How to Make Tempeh? | Fermented Foods | Industrial Microbiology

In this article we will discuss about the manufacturing process of tempeh.

Tempeh is the only Oriental fermented product that has been extensively investigated by the scientists in the West. It originated in Indonesia and is widely consumed in the region of Malaysia and Indonesia, but it was not known elsewhere. Tempeh, or tempe kedelee as it is called in Indonesia, is made by fermenting dehulled soybeans with a mold, Rhizopus; the mycelia bind the soybean cotyledons together in a cake-like product.

The fresh tempeh has a clean, yeasty odor, but does not have the beany flavor some people find unpleasant in whole soybeans. When fried in oil, it has a pleasant flavor, aroma, and texture that are familiar and highly acceptable even to people of the Western world. Unlike most of the other fermented soybean foods, which are usually used as flavoring agents or relishes, tempeh is used as a main dish and meat substitute in Indonesia.

Because of its high protein content and universally acceptable taste and texture, tem­peh is a potential source of low-cost protein. The tempeh fermentation is rather simple and quick. Traditionally, soybeans are soaked in tap water overnight until the hulls can be easily removed by hand. Some prefer to boil the soybeans for a few minutes to loosen the hulls and then to soak the beans overnight.

After dehulling, the beans are boiled with excess water, drained, and spread for surface drying. Small pieces of tempeh from a previous fermentation, or ragi tempeh (commercial starter), are mixed with the soybeans which are then wrapped in banana leaves and allowed to ferment at room temperature for 24 to 48 hr until the beans are covered with white mycelium and bound together as a cake. The cake is either sliced thin, dipped in a salt solution, and deep fat-fried in coconut oil, or cut into pieces and used in soups.

Biochemical and microbiological studies are necessary in order to under­stand the fermentation and to develop uniform, high quality products and well-defined, economically feasible processes to manufacture them. In the late 1950s, scientists at the New York Agricultural Experiment Station, Geneva, N.Y., and at the Northern Regional Research Center, Peoria III, began to study this century-old fermentation.

As a result, a pure culture fermentation method was developed on a laboratory scale. Changes in soybeans during fermentation and the nutritional value of tempeh were studied in detail. The physiology and biochemistry of the tempeh mold were also studied. More recently, a freeze-dried tempeh starter was developed so that the tempeh fermentation can be carried out at home as easily as bread making or yogurt fermentation.

The mold used for tempeh fermentation was reported earlier to be Rhizo­pus oryzae. We have received cultures isolated from different lots of tempeh in Indonesia, and we found that only Rhizopus could make tempeh in pure culture fermentation.

Of the 40 strains of Rhizopus received, 25 are R. oligosporus Saito; others are R. stolonifer (Ehren) Vuill, R. arrhizus Fischer, R. oryzae Went and Geerligs, R. formosaensis Nakazawa, and R. achlamydosporus Takeda. Apparently, R. oligosporus is the principal species used in Indonesia for tempeh fermentation; a strain identified as R. oligosporus Saito NRRL 2710 is one of the better producers of a good product.

This strain is characterized by sporangiospores showing no striations and being very irregular in shape under any condition of growth. The sporangiophores are short, un-branched, and arise opposite rhizoids that are much reduced in length and branching. All isolates show large numbers of chlamydospores.

The utilization of various carbon and nitrogen compounds by R. oligosporus Saito NRRL 2710 was investigated by Sorenson and Hesseltine (1966). They found that the principal carbohydrates of soybeans, i.e., stachyose, raffinose, and sucrose, are not utilized as sole sources of carbon, where­as common sugars such as glucose, fructose, galactose, and maltose sup­ported excellent growth, as does xylose.

Various vegetable oils can be substituted for, sugars as sources of carbon with excellent growth. Since the soybean sugars are not utilized by R. oligosporus, and since strong lipase activity has been reported for Rhizopus cultures used in tempeh fermenta­tion it is likely that lipid materials, and particularly fatty acids, are the primary sources of energy for the tempeh fermentation.

Ammonium salts and amino acids such as proline, glycine, aspartic acid, and leucine are excellent sources of nitrogen. Other amino acids are less suitable, and tryptophan supports no growth at all. However, the fungus does not depend upon the presence of any specific amino acid in the medium for growth. Sodium nitrate is not utilized as the sole source of nitrogen.

R. oligosporus is highly proteolytic, which is important in tempeh fer­mentation because of the high protein content of the substrate. Two proteo­lytic enzyme systems were observed one has an optimum pH at 3.0 and the other at 5.5. Both enzyme systems have maximum activities at 50°-55°C and are fairly stable at pH 3.0-6.0, but rapidly denatured at pH below 2 or above 7.

