From MicrobeWiki, the student-edited microbiology resource
The soybean, Glycine max, belongs to the family Leguminosae, which refers to the fruits of the flowering plants, legumes. Historical and geographical evidence indicates that the soybean originated from East Asia, specifically Northern China. Soybean has been cultivated and incorporated as food and medicine into the daily lives of the Chinese for the past 5,000 years.
Soybeans were first introduced to the United States in the 18th century. Only until the 1900s did soybean become an important fruit. Based on current statistics, 150 million metric tons of soybeans are produced. The major producers of soybean are currently the Unites States, Brazil, China, India and Argentina.(2) Demands of soybeans have been significantly increasing due to its nutritious and medicinal values.(1)
Fermentation of soybean products requires a yeast, bacteria, mold, or a combination of each. Usage of bacteria, mold, and yeast gives the fermented food a special flavors, texture and aroma. During the process of fermentation, the Chinese has transformed soybean into various types of soy foods. Soy paste, soy sauce, sufu, and stinky tofu are a few soy foods derived from China. These fermentation methods and soy food preparation were gradually introduced to other Asian countries, such as Japan, Vietnam, Indonesia, and India. Each country slightly modified their soybean product, producing soy food derivatives such as miso, tempeh, natto, hananatto and hawaijar. Fermentation is a very important process that allows the utilization of microorganisms to breakdown complex compounds to yield a unique tasting and aromatic foods. Not only does process of fermentation preserve foods, fermentation also improve digestibility by breaking down proteins within various foods and have been known to enrich substrates with nutritional essentials, such as vitamins, amino acids, and fatty acids.(1) Preservation of food, specifically in a brine (fermentation of these products uses a high salt content), is how many of these products were discovered when soy beans instead of meat or fish were used most likely during the vegetarian Buddhism movement (17).
Description of Soybean
Physical characteristics of soybean seed
Soybean is a hearty plant that can be easily grown. Soybean seeds are spherical to long ovals in shape. Although most soy seeds are yellow, soy seeds come in other various seed coat colors, such as: blue, green, dark brown, purplish black, or black. Soy seed varies in size, too. The seed coat of a mature soy seed is extremely hard and water resistant so that germ that is encased within the soy seed is protected. Damages to the seed coat inhibit the seed from germinating.
Chemical composition of the soybean seed
Soybean seeds are extremely high in protein content. On average, dry soybean contains roughly 40% protein, 35% carbohydrate, 20% soybean oil, and 5% ash (non-aqueous, metal oxides).(2) Therefore, soybean has the highest protein content among legume species. Soy protein is a heat-stable protein, thus allowing soy seeds to undergo high temperature cooking and fermentation, without destroying the entire chemical composition of the soybean.
The soluble carbohydrates in soybeans are made up of various saccharine: disaccharide sucrose, trisaccharide raffinose, and tetrasaccharide stachyose. These soluble carbohydrates can easily be broken by microbes down during fermentation to create a distinct flavor, odor and texture in soy products.
Other valuable components that are found in soybean are phospholipids, vitamins, minerals, and isoflavones.(2) Asia has referred to soybeans as the “miracle beans” and the “yellow jewel.”
Cultivation of Soybean
Soybeans are successfully grown in regions with high temperature. Since the soy protein is a heat-stable protein, high temperatures cannot easily destroy the seed itself. The optimum temperatures to cultivate soybeans are 20°C to 30°C (68°F to 86°F). Temperatures below 20°C and above 40°C can inhibit growth of the soybean plants. Soybeans can be grown in soil or sand, however, soil with high content of clay is not an optimal environment to grow soybeans. Soil with high organic contents allows soybeans to perform nitrogen fixation. By establishing a symbiotic relationship with the bacterium, Rhizobium japonicum, soybeans and R. japonicum, an aerobic microbe, can break down nitrogen gas from the atmosphere into ammonia, which is a nitrogen product that is usually low in the soil
Fermentation of Soybean
Stinky tofu, or chaotofu, is a traditional Chinese dish where tofu is fermented in a stinky brine to create a specialized flavor, color and odor. Stinky tofu, along with Chinese soy cheese called sufu, is block-type fermented soy foods. During fermentation of stinky tofu and sufu, sufu is made by utilizing molds, whereas bacteria aid in the fermentation process in stinky tofu.
