein et al (1998), and
these species belonged to: (a) Lb. acidophilus group; (b) Lb. casei
group; and (c) Lb. reuteri=Lb. fermentum group; most Lb.
acidophilus strains used in the manufacture of probiotic
fermented milks have been identified as Lb. johnsonii or
Lb. gasseri; both species are members of the Lb. acidophilus
group. Hence, the issue of health-promoting properties of
such products is questionable, and it cannot be assumed that
all strains of Lb. acidophilus have or possess the desirable
properties of probiotic microorganisms (Salminen et al,
1996b; Hamilton-Miller & Gibson, 1999; Ouwehand et al,
1999); furthermore, not all the commercial probiotic fer-
mented milk products disclose the strain or species used or
the actual numbers (Hamilton-Miller, 1999; Sanders & Huis
in’t Veld, 1999). In some instances, researchers have referred
to Lb. rhamnosus GG as Lb. acidophilus, Lb. casei subsp. casei
or Lb. paracasei subsp. paracasei.
Until the mid 1980s, the genus Bifidobacterium was classi-
fied as Lactobacillus spp. and currently 30 different species of
bifidobacteria have been identified. The six organisms that
have been used in dairy products are shown in Table 3 but,
according to Klein et al (1998), most of the strains used in
Table 4 A summary of some health-promoting activities in
humans associated with probiotic fermented milks
Action=effect Alleged health benefit
In digestive tract Active against Helicobacter pylori
Enhanced lactose digestion
Stimulation of the intestinal immunity
Stabilization of Crohn’s disease
Stimulation of intestinal peristalsis
On intestinal Improves balance between
microflora microbial populations
(eg increase in faecal bifidobacteria)
Decrease in faecal enzyme activity
Colonisation of the intestinal tract
Reduced carrier time for Salmonella spp.
On diarrhoea Prevention=treatment of
acute and of Rotavirus diarrhoeas
Prevention of antibiotic-induced diarrhoea
diarrhoea
Other effects Improved immunity to disease
Suppression of some cancers
Reduction in serum cholesterol
Reduction in hypertension
Adopted from Tamime and Robinson (1999).
European Journal of Clinical Nutrition
 Fermented milks — a review
AY Tamime
S8
probiotic fermented milks are B. animalis, although they
are declared on some labels as B. longum. B. animalis was
renamed B. lactis in the late 1990s (Meile et al, 1997), and
now it is re-classified as B. animalis (Cai et al, 2000). In my
view, the term ‘bifidus’ should not be used because it is old
terminology (Lactobacillus bifidus), and can cause some con-
fusion to the reader.
The 16S rRNA sequencing within the genus Entrococcus
has revealed the presence of three species groups: E. faecium
and E. durans are found in the first group, whilst E. faecalis
forms an individual line of descent. However, the pathogeni-
city of these organisms should not be overlooked when they
are used in probiotic fermented milk products.
Limited data are available on the use of the probiotic yeast
(Saccharomyces boullardii) in fermented milks (Lourens-
Hattingh & Viljoen, 2001a), and the most likely application
is Kefir and Koumiss making. Other non-lactic acid bacteria
that have been used in probiotic products around the world
include Bacillus cereus, Escherichia coli and Clostridum
butyricum (Shortt, 1999).
Therapeutic properties of bio-fermented milks
It is well established that the probiotic microorganisms
shown in Table 3 have therapeutic properties, and when
they are added with ‘traditional’ starter cultures during the
manufacture of fermented milks (see later), such products
have certain health-promoting activities in humans; Table 4
attempts to highlight the possible advantages. However, up-
to-date reports on some of the clinical=nutritional studies on
humans using probiotic fermented milks and their successful
applications in the treatment of certain human diseases,
include the following: (a) adherence of probiotic microor-
ganisms to human intestinal cells; (b) decreasing faecal
mutagenecity; (c) improvement of constipation; (d) produc-
tion of bacteriocins; (e) effects on superficial bladder cancer;
and (f) effect on the human salivary microflora (Mattila-
Sandholm & Salminen, 1998; Salminen et al, 1998b; Mattila-
Sandholm et al, 1999a,b; Ouwehand et al, 1999; Petti et al,
2001). Some selected studies on nutritional aspects relating
to humans have been reported by Oberhelman et al (1999),
Alander et al (1999), Lee et al (1999), Kar (1999), Ashar and

