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Appropriate starter culture technologies for small-scale

fermentation in developing countries

W.H. Holzapfel
IBM/IHT, BFE, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany
Accepted 28 December 2000
Modern food biotechnology has moved a long way since ancient times of empirical food fermentations. Preservation and
safeguarding of food are, however, still major objectives of fermentation. In addition, other aspects, such as wholesomeness,
acceptability and overall quality, have become increasingly important and valued features to consumers even in developing
countries where old traditions and cultural particularities in food fermentations are generally well maintained. Due to limitations
in infrastructure and existing low technologies, rural areas in most developing countries have not been able to keep abreast of
global developments toward industrialisation. At the same time, fermented foods play a major role in the diet of numerous
regions in Africa and Asia. In many traditional approaches, the advantages of some form of inoculation of a new batch, e.g. by
back-slopping or the repeated use of the same container (e.g. a calabash) is appreciated and generally practised. Still, the
benefits of small-scale starter culture application as a means of improved hygiene, safety and quality control, in support of
HACCP approaches, are not yet realised in small-scale fermentation operations. Approaches and considerations for the selection
of pure cultures for small-scale, low-tech applications may differ in some respects from the large-scale industrial approaches
practised since 100 years. Selection criteria should take account of the substrate, technical properties of the strain, food safety
requirements and quality expectations. Lack of experience in the application of starter cultures in small-scale operations and
under rural conditions presents a major obstacle but also an exciting challenge to food microbiologist and technologist. Culture
preservation, maintenance and distribution demand special logistic and economic considerations. Quality, safety and
acceptability of traditional fermented foods may be significantly improved through the use of starter cultures selected on the
basis of multifunctional considerations, also taking into account the probiotic concept and possibilities offered for improved
health benefits. D 2002 FAO-AGS. Published by Elsevier Science B.V. All rights reserved.
Keywords: Food preservation; Traditional fermentation; Functional properties; Lactic acid bacteria (LAB); Food safety; Selection criteria
1. Introduction
1.1. Food fermentation
Together with drying and salting, fermentation is
one of the oldest methods of food preservation. Its
importance in modern-day life is underlined by the
wide spectrum of fermented foods marketed both in
0168-1605/02/$ - see front matter D 2002 FAO-AGS. Published by Elsevier Science B.V. All rights reserved.
PII: S0168- 1605( 01) 00707- 3
Tel.: +49-721-6625-450; fax: +49-721-6625-453.
E-mail address: wilhelm.holzapfel@bfe.uni-karlsruhe.de
(W.H. Holzapfel).
International Journal of Food Microbiology 75 (2002) 197212
developing and industrialised countries, not only for
the benefit of preservation and safety, but also for their
highly appreciated sensory attributes. Fermented
foods are treasured as major dietary constituents in
numerous developing countries primarily because of
their keeping quality under ambient conditions, and
also for their safety and traditional acceptability.
As a technology, food fermentation dates back at
least 6000 years, and probably originated from micro-
bial interactions of an acceptable nature. Fermentation
has enabled our ancestors in temperate and cooler
regions to survive winter season and those in the
tropics to survive drought periods, by improving the
shelf life and safety of foods. Through the ages,
fermentation has had a major impact on nutritional
habits and traditions, on culture and on the commer-
cial distribution and storage of food. Traditional fer-
mentation process still serves as a substitute where
refrigeration or other means are not available for the
safekeeping of food.
Fermented foods can, in general, be described as
palatable and wholesome foods prepared from raw or
heated raw materials. They are generally appreciated
for attributes such as pleasant flavour, aroma, texture
and improved cooking and processing properties.
Microorganisms, by virtue of their metabolic activ-
ities, contribute to the development of characteristic
properties such as taste, aroma, visual appearance,
texture, shelf life and safety. Enzymes indigenous to
the raw materials may play a role in enhancing these
characteristics (Hammes, 1990). Through trial and
error, traditional skills have been developed for con-
trolling technical parameters during fermentation pro-
cesses. Experience has also shown that back-sloping,
or the inoculation of raw materials with a residue from
a previous batch, accelerates the initial phase of
fermentation and results in the promotion of desirable
changes during the fermentation process.
2. Development of concepts toward the use of
starter cultures
2.1. Definitions
A starter culture may be defined as a preparation or
material containing large numbers of variable micro-
organisms, which may be added to accelerate a fer-
mentation process. Being adapted to the substrate, a
typical starter facilitates improved control of a fer-
mentation process and predictability of its products
(Holzapfel, 1997). In addition, starter cultures facili-
tate control over the initial phase of a fermentation
2.2. Traditional approaches: spontaneous fermenta-
tions and back-slopping
Modern starter cultures are selected either as single
or multiple strains, specifically for their adaptation to a
substrate or raw material. Spontaneous fermentations,
i.e. processes initiated without the use of a starter
inoculum, have been applied in food preservation for
millennia and were elucidated through trial and error,
perhaps over thousands of years. The majority of
small-scale fermentations in developing countries and
even some industrial processes such as sauerkraut
fermentations are still conducted as spontaneous pro-
cesses. Various types of starter cultures and even back-
slopping are widely used in fermentation processes,
even in industrialised countries (Table 1). Spontaneous
fermentations typically result from the competitive
activities of a variety of contaminating microorgan-
isms. Those best adapted to the food substrate and to
technical control parameters, eventually dominate the
process. The production of metabolites (e.g. organic
acids) inhibitory to other contaminating microbes (e.g.
Enterobacteriaceae) may provide an additional ad-
vantage during fermentation. Bacteria typically domi-
nate the early stages of fermentation processes, owing
to their relatively high growth rate, followed by yeasts,
in substrates that are rich in fermentable sugars. In
numerous traditional processes, material from a pre-
vious successful batch is added to facilitate the initia-
tion of a new process. Through this practice of back-
slopping, the initial phase of the fermentation process
is shortened and the risk of fermentation failure re-
duced. Repeated use of back-slopping results in selec-
tion of the best-adapted strains, some of, which may
possess features that are desirable for use as starter
2.3. Inoculation to improve process control
Initiation of a spontaneous fermentation process
takes a relatively long time (2448 h), with high risk
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 198
for failure. During this early phase which is associ-
ated with the lag phase of microbial growth, contam-
inating microorganisms on raw materials, utensils and
from the environment, slowly increase in number and
compete for nutrients in order to produce metabolites.
