FEMS Microbiology Reviews 12 (1993) 253-272 253

© 1993 Federation of European Microbiological Societies 0168-6445/93/$15.00

Published by Elsevier

F E M S R E 00329

Selection criteria for lactic acid bacteria to be used

as starter cultures for various food commodities

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

H e r b e r t J. B u c k e n h i i s k e s

Gewi~rzmiiller, Stuttgart, FRG

Abstract: Lactic acid bacteria (LAB) are the most important bacteria used in food fermentations. Apart from general d e m a n d s for
starter cultures from the view of safety, technological effectiveness and economics, n u m e r o u s specific aspects have to be considered
when selecting strains for the different food fermentations. Therefore selection criteria for LAB depend on the type and the
desired characteristics of the final product, the desired metabolic activities, the characteristics of the raw materials and the applied
technology. Special selection criteria for LAB for use in the fermentation of sausages and vegetables as well as for the malolactic
fermentation in wine are discussed.

Key words: Starter cultures; Selection criteria; F e r m e n t e d sausage; F e r m e n t e d vegetables; Malolactic fermentation

Introduction tation, were mainly used to preserve foods of

Fermented foods
F e r m e n t e d foods are defined as palatable
products which are p r e p a r e d from raw or heated
raw materials and which acquire their character-
istic properties by a process in which microorgan-
isms are involved. In certain cases the endoge-
neous enzymes of the raw material play a decisive
role [1].
The origin of fermented foods seems to be
from the Orient and dates back to prehistoric
times. In the beginning, fermentation processes,
e.g. alcoholic, acetic acid and lactic acid fermen-
Correspondence to: H.J. Buckenhiiskes, Gewiirzmiiller, Post-
fach 30 04 80, D-70444 Stuttgart, FRG.
animal and plant origin. As a result of the devel-
opment of efficient heat sterilizing and refrigera-
tion systems, fermentation processes lost their
importance as preservation methods in the indus-
trialized countries.
However, fermentations are more than preser-
vation methods. Through the ages, people learned
more and more to control these processes and
fermented foods became an independent class of
foodstuffs. In Germany, approximately 25% of
the consumed victuals are fermented products [2].
As a regards taste, aroma, visual appearance,
texture, consistency, shelf life and safety, these
different products possess characteristic proper-
ties compared to the raw materials or to other
similar foods. The acidification of minced meat
during the production of dry sausages, for in-
stance, can be attained by the addition of glu-
254

c o n o - d e l t a - l a c t o n e or by f e r m e n t a t i o n . In both Starter cultures." from ancient to modern times

cases shelf life, safety a n d sliceability of the
sausage will be ac h i e v e d , but the original taste
and a r o m a can only be o b t a i n e d by f e r m e n t a t i o n .
In g e n e r a l , all classes of m i c r o o r g a n i s m s are
used for f o o d f e r m e n t a t i o n s , but in E u r o p e b a c t e -
ria and yeasts are m o r e c o m m o n than moulds.
A m o n g the bacteria, the lactic acid b a c t e r i a ( L A B )
are the most i m p o r t a n t . A c o m p i l a t i o n of those
N a t u r a l f e r m e n t a t i o n s are s p o n t a n e o u s pro-
cesses c a u s e d by m i c r o o r g a n i s m s d e r i v e d from
the raw m a t e r i a l s or the e n v i r o n m e n t . F o r m e r l y ,
p e o p l e w e r e i g n o r a n t c o n c e r n i n g the n a t u r e of
t h e expiring processes. But al r ead y in the earliest
times, artisanal and religious p r act i ces w e r e es-
t ab l i sh ed which w e r e very similar to the starter

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

foods which are p r o d u c e d by a process involving c u l t u r e s a p p l i e d today.
L A B is given in T a b l e 1. It can be seen that s o m e In the 1st c e n t u r y A .D ., Plinius the E l d e r
p r o c e s s e s are solely lactic acid f e r m e n t a t i o n s , d e s c r i b e d the p r e s e r v a t i o n of w h i t e c a b b a g e in
whilst o t h e r s are c o m b i n e d f e r m e n t a t i o n s . special e a r t h e n vessels, which w e r e used only for

Table 1
Fermented foods of the European market which are produced by a process involving lactic acid fermentation

Foodstuff Raw material Microorganisms used besides LAB ~'

BAC YEA MOU STA
Sauerkraut White cabbage +
Various vegetables Various vegetables +
Olives Green and black +
Vegetable juices Various vegetables ++
Soy products (yoghurt- Soy protein
or cheese-like) +++
Soy sauce Soybeans X

Sour dough Wheaten flour rye flour X ++

Kwass Malt, bread )4

Cacao ACB h X
Coffee X

Wine Grapes X +++

Beer (sour wort/ Malt
Berliner Weisse) × +++

Dry sausage Meat from various animals MIC c d

Curdled milk Milk +++

Yoghurt Milk +++
Butter Cream +++
Cheese Milk PRO ~ +++
Mouldy cheese Milk +++

~' BAC, bacteria; YEA, yeasts; MOU, moulds; STA, use of starter cultures. +, rarely; + +, often; + + +, always.
~ Acetic acid bacteria.
c Micrococci and staphylococci.
d Depends to the country of production.
" Propionibacteria.
 255

this p u r p o s e . T o d a t e , we a r e c e r t a i n t h a t u n d e r m i c r o o r g a n i s m s which a r e a p p l i e d with the inten-

t h e c o n d i t i o n s d e s c r i b e d by Plinius, t h e c a b b a g e
was f e r m e n t e d to s a u e r k r a u t by m i c r o o r g a n i s m s ,
which w e r e l o c a t e d in t h e p o r e s o f t h e vessels a n d
which p r o c e e d e d f r o m a f o r m e r f e r m e n t a t i o n .
E v e n in t h e s e days a n o t h e r p r i m i t i v e f o r m of
s t a r t e r a p p l i c a t i o n can b e o b s e r v e d d u r i n g p r e p a -
r a t i o n of several f e r m e n t e d b e v e r a g e s in s o m e
p a r t s of Africa. D u r i n g a religious c e r e m o n y p r e -
tion of m a k i n g use of t h e i r m i c r o b i a l m e t a b o l i s m
[1].
G e n e r a l l y , the p r e p a r a t i o n s u s e d as s t a r t e r s
t o d a y can be classified within t h r e e c a t e g o r i e s :
first, ' u n d e f i n e d c u l t u r e s ' which in t h e d a i r y in-
dustry a r e c a l l e d ' m i x e d - s t r a i n cultures'. T h e s e
s t a r t e r s a r e b a s e d on t h e use of f e r m e n t i n g sub-
strate, t a k e n from a s e l e c t e d p r o c e s s t h a t r e s u l t e d

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

c e d i n g t h e f e r m e n t a t i o n , t h e m e d i c i n e m a n dips
cult objects into the liquid, which again c o n t a i n
m i c r o o r g a n i s m s f r o m a p r e v i o u s batch.
Since the t i m e of P a s t e u r it has b e e n k n o w n
t h a t the n a t u r a l p r i n c i p l e o f f e r m e n t a t i o n d e -
p e n d s on a m u l t i t u d e o f d i f f e r e n t m i c r o o r g a n -
isms. O n t h e basis of this k n o w l e d g e t h e i d e a was
b o r n to s e a r c h for m i c r o o r g a n i s m s e s p e c i a l l y suit-
a b l e for p a r t i c u l a r food f e r m e n t a t i o n s . C h r i s t i a n
H a n s e n was the first to isolate a n d p r o p a g a t e
special yeasts to be u s e d in b r e w e r i e s . T h e first
s t a r t e r c u l t u r e was born.
F r o m a m o d e r n p o i n t of view, s t a r t e r c u l t u r e s
a r e d e f i n e d as p r e p a r a t i o n s which c o n t a i n living
in g o o d - q u a l i t y e n d p r o d u c t s . T h e s e s t a r t e r s a r e
also p r o p a g a t e d and d i s t r i b u t e d c o m m e r c i a l l y [1].
T h e two o t h e r c a t e g o r i e s of s t a r t e r s a r e r e p r e -
s e n t e d by ' s i n g l e - s t r a i n c u l t u r e s ' a n d ' m u l t i p l e -
strain c u l t u r e s ' , which c o n t a i n o n e o r m o r e de-
f i n e d strains, respectively.
In a d d i t i o n to true s t a r t e r cultures, s o - c a l l e d
' b a c k s l o p p i n g ' is p r a c t i s e d in f e r m e n t a t i o n tech-
nology. This p r o c e s s is b a s e d on the c o n t i n u o u s
i n o c u l a t i o n by f o o d s from a p r e v i o u s batch, a n d is
i n d u s t r i a l l y a p p l i e d to date.
T a b l e 2 gives an overview of the types of
s t a r t e r c u l t u r e s u s e d in the v a r i o u s fields of appli-
c a t i o n today.