The proteolytic enzyme having optimum pH at 3.0 has been purified and separated into 5 active fractions; crystalline enzymes were obtained from the 2 major fractions. In addition to high protease activity, the mold possesses strong lipase activity but low amylase activity and no detectable pectinase.

Pure culture methods of making tempeh with R. oligosporus were then developed. In many respects, the procedures are similar to those used in Indonesia. Hesseltine et al. (1963B) have carried out pure culture fermenta­tions in petri dishes, a testing procedure which proved to be very satisfacto­ry.

The preparation of soybeans for fermentation is the same as the tradi­tional manner. Later, Martinelli and Hesseltine (1964) introduced full-fat soybean grits for tempeh fermentation. Soybean cotyledons are mechanically cracked into 4 to 5 pieces. Since soybean grits absorb water easily, the soaking time can be reduced from more than 20 hr to 30 min.

Furthermore, since the hulls are removed mechanically in producing grits, much labor can be saved. The beans are boiled for 30 min, drained, cooled, and inoculated with spores of R. oligosporus, which have been grown on potato-dextro-agar slants at 28°C for 5-7 days.

The spore suspension is prepared by adding a few milliliters of sterilized distilled water to the slant. The inoculated beans are mixed, packed tightly into petri dishes, and placed in an incubator at 30°-31°C for about 20 hr. R. oligosporus does not require much aeration as do many other molds; as a matter of fact, too much- aeration may cause spore formation.

It is, therefore, important to pack the petri dishes tightly; even so, some sporulation may still occur at the edge of the dish, but it will not affect the product. This procedure can also be adopted for making tempeh either in shallow wooden or metal trays with perforated bottoms and covers or in perforated plastic bags and tubes.

Steinkraus and his coworkers (1960) suggested the use of 0.85% lactic acid as soaking water. The dehulled, soaked beans are also cooked in the acid solution. This treatment would bring the pH of the beans to a range of 4.0-5.0. At this pH range, the growth of contaminating bacteria will be inhibited, but not that of the tempeh mold. However, we have not encoun­tered bacterial growth in our process.

Because R. oligosporus produces an antibacterial agent and because this organism also has the unique characteristic of fast growth there is little chance for bacteria to gain ground before the tempeh fermentation is complete. Ko (1970) has further investigated this matter.

He purposely inoculated with different amounts of Escherichia coli, B. mycoides, Pseudomonas pyocyanea, Proteus sp. or P. cocovenenaus along with R. oligosporus in making tempeh as described by Hesseltine et al. (1963B). His results indicated that the fer­mentation is not interfered with by the presence of inoculated bacteria. Ko commented that prefermentation during soaking or addition of acid to the soaking water may not be very important in the process of tempeh fermen­tation.

The dehulling, soaking, washing, cooking, and fermenting steps em­ployed in the preparation of tempeh all contribute to loss of soybean constit­uents. Average values for these losses obtained from a number of tempeh preparations are given in Table 12.5. The total losses of solids range from 24.5-48.3%, depending on the variety and type of soybeans as well as the processes used.

The more significant differences in solid losses are in dehull­ing and cooking. Steinkraus et al. (1960) used an abrasive vegetable peeler to loosen the hull of the hydrated beans and found a loss of 17.1%, of which only 9.6% was due directly to the removal of hulls; whereas Smith et al. (1964) removed the hulls by hand and observed a loss of only 7.9%.

On the other hand, Smith et al. (1964) reported a much greater loss during cooking than Steinkraus et al. (1960). It can be explained, in part, by the fact that Steikraus et al. soaked and cooked the beans in an acid solution of pH 5, which is close to the isoelectric point of soybean proteins and may have reduced the leaching effect.

Although mechanically dehulled soybean grits are preferred for tempeh making, one disadvantage appears to be the greater solids losses during soaking. Further studies to determine the varietal effect of soybeans on the total solids losses in tempeh processing are warranted.

To prevent the loss of water soluble substances during preparation and cooking, soybeans were treated in a minimum amount of water, just enough to soak the beans thoroughly, or were sprayed with a certain volume of water before autoclaving. But Smith et al. (1964) found that when this procedure was followed, the tempeh showed less mold development and much sporulation.