Table 5 Some examples of probiotic fermented milk products available in different countries

Product Country Microorganismsa

I. Fermented milks
ABT, Biogarde1, Philus Germany Lb. acidophilus þ B. lactisb or bifidum þ S. thermophilus
UK
Denmark
Sweden
ACT4 Austria Similar to ABT þ Lb. casei
Vifit, Gefilus Germany Yogurtc þ B. bifidum þ Lb. acidophilus þ Lb. rhamnosus GG
UK
Netherlands
Belgium
Finland
d
SymBalance Switzerland Lb. casei þ Lb. reuterix þ Lb. acidophilus þ Bifidobacteria
Actimel France Yogurt þ Lb. casei strain Imunitasse
Biola Norway B. lactis þ Lb. rhamnosus þ Lb. acidophilus
Gaio Denmark S. thermophilus þ E. faecium
UK
Germany
Finland
BRA Sweden B. infantis þ Lb. reuteri þ Lb. acidophilus

II. Fermented milk beverages

Onaka He GG Japan Yogurt þ Lb. rhamnosus GG
Yakult Japan Lb. casei strain Shirota
Brazil
Some Asian countries
Caldus Japan B. longum þ Lb. acidophilus
Vitagen Malaysia Lb. acidophilus
Singapore
f
Aktifit Switzerland Similar to ABT þ Lb. rhamnosus GG
LC1 or LC1 GO Many European countries Lb. acidophilusg or Lb. helveticus
a
Lb. ¼ Lactobacillus; S. ¼ Streptococcus; B. ¼ Bifidobacterium; E. ¼ Enterococcus.
b
Currently it is reclassified as B. animalis (Cai et al, 2000).
c
Yogurt ¼ Lb. delbrueckii subsp. bulgaricus þ S. thermophilus.
d
Yogurt starter cultures have been found in the product rather than bifidobacteria (Sanders & Huis in’t Veld, 1999).
e
Lb. casei þ Lb. delbrueckii subsp. bulgaricus were the only organisms identified in the product (Sanders & Huis in’t Veld, 1999).
f
Lb. rhamnosus was the only organism present in the product (Sanders & Huis in’t Veld, 1999)
g
Lb. johnsonii þ S. thermophilus were found in the product (Sanders & Huis in’t Veld, 1999).
Data compiled from Tamime et al (1995), Tamime (1998), Sanders and Huis in’t Veld (1999) and Lee et al (1999).

European Journal of Clinical Nutrition


Prajpati (1999), Arunachalam (1999), Barone et al (2000),
Heyman (2000), Arunachalam et al (2000), Ahn et al (2000),
Chiang et al (2000), Meydani and Ha (2000), Armuzi et al
(2001), Collins (2001), Volpe et al (2001), Kawase et al (2001),
Perdigón et al (2001) and Reid et al (2001).
Another aspect, which has links with gut health, involves
prebiotics; they are defined as ‘non-digestible food ingredi-
ents’ that beneficially affect the host by selectively stimulat-
ing the growth and=or activity of one or limited number of
bacteria in the colon, that have the potential to improve the
host health’ (Gibson & Roberfroid, 1995). Most potential
prebiotic food ingredients are carbohydrate-based compo-
nents such as oligosaccharides (Hartemink, 1999; Gibson &
Angus, 2000). However, a mixture of probiotic microorgan-
isms and prebiotic food ingredient is known as synbiotic.
Commercial probiotic fermented milk products
Some of the critical aspects that can affect the quality of
fermented milks are: (a) good make which is attributed to
Fermented milks — a review
AY Tamime
S9
fast acid development using traditional starter cultures; acid
development of probiotic starter cultures is poor in compar-
ison with Lb. delbrueckii subsp. bulgaricus and S. thermophilus;
(b) stable product after manufacture where few undesirable
microorganisms can withstand the rapid acid development
or initiate growth at pH 4.6; and (c) the lactic and sour
flavour is the characteristic flavour acidity of majority of
traditional types of mesophilic and thermophilic fermented
milks, and the action of acid on the milk proteins (mainly
the casein) is important in texture formation of the product.
However, the heterofermentation by the bifidobacteria
results in lactic and acetic acid production; the latter acid
has a harshness which is undesirable for milk products
(Marshall & Tamime, 1997b).
As a consequence, the majority of commercially avail-
able probiotic fermented milks in different markets of the
world (ca 80 – 100) are made with a mixture of non-tra-
ditional and traditional starter cultures, and detailed exam-
ples have been reported by Hoier (1992), Tamime et al
(1995), Tamime & Marshall (1997), Holzapfel et al (1997)

