a 80% polish rate.
a 80% polish rate.
time is needed. Soybean that is boiled in water in a 1:4 ratio is easier to get soft and has a lighter color, but more than 10% of the soluble solids could be lost due to dissolution. In ambient steam cooking, it takes 4-8 hours to steam the soybean. However, under 1 kg/cm2 steam pressure (121°C), it needs only 45-50 min to cook (Figure 12). A rotatory pressure cooker takes only 20-30 min at 0.5-0.7 kg/cm2 (112-115°C). Recently, a modified continuous steam cooker has been used to cook at 1.5-2.0 kg/cm2 (127-135°C) for 2-5 min. The hardness of steam-cooked soybean should have breaking force lower than 500 g for a single kernel. Breaking force higher than 800 g results in a coarse-textured product, whereas breaking force of less than 300 g results in incomplete fermentation and a viscous product.
The cooking method influences the color of the products. Products with light color are made from boiled soybean because more water-soluble pigments are removed during boiling. To avoid enhancing the browning after cooking, immediate cooling of the cooked soybean to the temperature suitable for mixing (30 °C) is required. Vacuum cooling, coolair, or conveyer-type coolers are available to cool the soybeans rapidly rather than the old-fashioned method in which the soybeans are spread and cooled outside the cooking chamber. Such a cooling method needs longer time, and the soybeans are vulnerable to contamination with undesired microorganisms.
Cooled soybeans are further chopped to 5-6 mm diameter particle size. The smaller the size, the easier the digestion goes. Nevertheless, particles ground too fine will delay the fermentation process as well.
b. Treatment of Rice The degree of rice polishing depends on the type of miso to be processed. Light-color type miso requires rice with a higher degree of polishing (smaller polish rate value), whereas the red-type miso uses rice with lower degree of polishing. The polish rate of rice has to be lower than 93% for proper koji process; 90% is used in current production. The rice proceeds with washing, soaking, and draining steps. The rate of water absorption depends on the texture, polish rate, and water temperature. Rice with soft texture, immaturity, or a higher degree of polishing has a higher absorption rate. Generally, rice is soaked in water for 4 hrs to overnight to allow water to be absorbed evenly in the kernel, and the water absorption rate reaches 28% of original weight for complete starch gelatinization during steam cooking. Too little moisture uptake will produce steamed rice too hard for mycelia to penetrate inside the kernel. Too much moisture uptake will soften the cooked rice, which makes it difficult to process koji.
Rice in the washing and draining stage will lose some water-soluble components and potassium, about 30-50%. Some other ingredients, such as sodium, magnesium, phosphor, sugar, protein, amino acids and lipid will be lost partially while calcium and iron ions will be adsorbed. The purpose of draining are to (a) drain all the surface water to avoid surface stickiness and (b) allow tempering of the moisture. However, prolonged draining time under high temperature will result in rancidity, microbial spoilage, or red discoloration.
Steam cooking is used to gelatinize the starch, to dissolve protein, to help koji mold grow, and to sterilize. If the rice is undercooked, mycelia cannot penetrate inside the ungelatinized kernel resulting in koji with low enzyme activity. Properly cooked rice (a) should be full, elastic, soft inside and hard outside, and without stickiness on the surface and (b) have no white center inside the kernel. Cooked rice will contain 36-37% moisture.
A batch-type steam cooker takes 30-40 min at 1 kg/cm2 (121 °C) to cook the rice properly (Fig. 13). A continuous-type steam cooker uses a stainless conveyer to carry a layer of rice 20 cm thick and simultaneously supplies the stea from the bottom of the conveyer to cook for 25-30 min. For rice with low water absorption or hard texture, the
problem can be solved by steam cooking twice. Cooling can be achieved be either natural cooling or continuous air cooling to the temperature that is ready for koji processing. The temperature depends on the season, koji-processing method, status of koji bed, and the amount of koji piled up for germination. Cooling temperature sets at 3.5°C in the winter and at 3.2°C for the summer to accommodate the subsequent koji process.
c. Handling of Wheat Wheat can absorb moisture faster than rice, and it starts to take up moisture at the beginning of the washing stage. It is important to shorten the soaking time and to prevent uneven moisture distribution. Soaking time should be adjusted along with water temperature used. It takes more than 2 hr to drain the soaked wheat. Drained wheat weighs about 1.28-1.3 times its original weight and has about 35% moisture.