This phase can be shortened by inoculation either
through back-slopping or with the use of selected
starter cultures. Beneficial attributes of the substrate,
consumer expectations and technical requirements
dictate to a large extent the nature of the starter
culture to be used, i.e. single-strain vs. mixed-strain
2.4. Hygiene, safety and quality considerations in sup-
port of HACCP approaches in small-scale fermen-
tation processing
Fermentation is generally considered as a safe and
acceptable preservation technology for improving the
hygienic quality and safety of foods. Failure of
fermentation processes can, however, result in spoil-
age and/or the survival of pathogens, thereby creating
unexpected health risks in food products, which
would otherwise be considered safe. Inoculation with
starter cultures does not provide an absolute guarantee
against failure of fermentation processes, nor does it
eliminate health hazards associated with pathogens,
toxinogens, toxic components or residues. Metabolic
activities of desirable fermentation microorganisms
must be supported by observing the basic principles
of Good Manufacturing Practice (GMP). This implies
the maintenance and control of technical parameters
that ensure the desired outcome of the fermentation
process. Precautions should also be taken against the
introduction or transfer of potential health hazards or
factors that are potentially detrimental to quality dur-
ing the fermentation process.
The Hazard Analysis Critical Control Point system
(HACCP), presents a scientific and systematic ap-
proach for enhancing the safety of foods, from pri-
mary production to final consumption, through the
identification, evaluation and control of hazards that
are of significance for food safety (Amoa-Awua et al.,
1998; WHO, 1995). Application of HACCP to fer-
mentation is thoroughly covered elsewhere in this
publication (Motarjemi and Asante, 2002).
3. Selection of starter cultures and their application
in small-scale fermentations
3.1. Approaches and considerations for the selection
of pure cultures
Considerations for applying starter cultures at the
household level should take into account cultural
traditions, dietary habits and raw materials, which
differ across regions and continents. Lactic fermented
cereals produced by small-scale spontaneous solid- or
semisolid-state fermentations are widely accepted and
appreciated by consumers in most African countries.
In Southeast Asia, on the other hand, traditional
fermentations are reliant on moulds as the dominant
organism, and on legume food substrates such as soya
From a technical viewpoint, cost/benefit ratios,
logistical factors and the willingness of the small-scale
processor to accept new approaches is critical in any
assessment of the feasibility of introducing the use of
starter cultures in small-scale fermentations. In addi-
Table 1
Starter cultures, typical of industrialised countries and used in various fields of food fermentation
Foodstuff Single-strain cultures Multiple-strain cultures Mixed-strain cultures Back-slopping
Sauerkraut + +
Various vegetables +
Vegetable juices +
Soy products +
Sour dough + + + +
Wine + +
Dry sausage + + +
Dairy products + + + ( )
, not used; +, applied. Source: Buckenhuskes, 1993.
Brine from a previous fermentation.
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 199
tion, a minimum set of standards or quality parameters
for the handling and maintenance of these cultures
would need to be developed for use at the artisanal
level. The introduction of starter cultures should be
considered within the context of realistic prospects for:
information transfer minimal technical adjustments to
small-scale low-tech food fermentations, applica-
tion of the HACCP system and education and training.
The prospect of applying starter cultures will
become attractive to the small-scale processor only
if benefits, such as reduction of costs (e.g. energy),
reduced fermentation times, reduced risk of spoilage
(increased shelf-life), improved process control, im-
proved sensory quality (taste, aroma, visual appear-
ance, texture, consistency), improved safety attributes
(e.g. lower risk if diarrhoea, detoxification of cassava)
and reduced preparation procedures for the final pro-
duct, are perceived.
Some LAB and yeast strains associated with fer-
mented foods, are capable of degrading antinutritional
factors, such as phytic acid and phenolic compounds.
Incorporation of these organisms into starter cultures
may, therefore, to serve upgrade the nutritional value
of foods. Furthermore, selected strains may enhance
the general benefits of spontaneous fermentation such
as improved protein digestibility and micronutrient
bioavailability, and contribute more specifically to
biological enrichment through the biosynthesis of
vitamins and essential amino acids.
In recent times, there has been a considerable focus
on the inclusion of mycotoxin-degrading strains in
starter cultures. The use of mycotoxin contaminated
raw materials for fermentation in developing countries,
poses a special challenge for the selection of strains
that are capable of mycotoxin detoxification (Adegoke
et al., 1994; Smith et al., 1994; Westby et al., 1997;
Holzapfel et al., 1998). The probiotic properties of
strains involved in food fermentations are also being
studied in light of their potential contribution to the
improvement of general health and well being.
3.2. Selection criteria for starter culture development
Spontaneous food fermentations are neither pre-
dictable nor controllable. Pure cultures isolated from
mixed populations of traditional fermented foods
exhibit a diversity of metabolic activities, which vary
even among strains. These include differences in
growth rate, adaptation to a particular substrate, abili-
ty to degrade antinutritive factors, antimicrobial pro-
perties, flavour and quality attributes and competi-
tive growth behaviour in mixed cultures (Holzapfel,
1997). Single-and mixed-strain cultures must, there-
fore, be tested at the pilot scale, prior to their use in
small-scale operations.
According to Holzapfel (1997), the introduction of
starter cultures in traditional small-scale fermentations
should incorporate considerations for improving pro-
cessing conditions and product quality through: (i)
rapid accelerated metabolic activities (acidification or
alcohol production); (ii) improved and more predict-
able fermentation processes; (iii) desirable sensory
attributes; (iv) Improved safety and reduced hygienic
and toxicological risks.
Commercial starter cultures generally originate
either from food substrates or from the processes in
which they are applied. Environmental conditions,
back-slopping, adaptation and the repeated use of
specific utensils can contribute to the selection of
microbial populations typical of a fermentation proc-
ess. The selection of suitable starter strains should take
into account their interactions in mixed cultures, with
consideration for the behaviour of these strains under
defined conditions, and within the food substrate.
