b. Salt Either crude or refine salt is suitable for manufacturing douchi (Yinshi). The amount is about 20% of that for black soybeans.
c. Glutinous Rice In the manufacturing of yinyui, about 1-2% glutinous rice or Taiwanese rice is used. The purpose is to provide some stickiness in the final product. In the manufacturing of douchi only, about 0.3% of roasted (or pan-fried) wheat flour is used in the preparation of the starter.
d. Seed Starter (Koji) The seed starter is either Aspergillus oryzae or Aspergillus sojae. Asp. oryzae has higher amylase, acid protease, and acidic carboxyl peptidase activity, whereas Asp. sojae has higher endo-poly-galacturonase and glutaminase activity. The amount of spores used is 0.5% (w/w) that of black soybeans.
2. Raw Material Treatment a. Washing and Soaking of Black Soybeans Selected and weighed black soybeans are put in a perforated container and washed with water to remove the soil and dirt adhered to them. The beans are soaked in water for 3-4 hr (2-3 hr in the summer, and 4-5 hr in the winter) until the water absorbed by the beans is 1.8-2.0 times that of the weight of the beans (i.e., the soaked beans weigh 180-200 kg for 100 kg dried beans). Ferrous sulfate, used in the past to make the final product blacker and brighter with better appearance, is not recommended nowadays because of its toxicity.
b. Steaming of Black Soybeans After soaking, black soybeans are adequately drip-dried. Excessive moisture will create problems in the starter preparation stage. Soaked beans can be cooked with pressurized steaming, ambient pressure steaming, or closed-pot boiling and holding. For pressurized steaming, the drip-dried, beans are loaded into NK chambers for steaming at 113°C for 30 min or at 117°C for 20 min. For defatted black soybean flakes, the steaming process is at 131 °C for 5 minutes. Steaming of black soybeans is easier than of regular soybeans, and they cannot be overcooked. Extensive absorption of moisture will create problems at the starter preparation stage, producing undesirable odor and sticky compounds.
For ambient pressure steaming, soaked beans on trays are loaded or to the steamer for 2-3 layers. After steam appears from the top layer of beans, continue to steam for another 2 hr. The beans do not have to be cooked to too tender but have to be completely cooked, without the raw flavor when chewed.
For the boiling and holding method, soaked beans are put into the pot. The water added is about 90-100% of the volume of the beans. It is heated until fully boiling and then the lid is put on. Cooking of the beans is continued with medium heat for 70-90 min. The cooked beans are taken out and cooled on trays. The beans have to be turned over frequently to help evaporate the water to the point that they are somewhat dry and not sticky. These beans are then ready for making the starter.
3. Preparation of the Starter a. Mixing the Inoculum with the Beans The wheat flour is roasted (or pan-fried) to yellowish brown and let cool. Extensive holding of the roasted (or pan-fried) flour is not recommended as this flour can absorb moisture and be easily contaminated. Usually, 1-2 kg of flour is used for 100 kg black soybeans. The roasted (or pan-fried) flour is thoroughly mixed with the inoculum spore before mixing with the cooled beans. When the cooled bean is too soft or contains too much moisture, 3-4 kg of flour will be needed to adjust the moisture content of the mixture. When the beans are mixed with the inoculum, each bean has to have inoculum spores on its surface. Beans without the inoculum spores are easily spoiled due to contaminated bacteria. If wheat flour is not used, the amount of inoculum spores should be increased to 150 g for 100 kg of beans (0.15%) (Fig. 8).
b. Preparation of the Starter Koji In general, the procedures for making soy sauce can be followed (see Chapter 29). However, the incubation time will be extended to 5 days. This is longer than required for soy sauce making. For ancient methods, the duration is 7 days as seed starter is not used. The mixture is left in the sun for cooling (without temperature maintenance) and drying for a suitable period. This will adjust the moisture content and also allows for growth of other desirable microorganisms from the environment. At the end of this step, growth of Rhizopus and Mucor is usually visible.
At the end of the previous step, lots of spores are formed already. In the ancient procedure, lots of other microorganisms are present and create a bitter taste. A washing step to remove these spores is therefore needed. This step is not needed in the making of soy sauce using regular soybeans. However, when black soybeans are used for making yinyui-type soy sauce, the black soybeans are more tender and wheat flour is not used to adjust the moisture content of the raw material. Spores are inoculated directly onto the surface of the beans. The mycelia can enter the beans easily. Space between the two cotyledons will be full of mycelium and spores. Therefore, the spores on the surface of the beans will significantly affect the final product quality. If washing is not conducted before
loading into the crock for maturing, the mold odor and bitter taste will be too strong, affecting the flavor of the final product.