A bacterium is smaller than mold and their functions are significantly different. Molds are capable of multiplying under aerobic conditions. In these aerobic environments, molds produce amylases, proteases, and other hydrolases.(8) Bacteria, on the other hand, utilize and forms proteolytic enzymes made by microorganisms under anaerobic conditions. The proteolytic enzymes that are produced in the anaerobic, stinky brine partially hydrolyze the proteins in the tofu to make the soy proteins more digestible.(8) Intermediate metabolites, such as ammonia, are produced from the proteolytic enzymes, thus yielding an odorous brine or stinky brine.
The stinky brine that is used to make this distinct tofu come in different variations. The stinky brine’s ingredients can compose a combination of various vegetables and meats such as (a) amaranth leaves, bamboo shoots, and winter melon, (b) salted mustard brine with shrimp and salted egg brine, (c) fish, shrimp, and animal organs or (d) strong ammonia, which speeds up the overall fermentation process of stinky tofu.(8) Carried out in an open-fermentation process, the stinky brine allows the tofu that soaked in the brine to undergo a natural zymotic growth by producing a strong, stinky odor. The main bacterium that is involved in the fermentation of stinky tofu is the Bacillus sphaericus.
Bacillus sphaericus is an aerobic, spore-forming bacterium that is naturally found in soil.(9) B. sphaericus is part of the Bacillus family. B. sphaericus has a circular chromosome made up of 4,639,821 base pairs, with a 37% GC content and a two-copy plasmid (pBsph) of 177,642 base pairs, with a 33% GC content. Of all the 85 tRNA genes, each of the twenty of the amino acids are present as well as 10 rRNA operons in the chromosome.(9) B. sphaericus is a gram-positive bacterium, which contains a thick cell wall composed of peptidoglycan. The cell is rod-shaped and can form endospores. B. sphaericus is incapable of directly breaking down polysaccharide, thus requires an exclusive metabolic pathway that can utilize a wide variety of organic compounds and amino acids. B. sphaericus is capable of growing in the presence of oxygen, therefore utilizing oxygen as part of its aerobic cellular respiration.
After the stinky brine is inoculate with B. sphaericus, the ammonia content increases extensively due to the protein in the tofu is hydrolyzed by microbial proteases that forms amino acids.(8) Concurrently, deamination processes is carried out to form ammonia.(8) Throughout the fermentation process, the ammonia gradually increases due to the growth of more alkali-tolerant bacteria, which is more favorable over lactic acid bacteria. Due to the favorable growth of alkali-tolerant bacteria B. sphaericus, the cell count of the overall lactic acid bacteria decreases. With initial introduction of the bacterium, the pH drops from 6.5 to about 4.7 due to the production of lactic acid and to the growth of lactic acid bacteria. During the rest of the fermentation process, the pH increases slowly until it has reached pH of 7.5. The addition of B. sphaericus and lactic acid bacteria produces a meat-like texture and a unique flavor to the stinky tofu.
Miso is Japanese fermented soy bean paste or semisolid that can be served as a soup or used as a seasoning to heighten the flavor of meat and poultry. Miso is mainly made from soybean with addition of enzymes from rice, wheat, or soybean koji and salt. Koji processing utilizes a filamentous mold called Aspergillus oryzae as part of the fermentation process.
Miso comes in a variety of colors. Based on their colors, miso can be classified as white miso (butter color), red miso (reddish brown color), and light-color miso (light yellowish/ golden). The different colors of the miso are differentiated by their fermentation process.
The main components of miso are soybean, rice, or wheat, salt and water. The secondary components are koji, seasoning and nutritional enrichment ingredients, preservatives and ethanol. The soybean is rich in protein and lipids, which is suitable for miso processing. Rice is can be used miso processing because it has a high moisture uptake, low viscosity, and high rice koji enzymatic activity, which is needed to yield a strong sweet taste and aroma after the saccharization of saccharides that are abundant in soybean. Wheat is another alternative ingredient in miso processing because wheat is rich on glutamic acid, which results in stronger umami flavor, aroma and bolder miso color. Koji, or Aspergillus oryzae, is used as a starter mold, which contains medium-length hyphae with sporangiophores. Short hyphae produce stronger proteases while short hyphae produce stronger amylases.(10) As a result, sweeter miso uses A. oryzae with high amylase activity, while salty miso uses A. oryzae with high protease activity. The optimum temperature for A. oryzae growth is 30°C to 35°C, with a relative humidity of 95%. However, the optimal temperature for the digestion of protein and saccharides by the enzymes from miso koji are 45°C to 50°C for protease and 55°C to 60°C for amylase.(10) The optimal pH for A. oryzae is pH 6.0. As the pH decreases, the protease activity increases.