Other factors, which should be considered, include:
(i) competitive behaviour, viability and survival; (ii)
antagonism against pathogens and spoilage microbes;
(iv) the rate of acid or alcohol production; (v) organo-
leptic changes; (vi) primary metabolites of fermenta-
tion; (vii) degradation of antinutritive factors; (vii) de-
toxification; (viii) probiotic features (Holzapfel, 1997).
Modern approaches incorporate considerations for
technical safety and health-promoting features in the
selection of the most optimal strain(s) for a process.
Ideally, a multifunctional strain is targeted. The ration-
ale for such novel approaches is now discussed.
3.2.1. Technical considerations
Technical aspects of starter culture development
should incorporate considerations relevant to adoption
of the starter to the substrate, the rate of acid production,
fermentation metabolites (e.g. hetero- vs. homofermen-
tation) and the ability of single or mixed strain cultures
to produce desirable sensory qualities in the fermented
product. Numerous reports indicate that Lactobacillus
brevis, L. fermentum, L. plantarum, L. reuteri, Pedio-
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 200
coccus pentosaceus and P. acidilactici exhibit superior
performance in lactic fermented cereal and vegetable
products (Steinkraus, 1996, 1997; Holzapfel, 1997;
Lee, 1997; Oyewole, 1997). This is quite possibly the
case for root crops, while the initiation of milk fermen-
tations is typically associated with Lactococcus lactis,
followed by L. casei (paracasei) and other Lactoba-
cillus spp. during maturation. Several LAB are asso-
ciated with meat and fish fermentations. L. sakei and L.
curvatus (Hammes and Hertel, 1998) have been de-
termined to be superior starter cultures for meat fer-
mentations. Leuconostoc mesenteroides frequently
dominates the early stages of most spontaneous fer-
mentations. Plant materials containing fermentable
sugars provide suitable substrates for the yeast species
Saccharomyces, Candida, Torula and Hansenula.
Although the growth rate of these yeasts is lower than
that of bacteria, such as L. mesenteroides, strains of
Saccharomyces cerevisiae eventually dominate most
spontaneous alcoholic fermentations as in the produc-
tion of African opaque beers, palm wine and Asian
beverages, such as rice wines and Indonesian tape.
Indeed, most traditional fermentations results from the
combined metabolic activities of different types of
microorganisms. Hounhouigan et al. (1994, 1999)
reported a stimulating effect of the yeast Candida
krusei on L. fermentum and L. brevis during a mixed
starter culture fermentation of the fermented maize
product, mawe.
3.2.2. Antagonism
This is the combined effect of different biological
factors, resulting from metabolic activities of micro-
organisms and their competitive interactions. Fermen-
tations involving yeasts (alcoholic fermentations of
beer and palm wines), moulds (e.g. tempe fermenta-
tions), particular bacilli [alkaline fermentations e.g. as
for dawadawa (Nigeria and Ghana), soumbala (Bur-
kina Faso), natto (Japan) or kinema (Himalayas)] and
LAB are generally recognised as safe (GRAS).
Dominant microbes of these fermentations do not
appear to be associated with any health risks. These
beneficial microorganisms serve to some extent in
safeguarding against pathogens and spoilage organ-
isms. Lactic fermented foods in particular are consid-
ered to be safe and wholesome. On the other hand,
acidification to pH values of less than 4.2 constitutes a
major safety concern in fermented foods. Recent obser-
vations, however, confirm that a number of metabo-
lites, such as acetic acid (from heterofermentative
LAB), hydrogen peroxide and bacteriocins, produced
during the fermentation process, exhibit antimicrobial
properties which may contribute to the safety of lactic
fermented foods (Table 2). Organic acids, which show
strong antagonistic effects in the undissociated form at
lower pH values, are particularly effective in inhibiting
Gram-negative bacteria, such as pathogens. Bacteriocins. Bacteriocins are antimicrobial
substances of a proteinaceous nature that are active
against closely related bacteria. They exhibit a narrow
of activity but are not active against Gram-negative
bacteria. Proteolytic enzymes present in food substrate
are capable of inactivating bacteriocins. Bacteriocins
from LAB may be classified into three structural
groupings on the basis of their physico-chemical and
Table 2
Metabolic products of lactic acid bacteria which exhibit antimicrobial properties
Product Main target organisms
Organic acids
Lactic acid Putrefactive and Gram-negative bacteria, some fungi
Acetic acid Putrefactive bacteria, clostridia, some yeasts and fungi
Hydrogen peroxide Pathogens and spoilage organisms, especially in protein-rich foods
Low-molecular metabolites
Reuterin (3-OH-propionaldehyde) Wide spectrum of bacteria, moulds and yeasts
Diacetyl Gram-negative bacteria
Fatty acids Different bacteria
Nisin Some LAB and Gram-positive bacteria, notably endospore-formers
Other Gram-positive bacteria, inhibitory spectrum according to producer strain and bacteriocin type
Source: Holzapfel et al., 1995.
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 201
antimicrobial properties (Schillinger et al., 1995; Hol-
zapfel et al., 1995).
Though relatively uncommon in fermented foods
(Olasupo et al., 1994), bacteriocinogenic LAB strains
are of special interest in view of their possible
application in food safety assurance. Bacteriocino-
genic LAB have been shown to effectively inhibit
the growth of pathogens, such as Listeria monocyto-
genes, Staphylococcus aureus, Bacillus cereus and
Clostridium dificile, even under in situ conditions
(Holzapfel et al., 1995).
3.2.3. The lactic acid isomer
The lactic acid isomer produced during fermenta-
tion is typically related to the LAB species from
which it is produced. LAB genera, examples of which
include Streptococcus, Lactococcus, Enterococcus
and Carnobacterium produce > 90% of the L(+)-
isomer as an end product of sugar fermentation.
Leuconostoc spp. and L. delbrueckii (all subspecies)
on the other hand produce D( )-lactic acid. Lactic
acid isomers produced by lactobacilli and pediococci
are species specific. The L(+)-isomer is produced by
L. casei, for example, while a racemate (DL) is
produced for L. sakei, all heterofermentative lactoba-
cilli and practically all Weissella spp.