Development of starter cultures

General aspects
Table 2
G e n e r a l r e q u i r e m e n t s for s t a r t e r c u l t u r e s as
Types of starter cultures used in various fields of food fermen-
r e g a r d s safety, t e c h n o l o g i c a l effectiveness a n d
tation
e c o n o m i c s are s u m m a r i z e d in T a b l e 3. T h e par-
Foodstuff Single Multiple Mixed Back- ticular topics a r e self-evident a n d only t h e ques-
strain strain strain slopping
tion o f b i o g e n i c a m i n e f o r m a t i o n n e e d s to be
cultures cultures cultures
discussed in m o r e detail.
Sauerkraut + - _ + a
A n o t h e r g e n e r a l a s p e c t d e a l s with several po-
Various
vegetables + - - - tential health and nutritional benefits derived
Vegetable from s o m e species of L A B , e.g. i m p r o v e d nutri-
juices + - - - tional v a l u e of food, c o n t r o l of intestinal infec-
Soy-products + - - - tions, i m p r o v e d d i g e s t i o n o f lactose, c o n t r o l o f
Sour dough + + + + s o m e types o f c a n c e r a n d c o n t r o l of s e r u m
c h o l e s t e r o l levels [3-6]. H o w e v e r , in t h e field o f
Wine + + - -
f e r m e n t a t i o n s the discussion o f which now fol-
Dry sausage + + - + lows, n u t r i t i o n a l a n d h e a l t h a s p e c t s a r e c o n f i n e d
Dairy to special a p p l i c a t i o n s such as the p r o d u c t i o n o f
products + + + (- ) s o - c a l l e d L( + ) - p r o d u c t s o r t h e d e s t r u c t i o n o f the
, not used; +, applied. c y a n o g l y c o s i d e s in cassava f e r m e n t a t i o n (see be-
a Brine from a previous fermentation. low).
256
Table 3
General criteria for starter cultures after [2]
1 Safety
1.1 Starter organisms are not in possession of any pathogen
or toxic activity
1.2 The preparations are free from hygienic precarious infec-
tions or substances
"~ Technological effectiveness
2.1 Starter organisms dominate over the spontaneous mi-
croflora
2.2 The microorganisms perform the required metabolic ac-
tivity
2.3 The preparations are free from technological precarious
infections or substances
3 Economical aspects
3.1 The propagation must be feasible from the economical
point of view
3.2 The starter culture can be preserved by freezing or
freeze-drying with little practical loss of activity
3.3 The important properties are stable under defined stor-
age conditions for several months
3.4 The handling of the starter culture must be as easy as
possible
Biogenic amines
Biogenic amines have been defined as biologi-
cally active aliphatic, aromatic or heterocyclic or-
ganic bases of low molecular mass, which can be
formed and degraded during the metabolism of
man, animals, plants and microorganisms. Bio-
genic amines are in general either psychoactive or
vasoactive substances and are thereby involved in
many critical physiological functions in man and
animals.
The intestinal tract of mammals possesses an
efficient detoxification system in which the en-
zymes monoamine oxidase ( M A O ) and diamine
oxidase ( D A O ) play an important role. Therefore
the consumption of foods containing small
amounts of biogenic amines is not hazardous,
unless the detoxification system is inhibited or
genetically deficient. However, the oral intake of
high amounts may have toxicological effects. Pos-
sible symptoms are nausea, respiratory distress,
hot flushes, sweating, heart palpitation, headache,
a bright red rash, oral burning and hyper- or
hypotension [7].
In most foods, the majority of biogenic amines
are formed by decarboxylation of the correspond-
ing amino acid through substrate-specific en-
zymes derived from microorganisms [8].
Prerequisites for a considerable formation of
biogenic amines in foods are [3]: (i) The availabil-
ity of free amino acids, which may occur in the
food itself, but also be liberated from proteins as
a result of proteolytic activity. (it) The presence
Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022
of decarboxylase-positive microorganisms. Among
others, genera of Enterobacteriaceae and Bacil-
laceac as well as species of Lactobacillus, Pedio-
coccus and Streptococcus are reported to be ca-
pable of decarboxylating one or more amino acids.
(iii) Conditions that allow bacterial growth, de-
carboxylase synthesis and decarboxylase activity.
Considering these requirements, the occurrence
of biogenic amines must be expected in all fer-
mented products, especially in those which are
fermented spontaneously by an undefined mi-
croflora. The main amines found in higher con-
centrations in foods are histamine, tyramine, pu-
trescine and cadaverine. Fig. 1 shows examples of
the biogenic amines and their precursors which
were detected in lactic acid-fermented vegeta-
bles. The highest amounts reported in sauerkraut
are 104 ppm of histamine, 192 ppm of tyramine,
311 ppm of cadaverine and 550 ppm of putrescine
[8].
The kinetics of the formation of biogenic
amines in spontaneously fermented sauerkraut
(25-kg batches) are illustrated in Fig. 2A. Unfor-
tunately, the fermentation was not accompanied
by a detailed microbiological analysis. Details do
reveal, however, that the initial stage was domi-
nated by Leuconostoc mesenteroides and that a
strong propagation of Pediococcus species was
observed parallel to the major formation of his-
tamine. In a following batch the cabbage was
inoculated with 2 x 1()~' cfu g i. Lb. plantarum
(commercial starter culture). The effect of inocu-
lation on the formation of biogenic amines can be
seen from Fig. 2B. Within the initial stage, the
amount of histamine, tyramine and cadaverine
remained at the level of the spontaneously fer-
mented batch, but the amount of putrescine was
significantly reduced. Despite the inoculation with
Lb. plantarurn, L. mesenteroides dominated the
 257

initial phase of fermentation, but growth of Pe-
diococcus species was inhibited [14].
From this example it can be concluded that it
is strongly recommended to select LAB which fail
to produce biogenic amines [3].
ogy, the desired metabolic activities, and the de-
sired characteristics of the final product. These
special selection criteria shall be discussed below.
Meat products

The production of fermented sausages or raw,

Special fermented foods dry sausages has a long tradition and originates
from Mediterranean countries at the time of the

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

During the selection of starter cultures for Romans [10]. Although more than 700000 tonnes
special food commodities, numerous specific as- of fermented sausages are produced and con-
pects have to be considered, including the charac- sumed each year in Europe [11], the application
teristics of the raw materials, the applied technol- of LAB or other microorganisms in other fields of

COOH
I
D
HO CH 2 CH - NH 2 ----.I,,- HO - OH 2- CH 2- NH 2

Serine Ethanolamine )al

COOH
I
D
H2N (CH2) 3 - OH - NH 2 ----~" H2N - (OH2) 4 - NH 2

Ornithine Putrescine )al

COOH
I
D
H2N - (OH2] 4 - OH - NH 2 ----~ H2N - (CH 2)5 NH2

Lysine Cadaverine ) al

H
H2N (CH2)3- N - (CH 2 )4 NH2
Spermidine )al

~"'~N HCOOH _ D D. ~ ~ ' ' - / N H2

2
Phenylalanine Phenylethylamine )at

.~/~ COOH .D ~ ' ' ' ~ / N H2

HO~ NH2 H O ~
Tyroslne Tyramine )ar

I~'~-S COOH D t,. i~¢ ''~/ NH2

HN N NH 2 HN ~N

Histidine Histamine )he

Fig. 1. Biogenic amines and their precursors which were detected in lactic acid fermented vegetables [9]. ( D ) D e c a r b o x y l a t i o n . ( R )
Spermidine is formed by a reaction of putrescine with a propylamine residue which descends from methionine. ,,i), aliphatic; ,,r),
aromatic; h~), heterocyclic.
258

Table 4
Application of LAB in meat products [12]

Field Status Effect of LAB

Production of fermented sausages In general practical use Sensory quality;
Preservation;
Hygienic stability
Production of cooked meat products In practical use Sensory quality
Production of smoked h a m Pilot studies Standardizing meat quality

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

(counteract D F D condition);
(in use) Hygienic safety
Extension of shelf life of fresh meat and Pilot studies Prevent growth of a spoilage
meat products flora
Improving the hygienic status of meat prod- Research Inhibition of food pathogens,
ucts and of the health of c o n s u m e r s exerting probiotic effects

A
meat processing has remained a topic only of
mg amine/kg pH
minor importance to date. Table 4 gives a survey
300 Initial ] Intermediate I Final of possible applications of LAB which are under
.-- _st_a_ge..L. . . . stage_. . . . / ......... S.ta.ge_. . . . . . . . . . .
250
Putreseine
5,5 development in the laboratory or pilot studies.
200 . . . . . . . . . . . . . . . . . . . . . . . .

