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II. OVERVIEW
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1
Upgrading Traditional
Biotechnological Processes
M. 'I. R. Nout
TRADITIONAL FOOD FERMENTATION
The general aims of food technology are to exploit natural food
resources as efficiently and profitably as possible. Adequate and
economically sound processing, prolongation of shelf life by preserva-
tion and optimization of storage and handling, improvement of safety
and nutritive value, adequate and appropriate packaging, and maximum
consumer appeal are key prerequisites to achieving these aims.
Fermentation is one of the oldest methods of food processing. The
history of fermented foods has early records in Southeast Asia, where
China is regarded as the cradle of mold-fermented foods, and in Africa
where the Egyptians developed the concept of the combined brewery-
bakery. The early Egyptian beers were probably quite similar to some
of the traditional opaque sorghum, maize, or millet beers found in
various African countries today (11.
In technologically developed regions, the crafts of baking, brewing,
wine making, and dairying have evolved into the large-scale industrial
production of fermented consumer goods, including cheeses, cultured
milks, pickles, wines, beers, spirits, fermented meat products, and soy
sauces.
The introduction of such foreign "high-tech" fermented products to
tropical countries by early travelers, clergymen, and colonists was
followed by an accelerated demand during the early postindependence
period. Their high price ensured status, and their refined quality
guaranteed continued and increasing consumption.
In contrast, many of the traditional indigenous foods lack this image;
some may even be regarded as backward or poor people's food.
Factors contributing to such lack of appeal include inadequate grading
and cleaning of raw materials, crude handling and processing tech-
11
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12
FERMENTED FOODS
piques, and insufficient product protection due to lack of packaging.
Such unhygienic practices are easily translated into a fear of food-
borne diseases. From a nutritionist's point of view, many traditional
starchy staples are deficient in energy, protein, and vitamins. Variable
sensory characteristics (quality) and lack of durability (shelf life) reduce
convenience to the consumer: time needs to be spent selecting products
of adequate quality, whereas perishable products require frequent
purchasing and result in increased wastage. In addition, ungraded
heterogenous products, inconvenient unpacked bulk foods, or unattrac-
tive presentation inhibit consumers to develop regular purchasing
attitudes.
The contrast outlined here serves as a general guideline to the major
targets for upgrading the present status of traditional indigenous
fermented foods. The latter are part of the regional cultural heritage;
they are well known and accepted by consumers and consequently
provide an appropriate basis for development of a local food industry,
which not only preserves the agricultural produce but also stimulates
and supports agroindustrial development.
DECENTRALIZED SMALL-SCALE PROCESSES
In most African countries, 70 percent or more of the population lives
in rural areas. However, if the present trend in urbanization continues
(urban growth rates of 5 to 10 percent annually), 50 percent of the
African population will be living in cities by the year 2000. Governments
become increasingly aware that rural industrialization is a worthwhile
investment because it creates job opportunities, improves agricultural
productivity, and helps to check urbanization. But even at the present
urbanization rate, a rapidly increasing low-income population will be
located in urban areas. The resultant uncoupling in place and time of
primary production and food consumption necessitates the manufacture
of wholesome, low-cost, nutritious products that can withstand low-
hygiene handling.
Agro-allied industries are closely linked to regions of primary
production, and it is particularly in the field of food processing, with
low-cost perishable raw materials, that establishment of a rural network
of small-scale processing facilities is most appropriate. Home- or
village-scale enterprises require only modest capital investment, which
should be made available on a "soft loan" basis. Against this back-
ground, some basic process improvements that increase the appeal of
traditional fermented foods and that can be carried out by simple
means will be outlined (21.
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BIOTECHNOLOGICAL PROCESSES
BASIC PROCESSING OPERATIONS
13
In food manufacturing several operations are required to prepare
raw materials, handle and process them into products, and finally
prepare the finished product for distribution and sale by preservation
and/or packaging. One might think of sorting, grading, cleaning,
disinfection, grinding, or packaging. The establishment and success of
some indigenous enterprises in Nigeria and Kenya show that the appeal
and marketability of such products as beans, peas, gari, and spices,
formerly sold in bulk, increase significantly when they have "only"
been sorted, cleaned, graded, sometimes ground, labeled, and packaged
in simple polythene bags.
NUTRITIVE VALUE
The nutritive value of traditional fermented foods needs improve-
ment. The energy density of starch-based porridges is inadequate,
particularly when used for weaning purposes. Root crop- or cereal-
derived products have rather low protein contents, and the quality of
their protein is limited by the amount of lysine present. Various
antinutritional factors, including polyphenols, physic acid, trypsin
inhibitors, and lectins, are present in legumes and cereals.
Composite products (legume additions to starchy staples) offer an
opportunity to improve protein quantity and quality. Combinations of
simple unit operations, including roasting, germination, and fermenta-
tion, afford increased energy density in porridges and reduce antinutri-
tional factors considerably (31.