The nature of the lactic isomer is of concern, since
high levels of the D( )-lactic acid isomer are not
hydrolysed by LDH enzymes in humans and are, thus,
capable of causing acidosis. WHO recommendations
indicate a maximum daily intake of 100 mg/kg body
weight of this nonphysiological lactic acid isomer
(WHO, 1968). There are, however, no recommended
limitations for the intake of the L(+)-lactic acid isomer.
Consumption of 1 l of a fermented gruel containing
1% of DL-lactic acid, by an individual having a 50 kg
body weight, could potentially result in the intake of
the maximum recommended levels of this nonphysio-
logical acid. L(+)-lactic acid producing strains should,
therefore, be preferentially selected for the fermenta-
tion of beverages.
3.2.4. Antinutritive factors
Antinutritive components are of particular signifi-
cance in unbalanced diets, such as cereal-based diets.
Cereal staples (maize, sorghum and millet) which are
often admixed with legumes in order to upgrade their
protein content, contain a number of antinutritive fac-
tors. Protease and amylase inhibitors, polyphenols
(from millets and sorghum) and lectin-related haemag-
glutinin activities in legumes and tannins, adversely
affect the protein and starch availability of these foods.
Furthermore, the chelating properties of phytic acid
may significantly reduce the bioavailability of miner-
als, such as calcium, iron, magnesium and zinc. These
antinutritional factors, coupled with the lysine, trypto-
phane and methionine deficiencies in cereal proteins
contribute to malnutrition in developing countries
(Holzapfel, 1997). Fermentation may serve to improve
the nutritional value of cereal staples through the
reduction of antinutritive factors, as reported for a
number of foods of plant origin (Chavan and Kadam,
1989; Lorri, 1993; Mbugua et al., 1992). Proteinase inhibitors. Lactic fermentation
has been shown to lower the levels of proteinase inhi-
bitors in cereal porridges thereby increasing the avail-
ability of essential amino acids, such as lysine, leucine,
isoleucine, methionine and even tryptophane (Kazanas
and Fields, 1981; Mbugua, 1986; Nche, 1995). It is
effective in reducing proteinase inhibitors (e.g. trypsin
inhibitor) in legumes and tannins, and disulphide cross
linkages in sorghum prolamine proteins (Hamaker et
al., 1987; Khetarpaul and Chauhan, 1989). Combined
lactic and yeast fermentations have also been shown to
improve the protein digestibility of cereal porridges
(Grahamet al., 1986; Lorri, 1993; Mbugua et al., 1992)
and may, therefore, serve to improve the protein quality
of cereal grains. LAB strains, however, differ in their
ability to degrade trypsin inhibitor under defined con-
ditions. An approximately 50% reduction in trypsin
inhibitor activity was observed in our laboratories for L.
plantarumstrain 91 and Leuconostoc sp. 106 (Table 3),
isolated from Ghanaian fermented foods. A kinetic
study of L. mesenteroides 92 activity showed that a
significant decrease in trypsin inhibitor activity was
affected only during the stationary phase of growth,
indicating the importance of the length of the fermen-
tation process (Holzapfel, 1997). Phytic acid and tannins. Phytic acid and
tannins are antinutritive components typical of cereal
and legume foods. These compounds are of concern
since they may reduce both iron and mineral bioavail-
ability in cereal and legume-based diets. Essential steps
in traditional household-level processing, such as soak-
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 202
ing, germination and lactic fermentation, may contrib-
ute to a reduction of these inhibitors. Soaking has been
shown to activate endogenous phytases in most cereals
and legumes. Although lactic fermentation has also
been shown to reduce the phytate content of white
sorghum (Svanberg and Sandberg, 1988), non-tannin
containing cereals (Svanberg et al., 1993), maize
(Lopez et al., 1983), pearl millet (Mahajan and Chau-
han, 1987; Khetarpaul and Chauhan, 1989) and idli, a
fermented cereal legume product (Reddy et al., 1986),
phytic acid degrading ability is relatively rare among
pure LAB cultures. Some L. plantarum strains are,
however, capable of degrading phytic acid on incuba-
tion at 37 jC for 120 h (Holzapfel, 1997). Phytase
activity is not detectable for Bacillus spp. associated
with the fermentation of the African locust bean Parkia
biglobosa which is used in the preparation of iru or
dawadawa(soumbala) (Aderibigbe andOdunfa, 1990). Oligosaccharides. Raffinose, stachyose and
verbascose are oligosaccharides that typically occur in
legumes and cereals, and cause flatulence, diarrhoea
and indigestion in humans. These sugars possess a-D-
galactosidic bonds which are resistant to cooking and
small-scale processing, but which can be hydrolysed by
a-galactosidases produced by a number of moulds and
by bacteria associated both with the digestive tract and
with fermented foods. Extensive studies using both
pure and mixed cultures have shown that neither
sucrose nor raffinose is utilised by the tempe mould
Rhizopus oligosporus (Sorensen and Hesseltine, 1966)
although Shallenberger et al. (1967) determined sta-
chyose to be relatively slowly hydrolysed. Soaking
(Ogun et al., 1989) and germination (Abudu and
Akinyele, 1990; Trugo et al., 1990) were determined
to be important processing steps for reducing the
oligosaccharide content of legumes. The production
of a-galactosidase by LAB species, such as L. mesen-
teroides ssp. mesenteroides and ssp. dextranicum and
Weissella paramesenteroides, appears to be variable
(Milliere et al., 1989), whilst it appears to be a con-
stitutive property for L. fermentum, L. brevis, L. buch-
neri, L. cellobiosus and L. salivarius (Mital et al., 1973)
and is probably inducible in L. plantarum (ATCC
8014) (Ahrne and Molin, 1991). L. plantarum strains
isolated fromfermented Ghanaian maize products were
able to ferment raffinose, while strains of P. acidilactici
and P. pentosaceus were unable to do so (Table 4).
3.2.5. Degradation or inactivation of natural toxins
The degradation or inactivation of toxins by pure
cultures during fermentation has received considerable
attention in recent times. Naturally occurring toxins,
such as the cyanogenic glucosides (linamarin and
lotaustralin) in cassava, may cause severe intoxications
following the consumption of rawor unprocessed bitter
cassava (Holzapfel, 1997). Detoxification during the
fermentation of cassava is brought about primarily by
microbial activity (Westby and Choo, 1994), although
endogenous linamarinase enzymes present in cassava
play a significant role in the process. Lactobacillus spp.