The product also had an unpleasant odor and poor flavor. The presence of a water-soluble and heat-stable mold inhibitor in soybeans was suggested by Hesseltine et al. (1963A). Later, Wang and Hesseltine (1965) found that the water-soluble and heat-stable fraction of soybeans also inhibited the formation of proteolytic enzymes by R. oligosporus. There­fore, soaking and cooking of soybeans in excess water which is later dis­carded are essential in making tempeh.

The tempeh fermentation is characterized by its simplicity and rapidity. The lack of a suitable inoculum, however, could be a hindrance, since it is essential that the inoculum be pure and that spores have the ability to germinate immediately. Without these conditions, the fermentation process is almost inoperable.

Traditionally, small pieces of tempeh from a previous fermentation serve as inoculum. The fungus is then propagated mainly by means of fast-growing mycelia. This practice can lead to contamination by undesirable microorganisms, and the inability of my­celia to survive adverse temperatures and dehydration makes mycelia unsuitable for long-term preservation of their viability.

In the laboratory, preparing agar media for mass production of spores is expensive and time-consuming, not at all adapted either for industrial processes in advanced countries or for use in less industrially advanced countries. Steinkraus et al. (1965) used a powdered lyophilized tempeh mold to inoculate soybeans for a pilot-plant process for producing dehydrated tempeh.

They used 3 g of the lyophilized mold culture to each kilogram of precooked soybeans. The inoculum was grown as pure culture on sterilized, hydrated soybeans in 3 liter Fernbach flasks with 500 g of soaked beans per flask. The flasks, after autoclaving and cooking, were inoculated and incu­bated for 4 days at 37°C. The sporulated culture was freeze-dried and pulverized in a sterilized laboratory burr mill.

Wang et al. (1975) developed a tempeh inoculum having a high viable spore count that would maintain its viability for a long time with minimal attention. The spores of R. oligosporus Saito NRRL 2710 were made by fermenting rice at a 40% moisture level for 4-5 days at 32°C. The ferment­ing mass was made into slurry by blending with sterilized water and then was freeze-dried.

On a dry basis, the viable spore count per g of preparation was about 1 x 10⁹ before freeze-drying and 1 x 10⁸ after freeze-drying. When the freeze-dried preparation was kept in a closed plastic bag at 4°C up to 6 months, the spore counts showed typical experimental variations and were comparable to their original counts.

At room temperature, a significant decrease in viability was noted after 2 months (from 2 x 10⁷ to 1 x 10⁶); thereafter, no further decrease was observed. The bacterial count of the preparation was minimal; therefore, bacterial contamination was not found to be a problem either during the process of fermentation or in storage.

Wheat bran is also a good substrate for sporulation of R. oligo­sporus. When soybeans were used to prepare spores, an unpleasant odor often resulted after 4-5 days of fermentation, perhaps due to their high protein content. Among the substrates tested for spore production by R. oligosporus, wheat was the poorest. The stickiness of wheat substrate might have created an anaerobic condition unfavorable for sporulation.

We also found that poor growth and sporulation occurred when the ratio of water to substrate was below 4:10, but growth increased as the ratio in­creased. When the ratio of water to substrate was raised above 8:10, sporu­lation was significantly less, even though growth was abundant. Therefore, the moisture content of the substrate is of utmost importance in solid fermentations.

The amount of inoculum required to make satisfactory tempeh is signifi­cant because fermentation time becomes too critical if the amount of inocu­lum is too large. On the other hand, too small an amount of inoculum provides a chance for contaminating bacteria to grow. We recommend using 1 x 10⁶ R. oligosporus spores per 100 g of cooked soybeans.

In Indonesia, copra (pressed coconut cake) is sometimes used in tempeh fermentation; the product is then known as tempeh bongkrek. We have developed new tempeh-like products by fermenting cereal grains, such as wheat, oats, barley, rice, or mixtures of cereals and soybeans with Rhizopus.

Good tempeh can also be made from the water-insoluble fraction of soybeans which is the residue of making soybean milk and tofu, the two main food products derived from the water extraction of soybeans.

The moisture content of this water-insoluble fraction, however, must be reduced to less than 80% so that the texture appears crumbly before this fraction is suitable for fermentation. On a dry basis, this water-insoluble fraction contains 32% protein, which has the highest quality among several soybean fractions studied by Hackler et al. (1963); full-fat soybean flour, water- extract of soybeans, acid-precipitated curd, and whey protein.

Tempeh is perishable and usually is consumed the day it is made because the release of ammonia by enzymatic action causes the product to become obnoxious. Its shelf life, however, can be prolonged by various methods. In Indonesia, they cut the tempeh into slices which are then dried under the sun.