Steaming the soybean is required to prevent the growth of Bacillus subtilis contamination. B. subtilis can inhibit the growth of A. oryzae. After the soybean has been streamed to remove microorganisms that adhere to the surface of the soybean, salt and a brine, which is composed with yeast (Saccharomyces rouzii and Torulopsis versatilis) and a bacterium called Pediococcus halophilus, is mixed with the streamed soybean. P. halophilus is a essential in the miso processing. Pediococcus halophilus is an anaerobic, coccus-shaped, salt-tolerant bacterium.
P. halophilus. also known as Tetragenococcus halophilus, has a circular chromosome made up of 29,924 base pairs, with a 35% GC content. P. halophilus is gram-positive, non-motile, and non-spore forming. It is categorized as lactic acid bacteria, which are a group of bacteria that produces lactic acid as a metabolic end product of saccharide fermentation. P. halophilus can break down sugars, which are abundant in soybeans, by utilizing the enzyme glucose dehydrogenase. P. halophilus cannot grow in the presence of oxygen.
During the fermentation, the environment becomes suitable for salt-tolerant organisms, such as p. halophilus. The optimal temperature for bacterial growth is at 30°C. P. halophilus stops growing at 40°C. With introduction of the yeast and P. halophilus, the pH drops from 5.7 to 4.9-5.1. Throughout the fermentation process, the nitrogen concentration also increases. The desirable nitrogen concentration should be 1.51. It has been reported that yeast and P. halopohilus produced non-volatile amines, such as tyramine, histamine, and phenethlamine, which are not detected from the ingredients used to make miso.(2) The interactions between the various molds, yeast and bacteria results in acids reacting with alcohols to produce esters, which contributes to the miso’s aroma. Another interaction that occurs is that the color is produced by the interaction of the amino acids and sugars. Amino acids enhances flavor and darkens the color of miso.(2)
Traditionally made in Japan from steamed, whole soybeans (Glycine max (L)) and then fermented. It has a characteristic odor of ammonia and is covered in a so-called sticky substance (11). This viscous textured covering actually comes from poly-gamma-glutamic acid (12) or gamma-PGA within the biofilm covering the beans, a special characteristic of both the bacterium and the product (13). It is served on rice or noodles, or in salads, soups, or vegetables (17).
The production of Natto uses a unique bacterium Bacillus natto (previously classified as bacillus subtilis and is in fact a subspecies) that is a gram-positive rod that characterizes Natto because it produces a sticky/slimy covering that provides Natto with its texture . Bacillus Natto uses carbon sources such as glucose, fructose and sucrose, with sucrose in particular for production of Natto’s characteristic sticky substance (30% of the 20% of carbohydrate of a soybean is sucrose). Nitrogen sources are proteins and amino acids that soybeans possess. Bacillus Natto also requires biotin to germinate (Bacillus subtilis does not), which soybeans posses ample supply (60g/100g). (11)
Fermentation is done aerobically and because no further step is taken can be made in only a few days. First inoculation with bacteria (commercial is in spore form) at 80 degree Celsius, (although optimal germination temperature is about 40 degree Celsius this is for contamination purposes), and cooked for 30 minutes and then cooled to about 40-50 degree Celsius for multiplication. The spores are not heat-tolerant before germination, so cooling is important until they are formed. The beans are then transferred to a container, traditionally made of straw, although now bags with a perforated inner lining of polyethylene film or polystyrol containers are used. When Natto was first fermented this step of inoculation was skipped because the bacteria itself was introduced from the straw, a method that is still occasionally used today. After the initial heat shock to initiate germination, usually the beans will be at about 42 degrees Celsius with 85-90% relative humidity entering the incubator. It grows best in a neutral to slightly alkaline environment and increases from a pH of 6.4-6.8 (cooked soybean) to about 7.2-7.6 as the fermentation process takes place. An acidic substrate, pH of around 4.5, can actually have an inhibitory effect on growth. When the temperature of the soybeans reaches 48 degree Celsius a thin white film of bacteria is visible and the odor and sticky substance begin to appear. At 50 degree Celsius heating should be stopped. The humidity should be at 80-85% to ensure the production of soft enough beans (11).