(Amoa-Awua et al., 1996; Olasupo et al., 1997),
Bacillus spp. (Ejiofor and Okafor, 1981; Essers et al.,
1995; Amoa-Awua and Jakobsen, 1995) and yeasts and
moulds (Hahn, 1989, Essers et al., 1995) play an
important role in cassava processing. A comparison
of the effects of spontaneous fermentation, back-slop-
ping and the use of starter cultures for the reduction of
cyanogenic glucosides in cassava (Kimaryo et al.,
2000) revealed that all three types of fermentations
contributed significantly to the detoxification of cas-
sava, with a starter culture consisting of L. plantarum
strains giving the best results.
3.2.6. Mycotoxins
Mycotoxins, in particular aflatoxins and fumonisins
pose a major risk in stored cereals, which are raw
typical materials for traditional fermented foods in
most African countries (Holzapfel, 1997). Even with
strict regulations on maximum tolerable levels, control
Table 3
Degradation of trypsin inhibitor (TI) by lactic acid bacteria isolated
from aflata in Ghana
LAB isolate Reduction of TI (mg) Percent reduction
L. plantarum 91 2.41 48.0
L. fermentum 103 1.22 24.4
Pediococcus sp. 90 0.89 17.8
Pediococcus sp. 19 1.08 21.6
Leuconostoc sp. 106 2.68 53.6
Lactobacillus sp. 41 0.65 13.0
Laactobacillus sp. 17 1.86 37.2
Lactobacillus sp. 62 1.34 26.8
Investigations were conducted in a synthetic liquid medium
containing 5 mg TI/ml. Incubations were conducted at 30 jC for 5
days. The TI concentration was determined according to Kakade et
al. (1974) with synthetic benzoyl DL-arginine-p-nitro anilide as
substrate (Holzapfel, 1997).
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 203
mechanisms for mycotoxins in developing countries
are inadequate, and can neither be applied to agricul-
tural products sold in rural markets, nor to those used at
the household or small-scale level. Numerous reports,
although still controversial, indicate that some myco-
toxins may be degraded or inactivated during cereal
fermentations. Steinkraus (1983), reported reduced
aflatoxin B
) levels during Indonesian ontjom
fermentations, while Van Veen et al. (1968) observed
that the aflatoxin content of peanut press cake was
reduced by both the ontjommould, Neurospora and the
tempe mould, R. oligosporus.
Biological detoxification of AFB
through degra-
dation has, thus, far only been proven for Flavobac-
terium aurantiacum (Line et al., 1994; DSouza and
Brackett, 1998), an organism which appears to be
wrongly classified and which has now been identified
as Nocardia corynebacterioides, a member of the
Gram-positive phylogenetic group of the Actinomy-
cetales (Holzapfel et al., unpublished data). Studies
conducted under defined conditions using single LAB
strains isolated from Ghanaian kenkey (aflata) resulted
in reduced production of Alternaria toxin (Holzapfel,
1997). In addition, some authentic LAB strains and
others isolated from fermented Turkish foods (L.
plantarum and L. pentosus) were observed to reduce
patulin concentrations by >60% in semisynthetic
medium (Arici, 1997).
3.2.7. Biogenic amines
Biogenic amines are frequently produced by amino
acid decarboxylase positive microorganisms, during
fermentation. The occurrence of biogenic amines in
traditional fermented foods has, however, been re-
ported (Nout et al., 1994; Buckenhuskes et al., 1992).
Certain LAB, such as L. buchneri, have been shown
to produce biogenic amines, such as histamine, putres-
cine, tyramine and cadaverine, in fermented products
of plant and animal origin. A number of bacteria
associated with these fermentations, however, exhibit
the potential for degrading histamine and tyramine
through the production of mono- and di-amino-oxi-
dases (Leuschner et al., 1998).
3.3. Experience with the application of starter cultures
in small-scale operations
Experience gained in the field of traditional fer-
mentation technologies has shown that the addition of
malted grains to fermentation media increases the rate
of fermentation due to the endogenous amylolytic
activity of the grains. Fermentation processes may
also be accelerated through the addition of a starter
obtained from a previous fermentation batch (back-
slopping). In traditional back-slopping, inoculum
from a previous batch of fermented dough contains
large numbers of desirable microorganisms in an
active state, which are adapted to the substrate.
Inocula consisting of a portion of a fermenting
substrate may be preserved by dehydration (air- or
sundrying) and grinding into a powder. Dehydration
enhances the viability of microorganisms over rela-
tively long periods, provided the product is maintained
in the dehydrated state. Natural preservation of micro-
organisms can also be accomplished with the use of a
carrier, such as the porous material of a gourd, fermen-
Table 4
Raffinose fermentation by selected LAB strains isolated from fermented maize products of Ghana
Species Strain nos.
Acid production
Lactobacillus plantarum 1, 7, 8, 21, 31, 38, 43, 46, 47, 64, 65, 69 + +
Lactobacillus plantarum 37, 39, 44, 53, 55, 70, 91 +
Lactobacillus plantarum 58, 60, 68
Pediococcus pentosaceus 90
Pediococcus acidilactici 100
Leuconostoc mesenteriodes ssp. 92
Mesenteroides DSM 20343
+ +
Acid production: = negative; + = weak; + += strong. Qualitative fermentation tests were performed in MRS broth (pH 6.4) containing 1%
raffinose (instead of glucose) and 0.004% chlorophenol red as indicator. Incubation was conducted at 30 jC for 3 days (Holzapfel, 1997).
Origin of strains. Nos. 121, 92: from fermenting maize (Aflata) during Kenkey production. Nos. 3147: from fermenting maize/white
cowpeas mixture (70:30). Nos. 5370: from fermenting maize/red cowpeas mixture (70:30). No. 90: from Yakeyake after fermentation. No. 91:
from Agbelima, after cooking. No. 100: from Aflata (for Ga-Kenkey) after fermentation.
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 204
tation utensils or an inoculation belt, such as that
used in Ghana for the initiation of pito beer fermenta-
tions. Although such preservation methodologies are
common to many regions and probably have a long
tradition, they have not been adequately studied. Prod-
uct groups associated with traditional inoculation
methodologies are summarised in Tables 58.