The current practice is to use a semi-mechanical washer. The washer is filled with 2030 kg of water followed by 10-15 kg of the starter koji. The washer is manually rotated for 1-1.5 min to mix the water and the koji (Fig. 9). The mixture is decanted into perforated baskets and washed again rapidly with 4-5 kg of water by pouring the water through the beans from the top. The washed beans are drip-dried for 2 minutes. This procedure will lose 3-4% of the total nitrogen. The beans will now weigh 50% more as compared to the starter koji, or 1.5 times the original weight of the koji.
Twenty-five kilogram of washed koji is put into a 30 kg capacity perforated basket. Except during the summer, covering with cloth to maintain temperature is required. This incubation period is 3 hr in the summer and 8 hr in the winter, with holding period adjusted according to temperature in the environment. Heat will be generated from the upper and middle section of the beans. The beans are rinsed with 40-45°C water to allow second absorption of water. They are mixed and held to permit second heat generation. After this step, temperature of the bean koji is adjusted either by digging a hole to vent off the heat in the summer months or taking off the covering cloth in the winter. The temperature of the beans should be maintained below 38-40°C. Temperature too high will lower the activity of proteases in the koji. The complete process of incubation takes 6-8 hr in the summer and 16-24 hr in the winter months.
6. Crock Loading and Fermentation
After the incubation step, the koji is mixed thoroughly with 17% salt, and loaded into the crock followed by putting in another 3% salt on the top. The beans are pressed evenly. The crock is covered with the lid and sealed with a mixture of lime and sand. The sealed crock is left in the sun for the bean koji to mature. It takes about 1.5-2.0 months in the summer and 2-3 months in the winter to recover the yinyui or douchi. When the crock is opened, the undissolved salt is collected to avoid the salt getting into the Yinyui and making it too salty. Yinshi not soaked in the yinyui is recovered and transferred to another container. A hole is dug in the center of the yinshi to drip out the yinyui to the bottom. After some time, the yinyui will be dripped to the bottom and the dry yinshi is left at the top.
Another procedure is to layer the washed bean koji in the crock with a small amount of sweetened wine (sugar: rice wine ratio of 1:3). For the two top layers, salt in the amount of 20% of the washed bean koji is sprinkled in. The crock is filled to 80% full. The bean koji is pressed evenly and sealed in the crock. The crock is left in the sun for 1 month to mature. When the recovered product is too wet, it is sun-dried accordingly to adjust the moisture content.
When the bean koji is loaded to the crock, the temperature of the bean koji inside the crock is not that high when left in the sun. However, in the summer months, small amounts of the bean koji at the top and around the crock interior can reach a temperature of 42-43 °C. The rest can only reach 35-36 °C.
During the washing and incubation treatment, no salt is added. The enzymes are already working actively. When the bean koji is loaded into the crock, only a small amount of salt is added. Therefore, the fermentation (maturation) time is only 1.5 months in the summer, and 2-3 months in the winter. However, the longer the maturation, the better the flavor. When extended maturation is desirable, additional salt at the top of the bean koji is recommended to safeguard the process. Because the lid of the crock is sealed with a sand and lime mixture, the environment inside is not completely anaerobic. When the salt addition is not enough, the top 5 cm layer may be subject to microbial spoilage (Fig. 10).
7. Microbial Activities During Fermentation
At the time of loading the crock, the bean koji carries salt-tolerant bacteria, yeasts, and lactic acid bacteria at the level of 1.3 x 105/g, 3.0 x 106/g, and 2.2 x 106/g, respectively. After 14 days of fermentation, salt-tolerant bacteria can increase to 3 x 109/g and then decrease gradually to the 106/g range. With the gradual increase of salt-tolerant yeasts and lactic acid bacteria, the salt-tolerant bacteria decrease gradually to 3 x 104/g. The latter is at a lower level than the salt-tolerant yeasts, but still higher than the salt-tolerant lactic acid bacteria. At the end of fermentation, the salt-tolerant yeasts stay at a level of 2.2 x 106/g, and the salt-tolerant lactic bacteria at 6 x 103/g.
During fermentation, the enzymatic activities are as follows: lipase > amylase > protease. At the time of loading the crock, the enzyme activities for lipase, amylase, and protease are 1.4 x 105, 1.3 x 102 and 2.3 x 102 units/g mash, respectively. These enzymatic activities decrease with time of fermentation. This is especially true for protease. It decreases by one-half after 14 days of fermentation and stays at this level until the end of fermentation. During the whole fermentation process, lipase activity is about 1000 times more active than protease and amylase.