Fermented sausage
150 4,5 Fermented sausage is defined as comminuted
DH /
......~___HLs_ta_mine.. fat and meat, mixed with salt, curing agents,
100
sugar and spices, stuffed into casings, and sub-
50 ..... i....
~ -~-~am~rln: 3,5 jected to a fermentation process carried out by
microorganisms. Most fermented sausages are
0
10 15 20 25
tit ratable acidity Time ( d a y s )
approx. 11 g/ kg
S Meat l : Bacon i ~(~uring sa~

/
B
mg amine/kg pH
| Chills., ] I Frozen
: ' ~
Frozen
L , ui J

300 Initial I intermediate J Final

stage | stage | stage ~M;n~iing i r Cutting ~ ! Dosing
250 ................................................ 5,5
200 ............................................... ] Casings i ( Starter-~
culture j,

~wTreatment ' [Ferm

it=hmouldsjr ~ e n"Ta,"ion]i ' i/Ascorbic "~,
15050~
100 "#utrescine 3,5
4,5 \ acid /,

- : [yramlne
L,aoaverln e / Smoke
,\ /
i ~
i
Smoking ==
0 ~ , I , , Histamine
0 5 10 15 20 25 Ripening
titratable acidity Time ( d a y s )
approx. 12 g/kg

Fig. 2. Formation of biogenic amines during fermentation of ' Raw ~'

sauerkraut [14]. (A) s p o n t a n e o u s fermentation; (B) fermenta-
i sausage/i

tion after inoculation of Lactobacillus plantarum. Fig. 3. Flow sheet of raw sausage manufacture.

dried and can be stored with little or no refrigera-
tion. A flow sheet of the general course of the
manufacturing process is given in Fig. 3.
The main objectives of the fermentation and
ripening process are: (i) formation of the typical
red colour (nitrosomyoglobin); (ii) formation of
the characteristic fermentation flavour, (iii) devel-
opment of firmness and sliceability; (iv) inhibition
of pathogenic and spoilage bacteria; (v) shelf life.
These goals may be attained by a complex
interaction of microbiological, chemical and phys-
ical reactions. The spontaneous fermentation of
sausages is characterized by the participation of
L A B , catalase-positive cocci, yeasts and moulds.
However, the most important microorganisms are
the bacteria. Figure 4 presents a general overview
of the interactions influenced by the activity of
these organisms (see below).
These relations, considered most of the com-
mercially available starter cultures are mixtures
of LAB and catalase-positive cocci (Staphy-
lococci, micrococci or a blend of both species).
Yeasts are sometimes present in those cultures in
order to produce sausage with the so-called Ital-
I Lactic acid bacteria I
-------~ Cornpetitive flora
Drying
-T--
Decreaseof
aw-value
Inhibition of
undesirable
microorganisms
Shelf life
Fig. 4. Interactions during the fermentation of sausages caused by the action of LAB and catalase-positive cocci.
259
ian taste. Moulds are only used at the surface of
mould-ripened sausages which are predominantly
produced in Italy; Spain, France and Switzerland,
whereas most of the sausages produced in Ger-
many, Belgium or the Scandinavian countries are
smoked [11]. The same proportions can be found
regarding the general use of starter cultures.
Whereas starter cultures are very common in the
northern European countries, most of the
Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022
sausages are fermented spontaneously in the
south.
Given the investigations of the COST group
(COST = Cooperation in Science and Technol-
ogy; Concerted Action of the EEC, 1985-1989), it
can be concluded that better aroma, longer shelf
life, stronger antagonism against pathogens, faster
colour formation, and greater handling conve-
nience are the generally desired goals of using a
better starter [11].
Given this framework, it is possible to define
selection criteria for LAB for use in sausage
fermentation (Table 5).
The most important task of the LAB is the
formation of lactic acid from added carbohy-
Catalase-positive cocci
Reductase
Nitrate
reduction
--V-
Nitrite
Smell/Taste
Table 5
Selection criteria for lactic acid bacteria to be used in the
production of fermented sausage
+ Fast production of lactic acid
* Growth rate at differenl temperatures
Homofermentative species
Pcrsislcncc over the whole fermentation and ripening pro-
ccss
Nitrate reduction
Catalase positive
:~ l,actosc negative
, Formation of flawmr
* No formation of peroxide
No R~rmation of biogenic amines
* No formation of ropy slime
:, Tolerance or even synergy to other microbial coinponenls
of the statler
* Antagonism againsl pathogens
* Antagonism against technologically undesirable microor-
ganisms
, Factors tO inlprove tile nulri|[ona] value ot' t[ic sausages
[:conomical factors
. . . . . . . . . . . . . . . . . .
drates. F i g u r e 4 shows that the resulting d e c r e a s e
in p H causes various effects and interactions. T h e
most i m p o r t a n t effects arc:
(i) C o a g u l a t i o n of the m e a t proteins, w h e r e b y
the s a u s a g e b e c o m e s sliceable.
(ii) All r c a c t i o n s necessary for colour f o r m a -
tion are i m p r o v e d at low pH. T h e most i m p o r t a n t
step is the f o r m a t i o n of nitric oxide a c c o r d i n g to
the reaction:
3 H N O 2 ---, H N O ) + H 2 0 + 2 N O
T h e nitric oxide reacts with m y o g l o b i n to nitro-
somyoglobin, the s u b s t a n c e causing the c u r e d
m e a t colour.
(iii) I m p r o v e m e n t of the stability. T h e e n c a s e d
mixture of meat, fat a n d additives is highly per-
ishable since the c o n d i t i o n s are o p t i m a l for b a c t e -
rial growth. T h e s u b s t r a t e is c h a r a c t e r i z e d by a
rich c o n t e n t of n u t r i e n t s , growth factors a n d min-
erals, a w a t e r activity of a,, = 0.96-{).97 a n d a p H
of 5.6-5.9. A l t h o u g h a d d e d n i t r a t e or nitrite are
effective as a preservative, the stability s h o u l d be
r e a c h e d by d e c r e a s i n g pH, w a t e r activity and
r e d o x p o t e n t i a l in a relatively short time.
T o e n s u r e these r e q u i r e m e n t s , b o t h the growth
rate a n d the acid f o r m a t i o n u n d e r the c o n d i t i o n s
in the f e r m e n t i n g s u b s t r a t e a r e i m p o r t a n t selec-
tion criteria for LAB. A l t h o u g h the rate and the
a m o u n t of acid fl)rmation can be i n f l u e n c e d by
the t e m p e r a t u r e as well as the n a t u r e and quan-
tity of a d d e d sugars, t h e r e is a n e e d for species
with d i f f e r e n t growth and acidification p a t t e r n s .
This d e m a n d d e p e n d s on the various types of
f e r m e n t e d sausage ( a p p r o x i m a t e l y 330 different
types a r e p r o d u c e d in G e r m a n y alone), the alter-
Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022
native use of n i t r a t e or nitrite and on the a p p l i e d
technology. In the U n i t e d States, for instance,
s u m m e r sausage is f e r m e n t e d at high t e m p e r a -
tures up to 40°C and the sausages arc r e a d y
within 48 h. In contrast, in H u n g a r y , raw sausage
is t r a d i t i o n a l l y f e r m e n t e d at t e m p e r a t u r e s below
10°C until the w a t e r activity has r e a c h e d a valuc
b e t w e e n 0.93 and 0.92 [16].
T h e i m m e d i a t e a n d fast acid f o r m a t i o n at the
b e g i n n i n g of the t e r m e n t a t i o n is an essential re-
. . . . . . . . . . . . . . . . . . . . . . . . q u i r e m e n t but excessive acid f o r m a t i o n , often
a s s o c i a t e d with c o l o u r defects and s o m e t i m e s with
gas f o r m a t i o n , is o n e of the most i m p o r t a n t p r o b -
lems in sausage f e r m e n t a t i o n . T h e r e f o r e , the ex-
p e r t s c o n s u l t e d by the C O S T - g r o u p stated that
t h e r e is only a m a r g i n a l n e e d for strains with
i n c r e a s e d rate of acid p r o d u c t i o n c o m p a r e d to
the s t a r t e r s available t o d a y [11].
H e t e r o f e r m e n t a t i v e L A B are not suitable as
s t a r t e r s for sausage f e r m e n t a t i o n b e c a u s e higher
c o n c e n t r a t i o n s of acetic acid result in a p u n g e n t
off-flavour. In a d d i t i o n , the fl~rmation of higher
a m o u n t s of c a r b o n dioxide leads to the d e v e l o p -
m e n t of holes of d i f f e r e n t sizes. T h e r e f o r e . it has
b e e n p r o p o s e d to use the rate of C O , f o r m a t i o n
from glucose as an i m p o r t a n t negative selection
criterion [ 17].
N i t r a t e is used as a curing a g e n t in the p r o d u c -
tion of h m g - f e r m e n t e d sausage. To e n a b l e the
r e d d e n i n g p r o c e s s it is i n d i s p e n s a b l e to r e d u c e
nitrate to nitrite. U n d e r the c o n d i t i o n s in the
f e r m e n t i n g sausage, this r e a c t i o n is only possiblc
by a n i t r a t e r e d u c t a s e , which n o r m a l l y derives
from the M i c r o c o c c a c e a (see Fig. 4). Since the
d i s p r o p o r t i o n i n g of nitrite shown above leads to a
c e r t a i n a m o u n t of nitrate, this e n z y m e is even
necessary in s a u s a g e s that are m a n u f a c t u r e d with
nitrite.
N i t r a t e r e d u c t a s e s a n d even b e r n e - d e p e n d e n t

