Japanese sake is a clear, pale yellow, rice wine with an alcohol content of 15-16% or higher, a characteristic aroma, little acid, and slight sweetness.
25.7.1 INDUSTRIAL PRODUCTION
The industrial production of sake involves various steps. The essential steps are described in the following paragraphs:
Soybeans (100 kg)
Soaking (16-20 hr/15°C) Steaming (0.7 kg/cm2)
Cooling (37°C)
Packaging in unit packs
Fermentation within the packages (15-20 h/40-43°C)
NATTO (225 kg, 60% moisture content)
Pure culture of Bacillus subtilis
Figure 25. 3: Commercial production of natto
25.7.2 SELECTION/PREPARATION OF RICE
Rice of short-grained variety is considered best for sake manufacture. The rice should be finely polished to remove proteins, lipids and minerals. It is then washed and steeped in water (until 25-30% water uptake), drained (4-8 hours) and steamed (30-60 min). Steaming sterilizes and gelatinizes starch and the total water uptake amounts to 35-40%. The rice is cooled to 35°C for koji manufacture and 10°C for the preparation of moromi.
25.7.3 KOJI PREPARATION
The rice prepared as above is inoculated with spores from tane koji (at the rate of 60-100 g/100 kg prepared rice). Tane koji is prepared by culturing Aspergillus oryzae on soaked, steamed, polished rice for 5-6 days or until abundant sporulation. The inoculated rice is heaped on the floor of a room
INDUSTRIAL MICROBIOLOGY
with controlled humidity and held at 26-28°C. The internal temperature of the heap rises to 31-32°C.
After 10-12 hours, the mold spores germinate. The rice is mixed and after 20-24 hours the developing koji is placed in 15-45 kg capacity wooden boxes. Mixing is done every 6-8 hours to avoid overheating. After 40 hours, the temperature of the developing koji reaches 40-42°C, and the mycelia will cover the grains. The mycelia contain sufficient hydrolytic enzymes so that the koji can be used for saccharification of starch in the main mash.
25.7.4 PREPARATION OF KOONTOKA-MOTO
Koontoka-moto is a hot-mash, rapid-saccharification procedure used for the preparation of yeast starter (moto). After a 6-hr starch hydrolytic step at 56-60°C with koji amylases, the mash is cooled, acidified and filtered. The filtrate is used to grow pure sake culture.
Most recently, aerobically propagated compressed sake yeast has become available commercially and can be inoculated directly as 7% (w/w) of the total rice used in a moromi mash. Acidification of the mash is carried out with lactic acid. This method eliminates the necessity of preparing moto.
25.7.5 MAIN FERMENTATION
For the main fermentation mash (moromi), unsterilized koji, steamed rice and water are fermented in 6-20-kL tanks, each containing 1500-10,000 kg of rice. The yeast population in moromi is built up in a stepwise manner over a period of 3 days. Moto mash is combined with equal quantities of rice and water, reducing the yeast count by two-thirds. After 2 days at 12°C, the yeast population rises to 108/g and mash is diluted again by about one-half. The rice-koji-water mixture is added at 9-10°C to suppress the growth of contamination microorganisms. The following day, a third addition is made at 7-8°C, again reducing the yeast by one-half. In this way, the yeast population of 2.5×108 cells/g is reached after about 1 week of fermentation. Such stepwise fermentation permits careful temperature control, important in balancing saccharification and fermentation rates. With such control, ethanol concentration approaches 20% abv in 20-25 days.
The moromi tends to form a rather viscous foam that may occupy one-third of fermenter volume.
The mash is pressed, the liquor settled for 5-10 days, filtered, blended, and settled again for 30-40 days. Everything needs to be carried out at low temperature. The wine is pasteurized at 55-65°C and aged 13-18°C with or without activated carbon. Blending, dilution with water, filtration, and bottling marks the final steps of sake preparation. One metric ton of polished rice yields 3 kL of sake (20% abv) and 200-250 kg of residue (sake-kasu).
25.8 KINEMA
Kinema is a fermented soybean food product indigenous to Nepal. It is mostly prepared and consumed (and sometimes sold) by Limbus of eastern Nepal, especially in the hills. The preparation is limited to household level. Methods followed for its preparation are often subject to variation.
Some of the more important factors contributing to variation are: locality, convenience, availability of raw materials, and processing steps.
261 25.8.1 TRADITIONAL PROCESS OF KINEMA MAKING
Kinema preparation is a relatively simple process. The traditional process entails cooking of soybeans (white or brown), cooling to room temperature, mixing with a small amount of vegetable ash, wrapping banana leaves or rice straw, and leaving it for 2-3 days in a warm place for fermentation. The beans are usually mashed in wooden pestle or macerated with hands so as to split them apart. A well-fermented kinema has a slimy appearance, tends to form cake, and has a persistent nutty to musty flavor. The preparation has a very short shelf-life (2-3 days) at room temperature.