STABILIZATION OF NATURAL FERMENTATIONS BY INOCULUM
ENRICHMENT
Most traditional fermented products result from natural fermenta-
tions carried out under nonsterile conditions. The environment resulting
from the chemical composition of the raw materials fermentation
temperature, absence or presence of oxygen, and additives such as
salt and spices causes a gradual selection of microorganisms responsible
for the desired product characteristics.
The main advantage of natural fermentation processes is that they
are fitting to the rural situation, since they were in fact created by it.
Also, the consumer safety of several African fermented foods is
improved by lactic acid fermentation, which creates an environment
that is unfavorable to pathogenic Enterobacteriaceae and Bacillaceae.
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14
FERMENTED FOODS
In addition, the variety of microorganisms present in a fermented food
can create rich and full flavors that are hard to imitate when using pure
starter cultures under aseptic conditions.
However, natural fermentation processes tend to be difficult to
control if carried out at a larger scale; moreover, the presence of a
significant accompanying microflora can accelerate spoilage once the
fermentation is completed. Particularly with increased holding periods
between product fermentation and consumption when catering for
urban markets, uncontrolled fermentations under variable conditions
will cause unacceptable wastage by premature spoilage.-
Techniques to stabilize fermentations operating under nonsterile
conditions would therefore be appropriate in the control of natural
fermentations. For this purpose the use of pure culture starters,
obtained either by laboratory selection procedures or genetic engi-
neering, offers no realistic solutions because they are expensive and
require sterile processing conditions. A more feasible approach is to
exploit the ecological principle of inoculum enrichment by natural
selection. This can be achieved by the sourdough process, in which
some portion of one batch of fermented dough is used to inoculate
another batch. This practice is also referred to as `'back-slopping" or
inoculum enrichment. The resulting starters are active and should not
be stored but used in a continuous manner.
Sourdoughs from commercial sources, having been maintained by
daily or weekly transfers during 2 or more years, contain only two or
three microbial species, although they are exposed to a wide variety
of potential competitors and spoilage-causing microorganisms each
time the sourdough is mixed with fresh flour for a transfer. It can take
as long as 10 weeks of regular transfers before a sourdough population
becomes stabilized. Such populations could contain a yeast, Saccharo-
myces exiguous, and one or two Lactobacillus species, namely Lb.
brevis var. linderi II and Lb. sanfrancisco. Although the mechanism
of the stable coexistence of sourdough populations is not yet fully
understood, lack of competition for the same substrate might play an
important role. Other factors besides substrate competition, such as
antimicrobial substances produced by lactic acid bacteria, might play
an important role in the stability of such stable populations, obtained
by "back-slopping" (4~.
Similar experiments in the field of tempe manufacture showed that
the first stage of the tempe process soaking of soybeans can be
rendered more predictable in terms of acidification of the beans, by
simple inoculum enrichment. Depending on soaking temperatures,
stable soaking water populations were obtained after 30 to 60 daily
transfers, containing Leuconostoc spp. at 14° and 19°C, yeasts and
Lactobacillus spp. at 25°C, Lactobacillus spp. at 30°C, or Pediococcus
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BIOTECHNOLOGICAL PROCESSES
15
and Streptococcus spp. at 37° and 45°C. Tempe made with well-acidified
beans contained fewer undesirable microorganisms and was more
attractive (51.
Based on the same principle of inoculum enrichment, the intrinsic
microbiological safety of composite meals of cereals and legumes
can be improved significantly by lactic fermentation (6~. This offers
interesting possibilities in the manufacture of food for vulnerable
consumer groups, such as infants, malnourished patients, and the
elderly (7~.
Although development of such gradually evolved and stable fermen-
tation starters will be an attractive proposition for use in small-scale
fermentations under nonsterile conditions, they will not be the most
appropriate in all cases. This is exemplified by the sauerkraut (lactic acid
fermented cabbage) fermentation, during which flavor development is
determined by a succession of Leuconostoc and Lactobacillus species
occurring during the course of the fermentation. Practical experience
in the sauerkraut industry in the Netherlands has shown that carryover
of previous sauerkraut into a fresh batch of cabbage will cause a rapid
domination of homofermentative Lactobacillus spp., which should
normally only dominate during the final stage of fermentation. The
result is an excessively sour-tasting product that lacks the flavor
otherwise produced by the heterofermentative Leuconostoc and Lacto-
bacillus spp.
In the exercise of upgrading traditional food fermentation techniques,
it would therefore be worthwhile to investigate the effect of inoculum
enrichment on product characteristics and consumer acceptance.