and heme-independent nitrite reductases are re-
ported to be present in LAB strains involved in
meat fermentation. Whereas the reduction of ni-
trite by the heine-dependent nitrite reductase led
to ammonia, the heme-independent enzyme re-
leased dinitrogenic oxide (N20) and nitric oxide
(NO). Test fermentations using multiple-strain
cultures of Lb. pentosus, exhibiting nitrate reduc-
tase activity, and strains of Lb. sake and Lb.
farciminis, exhibiting nitrite reductase activities,
effected the reduction of nitrate and nitrite, even
when nitrate was used as a curing agent. How-
ever, the reaction was very slow and the sausages
failed the criteria of colour and flavour [18]. Cur-
ing in the absence of cocci is only conceivable
when LAB are available that possess significantly
higher nitrate reductase activities.
The majority of lactobacilli are capable of
forming hydrogen peroxide by oxidizing lactate.
In certain food fermentations this activity may
result in the inhibition of undesirable microor-
ganisms. However, in the case of meat products,
peroxides lead to discolouration since the sub-
stance attacks the heme pigments. Lb. sake and
Lb. curt,atus strains were found to be the domi-
nating flora in spontaneously fermented sausages,
and many strains of these species showed a rapid
hydrogen peroxide formation [19]. In meat fer-
mentation, LAB are preferred which possess none
or only little H 202-forming activity.
Another way to stabilize and protect the colour
is to destroy the peroxide formed by a catalase
normally derived from the Micrococcaceae. Since
the occurrence of catalase is known in LAB, e.g.
in strains of Lb. sake, screening for catalase-posi-
tive strains, is desirable.
Given the demand of flavour formation, it has
to be stated that only little is known about com-
pounds and biochemical activities in products with
poor and excellent flavour. Lipolysis and proteo-
lysis are considered to play a key role in the
development of aroma, but the importance of
lipolytic and proteolytic activities from LAB have
not yet been studied in fermenting sausage.
Bacteriophages have not been found to be a
problem in sausage fermentation. This may be
due to the fact that the absence of liquid will
preclude its dissemination. However, phages at-
261
tacking Lb. plantarum have been isolated which
caused a delay of 1 or 2 days in a sausage fermen-
tation experiment [20,21].
The search for starters possessing strongly an-
tagonistic effects against food-poisoning microor-
ganisms is of special interest. Those properties
were found in strains of Lb. plantarum, Lb. cur-
l,atus and Lb. sake, e.g. against Listeria monocy-
togenes [22] or Staphylococcus aureus [18]. How-
Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022
ever, it has to be pointed out that the effects are
restricted to a very limited number of microor-
ganisms. Furthermore, it was observed that strains
of Lb. eurl'atus and Lb. sake inhibit not only
food-poisoning microorganisms but also strains
that are often used in starter preparations, for
example against Micrococcus varians. In the lat-
ter case, nitrate reduction and the reddening
reaction were suppressed when such a combina-
tion was used [18]. Further information on the
genetics of bacteriocins produced by LAB can be
found in the contribution of T.R. K l a e n h a m m e r
elsewhere in this issue of FEMS Microbiology
Reviews.
Some strains of Lb. sake [23] are capable of
forming a ropy slime. A rate of up to 10 7 c f u g - I
for those microorganisms in the fermenting
sausages does not seem to have any negative
effect on the maturation or the colour and consis-
tency of the sausages. However it is assumed that
the processing facilities at the butchers can be
open to contamination through sausages contain-
ing those microorganisms. This may entail prob-
lems by 'cross-contamination', e.g. with cooked
meat products, if these are manufactured in the
same rooms or sliced using the same machines.
Therefore, it may be beneficial to use starters
which fail to produce ropy slime.
Vegetable fermentation
It is generally agreed among food scientists
that fermented plant products belong to the
" F o o d of the future". A world-wide increasing
interest can be observed especially for lactic acid
fermented fruit and vegetable products. An up-
to-date review concerning the situation of these
products in Europe was p r e p a r e d by the C O S T
group [24]. From this p a p e r it follows that a total
262

Table 6 The final product can either be distributed as

Lactic acid fermented vegetables available in the European a fresh product, packaged or unpackaged, or as a
market pasteurized product in cans or jars.
Artichoke Melons When vegetables are distributed as fermented
Capers Olives and unpasteurized products, it is important that
Carrots Red beets all fermentable sugars have been removed during
Cauliflower Red cabbage
the lactic acid fermentation. Otherwise, a sec-
Celery Sauerkraut (white cabbage)
Cucumbers Silver-skinned onions ondary fermentation by yeasts can occur which
Eggplants (Levant garlic) results in gaseous spoilage, brine turbidity [25]

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

Fungi (not specified) Swedes (Brassica napus) and probably in an alcoholic fermentation. In the
Green beans Tomato shaped paprika case of cucumber fermentation particularly, the
Green tomatoes (unripe (green and red)
CO 2 formation may result in bloater formation
fruits of Lycopersicon Turnips (Brassica rapa)
lycopersicum ) Waxy paprika [26].
Green paprika Whole cabbage (white cab- To date, pure lactic starter cultures are not
Lupinus beans bage) very common in European vegetable fermenta-
tion, although preparations are available on the
market. The major exceptions are the production

of 21 different fermented vegetables (Table 6), an LRaw Material /

unspecified number of variably composed veg- _ .... T
etable blends and several fermented vegetable Trimming/
Pickling/
juices are available in the market. At present, ._ Coring)a _
only three products are of real economical impor- I
tance: olives, sauerkraut and cucumbers. The re- Blanching/
Cooking/
ported production figures for 1985 were 510000, Peeling )a
220 000 and 45 000 tons, respectively [24].
A highly simplified flow sheet for manufactur- L Stitching
Shreddi)a_~
ng/I ~
~
-
bugars
~
ing lactic acid-fermented vegetables is given in
Fig. 5. The methods vary among vegetable or fruit Filling into ~_~..~tarter ~'~
It Water)a a Tank jr~_ ~:ulture)b~
commodities, depending on the properties of the j ....

commodity and the desired characteristics in the 1 J

final product. Given the fact that the majority of Fermentati°n
j i--~_ Salt 5
the vegetables are fermented by the naturally
occurring lactic flora, it is surprising which meth- l
ods of treatment are used prior to fermentation. Unpacking l
Fresh vegetables contain a numerous and var-
I
ied epiphytic microflora and an extremely small
population of LAB. For instance, the analysis of
B,anch,o0i
30 different samples of white cabbage from four
growing seasons showed that the flora is domi- =
I
Filling 1 i ann'n0 1
. . . .