We found that the most satisfactory way to keep tempeh is first to blanch the sliced tempeh to inactivate the mold and enzymes, and then to freeze it. Steinkraus et al. (1965) developed a pilot-plant process to dehydrate tempeh by a hot air dryer at 93°C for 90-120 min. However, hot air drying causes a reduction in soluble solids, including soluble nitrogen (Table 12.6) as reported by Steinkraus et al. (1960).

Whether or not the reduction in solubles represents a serious loss in nutritional value has yet to be deter­mined. Lyophilization was found to have less effect on soluble components. Iljas (1969) evaluated the acceptability and stability of tempeh preserved in a sealed can for 10 weeks.

There was no significant change in acceptability of the tempeh when the can was sealed and immediately stored at -29°C or when the can was filled with water, steam-vacuum sealed, heat-processed at 115°C for 20 min, and stored at room temperature. However, when tempeh was first air dried at 60°C for 10 hr and then sealed in a can which was stored at room temperature, acceptability of the tempeh tended to decrease as storage progressed. Another way to prolong the shelf life of tempeh is to defer the fermentation. Preinoculated beans are packaged, stored in the freezer, and allowed to ferment when needed.

The effects of R. oligosporus on soybeans have been studied by several investigators and reviewed by Hesseltine and Wang (1978) and Iljas et al. (1973). Steinkraus et al. (1960) found that the temperature of fermenting beans rises to above that of the incubators as fermentation progresses, but that it falls as the growth of mold subsides.

The pH increases steadily, presumably because of the protein breakdown. After 69 hr of incubation, soluble solids rise from 13 to 28%; soluble nitrogen also increases from 0.5 to 2.0%, whereas total nitrogen remains fairly constant, and reducing sub­stances slightly decrease, probably due to utilization by the mold. Similar changes were observed when wheat was fermented by R. oligosporus.

A decrease in ether-extractable substances of soy­beans after fermentation was reported by Murata et al. (1967) and Wang et al. (1968), indicating that the mold uses the soybean oil as its energy source. Wagenknecht et al. (1961) reported that one-third of the total ether-extractable soybean lipid is hydrolyzed by the mold after 69 hr of incubation, and among all the fatty acids, 40% of the linoleic acid is utilized by the mold.

Although the total nitrogen remains fairly constant during the fermenta­tion, free amino acids in tempeh increase. The amino acid composition of soybeans, on the other hand, is not significantly changed by fermentation. Perhaps the amount of mycelial protein present in tempeh is not high enough to alter greatly the amino acid composition of the soybeans, nor does the mold depend upon any specific amino acid for growth as suggested by Sorenson and Hesseltine (1966).

Niacin, riboflavin, pantothenic acid, and vitamin B₆ contents of soybeans increase after fermentation, whereas thiamin does not change significantly. Wang and Hesseltine (1966) also noticed in fermenting wheat with R. oligosporus that the amount of niacin and riboflavin of the wheat tempeh greatly exceeds that of unfermented wheat, while thiamin appears to be less. Apparently, R. oligo­sporus has a great synthetic capacity for niacin, riboflavin, pantothenic acid, and vitamin B₆, but not for thiamin.

R. oligosporus produces very little amylase. Since starch is seldom found in mature soybeans, it is not particularly important that this species produce amylase during tempeh fermentation. Lipase is produced by the mold to hydrolyze soybean lipids. Proteases are, perhaps, much more important enzymes in tempeh fermentation.

The ability of Rhizopus to produce proteolytic enzymes varies greatly between different strains of the same species as well as between species. The proteolytic enzyme systems have optimal pH at 3.0 and 5.5, with the pH 3.0 type predominating in submerged cultivation and pH 5.5 type predominating in tempeh fermentation.

Lipids in tempeh were found to be more resistant to autoxidation than those in control soybeans. The peroxide value of tempeh was 1.1, whereas that of control soybeans was 18.3 to 201.9. Ikehata et al. (1968) found that the peroxide value of lyophilized tempeh stored at 37°C for 5 months increased from 6 to 12 compared with 6 to 426 of unfermented soybeans.

The autoxidant activity of tempeh was further substantiated by Packett et al. (1971), who reported that corn oil containing 50% tempeh showed higher antioxidant potential than oil containing 25% tempeh, 0.01% α-tocopherol, or 0.03% α-tocopherol. In 1964, Gyorgy et al. isolated a new isoflavone from tempeh designated as “Factor 2,” which was then identified as 6,7,4′-trihydroxyisoflavone. 6,7,4′-Trihydroxyisoflavone was later chemically synthesized and proved to be a potent antioxidant for Vitamin A and for linoleate in aqueous solution at pH 7.4. However, when the isoflavone was mixed with soybean powder or soybean oil, it did not prevent their autoxidation.