Table 6 Some selected bacteriocins produced by dairy starter cultures and probiotic microflora

Genera=microorganisms Bacteriocins Molecular mass range (kDa)

I. Lactococcus
Lac. lactis subsp. lactis Lactostrepcin, Nisin, Lacticin, Bacteriocin, 1.3 – > 10
Dricin, Lactococcin
Lac. lactis subsp. lactis biovar diacetylactis Bacteriocin, Lactococcin, Lactocin, Diacetin 3.4 – 5.8
Lac. lactis subsp. cremoris Lactococcin, Bacteriocin, Diplococcin 3.4 – 8.5
II. Leuconostoc
Leu. mesenteroides subsp. mesenteroides Mesenterocin, Leucocin 2.5 – < 10
III. Streptococcus
S. thermophilus Bacteriocin, Thermophilin 4 – > 100
IV. Lactobacillus
Lb. acidophilus Lactacin, Acidophilin, Acidophilicin, 0.2 – 50
Acidocin, Acidolin, Bacteriocin
Lb. johnsonii Lactacin 2.5 – > 300
Lb. gasseri Bacteriocin, Gassericin 4.5 – 5.6
Lb. reuteri Reutericyclin, Reutericin 0.4 – > 200
Lb. casei Caseicin  41
Lb. plantarum Plantaricin 1 – 5.9
Lb. rhamnosus Bacteriocin < 1.0 – 14
a
Lb. delbrueckii subsp. Bulgarican NR
bulgaricus and subsp. lactis Lacticin, Lactobacillin NR
V. Enterococcus
E. faecalis Enteroccin 7.2
E. faecium Enterocin NR
VI. Bifidobacterium
Bifidobacterium spp. Bacteriocin 3.3 – 104
B. longum Novel protein
B. bifidum Bifidocin
VII. Pediococcus
P. acidilactici Pediocin 2.5 – 4.6
a
Data not reported.
After de Vuyst and Vandame (1994), Ali et al (1995), Maisnier-Patin et al (1996), Dave and Shah (1997), Marshall and Tamime
(1997a), McAuliffe et al (1998), Papathanasopoulos et al (1998), Anderssen et al (1998), Kawai et al (1998), Suma et al (1998), van
Reenen et al (1998), Yildirim and Johnson (1998), Farias et al (1999), Remiger et al (1999), Fujiwara et al (1999), Zhu et al (2000),
Gänzle et al (2000).

European Journal of Clinical Nutrition

 Fermented milks — a review
AY Tamime
S10
and Tamime (1998) (see Table 5); hence, probiotic micro-
organisms are used as therapeutic adjuncts in fermented
milk products (see also the review by Lourens-Hattingh &
Viljoen, 2001b).
Exopolysaccharides (EPS) production
Microbial EPS material is synthesized by certain strains of
lactic acid bacteria, and it can enhance the rheological
properties (ie minimize syneresis, improve the texture and
modify the structure) and sensory perception (ie firmness
and creaminess) of fermented milks and other dairy pro-
ducts. The topic has been extensively reviewed (IDF, 1998;
Cerning & Marshall, 1999), and illustrations of the micro-
structure of both mesophilic and thermophilic fermented
milks have been reported by Tamime and Marshall (1997)
and Tamime and Robinson (1999).
Quantitative and qualitative properties of EPS material are
influenced by the following aspects: (a) microbial strain; (b)
composition of the growth medium and=or growth factors;
and (c) processing conditions that include the growth tem-
perature and time of fermentation. In 2001, the scientific
papers presented at the first Symposium on EPS from Lactic
Acid Bacteria provided an up-to-date review of this field,
including their applications in the dairy industry and the
structures of EPS (Monsan et al, 2001; de Vuyst et al, 2001;
Boels et al, 2001; Jolly & Stingele, 2001; Degeest et al, 2001;
Duboc & Mollet, 2001; Marshall et al, 2001). However, some
of these EPS materials contain gluco- and=or fructo-oligosac-
charides and may generate short-chain fatty acids upon
hydrolysis in the intestinal tract by the colonic microflora,
and may have potential health (eg an anti-tumor effect,
cholesterol-lowering effects or immune-modulators effects)
and nutritional benefits as a prebiotic to the intestinal
microflora.
Bacteriocin production
Bacteriocins are antibacterial substances produced by
certain groups of microorganisms including probiotic and
lactic acid bacteria; they are also known as ‘colicin-like’
inhibitory substances. Incidentally, bacteriocins are usually
segregated from antibiotic compounds, and such naturally
produced metabolites inhibit the growth of undesirable
microorganisms in food and dairy products. In the case
of lactic acid bacteria, the bacteriocins produced could be
used to replace added chemical preservatives and, hence,
the shelf-life of the product could be extended by natural
means; however, these inhibitors can also affect certain
strains of starter cultures.
At present more than 70 different types of bacteriocins
have been identified as produced by lactic acid bacteria.
Table 6 summarizes some selected characteristics (for further
information refer to de Vuyst & Vandamme, 1994; Holzapfel
et al, 1995; Desmazeaud, 1994; Nes et al, 1996; Richard, 1996;
Marshall & Tamime, 1997a).
Partial characterization of the quality of
probiotic fermented milks
Many diversified probiotic dairy products are currently avail-
able to consumers in many markets, and some examples are:
pasteurized liquid milk, cheeses, ice-cream, milk powder feed
for infants and fermented milks (Tamime & Marshall, 1997).
However, fermented milk products are the most popular
vehicle used in the industry for the implantation of the
probiotic microflora in humans. In the mid 1990s, a colla-
borative research programme was established between the
Scottish Agricultural College (SAC) and the Hannah Research
Institute (HRI) to evaluate the quality (chemical composi-
tion, microbiological analysis, rheological properties and
sensory perception) of different types of newly developed