Interesting examples of mixed-culture dough inoc-
ula prepared either in the form of dried powders, flat
cakes or hard balls, are found in several Asian
countries where they are used for the inoculation of
starchy substrates in the production of alcoholic
beverages. These starters contain mixed cultures of
filamentous fungi (e.g. Amylomyces, Mucor, Rhizo-
pus, Actinomucor), yeasts (Saccharomyces spp.,
Pichia spp., Hansenula spp.) and LAB (species of
Lactobacillus and Pediococcus) (Tamang, 1998), and
are referred to by different names in accordance with
the location of production.
These old traditions in starter preparation, preser-
vation and distribution present an extremely valuables
basis for the development and application of other
types of starters in small-scale processing, and a
number of attractive challenges and benefits to the
entrepreneur: (i) the use of simple, inexpensive uten-
sils that are readily available; (ii) flexibility and sim-
plicity of maintenance and handling; (iii) minimal
losses due to fermentation failure; (iv) job creation
and income generation in rural areas; (v) handling and
storage at the household level; (vi) distribution and
sale in local markets.
3.4. Small-scale vs. large-scale industrial applica-
tions; single- vs. mixed-strain starters
Spontaneous fermentations typically result from the
competitive activities of different microorganisms.
Strains best adapted and with the highest growth rate
dominant during particular stages of the process. The
complexity and variability of microbial populations
associated with these fermentations is somewhat
reduced in back-slopping operations, where process-
ing conditions and continued recycling of a portion of
a previous batch, determines dominance of the best
adapted strains. Inoculations with a single-strain cul-
ture can eventually result in a mixed-strain fermenta-
Table 5
Starter cultures used for traditional dairy products
Product Country Raw materials Amount of inoculum Storage/starter application Microorganisms
Dahi India and
buffalo milk,
cow milk
12% (summer),
510% (winter)
Strains of one or
more of:
S. thermophilus,
L. cremoris,
L. delbruckii ssp.
L. acidophilus
Tairu Malaysia cow milk,
2.53.0% back-slopping thermophilic LAB
North Africa
cow milk,
camel milk
and wheat
1/3 (w/w) Laban zeer,
dried kishk
L. casei,
L. plantarum,
L. brevis,
Trahanas Greece,
sheep milk
and wheat
Addition on a
daily basis
S. thermophilus
L. delbruckii ssp.
bulgaricus or
L. fermentum
Kefir Russia
goat milk,
sheep milk,
cow milk
2530 g of
kefir grains
per 500 ml
kefir grains
L. brevis,
L. kefiranofaciens
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 205
tion if raw materials are not sterilised prior to in-
oculation and maintained axenic (free from foreign
microorganisms) throughout strict process control.
Single-strain cultures offer advantages of improv-
ing both process control and the predictability of me-
tabolic activities within the culture. They are, however,
relatively easily degraded by bacteriophage infection,
spontaneous mutation or through the loss of key phy-
siological properties (e.g. plasmid-mediated fermenta-
tion of lactose). Deterioration in culture performance
Table 6
Starter cultures applied in the preparation of traditional acid-leavened cereal and legume products
Product Country Raw materials Inoculum (starter) Storage conditions Microorganisms
Idli (Dosa) South India,
Sri Lanka
black gram,
Catso wheat,
maize, peas,
dried idli
L. mesenteroides,
E. faecalis,
S. cerevisiae,
Puto Philippines rice (a) leba dura
(ground slurry/
18 h, sugar),
(b) powdered
(a) neutralisation
of ground
(b) dehydration
L. mesenteroides,
E. faecalis,
S. cerevisiae
Injera (Ethiopia),
Anjeira (Sudan)
tef (Ethiopia),
(Sudan) or
other cereals
irsho (ersho)
cool place in home Candida and
other yeast spp.
Hopper (Appa) Sri Lanka rice or wheat
bakers yeast
or toddy
(fermented drink)
S. cerevisiae
toddy: mixed
Kisra Sudan sorghum vigorously
acetic acid,
A majority of these products are manufactured by spontaneous fermentation. Examples given refer to alternative options.
Lactic acid bacteria.
Table 7
Mixed starter cultures applied in the production of traditional acid- and acid-alcoholic fermented gruels
Product Country Raw materials Inoculum (starter) Storage conditions Microorganisms
Benin Maize previous
lactobacilli and
Mahewu Southern Africa Maize small portion
of whole-
wheat flour
(?) mainly
Kocho (a flour) Ethiopia ensete
(false banana)
(traditional beers)
west Africa
(maize millet)
Inoculation belt drying Lactobacilli,
Most of these products are still manufactured by spontaneous fermentation. Examples given refer to alternative options.
Lactic acid bacteria.
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 206
due to one or more of these effects adversely affects
the fermentation process. Modern equipment for the
preparation, handling and application of pure single-
strain cultures and for strict process control at all stages
of the fermentation, are not available or attainable in
most small-scale operations. In large-scale fermenta-
tions, on the other hand, consistent end product quality
is achieved over extended time periods through the
use of defined single-strain cultures and properly con-
trolled processes.
Mixed strain cultures, such as those of the ragi
type, on the other hand, are less susceptible to de-
terioration and are, thus, better suited to most small-
scale operations. Mixed strain cultures are relatively
unaffected by fluctuating conditions of handling,
storage and applications. In addition, they contribute
to a more complex sensory quality, whilst producing
favourable synergistic effects, such as the degrada-
tion of undesirable factors, flavour production and
accelerated ripening and maturation. Variation in
product quality with the use of mixed strain cultures
can be minimised through proper process control.
Although moulds play a minor role in the fermen-
tation of foods in Africa, they are of major importance
in Asian food fermentations. In Europe, traditional
mould-ripened foods are mainly restricted to blue-
mould (Penicillium roqueforti) and white-mould (P.
camemberti) cheeses and mould-ripened fermented
sausages (containing either P. nalgiovense or P. chrys-
ogenum) (Geisen, 1993).