These authors speculated that the insolubility of the isoflavone in the oil and the difficulty of disper­sion into soybean powder may be some of the reasons for its failure to prevent autoxidation. Therefore, the compound responsible for the antioxi­dant activity of tempeh has not yet been determined.

In the production of tempeh, soybeans are only partially cooked and they remain nearly as firm as the soaked beans. After fermentation the beans are soft and similar in texture to completely cooked soybeans. An earlier cytological study showed only slight penetration of the mycelia into the underlying tissue of the bean, suggesting that the digestion was mainly enzymatic.

However, a recent study revealed hyphae infiltration to a depth of 742 μm or about 25% of the average width of a soybean cotyledon. These authors speculated that the extreme depth of mycelial infiltration partially explains the rapid physical and chemical changes occurring during tempeh fermentation.

The hyphae may mechanically push the bean cells apart prior to, or in conjunction with, enzymatic digestion; thus, the beans become soft. Likewise, the penetration of enzymatic activity could also be enhanced, since the distance over which, diffusion of enzymes must occur is greatly reduced.

Indonesians consider tempeh to be a nourishing and easily digestible food. Van Veen and Schaefer (1950) observed beneficial effects of tempeh on patients with dysentery in the prison camps of World War II, and they suggested that tempeh was much easier to digest than soybeans.

However, animal feeding experiments have not substantiated this conclusion, even though more than half of the soybean protein, fat, and N-free extract could be solubilized by 72 hr fermentation. The protein efficiency ratio (PER) of tempeh is also not significantly different from that of the unfermented soybeans.

The cooking procedures, however, affect the nutritional value of tempeh; the PER value of tempeh significantly declined after more than 3 min of frying in oil; on the other hand, steaming up to 2 hr had no effect. The quality of tempeh protein can be improved by making tempeh from mixtures of cereals and soybeans. For example- the PER value of wheat-soybean (1:1) tempeh was comparable to that of casein.

The superior nutritive value of tempeh over unfermented soybeans has been noted by Gyorgy (1961) on animals fed low-protein diets. His results resemble those obtained with animals fed antibiotics added to their protein source. We found that R. oligosporus indeed produces an antibacterial agent during tempeh fermentation as well as in submerged culture.

The compound is especially active against some Gram-positive bacteria, including both microaerophilic and anaerobic bacteria, e.g., Strep­tococcus cremoris, Bacillus subtilis, Staphylococcus aureus, Clostridium perfringens, and C. sporogenes. The compound contains polypeptides hav­ing high carbohydrate content.

Its activity is not affected by pepsin or R. oligosporus proteases, is slightly decreased by trypsin and peptidase, but is rapidly inactivated by pronase. It is well established that antibiotics, in addition to minimizing infections, elicit growth-stimulating effects in ani­mals, especially those whose diets are deficient in any one of several vita­mins, proteins, or other growth factors.

Oriental people are constantly exposed to overwhelming sources of infection and their diets are frequently inadequate. Therefore, the finding of antibacterial agents produced by R. oligosporus may offer a clearer understanding of the value of tempeh in the diet of Indonesians, and, perhaps, of fermented foods in the diets of all Orientals.

Although stachyose and raffinose, known as flatulence factors, are not utilized as the sole source of carbon, stach­yose in soybeans was found to decrease as fermentation progressed. Calloway et al (1971) found that tempeh did not increase gas production over baseline values of healthy young men and caused a significant delay in the time of gas forming, suggesting temporary suppres­sion of intestinal bacteria. The delay could well bedue to the presence of the antibiotic substance produced by the mold Rhizopus.

Tempeh is used as a main dish in Indonesia. Total production data on tempeh are not available, but in the Province of Central Java alone, 35,100 MT of tempeh were made in 1972.

Because of the high acceptability of taste and texture, lack of beany flavor, nutritional advantages, and its simple, low-cost processing tech­niques, tempeh appears to be a good candidate for any country searching for a low-cost and high-protein food. With the recently increasing interest of vegetarians in foods of vegetable origin, tempeh consumption has been on an upsurge in the United States. In addition to tempeh-making as a home project, several commercial tempeh producers have been established. Tem­peh may soon be a regular item in the United States market.