During fermentation, the proteins in black soybean are hydrolyzed by the exoprotease from the mold amino acids. These amino acids are then hydrolyzed by the deaminase and decarboxylase into ammonia and amines (7). With the increase in fermentation time, the total nitrogen in the inyui increases significantly. This trend also applies to formaldehyde nitrogen and amino nitrogen. The hydrolytic rate of proteins can reach 63%, and the total nitrogen utilization rate can reach 69%. Total amino acids increase from 0.23% to 1.8%, with glutamic acid being the most significant, from 8.3% to 24.7%, followed by aspartic acid, from 3.3% to 15.6%.
In the carbohydrate category, reducing sugars increase from 0.7% to 3.16%, sucrose from 0.04% to 3.4%, and glucose from 0.02% to 2%. Reducing sugars increase at the beginning of the fermentation and then decrease gradually due to actions of the salttolerant lactic bacteria and yeasts. Also present are xylose and fructose, with fructose present at only detection level.
Changes in pH values are not that significant, between 5.2 and 5.8. However, the total acidity increases during fermentation, from 0.5% to 2.6%. Acetic and lactic acid increase from 0.10% to 0.23%, and 0.11% to 0.23%, respectively. Also detected are citric, malic, oxalic, and succinic acids.
Linoleic and oleic acids increase from 0.19% to 9.65%, and 0.23% to 4.50%, respectively.
Flavor-related compounds are 5 IMP, 5 UMP, and 5 CMP, with 5 IMP making up the largest amount, at 0.06-0.15%. Odor-related compounds include acids, alcohols, phenols, lactones, ketones, esters, aldehydes, furans, pyrans, thio compounds, and nitrogen-containing compounds. Isovaleric acid increases from 365 to 1836 ppb; isoamy lalcohol (from leucine) increases from 570 to 11244, phenyl ethyl alcohol (from phenyl-alanine) increases from 903 to 6836 ppb; n-hexanol increases from 53 to 537 ppb. Other compounds exist only as traces.
When the bean koji making process is reduced to 48 hr, there are higher activity rates of amylase, protease, and lipase. This is especially true for the wet fermentation process with addition of 45% water. The mash has a better hydrolysis rate for proteins and a higher total nitrogen utilization rate. After 3 months of fermentation, there is a higher protease activity, with subsequently more amino acids, and more deaminase and decarboxylase hydrolysate products such as isoamyl alcohol and phenyl ethyl alcohol. When the fermentation is extended to 5 months, the protein hydrolysis rate is 75%, and total nitrogen utilization rate is 77%. These are higher values as compared to values obtained from traditional methods. Total free amino acid content in wet fermentation is about the same as that in dry fermentation. However, there is less glutamate in wet fermentation with undetectable amounts of glutamate from the dry fermentation. Both fermentation processes produce more arginine.
When the incubation temperature for bean koji making is increased from 45 °C for 4 hr to 50-60 °C for 2 hr, the incubation time can be reduced. From the viewpoints of free fatty acids, nucleotide, and organic acids, products made from 20°C constant temperature incubation have better flavor. However, from the viewpoints of amylase, protease, and lipase activities, incubation at 45°C for 4 hr shows higher enzymatic activities. When the incubation temperature is raised to 50-60 °C, the incubation time can be reduced to 2-3 hr. From the viewpoint of flavor compounds, incubation at 45-50°C followed by sun-drying produce better-flavored products.
Douchi is a salt-containing, intermediate-moisture, fermented soybean product with storage stability. It contains rich amounts of protein, amino acids, unsaturated fatty acids, and flavor compounds. Its composition is listed in Table 3.
Douchi is packaged before retailing. The packaging can be large or small. Large packages are packed in a small bamboo container (in Taiwan), small packages in plastic bags. They are then wholesaled to retailers. Some douchi from China is also packed in ceramic jars.
Miso is Japanese fermented soybean paste or thick semisolid product. There are two types of miso, ordinary miso and processed miso. Miso is mainly made from soybean with the addition of enzymes from rice, wheat (barley or rye), or soybean koji and salt. The mixture ferments and ages to become a semisolid-type product. Some call it fermented soybean paste. Based on the koji materials, the ordinary miso can be further separated into rice miso, barley miso, and soybean miso (Table 4). Another classification is based on their
Table 3 Composition of Douchi
Amino Ammonia Total nitrogen nitrogen nitrogen F.N./ A.N./
Douchi-mash 12-13 1.1-1.2 1.0-1.1 0.3-0.4 3.1-3.2 30-36 11-14
Filtrate of mash liquid 12-13 1.0-1.1 1.0-1.1 0.3-0.5 2.6-2.7 38-41 19-17
Table 4 Miso Formulations and Composition
Materials Barley miso --Light/ Light Red/ Red/ - Soybean
Color/taste White sweet /salty sweet salty Sweet Salty miso
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