Cooking conditions for both rice and wheat are similar, usually 40-60 min or 30 min under pressurized. Cooked wheat has a light syrup (brown) color and is swollen evenly without surface stickiness. If wheat absorbs too much water or drains incompletely, surface stickiness will occur. Insufficient water absorption can result in ungelatinized white starch particles in the center. Steamed wheat contains 38% moisture.
3. Preparation of Miso Koji
The purposes of koji preparation are as follows:
1. To incubate koji mold to produce the amylase and protease for raw material digestion
2. To incubate koji mold for production of substances that promote growth of salttolerant microorganisms and to generate the precursor of miso flavor
3. To allow mycelia penetration into the structure of raw materials and breakdown the structure for enzymes to work
The optimal temperature for koji mold growth is 30-35°C along with a relative humidity of over 95%. Deviation from this koji temperature range will hinder mold growth and result in a bad koji due to undesirable microorganisms.
Miso koji mold should produce more saccharization-type amylase instead of the liquid-type amylase, especially for white miso or other high koji yield miso. Strong liquidtype amylase in koji mold gives a soft, viscous miso. Insufficient saccharization-type amylase will give a coarse texture with less sweet taste. More amylase is generated with koji produced at high temperature (40°C) than at low temperature (30°C). Therefore, koji mold should be incubated at 35-38 °C to have high amylase activity.
Based on optimal pH, there are three types of protease in koji culture: acid (pH 3.0), neutral (pH 6-7), and alkaline (pH 8-10). The protease activity increases as pH value decreases. Acid and neutral proteases affect the protein digestion in miso the most. The neutral protease, the most important one, acts at the mixing step at pH around 6.0. The best temperature for preparing koji with stronger protease activity is 30°C instead of 40 °C.
a. Starter Mold Starter mold for miso processes are prepared by multiplying koji mold culture. Barley flake and wheat bran in a 1:1 ratio are mixed with water (50% by weight of raw materials). The mixture is cooked for 30 min under 1 kg/cm2 pressure (121) followed by cooling to 28-30°C and inoculating with 0.1% long-hyphae koji mold. The starter mold is ready when spore count reaches 8x108/g after incubation at 28-30°C for 37 days.
Three to five hours after transferring koji mold, the spores start to germinate and the mycelia grow longer. Although the optimal temperature for fungi growth is 30-35°C after inoculation, the temperature needs to be adjusted to below 30° C to prevent contamination from undesired microorganisms. Mycelia grow actively after 18-34 hours of incubation. Their respiration demands much oxygen and generates CO2 and heat. It is necessary to supply oxygen and remove the CO2 as well as to decrease the temperature of starter mold.
b. Traditional Rice Koji Process The traditional rice koji process uses wooden koji trays and incubators. Cooked rice is cooled to 30-35°C and inoculated with the 0.1% starter mold. The well-mixed mixture is then covered with a wet towel and incubated at 30-35°C for 3-4 hr for spores to germinate. After 8-10 hr, the mixture starts to generate heat. The temperature reaches its peak after seeding for 18 hr. In order to prevent further increase in temperature and formation of aggregates, the incubated mixture needs to be broken up and blended four times approximately after seeding for 20 hr. The mixture is dispersed separately on several koji trays to avoid elevating temperature and to decrease CO2 concentration. Location of the koji trays should also be exchanged to provide even temperature distribution after the second blend. After incubating for 40 hr, the koji does not form additional spores. The koji is ready to be removed and cooled. Finished koji should be mixed with salt as a salted-koji to inhibit further generation of heat and moldy off-odor.
c. Mechanical Koji Process Compared with the traditional method, the mechanical koji process saves a lot time and labor. In recent years, the mechanical method has commonly been applied to replace the manual blending procedure. Air with proper temperature and relative humidity is introduced to pass through the surface or the center of the koji mixture and to exhaust the generated heat for temperature maintenance of the mixture. Two ventilation types are available for mechanical koji process: surface type and inner type. There are four inner types (steady-bed, shed, rotatory-drum, and high-pile) depending on the position of koji tray placed. Mechanical ventilation controls the temperature easier by maintaining vented air at 30-32 °C with minimal ventilation. After 28-30 hr of incubation, the piled mixture starts to generate a large quantity of heat. The mycelia lengthen and tangle with each other, making the ventilation difficult. Then manual blending is needed due to poor temperature adjustment. Saturated humid air with temperature adjusted to below 35°C is supplied until 2-3 hr before finished koji is ready.