nated by aerobic bacteria and yeasts, while LAB

normally present only between 0.15 and 1.5% of
/i ~--Pasteurizing 1
the total counts [15]. It is certain that the natural
flora is affected by methods of p r e t r e a t m e n t like i v___
nt~ ("Pasteurized ,er-~
trimming, peeling or blanching, but this does not
seem to have any significant effect on the fermen-
~t s J ~mented productsJ
Fig. 5. Flow sheet of the production of fermented vegetables.
tation processes. ~) depending on the species of vegetables; b) see text.
 263

Table 7 cultures according to the 'Lactoferment-process'

Lactic acid bacteria described to be used as starter cultures [27]. LAB recommended for juice fermentation
for the fermentation of vegetable juices [28] are summarized in Table 7.
It is supposed that in the future modern con-
Lactobacillus acidophUus
Lactobacillus bavaricus sumer demand and economical requirements will
Lactobacillus bifidus necessitate the development of controlled proce-
Lactobacillus breuis dures also in vegetable fermentation in order to
Lactobacillus casei provide safe products of a consistently high qual-
Lactobacillus delbrueckii
ity. Besides further advances in technology, the

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

Lactobacillus helveticus
Lactobacillus plantarum use of starter cultures will be a practical ap-
Lactobacillus salivarius proach to meet these demands, but because of
Lactobacillus xylosus the modest margins of vegetables and vegetable
Lactococcus lactis products.it is an open question whether the appli-
Leuconostoc mesenteroides
cation of starters is economically acceptable.
Starter cultures applied in vegetable fermenta-
tion must possess appropriate and specific at-
tributes depending on the properties of the fer-
of so-called L( + )-products, which are character- mented commodity and on the characteristics de-
ized by large amounts of L( + )-lactic acid pro- sired in the final product. Selection criteria for
duced by LAB and the manufacture of fermented starters to be used for the fermentation of
vegetable juices, except sauerkraut juice. The lat- sauerkraut, cucumbers, olives and vegetable juices
ter are predominantly fermented by using starter are summarized in Table 8.

Table 8
Selection criteria for lactic acid bacteria to be used for vegetable and vegetable juice fermentation (after [11,29,30]

Criteria Sauer- Cucum- Olives Vegetable

kraut bers juices a
Technologically relevant criteria
Rapid and predominant growth ++ ++ ++ +
Homofermentative metabolism - ++ ++ ++
Salt tolerance + ++ ++ 0
Acid production and tolerance ++ ++ ++ +
Inability to metabolize organic acids ++ ++ ++ +
Growth at low temperatures ++ ++ ++ 0
Few growth factors required 0 0 + 0
Tolerance of phenolic glycosides 0 0 ++ 0
Formation of dextrans
Pectinolytic activities
Formation of bacteriocins + + + 0
Bacteriophage resistance 0 + + ++

Sensorially relevant criteria

Heterofermentative q - q . -- m __

Formation of flavour precursors ++ ++ + 0

Nutritionally advantageous criteria

Reduction of nitrate and nitrite + 0 0 ++
Formation of L( + )lactate + + 0 ++
Formation of biogenic amines
+ + , important; + , advantageous; 0, not relevant; - , detrimental.
a Except sauerkraut juice.
 264

pH
Unfortunately, the fermentation of vegetables 42-
is difficult to control, primarily because of their A
shape, the large number of naturally occurring 4]
microorganisms and the variability in nutrient
content. 40

H e a t treatment or sterilization by other meth-

ods prior to fermentation is impossible in most 39-

cases, so that pure culture fermentation can only

be achieved in juice production. This means that 3,8-

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

selected LAB must grow rapidly and be highly
competitive in the environmental conditions un- 3.7-

der which the product is kept. These conditions

vary among different vegetables and affect above
all the content of sodium chloride (approximately
0.6-2.0% for sauerkraut [24], 5 - 1 0 % in the brine
of cucumbers [31]), fermentation temperature 25 50 75 100 125 150 175 2o0 days
(approx. 5-20°C for sauerkraut, 25-35°C for veg- Time of fermentation and storage
etable juices [28]), p H value upon starting fer- pH
mentation (approx. p H 5.9-6.5 for cabbage, maxi- 4
[]
mally p H 8.5 for olives) and the redox potential.
-1
Finally, however, growth of organisms other than
those inoculated may occur, which may have an
40
influence on the properties of the final product.
As an example, Fig. 6 shows the p H values of
39
sauerkraut fermented and stored in small pouches
(content approx. 560 g) following a method de- 8
scribed earlier [32]. The fermentation was started
at 19°C and after 7 days the products were cooled
to 4°C and stored. Every point on the curves in
Fig. 6 is the mean value of 4 - 1 0 different samples
and the vertical lines represent the maximum and
minimum values. The samples in Fig. 6A were
spontaneously fermented and those in Fig. 6B
:>5 50 75 100 125 150 175 200 days
were inoculated with 10 7 cfu g I Leuconostoc
Time of fermentation and storage
mesenteroides. It can be seen that compared to
Fig. 6. Fermentation of sauerkraut in small pouches. (A)
the spontaneous fermentation the use of starter spontaneous fermentation; (B) fermentation after inoculation
cultures resulted in mild products of high uni- of Leuconostoc mesenteroides.
formity, but during storage the inoculated organ-
isms were overgrown mainly by Lb. plantarum,
which resulted in a further decrease of pH and moved or destroyed by microorganisms during
the uniformity between the different samples as- fermentation processes.
similated to those of the spontaneously fer- For instance, roots of cassava (Manihot escu-
mented products [15,32]. To date, growth of Lb. lenta Crantz) contain large amounts of cyanogly-
plantarum or other LAB can only be prevented cosides, yet cassava represents the staple food of
by pasteurizing the fermented sauerkraut at a over 500 million people in developing countries.
previously defined degree of acidity. Because of the high level of toxicity, most pro-
Several plants possess naturally occurring un- cesses for preparing traditional cassava-based
desirable substances or toxins which can be re- foods include more or less intensive stages of
 265

CH2OH CH3
I H3C
H) 0 O--C--CN CH3
\
Linamarase =.. r
CH3
,w HO--C--CN c.=o
Glucose I - HCN /
CH3 H3C
H OH

Linamarin Acetone Acetone

cyanohydrin

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

Fig. 7. Hydrolysisof linamarin.

fermentation to eliminate these substances phytate content of fermenting lupins (Lupinus