MULTISTRAIN DEHYDRATED STARTER
A different tool to stabilize fermentations under nonsterile conditions
is the use of multistrain dehydrated starters, which can be stored at
ambient temperatures, enabling more flexibility. Such homemade
starters are widely used in several Asian food fermentations. Examples
are the manufacture of tempe (mainly from soybeans) and tape (from
glutinous rice or cassava). Indonesian traditional tempe starters (usar)
are essentially molded hibiscus leaves that carry a multitude of molds,
dominated by Rhizopus spp., including the Rh. oryzae and Rh.
microsporus varieties. Instead of using usar, Indonesian tempe produc-
tion is increasingly carried out with factory-prepared "pure" starters
consisting of granulated cassava or soybean fiber carrying a mixed
population of Rhizopus species (51. These starters are more homoge-
nous and their dosage is convenient, but because they are manufactured
under nonsterile conditions, some are heavily contaminated with
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16
FERMENTED FOODS
spoilage-causing bacteria and yeasts. This requires quality monitoring
of the inoculum and of the fermentation process in which it is used.
Other examples of durable home-prepared starter materials used in
Asian food fermentations are Indonesian ragi and Vietnamese men
tablets (81. Depending on their specific purpose, these dehydrated
tablets, prepared from fermented rice flour, contain mixed populations
of yeasts, molds, and bacteria. Ragi tablets can be stored up to 6
months and constitute a convenient starter material for application in
home and small-scale industrial fermentations of rice or cassava, for
example.
Especially in the fermentation of neutral pH, protein-rich substrates,
such as legumes, one should be extremely careful with the use of
substandard inoculum. If the process lacks factors that control micro-
bial development, pathogens may survive or produce toxins in such
products. Tempe manufacture is a good example of a process with
intrinsic safety. The preliminary soaking of the beans results in an
acidification that inhibits the multiplication of bacterial contaminants
during the mold fermentation stage. Also, antimicrobial substances of
Rhizopus oligosporus would play a protective role against outgrowth
of several genera of microorganisms. Moreover, near-anaerobic condi-
tions and microbial competition during the fermentation stage, and the
usual cooking or frying of tempe prior to consumption, strongly reduce
the chances of food-borne illness (51.
Nevertheless, the introduction of fermentation processes in regions
where they are not traditionally mastered requires adequate guidance,
supervised processing, and monitoring of product safety.
ENZYME PRODUCTION BY KOJ/ TECHNIQUE
Not only microorganisms but also enzymes play an important role
in the manufacture of traditional fermentation processes. In cassava
processing the naturally occurring enzyme linamarase is able to
degrade potentially toxic cyanogenic glycosides (e.g., linamarin). This
enzymatic detoxification has always been an integral part of traditional
cassava fermentations, such as in Sari and lalun. Under certain
conditions the detoxification of linamarin is accelerated by linamarase
addition (91. It is conceivable that there will be commercial applications
for the enzymatic process of linamarin decomposition, which could be
used to detoxify cassava without having to ferment it; the result would
be a neutral and bland-havored product.
Enzyme sources for African traditional beer brewing are mostly
germinated sorghum and millet varieties, whereas sorghum and millet
malts possess adequate diastatic power with cx-amylase, resulting in
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BIOTECHNOLOGICAL PROCESSES
17
poor conversion of dextrins into maltose (101. The availability of cheap
technical-grade B-amylase preparations could lead to the development
of novel brewing processes utilizing home-grown starch sources instead
of imported barley malt.
In East Asia, koji is used as a source of enzymes in the manufacture
of soy sauce and rice wine. Koji is obtained by solid-substrate
fermentation of cereals or soybeans with fungi (e.g., Aspergillus o~yzoe
and Asp. soyne). Depending on the particular substrate to be degraded,
selected strains of molds are used, often as mixed cultures. Their
enzymes include amylases, proteases, and cellulolytic enzymes. During
fermentation the enzymes are accumulated into the koji. The enzymes
produced are subsequently extracted from the koji using brine solutions.
Koji fermentations are carried out in East Asia at a small home scale,
as well as in the large-scale industrial manufacture of soy sauce
and rice wine ( 1 1 ). Although mycotoxin-producing molds such as
Aspergillus Jqavus and Asp. parasitious occur in koji as natural
contaminations, they have not been observed to produce aflatoxins
under the given conditions.
The principle of fungal solid-substrate fermentation may be used to
prepare enzyme concentrations for conversion of starch, detoxification
of cyanogenic glycosides, and other applications.
DRY MATTER BALANCE
Food fermentation is advantageously used for food preservation and
to obtain desirable flavor and digestibility. However, some processes
are rather wasteful. For instance, prolonged soaking and microbial
respiration of organic matter may lead to considerable losses of valuable
raw material dry matter. Examples can be found in the traditional
process of ogi manufacture (fermented maize cake) and the tempe
process, during which up to 30 percent of the raw material may be lost
by leaching during soaking steps. Encouraging research has been
carried out by Banigo et al. ( 12) in the field of Nigerian ogi manufacture,
aimed at reducing these raw material losses by omitting soaking stages.
It would certainly be worthwhile to investigate dry matter balances of
traditional fermentations with a view to reducing losses of raw material
by implementing "dry" instead of"wet" processing.
IMPLEMENTATION
No matter how much research is carried out on improved traditional
processes or novel products, the ultimate aim is implementation.