d. Wheat Koji Wheat generates more heat than rice due to the higher content of protein, mineral, and vitamins. Koji mold will not be able to grow due to easier moisture evaporation on the wheat surface. High humidity should be maintained at the beginning of koji preparation. Ventilation of humid air should be coordinated with the corresponding temperature of koji in mechanical koji preparation. In order to have a koji with strong amylase, lighter color, and a high production rate, we need to control the temperature at 36-38°C for the first 10-18 hr, followed by adjustment to 30-32°C. The koji for red-type miso has high protease and amylase activities, and the koji temperature is controlled at 2931 °C. With its high moisture content and stickiness, wheat koji is more easily contaminated by Neurospora than rice koji.
e. Soybean Koji Steam-cooked soybean requires mechanical kneading into a miso (soybean) ball at 60° C. With 50% water content, steam-cooked soybean is prone to B. subtilis contamination. Facultative anaerobic lactic acid bacteria grow inside the soybean ball. This lowers the pH to prevent B. subtilis growth, and the soybean ball becomes a suitable environment for fungi to multiply safely. The size of the soybean ball depends on the size of the device available, with a large ball more than 45 mm in diameter and the small ball 19-24 mm in diameter. Small soybean balls are commonly used for miso making.
Fungi growing on the surface or surface layer of large soybean balls result in less enzyme activity. Because lactic acid bacteria can grow inside the soybean ball, more lactic acid is obtained from a larger ball. A small soybean ball has a faster aging rate and a darker color than a large ball. It is important to avoid high temperature at the early stage of soybean koji preparation and to blend the moist soybean ball with starter mold thoroughly to control possible B. subtilis contamination.
f. Storage of Prepared Finished Koji To prevent further heat generation, the finished koji is mixed immediately with one-third of the total salt required for the formulation. Normally, the salted koji should be used in 2 days due to gradual loss of enzyme activity.
The criteria for a good finished koji are as follows:
1. Possession of proper enzyme activity
2. No contamination from undesirable microorganisms
3. Good mycelial growth into all rice kernels
4. Bright and without browning in appearance
5. Possession of miso aroma without off-odor
Depending on the types of miso, the level of each criterion may be varied. For example, light-color miso requires no browning in appearance and high amylase activity, whereas red-color salty-type miso needs high protease activity.
While the cooked soybean is cooled, salted koji, salt and sterilized brine with some yeast, lactic acid bacteria, and vitamins are added. Then the mixture is chopped through a 6 mm (for granular-type miso) or 1-2 mm (for fine-type miso) particle size sieve plate and put into a fermentation container for further fermentation and aging (Fig. 14). Barrel-type mixer (Fig. 15), automatical weighing mixer, screw-type mixer, or rotary drum-type mixer are used for large-quantity of mixture; the small quantity can be mixed in a tank manually.
Figure 15 Barrel mixer.
Light-color type miso requires the salted koji mixed with cooked soybean while the cooked soybean is still warm. The key factor in mixing is thorough mixing. The salt concentration should not deviate for more than 0.5% in the mixed mash. The temperature of mixed mash, an influencing factor for the aging process, will affect its enzyme activity, microbial growth, or chemical reaction. For sweet-type miso, mixing is carried out at 50°C to inhibit microbial growth and to pursue high enzyme activity. If the mixing temperature is below 30 C, the aging process will be in trouble. Salty-type miso requires fermented aroma and therefore is mixed at 28-32 °C in warm fermentation method or at 20 °C in natural fermentation method (Fig. 16). In general, adjustment of the mixing temperature relies on the temperature of the cooked soybeans. The end temperature of the mixture, except for the high temperature mixing, is usually maintained at 20-25°C, or even at 10°C, which gives better-quality miso.
Addition of sterilized water can adjust the water content and the texture of miso. It also assists the fermentation and aging processes. The amount of water needed depends on the moisture of cooked soybean and miso koji. It can be calculated as follows:
Amount of sterilized water = Total amount in mixing — (Cooked soybean + finished koji + salt)
. . . (Total solid of cooked soybean, finished koji and salt)
Amount of miso mixture =---——------—--r-x 100
(100 — added water percentage by total weight)
The amount of moisture can affect the microbial growth or aging process even though the mixture have the same amount of salt. The relationship between the moisture and the amount of salt can be expressed as follows:
Concentration of salt by total water (%) = ——- Salt(/o)-, .„,, x 100
The concentration of salt by total water in salty miso is 21-22% when soybean and koji are in a 1: 5-7 ratio and it is 20-21% when koji ratio is 8-10.