[34,35], but additionally for starch breakdown,
acidification and flavour development [33]. The
most important cyanoglycoside is linamarin (96%
of the total content) which can be destroyed in
the presence of linamarase, released from the
plant cells when the structure is damaged. Lina-
marase is a /3-glycosidase which catalyses the
hydrolysis of linamarin into glucose and acetone
cyanohydrin (Fig. 7). It would appear that the
amount of plant-origin enzyme or the acid pH
conditions caused by the fermentation do not
permit the complete breakdown of linamarin.
Therefore, ten lactic acid bacteria were screened
for linamarase activity after culture on MRS cel-
lobiose. Six of the tested strains displayed linama-
rase activity, including strains of Lb. plantarum,
Streptococcus lactic, Leuconostoc mesenteroides
and Pediococcus pentosaceus. The strongest lina-
marase activity was measured in Lb. plantarum,
especially in strain A6 which was isolated from
retted cassava. In order to determine the most
suitable carbon substrate for maximum linama-
rase production, it was found that linamarase
seems to be a constitutive enzyme in Lb. plan-
tarum A6 and that the amount of enzyme can be
increased by induction (Table 9) [35].
Furthermore, useful reductions in oligosaccha-
ride and phytate concentrations were observed in
fermenting peas, lentils and chick peas with a
mixture of two strains of Lb. acidophilus. Strains
of Lb. buchneri and Lb. fermenti were useful as
well, but they adversely affected contents of ly-
sine, methionine and riboflavin [36]. In an addi-
tional investigation, growth of Lb. acidophilus
and Lb. buchneri was related to decreases in
albus cv. Multolupa)[37].
The softening of fermenting or fermented veg-
etables is one of the most important economical
problems of the pickle manufacturers. The soft-
ening process can be caused by a multitude of
factors, depending on the raw material as well as
on the applied technology. Up till now it is not
clear whether strains of LAB are significantly
involved in these softening processes, lndeed, the
presence of a pectinesterase and an endopoly-
galacturonase activity was found to be present in
a strain of Lb. plantarum [38,39], but to date this
has not been confirmed by other researchers.
Given the assumption that selected strains of
Leuconostoc mesenteroides are suitable to im-
prove p r o d u c t quality and uniformity of
sauerkraut [15,41], research was undertaken to
find bacteriocin-producing strains with a selective
advantage over competing but sensitive strains.
Since these investigations failed, a new approach
Table 9
Linamarase activities of Lactobacillusplantarum A6 cultured
on different carbon sources [35]
Carbon source Linamarase
(U (g dry biomass)- t)
Cellobiose 35.5
Melibiose 12.1
Lactose 6.2
Starch 5.6
Sucrose 3.4
Maltose 1.5
Glucose 1.5
266
was developed using a paired starter-culture sys-
tem, consisting of a nisin-resistant L. mesen-
teroides strain and a nisin-producing Lactococcus
lactis strain, both of which were isolated from
c o m m e r c i a l s a u e r k r a u t f e r m e n t a t i o n s [40].
W h e t h e r the presence of nisin can actually pro-
mote and extend the dominance of heterofermen-
tative L. mesenteroides in a natural fermentation
has still to be proven [41].
The production of table olives, particularly of
green table olives, involves a lye treatment in
order to destroy a part of the extremely bitter-
tasting oleuropein, followed by a washing step to
remove excessive lye (for technological details see
[42,43]). The lye treatment leads to a relatively
high starting pH, a loss in nutrients and forma-
tion of some antimicrobial substances derived
from oleuropein. In addition, the fermentation is
influenced by the more or less high content of
phenolic glycosides, particularly in black olives.
To date, bacteriophages have not been found
to be a problem in vegetable fermentation. This
may be due to the fact that pure-culture fermen-
tations have only seldomly been used. Thus, in
the event that a starter is infected with phage,
strains of the naturally occurring lactic flora will
take over and carry out the fermentation. How-
ever, when pure cultures are used extensively for
vegetable fermentations, bacteriophage infections
could become a problem, especially in cases where
large pieces, like cucumbers, are fermented in a
brine. However, in the fermentation of products
like sauerkraut or silages, the absence of liquid
will preclude the dissemination of the phage.
Malolactic fermentation in wine
Red and white wines are commonly manufac-
tured by alcoholic fermentation of musts pre-
pared from grapes of defined varieties of Vitis
vinifera. In the cooler winegrowing regions, matu-
ration of the grapes occurs very late in the last
weeks of September to October, resulting in wines
with high acidity and low pH. In cooler years,
these wines are often tart; therefore acid reduc-
tion by chemical or biotechnological means is a
common procedure in wine technology. The lat-
ter method, known as malolactic fermentation
CO0 - COO -
i I
OH-- C--H + H+ b.~ H-- C--OH + CO 2
I
H -- C -- H CH 3
CO0 -
Fig. 8. T r a n s f o r m a t i o n o f m a l i c a c i d to lactic acid a n d c a r b o n
dioxide.
Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022
(MLF), was discovered at the end of the 19th
century by Muller-Thurgau. He described the
transformation of L-malic acid to L-lactic acid and
carbon dioxide by LAB (Fig. 8). Malolactic fer-
mentation occurs naturally towards the end of or
after alcoholic fermentation. It may be delayed or
be absent due to inappropriate pH, temperature
or SO 2 and alcohol content, or even by phage
[44].
Since the beginning of the 1980s, commercial
starter cultures for the induction of MLF have
been available, consisting of strains of Lb. plan-
tarum, Lb. hilgardii and Leuconostoc oenos as
single- or multiple-strain preparations [1]. Malo-
lactic fermentation is more commonly used in red
wines but recently also increasingly in white wines
[45].
The advantages of inducing malolactic fermen-
tation by inoculation include the better control
over time of onset and rate of completion of
M L F as well as over the strain of LAB that
carries out the process [44]. The latter point is
significant because malolactic fermentation is not
only a process of acid reduction. Depending on
the LAB involved, the following advantages could
be obtained:
(i) contribution to the biological stability [46];
(ii) flavour modification;
(iii) contribution to complexity (structure,
body);
(iv) decrease in organic N-compounds, result-
ing in better storage stability [1];
(v) decrease in aldehydes and ketones, per-
mitting a limited application of SO 2 [47].
To meet all the different above-mentioned re-
quirements, three groups of selection criteria were
defined to develop or improve starter cultures for
 267

Table 10 the induction of malolactic fermentation in wine

Selection criteria for lactic acid bacteria for induction malo- (Table 10).
lactic fermentation in wine ~ Today, active starter cultures are available.
Most of them consist of strains of LAB, prefer-
1st order criteria
ably L e u c o n o s t o c oenos, with high malolactic ac-
* resistant to low pH
* resistant to ethanol tivity and high tolerance to low p H and ethanol.
* tolerant to low temp eratures The wine conditions necessary to induce a MLF
* limited metabolism of hexoses and pentoses are summarized in Table 11.
2nd order criteria The cultures are offered as fresh, frozen or

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

* count of living organisms after propagation in a standard-
ized medium
* time for propagation in a standardized medium
* yield by propagation in a standardized medium
* kinetics of survival in a standardized wine
* kinetics of malate degradation in a tartraic buffer (pH 4.5)
and in a standardized wine
3rd order criteria
* limited interactions with yeasts of the alcoholic fermenta-
tion
* limited interactions with other lactic acid bacteria
* phage resistance
* resistant to SO2
* resistant to pesticides
* no formation of biogenic amines
* citrate metabolism under aerobe and anaerobe conditions
* potential to form diacetyl and acetoine
* limited potential to form volatile acids from hexoses and
pentoses
* probably no formation of acetic acid
* limited metabolism of organic acids of the wine (e.g. succi-
nate)
* no glycerine degration
* sensorial alterations of the wine
a Krieger, S.A., personal communication.
freeze-dried preparations, but from a commercial
point of view freeze-dried cultures are definitely
preferred. This is due to the fact that fresh starter
cultures have to be produced and sold directly in
the winegrowing regions. In the case of frozen
starters, long-distance distribution is very difficult
because the maintenance of the specified temper-
atures is hard to guarantee.
Especially for smaller wineries, easy handling
of the starter cultures is essential because there
are no microbiologists present to perform precul-
turing or reactivation. However, still the direct
inoculation of the cultures into wine can fail due
to a sharp decrease in the number of viable
organisms, mainly as freeze-dried preparations of
L e u c o n o s t o c oenos strains [45]. Studies were per-
formed to obtain a better adaptation of the bac-
teria to the hostile wine environment. The results
led to the suggestion to preculture the malolactic
starter cultures, as according to the procedure
given in Fig. 9. The procedure described is spe-
cific for the reactivation of Vino-starter cultures

Table 11
Conditions necessary to induce malolactic fermentation in wine

Wine type Condition Limiting conditions Normal conditions Ideal conditions

White wine Temperature < 15°C 16-18°C > 18°C
pH < 3.1 3.2 > 3.2
Alcohol 13.5% 12.5-13.5% < 12.5%
Total SO 2 > 30 ppm 15-30 ppm No SO 2
Inoculation During active At the end of After yeast fermentation
yeast fermentation alcoholic fermentation when wine is still turbid

Red wine Temperature < 15°C 16-18°C > 18°C

pH < 3.1 3.1 > 3.1
Alcohol 14% 13-14% < 13%
Total SO 2 > 30 ppm 15-30 ppm No SO 2
268

(Condimenta, Stuttgart)which contain 2 - 5 × 10 j] strain collection ]-~ pre-propagation control ]

cfu g J. During reactivation, the bacterial culture ' I
L_cult ..... dium~ [__9 . . . . lum ] I control and regulation ]
increases from approximately 5 × 10 ") to 5 × 10 ~
cfu ml-~. This means an inoculation of the wine
with 5 × 10 7 cfu ml ~. The main component of I st°r"i"ng Or°"agat'°n i<
the reactivation mixture is a specially selected I
yeast hydrolysate. I wast wa,e _ so aratio° I
The development of malolactic starters which
I
I o,st , ....
can be applied without any pretreatment is one of I

Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022

the most important requirements for comprehen- I .oe.,ree.,no 1
sive acceptance in the wineries. I
I 'r°eze- r '°g I
I
L storing I
Production of starter cultures [
control ]
__T
To meet the requirements of the modern food L s,an~arOiziog ]
industry, starter cultures are produced in high-
1 . . . . i. . . . terial }~_. mixing
engineering biotechnical plants under strictly de-
fined conditions. Figure 10 gives a general I
overview of the production of freeze-dried starter
cultures for sausage fermentation, but because of L__ starter c u l t u r e ~ - - ~ _ control 1

the complexity it is impossible to discuss the Y

[ f...... torage ]
process here in detail. The propagation of the
Fig. 10. I.arge-scale production of starter cultures.
microorganisms may occur by traditional batch

fermentation or by semi-continuous or continu-

1 ous fermentation with or without cell recycle.
50 I grape juice l 50 i water or wine dissolve bacteria
[);is;fo~Jrl/( d
~r he lted ] + reactivation
mixture
he,it 1o 65 C
(100 - 200 g)
in I I water
(ca. 22 C)
60 min
However, the method of propagation exerts an
influence on the productivity of the installed fer-
menter capacity, the counts of surviving microor-
ganisms after separation and lyophilisation as well
mix, as on the physiological activities of the produced
adjust to pH 4 0
cool 10 25 C cells. For detailed information see Metz [50].
Freeze drying is a very expensive step in the
production of starter cultures, but in the fields of
rehydrated
iuice/water/wine-n utrient-mix bacteria application discussed here, freeze-dried starters
solution are undoubtedly preferred. Frozen-starter prepa-
rations are on the market, but the argued advan-
mix after cooling
tage of faster activation in the fermenting sub-
bacteria preculture strate has no practical relevance. The most im-
48 to 72 hours at
ca. 22 'C portant advantages of the freeze-dried starter
(s~rong CO, development
foam island bn the surface preparations are the excellent storage stability
SaLlerkraut odour)
and the easy handling during storage, distribution
and application. Desirable storage conditions for
1 freeze-dried starters are freezing temperatures
bacteria solution inoculate ]
into 10.000 I wine
l below - 1 5 ° C and the absence of oxygen and
Fig. 9. Reactivation of freeze-dried starter cultures for the humidity. Under these conditions, the optimal
induction of malolactic fermentation. activity of the starter culture will be guaranteed

for 8 months (Gewiirzmfiller, Stuttgart). Higher
temperatures of up to 20°C for a few days and
following a repeated storage at freezing tempera-
tures has no significant influence on the stability,
so that freeze-dried starter cultures may be dis-
tributed without cooling.
Concluding r e m a r k s
Lactic acid-fermented foods have proven their
wholesome value for thousands of years and are
accepted without restrictions by the consumer.
The LAB involved possess numerous metabolic
properties which are responsible for their suc-
cessful application as starter cultures in various
fields of food production.
Whereas the use of starter cultures in veg-
etable fermentation until now is limited to very
special applications, the controlled induction of
MLF in wine is more and more accepted even in
the more conservative winegrowing regions. In
sausage fermentation, there is no doubt that the
use of starter cultures ensures that the aims of
the fermentation process are achieved more safely
than in the traditional fermentation by the natu-
ral microflora.
Despite all the efforts and progress in the field
of application discussed here, however, we are far
removed from having a complete understanding
of the interrelationships between microbiology,
technology and internal and external factors in-
fluencing the fermentation processes. For further
selection and improvement of starter cultures we
need better knowledge of the ecological factors in
the fermenting substrates, an improved under-
standing of the role of microorganisms in the
formation of aroma, flavour, texture and appear-
ance of the foods, and, last but not least, about
the genetics of LAB involved in these fermenta-
tions.
Most of the physiological properties which
hitherto are recognized to be essential for the
different food fermentations are present in the
various species of lactobacilli. Therefore, it is a
task to combine or enhance the desired proper-
ties. This can be achieved by using multiple-strain
cultures or by the construction of new strains
269
using recombinant D N A technology. Several of
the desired properties are plasmid-encoded traits,
which facilitates genetic manipulation. However,
the use of genetically modified microorganisms in
food production is a controversial issue in public
opinion. In the fields of application discussed
here this situation is of special interest, because
normally the final products will contain living
microorganisms. Regardless of the legal implica-
Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022
tions at this time in Germany, it is inconceivable
to apply genetically modified microorganisms in
food production. Given this situation and know-
ing there is a pool of naturally available strains of
LAB, even experts ask the question whether ge-
netic engineering is required for improving starter
cultures within the next years [51].
References
1 H a m m e s , W.P. (1990) Bacterial starter cultures in rood
production. Food Biotechnol. 4, 383-397.
2 Holzapfel, W.H. and Hammes, W.P. (1989) Die Bedeu-
tung moderner biotechnologischer Methoden fiir die
Lebensmittelherstellung. Biotechnologie in der Agrar- und
Erniihrungswirtschaft Berichte fiber Landwirtschaft. 201.
Sonderheft. Verlag Paul Parey, H a m b u r g und Berlin.
3 Huis in't Veld, J.H.J., Hose, H., Schaafsma, G.J., Silla, H.
and Smith, J.E. (1990) Health aspects of food biotechnol-
ogy. In: Processing and Quality of Foods, Vol. 2. Food
Biotechnology: Avenues to Healthy and Nutritious Prod-
ucts (Zeuthen, P., Cheftel, J.C., Eriksson, C., Gormley,
T.R., Linko, P. and Paulus, K., Eds.), Elsevier Applied
Science, London and New York.
4 Marshall, V.M.E. and Henry, C.J.K. (1990) Nutritional
benefits of food b i o t e c h n o l o g y - - T h e micro-organisms'
contribution. In: Processing and Quality of Foods. Vol. 2.
Food Biotechnology: Avenues to Healthy and Nutritious
Products (Zeuthen, P., Cheftel, J.C., Eriksson, C., Gorm-
[ey, T.R., Linko, P. and Paulus, K., Eds.), Elsevier Applied
Science, London and New York.
5 Gilliland, S.E. (1990) Health and nutritional benefits from
lactic acid bacteria. FEMS Microbiol. Rev. 87, 175-188.
6 PooI-Zobel, B.L., Mfinzner, R., Neudecker, C. and
Holzapfel, W. (1992) Antimutagenic and antigenotoxic ef-
fects of lactic acid bacteria. In: Les bact&ies [actiques.
Actes du Colloque L A C T I C 91. Adria Normandie. Centre
de Publication de l'Universit~ de Caen, France.
7 Rice, S.I., Eitenmiller, R.R. and Koehler, P.E. (1976)
Biologically active amines in food: A review. J. Milk Food
Technol. 39, 353 358.
8 Ten Brink, B., Damink, C., Joosten, H.M.L.J. and Huis
in't Veld, J.H.J. (1990) Occurrence and formation of bio-
270
logically active amines in foods. Int. J. Food Microbiol. 11,
73-84.
9 Buckenhiiskes, H.J., Sabatke, I. and Gierschner, K. (1992)
Zur Frage des Vorkommens Biogener Amine in milch-
sauer fermentiertem Gemiise. Critical Review. Die indus-
trielle Obst- und Gemiiseverwertung 77, 255-263.
10 Liicke, F.-K. (1985) Fermented sausages. In: Microbiology
of Fermented Foods (Wood, B.J.B., Ed.), Elsevier Applied
Science, London and New York.
11 Liicke, F.-K. Briimmer, J.-M., Buckenhiiskes, H.J., Gar-
rido Fernandez, A., Rodrigo, M. and Smith, J.E. (1990)
Starter culture development. In: Processing and Quality of
Foods. Vol. 2. Food Biotechnology: Avenues to Healthy
and Nutritious Products (Zeuthen, P., Cheftel, J.C., Eriks-
son, C., Gormley, T.R., Linko, P. and Paulus, K., Eds.),
Elsevier Applied Science, London and New York.
12 Hammes, W.P. (1992) The application of lactic acid bacte-
ria in meat fermentation. In: Les Bact~ries Lactiques.
Actes du Colloque LACTIC 91. Adria Normandie. Centre
de Publication de l'Universit~ de Caen, France.
13 Buckenhiiskes, H. (1991) Starterkulturen fiir die Roh-
wurstproduktion--eine Standortbestimmung. Fleisch 45,
163-172.
14 Kiinsch, U., Schfirer, H. and Temperli, A. (1989) Biogene
Amine als Qualit~itsindikator von Sauerkraut. XXIV.
Tagung der Deutschen Gesellschaft fiir Qualit~itsfor-
schung, Kiel.
15 Schneider, M. (1988) Zur Mikrobiologie von Sauerkraut
bei der Vergfirung in verkaufsfertigen Kleinbeh~iltern. Dis-
sertation Hohenheim University, Stuttgart.
16 lncze, K. (1986) Technologie und Mikrobiologie der un-
garischen Salami. Fleischwirtschaft 66, 1305-1311.
17 Curic, M., Obradovic, D., Oljacic, E. and Kosic, M. (1992)
Criteria for selection of lactobacilli for starters for fer-
mented sausages. Tehnologija Mesa 33, 28-30.
18 Hammes, W.P., Bantleon, A. and Min, S. (1990) Lactic
acid bacteria in meat fermentation. FEMS Microbiol. Rev.
87, 165-174.
19 Liicke, F.-K., Popp, J. and Kreutzer, R. (1986) Bildung von
Wasserstoffperoxid durch Laktobazillen aus Rohwurst und
Briihwurstaufscbnitt. Chem. Mikrobiol. Technol. Lebensm.
10, 78-81.
20 Nes, I.F. and Sorheim, O. (1984) Effect of infection of a
bacteriophage in a starter culture during the production of
salami dry sausage: A model study. J. Food Sci. 49, 337
340.
21 Trevors, K.E., Holley, R.A. and Kempton, A.G. (1984)
Effect of bacteriophage on the activity of lactic acid starter
cultures used in the production of fermented sausage. J.
Food Sci. 49, 650-653.
22 Schillinger, U. and Liicke, F.-K. (1989) Einsatz von
Milschs~iurebakterien als Schutzkulturen bei Fleischer-
zeugnissen. Fleischwirtschaft 69, 1581-1585.
23 M~ikel~i, P.M. (1992) Fermented sausage as a contamina-
tion source of ropy slime-producing lactic acid bacteria. J.
Food Protect. 55, 48-51.
24 Buckenhiiskes, H., Aabye Jensen, H., Andersson, R., Gar-
rido Fernfindez, A. and Rodrigo, M. (1990) Fermented
vegetables. In: Processing and Quality of Foods. Vol. 2.
Food Biotechnology: Avenues to Healthy and Nutritious
Products (Zeuthen, P., Cheftel, J.C., Eriksson, C., Gorm-
ley, T.R., LInko, P. and Paulus, K., Eds.), Elsevier Applied
Science, London and New York.
25 Fleming, H.P., McFeeters, R.F. and Thompson, R.L. (1983)
Test for susceptibility of fermented vegetables to sec-
ondary fermentation. J. Food Sci. 48, 982-983.
26 Daeschl, M.A. and Fleming, tt.P. (1984) Selection of lactic
acid bacteria for use in vegetable fermentations. Food
Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022
Microbiol. 1,303-313.
27 Kardos, E. (1975) Herstellung und Haltbarmachung von
GemiisesMten. Flfissiges Obst 42, 488--497.
28 Buckenhiiskes, H. and Hammes, W.P. (1990) Starterkul-
turen bei der Verarbeitung von Obst und Gemiisc. Bio-
Engineering 6, 34-42.
29 Buckenhiiskes, H.J. (1992) Advances in vegetable fermen-
tation. In: Les Bactdries Lactiques. Actes du Colloque
LACTIC 91. Adria Normandie. Centre de Publication de
I'Universitd de Caen, France.
30 Daeschl, M.A., Andersson, R.E. and Fleming, H.P. (1987)
Microbial ecology of fermenting plant materials. FEMS
Microbiol. Rev. 46, 357-367.
31 Rodrigo, M. and Lazaro, M.J. (1990) Controlled fermenta-
tion of cucumbers. In: Processing and Quality of Foods.
Vol. 2. Food Biotechnology: Avenues to Healthy and Nu-
tritious Products (Zeuthen, P., Cheftel, J.C., Eriksson, C.,
Gormley, T.R., Linko, P. and Paulus, K., Eds.), Elsevier
Applied Science, London and New York.
32 Buckenhfiskes, It. (1984) Untersuchungen der technolog-
ischen Rahmenbedingungen fiir ein neues Verfahren zur
Herstellung von Sauerkraut in Kleinbeh~iltern. Disserta-
tion ttohenheim University, Stuttgart.
33 Oyewole, O.B. (1991) Fermentation of cassava for lafun
and fufu production in Nigeria, Food Lab. News 7, 29-31.
34 Westby, A. (1991) Strategies for research into the mecha-
nisms of cyanide reduction during the fermentation of
cassava. Food Lab. News 7, 24-28.
35 Giraud, E., Gosselin, L. and Raimbault, M. (1992) Degra-
dation of cassava linamarin by lactic acid bacteria.
Biotechnol. Lett. 14, 593-598.
36 Camacho, L., Sierra, C., Marcus, D., Guzman, E., An-
drade, M., Campos, R., Diaz, N., Parraguez, M. and
Trugo, L. (1991) Nutritional improvements of commonly-
consumed vegetables fermented by cultures of Lactobacil-
lus spp. Alimentos 16, 5-[1.
37 Camacho, L., Sierra, C., Marcus, D., Guzman, E., An-
drade, M., Campos, R., Von B~ier, D. and Trugo, L. (1991)
Nutritional quality of lupine (Lupinus albus cv. Multolupa)
as affected by lactic acid fermentation. Int. J. Food Micro-
biol. 14, 277-286.
38 Sakellaris, G. and Evangelopoulos, A.E. (1989) Produc-
tion, purification and characterization of extracellular
pectinesterase from Lactobacillus plantarurn (str. Ba 11).
Biotechnol. Appl. Biochem. 11, 503-507.
39 Sakellaris, G., Nikolaropoulos, S. and Evangelopoulos,