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18
FERMENTED FOODS
Unfortunately, a wide gap exists between research data published in
scientific journals and the practice of food processing. Much attention
should be given to the extent of usefulness of new products to the end
user. To this effect, not only should the sensory, nutritional, and other
quality characteristics of newly developed products or processes be
taken into account, but they should also be integrated with sound price
calculations, market surveys, and extension efforts. Only a competitive
process has good chances of being implemented.
In conclusion, the importance of a business-oriented approach and
close contact between researchers and food processors, working
together toward mutual benefit, must be stressed.
REFERENCES
1. Hesseltine, C. W. 1981. Future of fermented foods. Process
Biochemistry 16:2-13.
2. Bruinsma, D. H., and M. J. R. Nout. 1990. Choice of technology
in food processing for rural development. Paper presented at the
symposium `'Technology and Rural Change in Sub-Saharan Africa,"
Sussex University, Brighton, U.K., Sept. 27-30, 1989. In: Rural
Households in Emerging Societies: Technology and Change in Sub-
Saharan Africa. M. Haswell, and D. Hunt (Eds.~. New York: Berg
Publishers.
3. Nout, M. J. R. 1990. Fermentation of infant food. Food Labora-
tory News 6~2120:10-12.
4. Spicher, G. 1986. Sour dough fermentation. Chemie Mikrobiolo-
gie Technologie der Lebensmittel 10~3/4~:65-77.
5. Nout, M. J. R., and F. M. Rombouts. 1990. Recent developments
in tempe research. Journal of Applied Bacteriology 69~51:609-633.
6. Nout, M. J. R. 1991. Ecology of accelerated natural lactic
fermentation of sorghum-based infant food formulas. International
Journal of Food Microbiology 12~2/31:217-224.
7. Mensah, P., A. M. Tomkins, B. S. Drasar, and T. J. Harrison.
1991. Antimicrobial effect of fermented Ghanaian maize dough. Journal
of Applied Bacteriology 70~3~:203-210.
8. Hesseltine, C. W., R. Rogers, and F. G. Winarno. 1988.
Microbiological studies on amylolytic Oriental fermentation starters.
Mycopathologia 101~31:141-155.
9. Ikediobi, C. O., and E. Onyike. 1982. The use of linamarase in
gari production. Process Biochemistry 17:2-5.
10. Nout, M. J. R., and B. J. Davies. 1982. Malting characteristics
of finger millet, sorghum and barley. Journal of the Institute of Brewing
88: 157-163.
.
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BIOTECHNOLOGICAL PROCESSES
19
11. Fukushima, D. 1989. Industrialization of fermented soy sauce
production centering around Japanese shoyu. Pp. 1-88 in: Industrializa-
tion of Indigenous Fermented Foods. K. H. Steinkraus (Ed.~. New
York: Marcel Dekker, Inc.
12. Banigo, E. O. I., J. M. de Man, and C. L. Duitschaever. 1974.
Utilization of high-lysine corn for the manufacture of ogi using a new,
improved processing system. Cereal Chemistry 51:559-572.
OCR for page 48
48
FERMENTED FOODS
been isolated and identified in sourdough leaven. The lactobacillus
species has a preference for maltose and uses the maltose phosphorylase
pathway to metabolize the sugar, whereas T. holmii grows on glucose
but not on maltose, so that both develop in a dough where the amylases
hydrolyze starch to maltose.
The basic biochemical changes that occur in sourdough bread
fermentation are (1) acidification of the dough with lactic and acetic
acids produced by the lactobacilli and (2) leavening of the dough with
carbon dioxide produced by the yeast and the lactobacilli. Typical
flavor and aroma development can be traced to biochemical activities
of both lactobacilli and yeasts. The chewy characteristic of sourdough
bread may be due to the production of bacterial polysaccharides by
the lactobacilli.
NIGERIAN OGI (KENYAN Udl)
Nigerian ogi is a smooth-textured, sour porridge with a flavor
resembling that of yogurt. It is made by lactic acid fermentation of
corn, sorghum, or millet. Soybeans may be added to improve nutritive
value. Ogi has a solids content of about 8 percent. The cooked gel-
like porridge is known as "pap."
The first step in the fermentation is steeping of the cleaned grain for
1 to 3 days. During this time the desirable microorganisms develop
and are selected. The grain is then ground with water and filtered to
remove coarse particles. After steeping, the pH should be 4.3. Optimum
pH for ogi is 3.6 to 3.7. The concentration of lactic acids may reach
0.65 percent and that of acetic acid 0.11 percent during fermentation.
If the pH falls to 3.5, it is less acceptable.
Ogi is a naturally fermented product. A wide variety of molds,
yeasts, and bacteria are present initially. Lb. plantarum appears to be
the essential microorganism in the fermentation. Following depletion
of the fermentable sugars, it is able to utilize dextrins from the corn.
