The optimum germination temperature for B. natto spores is about 40 °C. The spores can still germinate after storage at 0°C for one year. In actual practice, the cooked soybeans are inoculated at about 80 °C. Fortunately, the B. natto spores are heat-tolerant and can survive this exposure to high temperature. This heat-shock helps induce the dominant spores to germinate. After exposure to 100°C for 3 mins, or 80°C for 30 mins, the spores start to germinate. It is important that the beans be immediately cooled to about 40-50 °C for proper germination and multiplication. Overexposure to high temperature such as 100 °C for 30 min, at this stage will eliminate the germination capability of the spores. The germinate spores also are not heat-tolerant. The bacteria will not grow at temperatures higher than 55°C. Growth at 20°C is only one-tenth that at 40°C.
Growth of B. natto is also affected by pH of the substrate. It grows best in a neutral or slightly alkaline environment. Cooked soybean has a pH of 6.4 to 6.8 and increases to
7.2 to 7.6 as fermentation progresses. A pH value of 4.5 has significant inhibitory effect in growth of B. natto.
Oxygen also affects the growth of B. natto. In actual practice, carbon dioxide concentration in the fermentation/incubation room can be over 15%. However, it has little effect on the maturation of the natto soybeans.
The B. natto bacterium grows easily on cooked soybeans. It can also grow on other animal or plant foods. However, it grows better on plant protein foods and produces more sticky substance.
Carbon sources such as glucose, fructose, and sucrose can be used easily by B. natto for growth. Sucrose is also needed for the production of sticky substance. Soybean contains about 20% carbohydrates, of which 30% is sucrose. This is plenty for the growth of B. natto.
The nitrogen source for growth of B. natto is proteins and amino acids. B. natto can easily utilize glutamic acid, arginine, aspartic acid, and proline but not threonine, tryp-ophan, phenylalanine, and methionine. In soybeans, there are more amino acids that B. natto can utilize than ones it cannot.
Bacillus sp. such as B. subtilis, B. megaterium, B. cereus have no requirement for biotin for growth; however, other Bacillus sp., such as B. natto, B. mycoides, B. pumilus, and B. coagulans, do. B. natto does not germinate without biotin. It requires at least 0.1% (0.001 ppm) for minimal growth and 1% (0.01 ppm) for proper growth. Soybeans have 60 g biotin per 100 g. Other B vitamins are abundant in soybeans and they are also beneficial to B. natto growth (2).
Fermentation of natto is greatly affected by the temperature of soybeans entering the incubator and the temperature and relative humidity of the incubator thereafter. When the initial temperature soybean is cooled below 40 °C, germination of B. natto will be delayed and the time to increase bean temperature will be extended. This is the critical time when contamination can occur most easily. It should be shortened by all means to ensure safe fermentation. Therefore, the actual practice is to have the beans at about 42 °C when entering the incubator with 85-90% relative humidity. After 2 hr, the B. natto spores will germinate and produce heat. At 6-8 hr, maximum heat is generated from fermentation.
Temperature of the fermenting beans will reach about 48 °C. Thin, white film of the bacteria is visible. The characteristic odor of natto and sticky substance start to develop. When the temperature of fermenting beans reaches 50 °C, supplemental heating should be discontinued. Relative humidity too low will produce hard natto beans. Therefore, relative humidity in the incubator should be maintained at 80-85% and temperature at 50°C. The maturation period is about 16-18 hr. At the end of this period, the natto beans should be taken out from the incubator to cool down. They should be delivered for marketing the same day. If it is necessary to delay the delivery, holding the product at 5-10°C is necessary.
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