The biggest change during Natto formation is the reducing sugars decrease by about 15%. Also, about 50-60% of soybean proteins are hydrolyzed to water-soluble nitrogenous compounds (also a reason that Natto can be digested more easily). Glutamic acid is mainly responsible for the umami taste. Organic acids (highest concentrations being acetic and lactic acids) also contribute to the flavor. The smell is even more complex as it is characterized by ten main volatile compounds (down from about 300 isolated). Diacetyl and pyrazines are thought to be that of the preferred aroma, where iso-valeric acid and iso-butyric acid are responsible for the unpleasant smells that mask the pleasant aroma (13). There is new evidence suggesting that the ammonia smell is actually caused by a secondary (excess) reaction that takes place if the Natto is fermented longer than it should or storage in a warm area. It is believed to have many complex ammonia releasing reactions, but one of the main reactions is the deamination of glutamate (13). Natto bacteria produce mainly proteases, but other enzymes (amylase, lipase, and cellulase) are active (11).
This variety of Japanese fermented intermediate-moisture soybean belongs to a group that is sometimes called soy nuggets. This variety of fermented products is mostly used in soups and as a flavoring (2). Although it shares it’s name with Natto the production is actually more similar to miso/soy sauce in that it is made by fermenting cooked soybeans with koji mold using Aspergullus oryzae and wheat as a culture. After the mold is incubated to form the koji starter it is mixed with the soybeans and fermented at 30-33 degree Celsius for 50 hours. After fermentation the beans are sundried to reduce the moisture to about 22 %. The dried beans are then in 15 degrees Baume brine for 8 months. Soy sauce can also be used as brine. The finished product has a strong umami flavor and a pH of 5.10-5.15, which also makes this form have a longer shelf life (11).
Hawaijar is a traditional fermented soybean indigenous to Manipur valley, India similar to Natto in that it is alkaline fermented using whole soybeans. The biggest difference between the two being that Natto uses a pure starter strain (of a subspecies that hawaijar uses) and Hawaijar uses naturally occurring microorganisms. This natural cocktail of microorganisms mainly results because the production of Hawaijar is currently done in people's houses and sold in small shops, without a way to monitor the product. Because of this variability and lack of control in starter organisms as well as varying methodologies, fermentation time, and temperature the quality is quite varied. Frequently the fermentation is unsuccessful or poor quality due to possible competition by native fermenting microflora (check out paper to find out what these are). Despite this inconsistency in quality the bacterial species present is very similar due most likely to the place of collection/production (which falls under the Indo-Burma biodiversity hotspot). Traditional Hawaijar is distinguished by it’s high pH (8.0-8.2) and stickiness and and dominant ammonia odor. Also unlike Natto, Hawaijar is not commercially produced, but is sold in small shops with product from people’s homes. The people of the Manipur valley would like to begin to commercial production, however the necessity for a more uniform product is crucial for marketability and safety (14).
Traditionally, Hawaijar is prepared by 1) Boiling soybean seeds (Glycine max (L)), and washed with hot water 2) Packed tightly in a bamboo basket layered with banana (Musa spp.) leaves or Asse Heibong (Ficus Hispida) 3) The baskets are incubated near an earthen oven or covered with gunny bags to achieve above ambient temperature. 4) After 3-5 days it will be ready (with high quality products having mucilage fiber production and dark brown color and distinct odor) (14).
Bacillus spp. (gram positive, catalase positive, motile, and endospore forming rods) are the bacteria responsible for fermentation, specifically Bacillus subtilis and Bacillus cereus and Bacillus licheniformis (present in very high numbers). There are also present in smaller numbers Staphylocuccus spp. (cocci) responsible for some fermenting. Other bacteria present in very small numbers (and not all products) are P. rettgeri and Alcaligenes spp. (14). Not much is known at this time about Hawaijar as it has never been commercialized and only recently been characterized relating to the specific microorganisms present (14). A next step would be the research in what each bacterium specifically does and what compounds are responsible for distinct characteristics.
Within this mixture of bacteria there are some potentially alarming microorganisms such as S. aureus and enterobacteriaceae. B. cereus has also been associated with types of food poisoning, however unless the Hawaijar is cooked as a curry its pathogenesis is normally inhibited by B.subtilis most likely by competition. An interesting finding was that there was a lower presence of pathogenic bacteria when traditional leaves were used versus banana, and one that scientists have indicated addressing in further research (14).