Saccharomyces cerevisiae and Candida mycoderma contribute to the
pleasant flavor.
NIGERIAN GARI
Nigerian gari is a granular starchy food made from cassava (Manihot
utilissima or M. esculenta) by lactic acid fermentation of the grated
pulp, followed by dry-heat treatment to gelatinize and semidextrinize
the starch, which is followed by drying. Cassava tubers are washed,
peeled, and grated. An inoculum of 3-day-old cassavajuice or fermented
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LACTIC ACID FERMENTATIONS
49
mash liquor is added. The pulp is placed in a cloth bag, excess water
is squeezed out, and the pulp undergoes an anaerobic acid fermentation
for 12 to 96 hours. Optimum temperature is 35°C. When the phi of
the mash reaches 4.0, with about 0.85 percent total acid (as lactic acid),
the gari has the desired sour flavor and a characteristic aroma. In
village processes, further moisture may be removed, and the pulp is
then toasted (semidextrinized) in shallow iron pots and dried to less
than 20 percent moisture. Village-processed gari has a carbohydrate
content of about 82 percent with 0.9 percent protein. Lactic, acetic,
propionic, succinic, and pyruvic acids have been identified in Sari,
with aldehydes and esters providing the aroma.
For consumption the gari is added to boiling water, in which it
increases in volume by 300 percent to yield a semisolid plastic dough.
The stiff porridge is rolled into a ball (10 to 30 grams wet weight) with
the fingers and dipped into stew.
PHILIPPINE BALAO BALAO
Balao balao is a lactic acid fermented rice-shrimp mixture, generally
prepared by blending cooked rice, whole raw shrimp, and solar salt
and then allowing the mixture to ferment for several days or weeks,
depending on the salt content. The chitinous shell becomes soft, and
when the fermented product is cooked, the whole shrimp can be eaten.
With a salt concentration of 3 percent added to the rice-shrimp
mixture, the pH falls to an organoleptically desirable value of 4.08,
with titratable acidity reaching 1.32 percent acid (as lactic acid) in 4
days.
Balao balao made with 3 percent salt is best in color, odor, flavor,
texture, and general acceptability and is the least salty. Balao balao
offers a basic method of preservation for cereal-shrimp-fish mixtures.
When properly packed to exclude air, sufficient acid is produced to
preserve the products without resorting to high-temperature cooking.
MEXICAN PULQUE
Pulque is a white, acidic, alcoholic beverage made by fermentation
of juice of Agave species, mainly A. atrovirens or A. americana, the
century plants. It has been a national Mexican drink since the time of
the Aztecs. Pulque plays an important role in the nutrition of low-
income people in the semiarid regions of Mexico. The essential
microorganisms in the pulque fermentation are Lb. plantarum, a
heterofermentative Leuconostoc, Sac. cerevisiae, and Zymomonas
mobilis.
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FERMENTED FOODS
The heterofermentative Leuconostoc plays the essential role of
producing dextrans, which contribute a characteristic viscosity to
pulque and also increase the acidity of the agave juice very rapidly,
inhibiting growth of other less desirable bacteria. Lb. plantarum
contributes to the final acidity of pulque. Sac. cerevisiae appears to
be a major producer of ethanol, but Z. mobilis is considered to be
the most important ethanol producer in pulque. Under anaerobic
conditions, Zymomonas transforms 45 percent of the glucose to
ethanol and carbon dioxide. It also produces some acetic acid,
acetylmethylcarbinol, and some slime gums, which may contribute to
the viscous nature of traditional pulque.
Soluble solids in the fresh agave juice decrease from 25-30 percent
to 6.0 percent in pulque. The pH falls from 7.4 to 3.5-4.0. Total acid
increases from 0.03 percent to 0.4-0.7 percent (as lactic acid). Sucrose
decreases from 18.6 percent to less than 1 percent. Ethanol increases
from O percent to 4-6 percent (v/v). The B vitamins are present in
nutritionally important quantities, with ranges reported as follows (in
milligrams per 100 grams): thiamine, 5 to 29; niacin, 54 to 515;
riboflavin, 18 to 33; pantothenic acid, 60 to 335; p-aminobenzoic acid,
10 to 12; pyridoxine, 14 to 23; and biotin, 9 to 32.
EGYPTIAN KISHK, GREEK TRAHANAS, AND TURKISH
TARHANAS
Egyptian kishk, Greek trahanas, and Turkish tarhanas are mixtures
of sheep's milk yogurts and parboiled wheat. Tomato, tomato paste,
or onion are sometimes added. In all cases the milk or buttermilk
undergoes a typical lactic acid fermentation in which the pH ranges
from 3.5 to 3.8 and titratable acidity is 1.3 to 1.8 percent (as lactic
acid). Proportions of wheat to yogurt range from 2:1 to 1:3. The wheat
is parboiled at some stage in the process. In its simplest form the wheat
is added directly to the yogurt and the mixture is boiled until the wheat
has absorbed the free moisture. The mixture is cooled and formed into
biscuits that are sun dried. If the wheat is ground prior to mixing with
the yogurt, the fines are discarded because they harden the final
product.
