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 combinations 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 Leenwenhoek 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 pureculture of fungi, and in 1878 Koch obtained pure cultures of pathogenic bacteria. The objective of both Brefeld's and Koch's 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 mixedculture fermentations of the Orient. Among those described were Chinese yeast, koji, 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 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 hulgaricus. Driessen (2) demonstrated that when these species were grown separately, 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 microorganism may produce needed growth factors or essential growth compounds such as carbon or nitrogen sources beneficial to a second microorganism. 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 oryzue 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 microorganisms 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 complement each other and work to the exclusion of unwanted microorganisms. 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.

· Mixed cultures permit better utilization of the substrate. The substrate for fermented food is always a complex mixture of carbohydrates, 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 mixedculture 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 pureculture commercial fermentations involving bacteria and actinomycetes, 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 phageresistant 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 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 fermentations, 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 (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 Aspergill`'s oryzue and A. soyue. The inoculum consists of several mold strains combined, with each strain producing a desired enzyme(s). The molded rice is called l~oji 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 koji is harvested before mold sporulation starts, usually in I or 2 days. The Hot 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 hoji with molds, the paste is placed in large concrete or wooden tanks for the second fermentation. The inoculum consists of osmophilic yeasts Saccharomyces rouxli and Ca'~dida 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 pergram. 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 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.