In the past decade, health-promoting effects of soy foods has been observed through various medical research. Ingestion of soybean and soy products has shown a reduction in cholesterol level, preventing certain cancers, improving vascular health, sustaining bone mineral density and mitigating and delaying of menopausal symptoms in women.(2-7)
One interesting area of research related to Bacillus sphaericus and its use as mosquito control agent. This is done in the larval stage made possible by a receptor present in the gut of mosquito larval for a binary toxin, the Bin toxin produced by the bacteria. The toxin specifically affect species of Anopheles, Culex, and Aedes, which are implicated as vectors in various infectious diseases such as malaria, West Nile virus, chikungunya, and yellow fever. The mode of action from this bacterium differs from that of chemical agents, and has been able to successfully combat insecticidal resistance in Anopheles mosquitoes and has been able to reduce incidence of filiariasis and malaria. This toxin is produced during sporulation and composed of two proteins BinA (binding component) and BinB (toxic component) both of which are necessary for anti-mosquito activity. Although this B. sphaericus has been used for years effectively, characterization for the binding site and receptor could possibly play roles is a wider range of action and a higher concentration for future mosquito control. (15)
Another direction of research using Bacillus sphaericus is in the unlikely field of construction of concrete surface treatments. Conventional methods of surface treatment to create a sort of sealant from water and other substances deleterious to the upkeep of concrete are very harmful to the environment in the production of these substances as well as need to be constantly maintained. Currently research is testing bacterial induced carbonate mineralization as an environmentally friendly way to protect concrete and other stone. Although other bacteria can be used, a pure culture of B. sphaericus was the most effective and the least amount of water penetration. The bacteria are able to induce precipitation of calcium carbonate (crystals) by the production of a urease enzyme that catalyzes the hydrolysis of urea to CO2 and ammonia (similar to ammonia production in stinky tofu), which raises the pH and carbonate concentration of the surrounding environment. A pure culture of B. sphaericus was found to be comparable to conventional methods in capillary water uptake, gas permeability, and chromatic analysis and a promising method for protection of a variety of surfaces.(16)
Although it is commonly known that soybeans have health benefits, including one of the highest content of protein for a legume (17) are are a well rounded good good to eat, there are undesirable compounds present including trypsin inhibitors, lectins, flatulence-producing compounds and well as being hard to digest. Infants are especially susceptible to these compounds, and soy meal use in baby food is controversial for this reason. A way to eliminate the majority of these effects is by fermenting soybeans to produce different foods that have similar compositions. Fermenting provides a higher calcium and B and A vitamin content, as well as reduced trypsin inhibitors and higher protein and anti-cancer effects. The positive compounds in soy are also more easily digested and absorbed following fermentation. A specific study was done to analyze these effects using aspergillus oryzae and chracterizing he quantity and quality of nutrients pre and post fermentation. They found the above to be true as well as containing probiotic effects and suitable for use in animal feeds (including young) and all of the above for human consumption. Further research could be done on the specifics of each fermented food, but with this study they demonstrated a wide array of health effects, and the incredible benefit of fermentation. (18)
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9. Hu, Xiaomin et al. “Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison of Those Closely related to Bacillus Species.” Journal of Bacteriology 190.8 (2008): 2892 – 2902.
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12. Murooka, Y. and Yamshita, M. Traditional healthful fermented products of Japan. J Ind Microbiol Biotechnol. 35: 791-798, 2008
13. Kada, S. Yabusaki, M. Kaga, T. Ashida, H. and Yoshida, K. Identification of Two Major Ammonia-Releasing Reactions Involved in Secondary Natto Fermentation. Biosci. Biotechnol. Biochem. 72: 1869-1876, 2008.
14. Jeyaram, K. Mohendro, W.S. Premarani, T. Ranjita, A.D. Selina, K.C., Talukdar, N.C. and Rohinkikumar, M.S. Molecular identification of dominant microflora associated with ‘Hawaijar’- A traditional fermented soybean (Glycine max (L.)) food of Manipur, India. International Journal of Food Microbiology. 122: 259-268, 2008.
15. Opota, O. Jean-Francois, C. Warot, S. Pauron, D. and Darboux, I. Identification and characterization of the receptor for the Bacillus sphaericus binary toxin in the malaria vector mosquito, Anopheles gambiae. Comparative Biochemistry and Physiology. Part B 149: 419-427, 2008
16. De Muynck, W. Cox, K. De Belie, and N. Verstraete, W. Bacterial carbonate precipitation as an alternative surface treatment for concrete. Construction and Building Materials. 22: 875-885, 2008
17. McGee, Harold. "On Food and Cooking the science and lore of the kitchen." New York: Scribner, 2004.
18. Hong, J.K. Lee, C.H. Kim, S.W. Aspergillus oryzae GB-107 Fermentation Improves Nutritional Quality of Food Soybeans and Food soybean Meals. Journal of medicinal food. 4: 430-435, 2004.
Edited by [Amelia Cline and Mimi Van Dang], students of Rachel Larsen