In Egypt the principal microorganisms reported in kishk are the
heterofermentative Lb. brevis and the homofermentative Lb. cased and
Lb. plantarum. In Cyprus sheep's milk yogurt contains principally S.
thermophilus and Lb. bulgaricus. Dried kishk and trahanas are not
hygroscopic and can be stored in open jars for several years without
deterioration. They also are well balanced nutritionally.
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LACTIC ACID FERMENTATIONS
OTHER FOODS
51
Lactic acid fermentation also plays an essential role in the production
of Indonesian tempe, a vegetable (soybean) protein meat substitute the
texture of which is provided by mycelium of Rhizopus oligosporus,
which overgrows and knits the soaked, partially cooked cotyledons
into compact cakes that can be sliced thinly and deep fried or cut into
chunks and used in soups in place of meat. The essential part played
by lactobacilli occurs during the initial soaking when the pH falls from
about 6.5 to between 4.5 and 5.0. The lower pH facilitates growth of
the mold and prevents development of undesirable bacteria that might
spoil the tempe.
In Chinese soy sauce (Japanese shoyuJ and Japanese miso and
related meat-flavored, amino acid peptide sauces and pastes, the
essential microorganism for amylolytic, proteolytic hydrolysis of the
soybean-wheat or soybean-rice or barley substrates is Aspergillus
oryzae. Following overgrowth of the substrate by the mold, the koji is
subsequently allowed to ferment in approximately 19 percent salt brine
for the sauces and 6 to 13 percent salt for the pastes. Lactobacilli
grow and lower the pH to about 4.5, which then allows the osmophilic
yeast Sac. rouxii to grow and produce some ethanol. The ethanol
combines with organic acid in the substrate, producing esters that
contribute to the agreeable havor and aroma.
Given the fact that these acid fermentation techniques are simple,
effective, and inexpensive, their application in developing countries
should be encouraged.
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6
Mixed-Culture Fermentations
Clifford W. Hesseltine
Mixed-culture fermentations are those in which the inoculum always
consists of two or more organisms. Mixed cultures can consist of
known species to the exclusion of all others, or they may be composed
of mixtures of unknown species. The mixed cultures may be all of one
microbial group all bacteria or they may consist of a mixture of
organisms of fungi and bacteria or fungi and yeasts or other combina-
tions in which the components are quite unrelated. All of these
combinations are encountered in Oriental food fermentations.
The earliest studies of microorganisms were those made on mixed
cultures by van Leeuwenhoek in 1684. Micheli, working with fungi in
1718, reported his observations on the germination of mold spores on
cut surfaces of melons and quinces. In 1875 Brefeld obtained pure-
culture of fungi, and in 1878 Koch obtained pure cultures of pathogenic
bacteria. The objective of both Brefeld's and Kochts studies was to
identify pathogenic microorganisms. They wanted to prove what
organism was responsible for a particular disease. Thus, part of Koch's
fame rests on his discovery of the cause of tuberculosis.
An early paper on mixed-culture food fermentation was an address
by Macfadyen (1) at the Institute of Brewing, in London, in 1903
entitled, "The Symbiotic Fermentations," in which he referred to
mixed-culture fermentations as "mixed infections." Probably this
expression reflected his being a member of the Jenner Institute of
Preventive Medicine. About half of his lecture was devoted to mixed-
culture fermentations of the Orient. Among those described were
Chinese yeast, koVi, Tonkin yeast, and ragi.
Mixed cultures are the rule in nature; therefore, one would expect
this condition to be the rule in fermented foods of relatively ancient
origin. Soil, for example, is a mixed-organism environment with
protozoa, bacteria, fungi, and algae growing in various numbers and
kinds, depending on the nutrients available, the temperature, and the
s2
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MIXED-CULTURE FERMENTATIONS
53
pH of the soil. Soil microorganisms relate to each other some as
parasites on others, some forming substances essential to others for
growth, and some having no effect on each other.
ADVANTAGES
Mixed-culture fermentations offer a number of advantages over
conventional single-culture fermentations:
· Product yield may be higher. Yogurt is made by the fermentation
of milk with Streptococcus thermophilus and Lactobacillus bulgaricus.
Driessen (2) demonstrated that when these species were grown sepa-
rately, 24 mmol and 20 mmol, respectively, of acid were produced;
together, with the same amount of inoculum, a yield of 74 mmol was
obtained. The number of S. thermophilus cells increased from 500 x
106 per milliliter to 880 x 106 per milliliter with L. bulgaricus.
· The growth rate may be higher. In a mixed culture one microorgan-
ism may produce needed growth factors or essential growth compounds
such as carbon or nitrogen sources beneficial to a second microorgan-
ism. It may alter the pH of the medium, thereby improving the activity
of one or more enzymes. Even the temperature may be elevated and
promote growth of a second microbe.