A.E. (1989) Purification and characterization of extracellu-
lar polygalacturonase from Lactobacillus plantarum strain
BA 11 J. Appl. Bacteriol. 7, 77-85.
40 Harris, L.J., Fleming, H.P. and Klaenhammer, T.R. (1992)
Characterization of two nisin-producing Lactococcus lactis
subsp, lactis strains isolated from a commercial sauerkraut
fermentation. Appl. Environ. Microbiol. 58, 1477-1483.
41 Harris, L.J., Fleming, H.P. and Klaenhammer, T.R. (1992)
Novel paired starter culture system for sauerkraut, consist-
ing of a nisin-resistant Leuconostoc mesenteroides strain
and a nisin-producing Lactococcus lactis strain. Appl.
Environ. Microbiol. 58, 1484-1489.
42 De Castro, A. and Garrido Fernandez, A. (1992) Fermen-
tation of table olives by lactic acid bacteria: perspectives.
In: Les Bact6ries Lactiques. Actes du Colloque LACTIC
91. Adria Normandie. Centre de Publication de I'Uni-
versit~ de Caen, France.
43 Garrido Fernandez, A. (1990) Controlled fermentation of
green table olives. In: Processing and Quality of Foods.
Vol. 2. Food Biotechnology: Avenues to Healthy and Nu-
tritious Products (Zeuthen, P., Cheftel, J.C., Eriksson, C.,
Gormley, T.R., Linko, P. and Paulus, K., Eds.), Elsevier
Applied Science, London and New York.
271
44 Uonvaud-Funel, A. (1992) La fermentation malolactique.
In: Les Bact~ries Lactiques. Actes du Colloque LACTIC
91. Adria Normandie. Centre de Publication de I'Uni-
versit6 de Caen, France.
45 Krieger, S.A., Hammes, W.P. and Henick-Kling, T. (1990)
Management of malolactic fermentation using starter cul-
tures. Vineyard Winery Management 16, 45-50.
46 Kunkee, R.E. (1974) Malolactic fermentation in winemak-
ing. In: The Yeasts. Vol. 3. Yeast Technology (Rose, A.H.
and Harrison, J.S., Eds.), American Chemical Society,
Washington, DC.
Downloaded from https://academic.oup.com/femsre/article/12/1-3/253/495258 by guest on 09 February 2022
47 Krieger, S.A. (1991) Neue Erfahrungen bei der Einleitung
des biologischen S~iureabbaus in Wein mit Starterkul-
turen. Deutsche Weinbau 25/26, 987-992.
48 Reference omitted.
49 Lindner, E. (1979) Toxikologie der Nahrungsmittel. Georg
Thieme Verlag, Stuttgart.
50 Metz, M. (1992) Grosstechnische Fermentation von Lac-
tobaciUus curt,atus und Pediococcus pentosaceus fiJr den
Einsatz als Starterkulturen in der fleischverarbeitenden
Industrie. Dissertation Hohenheim University, Stuttgart.
51 Metz, M. (1993) Starterkulturen--lndustrielle Herstellung
fiir die Fleischwirtschaft. Fleischwirtschaft 73, 220-227.