Plant materials containing fermentable sugars pro-
vide suitable substrates for yeast species of Saccharo-
myces, Candida, Torula, Hansenula and others. These
yeasts, in particular Saccharomyces spp., are typically
associated with spontaneous alcoholic fermentations,
such as in the production of African opaque beers, palm
wine and Asian types of beverages, such as rice wines,
palm wines and Indonesian tape. Spontaneous alco-
holic fermentation of palm wines and most opaque
cereal beers. Selected strains of S. cerevisiae are used
for the industrial production of both western style and
traditional African beers. Dehydrated yeast, which is
mainly applied in bread making, is readily available on
the market throughout Africa. It is also applied in
small-scale beer brewing (Tables 6 and 7).
LAB are major importance among bacteria associ-
ated with traditional fermented foods. The largest
spectrum and richest variety of lactic fermented foods
is probably found in Africa. The association of LAB
with the human environment and their beneficial inter-
actions, both in food and in the human intestinal tract,
combined with the long tradition of lactic fermented
foods in many cultures, have led to the conclusion that
these foods may be generally recognised as safe
(GRAS). Several factors must be taken into consid-
eration when evaluating the use of LAB as starters
Table 8
Ragi-type starter cultures
Product Country Raw materials Inoculum (starter) Storage conditions Microorganisms
Tape ketan Indonesia
rice Ragi Dehydrated
(air- or
yeasts and LAB
other cereals
Krachae Thailand (bran)
rice millet
Lao-Chao China,
Tapai perlert
Rice wine
Nalyoia rice
ragi tapai
ragi samsu
Tapai ubi
tape kerccella
Baseam Ragi
Sources: Steinkraus (1996), Tamang (1998) and Merican and Quee-Lan (1989).
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 207
(Holzapfel, 1997). (i) Not all LAB are of equal techni-
cal and practical importance in food fermentations. (ii)
Lactobacillus (both homo- and heterofermentative),
Leuconostoc and, to a lesser extent, Pediococcus,
Lactococcus, Enterococcus and Weissella are the gen-
era which generally occur in traditional fermented
foods. (iii) The genus Bifidobacterium, although phy-
logenetically not related to LAB, is often grouped as
part of the LAB for its probiotic functions (Reddy and
Rivenson, 1993; Salminen et al., 1996; Holzapfel et al.,
1998). (iv) With the exception of S. thermophilus,
species of the genus Streptococcus are generally
regarded as pathogens. (v) Suitable cultures for fer-
mentation must be selected at the strain level since not
all strain of a species are equally suitable for use as
starters, nor are all equally well adapted to a food
substrate. (vi) A number of industrial lactic food
fermentations (e.g. the production of sauerkraut and
dill cucumbers) are spontaneous although technically
well controlled.
The association of certain strains of Enterococcus
faecium, E. faecalis and L. rhamnosus, with excep-
tional cases of endocarditis, should be no absolute
reason to disqualify the use of food grade strains of
these and other LAB species from their potential use in
food fermentations and even as probiotics. LAB starter
cultures are not yet commercially available for the
small-scale fermentation of traditional African foods.
Making them available to the small-scale processor,
however, poses a great challenge to both the food
microbiologist and the potential entrepreneur.
4. Handling, maintenance and distribution of
starter cultures for small-scale fermentations
4.1. Traditions in the conventional handling, preser-
vation and application of starter cultures
Perhaps the oldest traditions in the preparation,
handling and distribution of starter cultures are to be
found in the different regions of Asia (Lee and Fujio,
1999). This is particularly true for the mixed-culture
dough inocula, such as the ragi-type starter cultures
which have been used for centuries in the production
of a variety of sweet and sour alcoholic beverages and
pastes (Steinkraus, 1997; Tamang, 1998) (Table 8).
Although ragi production does not incorporate the use
of specialised equipment, ragi formulations are main-
tained proprietary by manufacturers. Powdered ragi
from a previous batch is sprinkled as an inoculum
over the paste prepared from rice flour and water and
moulded into a ball. Inoculated balls are placed on
bamboo trays and either covered with muslin cloth
(Malaysia) or with ferns (Himalayas). Microbial
growth takes place over a 25-day period under am-
bient conditions, during which gradual desiccation of
the rice balls occurs. Slow drying of ragi balls during
the rainy season, results in large numbers of Mucor
and Rhizopus spp. (Merican and Quee-Lan, 1989). In
some countries of the Near East (Egypt, Iraq) and
North Africa dried kishk or laban beer is used as an
inoculum for kishk and kuschuk production (Table 5).
Relatively little information is available on starter
culture traditions in Sub-Saharan Africa. The use of
back-slopping approaches for inoculation are, how-
ever, widespread in that region. One example of a
preserved starter is the inoculation belt, typical of
Ghana and some countries in West Africa. The inert
surface of the belt or woven rope, which consists of
flax of hennep, facilitates the preservation of essential
microorganisms during drying and storage. Sundrying
may destroy some microorganisms and thereby reduce
viable numbers, while slow and insufficient airdrying
during the rainy season may result in contamination
and poor quality starters. The shelf life of dehydrated
starters may be enhanced by storage in an airtight
Bakers yeast is used worldwide in bread baking. It
is also applied in brewing and the production of wine
at the household level. Bakers yeast is commonly used
in the fermentation of sorghum and other cereal beers
in Africa (Holzapfel, 1989).
The predominance of lactic food fermentations in
Africa and the contribution of these fermentations to
food safety deserve special attention. Low expected
turnover, distribution over extensive areas, and so-
phisticated methods for propagation and preservation
are factors which complicate LAB starter culture
development. Studies into the application of selected
LAB strains for traditional fermentations, however,
suggest sufficient potential for the use of LAB starters
in small-scale food fermentations in Africa (Holzap-
fel, 1997). (i) The traditional starter kudeme is used as
an inoculant in West Africa, for preparing the ferment-
ing cassava dough, agbelima (Amoa-Awua and Ja-
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 208
kobsen, 1996). (ii) A stable LAB-enriched starter
dough, aflata applied in the preparation of the fer-
mented maize-meal product, kenkey, produces accel-
erated acidification and contains a natural selection of
acid resistant strains (Nout et al., 1989; Nche et al.,
1994). Cabinet and drum-drying of this dough from a
54% to a 10% moisture content, was shown to en-
hance the viability of LAB strains (Nche et al., 1994),
thereby indicating the potential for its extended stor-
age and distribution as a starter. (iii) Addition of
(chalk) to fermenting substrates increases
retention of the metabolic activities of LAB. CaCO
confers this protective effect primarily through the
neutralisation of lactic acid. It is not, however, effec-
tive in inhibiting undesirable microbes, such as yeasts
and moulds. (iv) fermenting L. plantarum, isolated
from kivunde, a traditional product of the Western and
North-Western Regions of Tanzania produced safe
cyanide levels < 10 mg/kg during cassava fermenta-
tions (Kimaryo et al., 2000). Kivunde is typically
formed into small balls, (35 cm in diameter) and
air-dried, thereby preserving viable strains over ex-
tended periods.