· Mixed cultures are able to bring about multistep transformations
that would be impossible for a single microorganism. Examples are
the miso and shoyu fermentations in which Aspergillus oryzae strains
are used to make koVi. Koji produces amylases and proteases, which
break down the starch in rice and proteins in soybeans. In the miso
and shoyu fermentations, these compounds are then acted on by lactic
acid bacteria and yeast to produce flavor compounds and alcohol.
· In some mixed cultures a remarkably stable association of microor-
ganisms may occur. Even when a mixture of cultures is prepared by
untrained individuals working under unsanitary conditions, such as in
ragi, mixtures of the same fungi, yeasts, and bacteria remain together
even after years of subculture. Probably the steps in making the starter
were established by trial and error, and the process conditions were
such that this mixture could compete against all contaminants.
· Compounds made by a mixture of microorganisms often comple-
ment each other and work to the exclusion of unwanted microorgan-
isms. For example, in some food fermentations yeast will produce
alcohol and lactic acid bacteria will produce lactic acid and other
organic acids and change the environment from aerobic to anaerobic.
Inhibiting compounds are thus formed, the pH is lowered, and anaerobic
conditions are developed that exclude most undesirable molds and
bacteria.
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FERMENTED FOODS
· Mixed cultures permit better utilization of the substrate. The
substrate for fermented food is always a complex mixture of carbohy-
drates, proteins, and fats. Mixed cultures possess a wider range of
enzymes and are able to attack a greater variety of compounds.
Likewise, with proper strain selection they are better able to change
or destroy toxic or noxious compounds that may be in the fermentation
substrate.
· Mixed cultures can be maintained indefinitely by unskilled people
with a minimum of training. If the environmental conditions can be
maintained (i.e., temperature, mass of fermenting substrate, length of
fermentation, and kind of substrate), it is easy to maintain a mixed-
culture inoculum indefinitely and to carry out repeated successful
fermentations.
· Mixed cultures offer more protection against contamination. In
mixed-culture fermentations phage infections are reduced. In pure-
culture commercial fermentations involving bacteria and actinomy-
cetes, invariably an epidemic of phage infections occurs, and the
infection can completely shut down production. Since mixed cultures
have a wider genetic base of resistance to phage, failures do not occur,
often because if one strain is wiped out, a second or third phage-
resistant strain in the inoculum will take over and continue the
fermentation. In such processes, especially with a heavy inoculum
of selected strains, contamination does not occur even when the
fermentations are carried out in open pans or tanks.
· Mixed-culture fermentations enable the utilization of cheap and
impure substrates. In any practical fermentation the cheapest substrate
is always used, and this will often be a mixture of several materials.
For example, in the processing of biomass, a mixed culture is desirable
that attacks not only the cellulose but also starch and sugar. Cellulolytic
fungi along with starch- and sugar-utilizing yeasts would give a more
efficient process, producing more product in a shorter time.
· Mixed cultures can provide necessary nutrients for optimal
performance. Many microorganisms, such as the cheese bacteria,
which might be suitable for production of a fermentation product,
require growth factors to achieve optimum growth rates. To add the
proper vitamins to production adds complications and expense to the
process. Thus, the addition of a symbiotic species that supplies the
growth factors is a definite advantage.
DISADVANTAGES
Mixed-culture fermentations also have some disadvantages.
· Scientific study of mixed cultures is difficult. Obviously, it is more
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MIXED-CULTURE FERMENTATIONS
55
difficult to study the fermentation if more than one microorganism is
involved. That is why most biochemical studies are conducted as
single-culture fermentations because one variable is eliminated.
· Defining the product and the microorganisms employed becomes
more involved in patent and regulatory procedures.
· Contamination of the fermentation is more difficult to detect and
control.
· When two or three pure cultures are mixed together, it requires
more time and space to produce several sets of inocula rather than just
one.
· One of the worst problems in mixed-culture fermentation is the
control of the optimum balance among the microorganisms involved.
This can, however, be overcome if the behavior of the microorganisms
is understood and this information is applied to their control.
The balance of organisms brings up the problem of the storage and
maintenance of the cultures. Lyophilization presents difficulties
because in the freeze-drying process the killing of different strains'
cells will be unequal. It is also difficult, if not impossible, to grow a
mixed culture from liquid medium in contrast to typical fermentations
on solid mediums, without the culture undergoing radical shifts in
population numbers. According to Harrison (3), the best way to
preserve mixed cultures is to store the whole liquid culture in liquid
nitrogen below -80°C. The culture, when removed from the frozen
state, should be started in a small amount of the production medium
and checked for the desired fermentation product and the normal
fermentation time. Subcultures of this initial fermentation, if it is
satisfactory, may then be used to start production fermentations.