Table 7 The microorganisms used in commercial mixed starter cultures

Bifidobacterium spp. Lactobacillus spp.

Streptococcus
Code Supplier Typea bifidum lactisb longum infantis acidophilus bulgaricusc thermophilus

AB Chr. Hansen FD P P P
AC=BL Rhodia FD P P P
DVB-100 SBId FD P P P
MSK 2 Visby DF P P P
e
ABT 1 Chr. Hansen FD P P P P
ABT 3 Chr. Hansen FD P P P P
e
MSKB 2 Visby FD P P P P
MY 087f Rhodia FD P P
a
FD ¼ freeze dried, DF ¼ deep frozen.
b
Currently it is reclassified as B. animalis (see text).
c
Lb. delbrueckii subsp. bulgaricus.
d
SBI ¼ Systems Bio-Industries Ltd.
e
The starter cultures produces exopolysaccharide (EPS) material
f
This is a yoghurt starter culture, which is used as a control.
The symbol (P) denotes the presence of the microorganism in the starter culture.

European Journal of Clinical Nutrition


Figure 2 Sensory space maps for probiotic fermented milks, factor
scores on first two dimensions are shown. For starter cultures symbols
refer to Table 7. After La Torre (1997).
fermented milk products (eg vegetable oil yoghurt, kishk,
yoghurt made with fat substitutes, kefir and probiotic fer-
mented milks). In this paper, some aspects of the quality of
probiotics fermented milks will be reported, and for further
details refer to La Torre (1997), Hartley et al (2001) and La
Torre et al (2002).
In the first instance, single strains of Bifidobacterium spp.
(B. adolescentis NCFB 2230, B. bifidum NCFB 2715, B. breve
NCFB 2258, B. infantis NCFB 2205 and B. longum NCFB 2259,
were obtained from National Collection of Food Bacteria;
B. longum BB 46 and B. bifidum BB11 – B. longum BBL and BL
Figure 3 Interpretation of sensory results for fermented milk products. After La Torre (1997).
Fermented milks — a review
AY Tamime
S11
and B. infantis 410 – B. longum 2 and 913 were obtained from
Chr. Hansen, Rhodia and Visby, respectively) were used to
manufacture fermented milks. The quality of the products
made with the NCFB strains did not satisfy the minimum
requirements, the pH values were borderline (eg between 4.8
and 5.4), excessive whey separation was evident, the coagu-
lum was extremely week and, in some cases, the odour and
taste were unpleasant. However, slight improvement in the
quality of the product was evident, especially when the milk
bases were fortified with Raftaline1 or Jerusalem artichoke,
but the overall pHs ranged between 4.79 and 5.91 after 24 h
of incubation (with the exception of strains of B. longum BBL
and 2; pH  4.6). As the results were not satisfactory, it was
decided that commercial mixed blends of starter cultures
containing lactic acid bacteria and different Bifidobacterium
species would be used (see Table 7).
The quality of the fermented milks was superior to those
made with single strains of bifidobacteria, and the results
could be summarized as follows:

Acidification of the milk base (total solids, protein and fat
were 14.5, 5.5 and 1.5 g=100 g, respectively) ranged
between 6 and 10 h or 13 and 22 h for pH  4.4.