These starter cultures may find application and
might serve to improve small-scale fermentations
even in rural areas, upon identification and selection
of suitable strains. Such approaches will, however,
only be successful if the basic principles of good pro-
cessing practices (GMP) are observed.
4.2. Logistical requirements, pragmatic approaches
and economic considerations
Apart from experience with the traditional use of
mixed culture inoculants of the ragi type in Asia,
relatively little is known about the economics and
practical constraints of starter culture supply and
distribution to small-scale processors in developing
countries. Infrastructure required for the manufacture,
distribution and storage (e.g. by refrigeration) of
starters on a continuous basis is generally available
in urban areas. This is not however the case in most
rural areas of developing countries. Information on the
logistics of starter culture distribution in developing
countries is, therefore, required. Research priorities
for starter culture development were extensively
reviewed elsewhere in this publication (Motarjemi
and Asante, 2002). Four major focal areas for research
on starter cultures were recommended by WHO and
FAO (FAO/WHO, 1996). (i) Assessment of the need
for, and feasibility of, using starter cultures. (ii)
Establishment of an appropriate level of starter culture
technology. (iii) Development of appropriate starter
culture delivery mechanisms. (iv) Selection of micro-
organisms with desirable properties.
5. Approaches toward starter cultures with
improved properties
5.1. Multifunctional considerations
On the basis of all of the foregoing discussion, it is
clear that the selection of starter cultures should take
account of properties beyond those of acid (by LAB)
or alcohol production (by yeasts). Prospects for
improving the safety, shelf life, sensory character-
istics, nutritional value and even health-promoting
properties of fermented foods by multifunctional
starter cultures, have become a reality in our time.
Modern selection techniques (Zhong et al., 1998) and
the application of molecular biological tools have
been covered elsewhere in this publication (Valyasevi
and Rolle, 2002). These tools and techniques provide
improved means for obtaining the most suitable
microbial strains for each specific substrate and sit-
uation and have opened up opportunities for the
development of tailor-made starter cultures.
5.2. Genetic improvements: pro and contra
It is doubtful whether the most modern techniques
and selection procedures would result in a multifunc-
tional strain having all desirable metabolic features.
Selected strains may be improved through the applica-
tion of genetic technologies. Recombinant DNA tech-
nology may be applied in the production of tailor-made
starter cultures which would meet technical and meta-
bolic requirements necessary for a specific fermen-
tation (e.g. accelerated acid production, improved
wholesomeness, health-promoting properties, overpro-
duction of bacteriocins or of particular enzymes ne-
cessary for degradation of undesirable factors). Unde-
sirable properties, such as mycotoxin or antibiotic
production by food-grade moulds, may be eliminated
by techniques, such as gene disruption (Geisen and
W.H. Holzapfel / International Journal of Food Microbiology 75 (2002) 197212 209
Holzapfel, 1996; Hammes and Vogel, 1990). A large
number of such optimised cultures already exist. Reg-
ulatory issues however preclude their use.
6. Conclusions
Knowledge on traditional fermentations is rapidly
increasing. Studies on microbial dynamics, substrate-
related interactions and metabolic activities of differ-
ent microbial groups, on key enzymes and on the role
of technical and other process parameters, have pro-
vided a firm basis for improvement of traditional,
small-scale and household level fermentation pro-
cesses. In addition, an understanding and application
of HACCP principles and the observance of GMP are
of vital importance. The introduction of appropriate
starter culture techniques may constitute one major
step towards improved safety, quality and security of
traditional small-scale fermentation. Artisanal level
starter culture traditions in Asia have proven feasible
over generations, and may serve as model for appli-
cation in other regions.
Focused studies toward the introduction of starter
cultures for small-scale fermentations seem more than
justified, and in fact deserve the highest priority. A
number of aspects of relevance to starter culture
production should, therefore, receive special attention.
As examples, the following should be mentioned. (i)
Information on inhibition kinetics of food-borne
pathogens by typical LAB cultures under practical,
product-specific conditions. (ii) Viability and survival
of sublethally injured enteropathogens in fermented
foods and their potential pathogenicity. (iii) The role
of bacteriocin producing LAB strains as an additional
safety factor against Gram-positive pathogens (e.g. S.
aureus) and improvement of inhibitory action, e.g. by
synergistic effects among different antimicrobial
agents and physico-chemical factors related to the
food substrate. (iv) information on the typical con-
centrations of D( )-lactic acid in traditional fer-
mented gruels and beverages (e.g. traditional beer
types), the average daily intake of such beverages,
and the relevance of D( )-lactic acid producing LAB
strains in foods typically consumed in large quantities.
(v) Growth patterns, stability and acidification poten-
tial of L(+)-lactate producing strains during small-
scale operations. (vi) Amino acid decarboxylase activ-
ity as a negative selection feature for potential starter
cultures, and mono-amino-oxidase activities as a pos-
itive selection criterion, with the aim of reducing
levels of biogenic amines in selected fermented foods.
(vii) Potential for the fermentative detoxification of
mycotoxins, approaches for strain selection and tox-
icological studies on possible degradation products.
(viii) Studies on functional (probiotic) properties of
LAB strains involved in traditional fermented foods,
development of in vitro selection methods for pro-
biotic strains, and studies on growth behaviour and
technical performance of probiotic strains.
Some data presented have been generated from
research projects that were financially supported by
the German Ministry of Nutrition, Agriculture and
Forestries (BMVEL) and the European Commission
(STD projects TS2.0267 and TS3 * CT-940344).
These are gratefully acknowledged.
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