| Applications of biotechnology to traditional fermented foods |
Keith H. Steinkraus
Lactic acid bacteria perform an essential role in the preservation and production of wholesome foods. The lactic acid fermentations are generally inexpensive, and often little or no heat is required in their preparation, making them fuel efficient as well. Foods fermented with lactic acid play an important role in feeding the world's population on every continent.
Lactic acid bacteria perform this essential function in preserving and producing a wide range of foods: fermented fresh vegetables such as cabbage (sauerkraut, Korean kimchi); cucumbers (pickles); fermented cereal yogurt (Nigerian ogi, Kenyan uji); sourdough bread and bread-like products made without wheat or rye flours (Indian idli, Philippine puto); fermented milks (yogurts and cheeses); fermented milk-wheat mixtures (Egyptian kishk, Greek trahanas); protein-rich vegetable protein meat substitutes (Indonesian tempe); amino acid/peptide meat-flavored sauces and pastes produced by fermentation of cereals and legumes (Japanese miso, Chinese soy sauce); fermented cereal-fish-shrimp mixtures (Philippine balao balao and burong dalag); and fermented meats (e.g., salami).
Lactic acid bacteria are generally fastidious on artificial media, but they grow readily in most food substrates and lower the pH rapidly to a point where competing organisms are no longer able to grow. Leuconostocs and lactic streptococci generally lower the pH to about 4.0 to 4.5, and some of the lactobacilli and pedicocci to about pH 3.5, before inhibiting their own growth.
In addition to producing lactic acid, lactobacilli also have the ability to produce hydrogen peroxide through oxidation of reduced nicotinamide adenine dinucleotide (NADH) by flavin nucleotide, which reacts rapidly with gaseous oxygen. Flavoproteins, such as glucose oxidase, also generate hydrogen peroxide and produce an antibiotic effect on other organisms that might cause food spoilage; the lactobacilli themselves are relatively resistant to hydrogen peroxide.
Streptococcus lactis produces the polypeptide antibiotic nisin, active against gram-positive organisms, including S. cremoris, which in turn produces the antibiotic diplococcin, active against gram-positive organisms such as S. lactis. Thus, these two organisms compete in the fermentation of milk products while inhibiting growth of other gram-positive bacteria.
Carbon dioxide produced by heterofermentative lactobacilli also has a preservative effect in foods, resulting, among others, from its flushing action and leading to anaerobiosis if the substrate is properly protected.
Brining and lactic acid fermentation continue to be highly desirable methods of processing and preserving vegetables because they are of low cost, have low energy requirements for both processing and preparing foods for consumption, and yield highly acceptable and diversified flavors. Depending on the salt concentration, salting directs the subsequent course of the fermentation, limiting the amount of pectinolytic and proteolytic hydrolysis that occurs, thereby controlling softening and preventing putrefaction. Lactic acid fermentations have other distinct advantages in that the foods become resistant to microbial spoilage and toxin development. Acid fermentations also modify the flavor of the original ingredients and often improve nutritive value.
Because canned or frozen foods are mostly unavailable or too expensive for hundreds of millions of the world's economically deprived and hungry people, acid fermentation combined with salting remains one of the most practical methods of preservation, often enhancing the organoleptic and nutritional qualities of fresh vegetables, cereal gruels, and milk-cereal mixtures.
Lactic acid fermentation of cabbage and other vegetables is a common way of preserving fresh vegetables in the western world, China, and Korea (where kimchi is a staple in the diet). It is a simple way of preserving food: the raw vegetable is sliced or shredded, and approximately 2 percent salt is added. The salt extracts liquid from the vegetable, serving as a substrate for the growth of lactic acid bacteria. Anaerobic conditions should be maintained, insofar as possible, to prevent the growth of microorganisms that might cause spoilage.