Kinema preparation does not require any addition of microbial culture: it is a spontaneous fermentation. The microbial flora present in the banana leaves or rice straw (sometimes other leaves are also used) act as an inoculum. Vegetable ash, which is so often considered an essential ingredient in kinema making, may not be indispensable for the fermentation itself but can be considered desirable in that it may furnish certain minerals to the organisms. The condition also becomes alkaline, which favors fermentation. The use of kinema can also be related to the development of characteristic taste in kinema. See figure 25.4 for an outline of traditional kinema making.
Kinema falls in a position somewhere between the two familiar oriental fermented soybean products, namely, tempeh kedele and natto. Tempeh is a traditional mold-fermented food native to Indonesia. The most popular type of tempeh is produced from soybeans and is known as tempeh kedele. Natto is bacterium-fermented soybean food product of Japan.
It is obvious, because of the nature of fermentation, the quality of kinema never remains consistent. Since rice straw as well as banana leaf is used as the source of inoculum the quality of the final product can only be as good as the quality (and relative proportions) of microorganism present in the source. The final product has the characteristics of natto as well as tempeh but since the fermentation produces a more ammoniacal odor, the characteristics of tempeh are usually masked. Overall, kinema more resembles natto than tempeh. The sticky substance present in kinema has been identified as exopolypeptides of D-isomeric glutamic acid having γ-glutamyl peptide bonds. The organism, according to T.B. Karki (1994), is: Bacillus subtilis, Enterococcus faecium, Candida parapsilosis, and Geotrichum candidum
Natto, tempeh kedele, and kinema have many things in common, for instance:
1. Soybean is the basic raw material for preparation 2. They are traditional fermented foods of the orient
3. Traditional methods of preparation depend on spontaneous fermentation Of the differences, the more important ones are as under:
Table 25. 5: Fundamental difference between natto, kinema and tempeh
Parameter Natto Tempeh kedele Kinema
Organism Bacteria Mold Mixed flora
Flavor/odor Musty to ammoniacal Nutty Nutty to musty Use Condiment, in soups, etc. Staple food,
usually deep fried
Condiments, in soups, curry, and as chutney substitute
Incubation
temperature 40-43°C 35°C 30-40°C
262 25.8.2 NUTRITIONAL SIGNIFICANCE
Soybeans are fermented not primarily for preservation. In fact, the fermented product has a very short shelf-life. Flavor development is the principal reason of such fermentations. Nevertheless, among other things, fermentation of soybeans also leads to following advantages:
1. Leaches out during cooking flatulent principles like stachyose and raffinose
2. Decreases/destroys antinutritional factors like trypsin inhibitors, lectins and phytic acid 3. Increases soluble nitrogenous substances
Whole soybean Soaking overnight Draining excess water Cooking in fresh water
Draining dry Mashing/macerating Wrapping in banana leaves Packing in bamboo baskets Storing at 35-40°C/2-3 days
(near the fire place) KINEMA
Vegetable ash
Figure 25. 4: Traditional preparation of kinema
25.8.3 PALATABILITY
Palatability of fermented soybean products is largely a matter of food habit. What can be mouth-watering to one can be offensive, or even revolting to another. For a beginner, natto and kinema can be revolting but for a habitual consumer the same flavor can be highly appetizing. Tempeh kedele has a much milder and nutty flavor and is being slowly accepted in the West also.
25.9 TEMPEH
Tempeh is an oriental mold-fermented food indigenous to Indonesia. The most popular type of tempeh is produced from soybeans (preferably white) and is known as tempeh kedele.
25.9.1 TEMPEH ORGANISM
The fermentation is invariably a mixed culture of molds, yeasts, and bacteria but the most important component appears to be Rhizopus oligosporus, although other Rhizopus species and Mucor are also often isolated. One of the better producers of tempeh has been identified by Hasseltine et al (1963) as Rhizopus oligosporus Saito NRRL2710.
TRADITIONAL FERMENTED FOODS 25.9.2 METABOLIC CHARACTERISTICS OF THE ORGANISM
Rhizopus oligosporus Saito NRRL2710 has a low amylase activity but high protease and lipase activity. Fatty acids in soybeans are the principal source of carbon and energy. Stachyose and raffinose are not utilized but common sugars such as glucose, fructose, etc., support excellent growth. By virtue of high protease activity, the organism can hydrolyze and utilize soybean proteins for nitrogen source. Proline, glycine, aspartic acid and leucine are excellent sources of nitrogen but tryptophan supports no growth at all. It can utilize ammonium salts but not sodium nitrate.