Organic acid profiling indicated that the starter cultures
produced appreciable quantities of lactic acid, and only
two cultures (AB and AC=BL) produced the high levels of
acetic acid (3229 and 2794 mg=g, respectively; figures are
averages of three trials and of fresh and stored products).

The viable cell counts in fresh and stored products for
bifidobacteria varied (eg two cultures (AB and AC=BL)
showed an increase, three cultures (DVB-100, ABT 3 and
MSKB 2) remained constant, and two cultures (ABT 1 and
MSK 2) exhibited a slight decline); Lb. acidophilus
increased during production but, in some products (DVB-
100, MSK 2, ABT 1 and 3 and MSKB 2), the counts were
European Journal of Clinical Nutrition
 Fermented milks — a review
AY Tamime
S12
decreased after storage; S. thermophilus counts of all starter
cultures showed a remarkable increase during production
and survival during storage.

Syneresis values of fermented milks ranged between 2.5
and 3.0 ml for fresh products and decreased to 1.5 – 2.5 ml
after 20 days’ storage at 5 C; the rate of decrease was
dependent on the starter culture used.

The firmness measurements of all fermented milks
increased over time, and the rate was independent
of the starter culture used; after the storage period, the
firmness values ranged between 1.8 and 2.5 N for the EPS
cultures MSKB 2 and ABT 1, respectively.

Sensory analysis indicated the following: (a) ‘acid’,
‘creamy’ and=or ‘other’ characters (for the odour, flavour
and after-taste attributes) were significant (P < 0.001); (b)
the flavour character ‘other’ was identified to the pre-
sence of a high level of acetic acid produced by two
starter cultures (AB and AC=BL); (c) analysis of variance
accounted for over 95% of the first three factors, explain-
ing 52.0, 30.7 and 12.9%, respectively; and (d) sensory
space maps were constructed that grouped the starter
cultures into different clusters based on the identified
significant attributes (see Figure 2). Factors 1 and 2 clearly
separated the cultures AB, AC=BL and MSK 2 from the
others, and cultures MY 087 and MSKB 2 were slightly
separated from the grouping of ABT 1, ABT 3 and DVB-
100 cultures. Separation on factor 1 was mainly asso-
ciated with differences in acid character, whilst factor
2 resolved the fermented milk products in terms of
the contrast between ‘creamy=sweet’ odour and ‘other’
(ie vinegar) flavour (see Figure 3).

It is possible to conclude that the level of metabolites
(mainly acetic acid) produced by the bacterial strain(s)
can influence the organoleptic assessment.
Conclusion
It is evident that fermented milks can be successfully pro-
duced using different blends of lactic acid bacteria and
probiotic microorganisms. Over the past century, volumi-
nous scientific knowledge has been well established regard-
ing the technological aspects of fermented milks, including
the physiology of starter cultures and related probiotic
microflora. However, over the coming years the possible
research areas may include the following aspects:

appropriate international definition(s) of yoghurt and
other fermented milks including probiotic products;

more properly designed clinical studies to establish the
proper health benefits to humans; in vitro results cannot
be found always in vivo, and observations reported in
animals cannot be translated directly to humans; there
are problems in generalizing the results given the large
number of types of microorganisms used and, in some
instances, the count of probiotic microorganisms in clin-
ical studies is not reported; clinical studies should be
European Journal of Clinical Nutrition
conducted on humans of different races in different
countries to properly substantiate the health benefits to
humans in general;

how the starter culture polymerizes lactose in milk to
produce exopolysaccharide material;

what induces the microorganisms to cyclize the
peptide(s) to secrete bacteriocins and what their mechan-
ism(s) for controlling pathogens in the gut is (are);

the pathogenicity of certain cultures requires further
clearance.
Acknowledgements
The author is grateful for the invitation to participate in the
conference and the funding provided by Instituto Danone
Mexico.
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