The sequence of organisms that develop in a typical sauerkraut fermentation is as follows: Leuconostoc mesenteroides initiates the growth in the shredded cabbage over a wide range of temperatures and salt concentrations. It produces carbon dioxide and lactic and acetic acids, which quickly lower the pH, thereby inhibiting development of undesirable microorganisms that might destroy crispness. The carbon dioxide produced replaces the air and facilitates the anaerobiosis required for the fermentation. The fermentation is completed in sequence by Lactobacillus brevis and Lb. plantarum. Lb. plantarum is responsible for the high acidity. If the fermentation temperature or salt concentration is high, Pecicoccus cerevisiae develops and contributes to acid production.
As would be expected, the rate of completion of the fermentation depends on the temperature and salt concentration. At 7.5°C fermentation is very slow: under these circumstances, L. mesenteroides grows slowly, attaining an acidity of 0.4 percent in about 10 days and an acidity of 0.8 to 0.9 percent in a month. Lactobacilli and pediococci cannot grow well at this temperature, and the fermentation may not be completed for 6 months. At 18°C a total acidity (as lactic acid) of 1.7 to 2.3 percent will be reached, with an acetic to lactic acid ratio of 1:4, in about 20 days. At 32°C a similar activity will be reached in 8 to 10 days, with most of the acid being lactic acid produced by the homofermentative bacteria Lb. plantarum and P. cerevesiae.
Increasing the salt concentration to 3.5 percent results in 90 percent inhibition of growth and acid production for both L. mesenteroides and Lb. brevis. The ratio of nonvolatile to volatile acid produced has a marked effect on flavor, Lb. brevis producing a harsh, vinegar-like flavor and L. mesenteroides a mild, pleasantly aromatic flavor. The homofermenters Lb. plantarum and P. cerevesiae yield unacceptable products.
Korean kimchi differs from sauerkraut in two respects: it has, optimally, much less acid and it is carbonated. Chinese cabbage and radish are the major substrates; garlic, green onion, ginger, leaf mustard, hot pepper, parsley, and carrot are minor ingredients.
Kimchi is available year-round, is served three times daily, and is a diet staple along with cooked rice and certain side dishes. It accounts for about an eighth of the total daily food intake of an adult. Its popularity is largely due to its carbonation derived from fermentation with natural microflora.
Salting of the cabbage can be done at 5 to 7 percent salinity for 12 hours or 15 percent salinity for 3 to 7 hours, followed by rinsing and draining. Optimum salt concentration during kimchi fermentation is approximately 3 percent. Lower temperatures (about 10°C) are preferred to temperatures above 20°C. Optimum acidity of kimchi is 0.4 to 0.8 percent lactic acid with a pH between 4.2 and 4.5; higher acidity makes it unacceptable. Organisms isolated from kimchi include L. mesenteroides, S. faecilis, Lb. brevis, Lb. plantarum, and P. cerevesiae.
Pickling of cucumbers and other vegetables is widely practiced today. Although a variety of techniques are used, placing cucumbers in a 5 percent salt brine is a satisfactory method. The cucumbers absorb salt until there is an equilibrium between the salt in the cucumbers and the brine. Acidity reaches 0.6 to 1.0 (as lactic acid) with a pH of 3.4 to 3.6 in about 2 weeks, depending on the temperature.
In Malaysia the most common vegetables pickled are cucumbers, ginger, onion, leek, chili, bamboo shoots, and leafy tropical vegetables like mustard leaves. Young unripe fruits commonly pickled include mangoes, papaya, pineapple, and lime. In Egypt carrots, cucumbers, turnips, cauliflower, green and black olives, onions, and hot and sweet peppers are among the vegetables pickled. They are used as appetizers and served with practically every meal.
INDIAN IDLI AND DOSA
Indian idli is a small, white, acidic, leavened, steam-cooked cake made by lactic fermentation of a thick batter made from polished rice and dehulled black gram dhal, a pulse (Phaseolus mungo). The cakes are soft, moist, and spongy and have a pleasant sour flavor. Dosa, a closely related product, is made from the same ingredients, both finely ground. The batter is generally thinner, and dosa is fried like a pancake.