FUTURE
Mixed-culture fermentations will continue to be used in traditional
processes such as soybean and dairy fermentations. As noted above,
the extensive uses of mixed-culture fermentations for dairy and meat
products are well known as to the type of cultures used and the
fermentation process. However, there are a large number of food
fermentations based on plant substrates such as rice, wheat, corn,
soybeans, and peanuts in which mixed cultures of microorganisms are
used and will continue to be used
One example of the complex sequential interaction of two fermenta-
tions, and which employs fungi, yeast, and bacteria, is the manufacture
of miso. This Oriental food fermentation product is based on the
fermentation of soybeans, rice, and salt to make a paste-like fermented
food. Miso is used as a flavoring agent and as a base for miso soup.
There are many types of miso, ranging from a yellow sweet miso
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FERMENTED FOODS
(prepared by a quick fermentation) to a dark, highly flavored miso.
The type depends on the amount of salt, the ratio of cereals to
soybeans, and the duration of the fermentation.
The miso fermentation begins with the molding of sterile, moist,
cooked rice that is inoculated with dry spores of Aspergillus oryzoe
and A. soyae. The inoculum consists of several mold strains combined,
with each strain producing a desired enzymefs). The molded rice is
called koVi and is made to produce enzymes to act on the soybean
proteins, fats, and carbohydrates in the subsequent fermentation.
After the rice is thoroughly molded, which is accomplished by
breaking the koji and mixing, the koVi is harvested before mold
sporulation starts, usually in 1 or 2 days. The koji is mixed with salt
and soaked and steamed soybeans. This mixture is inoculated with a
new set of microorganisms, and the four ingredients are now mashed
and mixed. After the production of koji with molds, the paste is placed
in large concrete or wooden tanks for the second fermentation. The
inoculum consists of osmophilic yeasts Saccharomyces rouxii and
Candida versatilis and one or more strains of lactic acid bacteria,
typically Pediococcus pentosaceus and P. halophilus (4~. Conditions in
the fermentation tanks are anaerobic or nearly so, with the temperature
maintained at 30°C. The fermentation is allowed to proceed for varying
lengths of time, depending on the type of miso desired, but it is typically
1 to 3 months. The fermenting mash is usually mixed several times,
and liquid forms on the top of the fermenting mash.
The initial inoculum is about 105 microorganisms per gram. Typically,
3,300 kg of miso with a moisture level of 48 percent is obtained when
1,000 kg of soybeans, 600 kg of rice, and 430 kg of salt are used. When
the second fermentation is completed, aging is allowed to take place.
A number of other mixed-culture fermentations are similar to the miso
process, including shoyu (soy sauce) and sake (rice wine).
A legitimate question can be asked as to the future prospects for the
use of mixed cultures in food fermentations. What will be the effect
of genetic engineering on the use of mixed cultures? Would engineered
organisms be able to compete in mixed culture? Many laboratories are
busy introducing new desirable genetic material into a second organism.
The characteristics being transferred may come from such diverse
organisms as mammals and bacteria and may be transferred from
animals to bacteria. In general, the objective of this work involves
introduction of one desirable character, not a number. For instance,
strains of Escherichia cold have been engineered to produce insulin.
However, I suspect that it may be a long time, if ever, before a single
organism can produce the multitude of flavors found in foods such as
cheeses, soy sauce, miso, and other fermented foods used primarily
as condiments. The reason for this is the fact that a flavoring agent
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MIXED-CULTURE FERMENTATIONS
57
such as shoyu contains literally hundreds of compounds produced by
the microorganisms, products from the action of enzymes on the
substrate, and compounds formed by the nonenzymatic interactions
of the products with the original substrate compounds.
To put such a combination of genes for all these flavors into one
microorganism would, at present, be almost impossible. Second, the
cost of producing the food, which is relatively inexpensive as now
produced, would become economically prohibitive. The use of mixed
cultures in making fermented foods from milk, meat, cereals, and
legumes will continue to be the direction in the future.
Harrison (3), in his summary of the future prospects of mixed-culture
fermentations, very succinctly concluded as follows:
No claim'for novelty can be made for mixed cultures: They form
the basis of the most ancient fermentation processes. With the
exploitation of monocultures having been pushed to its limits it is
perhaps time to reappraise the potential of mixed culture systems.
They provide a means of combining the genetic properties of
species without the expense and dangers inherent in genetic
engineering which, in general terms, aims at the same effect.
REFERENCES
1. Macfadyen, A. 1903. The symbiotic fermentations. Journal of the
Federal Institutes of Brewing 9:2-15.
2. Driessen, F. M. 1981. Protocooperation of yogurt bacteria in
continuous culture. Pp. 99-120 in: Mixed Culture Fermentations. M.
E. Bushell and J. H. Slater, Eds. London: Academic Press.
3. Harrison, D. E. F. 1978. Mixed cultures in industrial fermentation
processes. Advances in Applied Microbiology 24:129-164.
4. Hesseltine, C. W. 1983. Microbiology of oriental fermented foods.
Annual Reviews of Microbiology 37:575-601.
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Representative terms from entire chapter:
lactic acid