Bacterial contamination is generally not encountered. Because the organism produces an antibacterial agent, and also because it has the unique characteristic of fast growth rate, there is little chance for bacteria to gain ground before the tempeh fermentation is complete.
Commercial tempeh production process
Commercial tempeh preparation starts with dehulled, full-fat soybeans or soybean grits. Grits yield better quality tempeh. The soaking time is usually 30 min to an hour but some investigators consider this step superfluous. Commercial fermentations use pure culture in regulated amounts. Banana leaves may be optional (see figure 25.5).
25.9.3 PROCESSING LOSS
The dehulling, soaking, washing, cooking, and fermenting steps employed in the preparation of tempeh all contribute to loss of soybean constituents. The total losses of solids range from 24 to 48%, depending on the variety and type of soybeans as well as the process used. The more significant losses are in dehulling and cooking. Losses can be minimized by using less water during cooking but the fermentation as well as the quality of tempeh will not be sound. In such cases, the tempeh shows less mold development and much sporulation (and therefore discoloration). The flavor and odor are also unpleasant and poor. The factor responsible for this is the presence of heat-stable and water-soluble mold inhibitor in soybeans. This factor also inhibits the formation of proteolytic enzymes by Rhizopus oligosporus. Therefore, soaking and cooking of soybeans in excess water (which is discarded later) are essential to tempeh making.
25.9.4 MOLD MANAGEMENT
Traditionally, small pieces of tempeh from previous fermentation serve as inoculum. The fungus is then propagated mainly by means of fast-growing mycelia. The disadvantage of this method is that it can lead to contamination by undesirable microorganisms. Moreover, the inability of mycelia to survive adverse temperatures and dehydration makes mycelia unsuitable for long-term preservation. For long-long-term storage, either lyophilized cultures or other suitable modifications are prepared. Spores may be produced in rice, soybeans, or wheat substrate.
Soybean as a substrate results in unpleasant flavor and odor while wheat bran results in poor sporulation. Fermentation in rice of 40% moisture level for 4-5 days at 32°C produces good growth and spores. The whole can be made into slurry by blending with sterilized water and then freeze-dried. The spores show comparable viability even after storage for 6 months in plastic packages at 4°C.
25.9.5 INOCULUM SIZE
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
INDUSTRIAL MICROBIOLOGY
hand, too small an amount of inoculum provides chance for contaminating bacteria to grow. A level of 106 Rhizopus oligosporus spores per 100 gram of cooked soybeans seems appropriate.
SOYBEAN
[full-fat, 1000 kg (900 kg dry basis)]
Soaking (30-60 min)
Draining Cooking (with excess water)
Draining Spreading/Cooling/Surface -drying
Inoculation Packing (in shallow trays o r small packs)
Fermentation (30-35°C/24-36hrs) TEMPEH KEDELE CAKE
[51 kg dry basis)
Water
Tempeh starter (ragi):
106spores/100g cooked soybean
Figure 25. 5: Commercial preparation of tempeh kedele
25.9.6 SHELF-LIFE OF TEMPEH
Fresh tempeh has a shelf-life of only one to two days as sporulation of mold discolors the product and a rich ammoniacal odor develops as proteolysis proceeds. The release of ammonia makes the product noxious. Its shelf-life, however, can be prolonged by various methods. In Indonesia, it is cut into slices and sun-dried. An alternative method could be to first blanch the sliced tempeh to inactivate the mold and enzymes and then freeze it.
25.9.7 PALATABILITY
Fresh tempeh has a pleasant nutty flavor and odor. It is free from beany flavor and odor of raw soybeans and is therefore highly palatable. It is the only Oriental fermented product that has been extensively investigated in the West. Many countries in the West have now begun to consume tempeh.
25.9.8 NUTRITIONAL VALUE
Tempeh has a superior nutritional value over unfermented soybeans. Undesirable soy components such as flatulence factors and trypsin inhibitors, etc., are removed and/or destroyed during cooking and draining. Lipids become more resistant to autoxidation. Niacin, riboflavin, Vit. B6, and pantothenic acid increase after fermentation. The food also contains beneficial antibacterial components. At any rate, it is a potential source of low-cost protein that can have significant role in solving protein malnutrition.
265 25.9.9 CONSUMPTION PATTERN
Tempeh, in its various forms and types, is consumed in Java, Indonesia, and some other Oriental and western countries. In Indonesia, the annual production of tempeh is over 80,000MT, which accounts for about 14% of the total soybeans produced there. Indonesians consider tempeh to be a nourishing and easily digestible food. They use it as a main dish. In Java, the per capita daily consumption of fresh tempeh is in the order of 20-120 gram.
The food is consumed in a variety of ways. The more common ways are:
1. Slicing, dipping in salt and deep frying in coconut oil 2. Including pieces of tempeh in soups