Idli fermentation is a process by which leavened bread-like products can be made from cereals other than wheat or rye and without yeast. The initial step in the fermentation is to wash both rice and black gram dhal. They are then soaked for 5 to 10 hours and drained. The coarsely ground rice and black gram are then combined with water and I percent salt to make a thick batter. The batter is fermented in a warm place (30 to 32°C) overnight, during which time acidification and leavening occur. The batter is then placed in small cups and steamed or fried as a pancake. The proportions of rice to black gram vary from 4:1 to 1:4, depending on the relative cost on the market.
Idli and dosa are both products of natural lactic acid fermentation. L. mensenteroides and S. faecalis develop during soaking, then continue to multiply following grinding. Each eventually reaches more than 1 x 109 cells per gram, 11 to 13 hours after formation of the batter. These two species predominate until 23 hours following batter formation. Practically all batters would be steamed by then. If a batter is further incubated, the lactobacilli and streptococci decrease in numbers and P. cerevisiae develops. L. mesenteroides is the microorganism essential for leavening of the batter and, along with S.faecalis, is also responsible for acid production. Both functions are essential for producing a satisfactory idli.
In idli made with a 1:1 ratio of black gram to rice, batter volume increased about 47 percent 12 to 15 hours after incubation at 30°C. The pH fell to 4.5 and total acidity rose to 2.8 percent (as lactic acid). Using a 1:2 ratio of black gram to rice, batter volume increased 113 percent and acidity rose to 2.2 percent in 20 hours at 29°C. Reducing sugars (as glucose) showed a steady decrease from 3.3 milligrams per gram of dry ingredients to 0.8 milligrams per gram in 20 hours, reflecting their utilization for acid and gas production. Soluble solids increased, whereas soluble nitrogen decreased. Flatulence-causing oligosaccharides, such as stachyose and raffinose, are completely hydrolyzed.
A 60 percent increase in methionine has been reported during fermentation. The increase would be of considerable nutritional importance if true, but the results conflict with earlier findings. Thiamine and riboflavin increases during fermentation and phytate phosphorous decreases have also been reported.
Philippine puto is a leavened steamed rice cake made from year-old rice grains that are soaked, ground with water, and allowed to undergo a natural acid and gas fermentation. Part of the acid is neutralized with sodium hydroxide during the last stage of fermentation. Puto is closely related to Indian idli, except that it contains no legume.
SOURDOUGH BREADS AND RELATED FERMENTATIONS
There is a close relationship between yeasts and lactic acid bacteria in sourdough breads, soy sauce, miso, and kefir. Sourdough leaven contains both yeasts and lactobacilli. The method of preparing such leavens is ancient. Wheat, rye, or other cereal grain flour is mixed with water and incubated for a few days in a warm place. Initially, a wide range of microorganisms develop, but eventually the lactic acid bacteria predominate because of their acid production. Yeasts also can survive, because they tolerate acid well. More flour is added to make a dough. This dough is then subdivided and used to make a batch of bread, while the rest of the dough is kept for future bread making. Wherever sourdough leavens have been studied, the organisms found have been similar.
The essential microorganisms in sourdough are a Lactobacillus sp. and a yeast, Torulopsis holmii. Saccharomyces inusitatus also has 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 UJI)
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 I 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 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 cassava juice or fermented 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 pH 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 gari, 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.
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.
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 I percent. Ethanol increases from 0 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.
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 shoyu) 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 oryzue. Following overgrowth of the substrate by the mold, the koVi 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. rouxli to grow and produce some ethanol. The ethanol combines with organic acid in the substrate, producing esters that contribute to the agreeable flavor and aroma.
Given the fact that these acid fermentation techniques are simple, effective, and inexpensive, their application in developing countries should be encouraged.