A clean plastic cloth is used to cover the top of the fermentation tank, and a piece of rock 20-30% of total mixture weight on top of it ensures that the miso is under anaerobic condition. The rock weight is usually heavy to press out enough fermentation liquid for covering the surface of the miso.
The salt-tolerant microorganisms used in the mixture for enhancing the fermentation can be yeast such as Saccharomyces rouxii, Torulopsis versatilis, and the lactic acid-producing bacteria Pediococcus halophilus. Antagonism occurs between yeast and lactic acid bacteria, and the growth of lactic acid bacteria can be hindered by yeast. Therefore, the amount of lactic acid bacteria used (106 cell/g) should be 10 times higher than the yeast (105 cell/g).
5. Fermentation Management
The purposes of miso aging are as follows:
1. To utilize enzymes from miso koji to disintegrate the raw materials, and
2. To utilize the fermentation power of salt-tolerant microorganisms (yeast and lactic acid-producing bacteria) in which chemical degradation or synthesis of fermented products can generate color as well as flavor at early stage of fermentation and aging.
After mixing, the first stage of fermentation, primarily enzymatic reactions, lasts for about 10 days and the environment is suitable for growth of salt-tolerant microorganisms. The optimal temperature for the digestion of protein and carbohydrates by the enzymes from miso koji are 45-50°C for protease, and 55-60°C for amylase. Therefore, sweet-type miso, which relies on enzymatic reactions rather than fermentation, has optimal temperature around 55-60°C. However, the aging process at such a high temperature kills yeast and lactic acid bacteria and causes dark color and burnt off-flavor.
Some microorganisms grow in miso koji and the surrounding environment, and are added microorganisms for fermentation. Most of these microorganisms are not salt-
tolerant, cannot grow under the 20% concentration of brine and will die out gradually in a week. Then the remaining salt-tolerant microorganisms begin to grow. For those desirable yeast and lactic acid bacteria in miso, the optimal temperature is about 30°C they stop growing at 40 °C.
A salty-type miso requires both enzymatic reaction and fermentation. The optimal temperature for the microorganism growth and aging is about 30 °C. For example, a fermentation for salty-type miso containing 12-13% salt should be mixed at 25-30 °C and maintained at this temperature for 7-15 days for lactic acid bacteria to grow and for lowering the pH below 5.5, and to build an suitable environment for the yeasts. Saccharomyces rouxii produces some flavor components such as ethyl alcohol and amyl alcohol. Torulopsis versatilis can convert lignin into ferulaic acid and further into 4-ethylguaiacol, a flavor component. During this period, the temperature of miso rises slowly to 30-35°C. Temperature is maintained for 15-30 days, followed by a decrease in miso temperature to 20-25 °C for 40-60 days for post-aging. The post-aging process is carried out under lower temperatures to allow slow miso color development and to temper all the flavor components. When a balanced flavor is developed from the blending of sweet taste from sugars, salty taste from salt, acid taste from organic acids, and umami taste from amino acids, the aging is complete. A browning reaction from amino carbonyl reaction is also developed simultaneously. The control points for fermentation are the temperature programming as well as blending technique, frequency, and schedule.
Natural fermentation of miso does not apply any thermal treatment nor temperature control, but sometimes long-term aging proceeds in the manually adjustable fermentation room. Blending procedure can assure homogeneous fermentation. At least one blending is required after 2-3 weeks of mixing due to vigorous fermentation, and later another one or two blendings can be helpful. Blending can evenly maintain the temperature of the mixture and homogenize the mixture, as well as supply the oxygen for yeast to grow.
The quality of miso can be evaluated using a sensory method or chemical determination. Color can be judged visually or determined by colorimeter. The color brightness decreases and redness increases as the aging proceeds. Acidity I is the freshness-related acid determination. The pH value of raw materials at an early stage of mixing is 5.7-5.8. Protein disintegration produces amino acids and organic acids, which increases titratable acidity I. The pH values become 4.9-5.1 at the end of aging. Protein solubility is defined as the ratio of total nitrogen and water-soluble nitrogen, and protein digestibility is defined as the ratio of total nitrogen and formol (amino group nitrogen) nitrogen. The higher the degree of aging process, the higher the values of protein solubility and digestibility are determined. Both values increase significantly at early stage of aging. The values stabilized 30-40 days from the beginning of aging. After 50 days, protein solubility and protein digestibility are about 60% and 25%, respectively. Free amino acids (glutamic acid and aspartic acid) can also be measured. Alcohol and lactic acid contents vary among different types of miso. Alcohol and lactic acid content should be higher than 0.2% in yeast-related fermentation. If the values are below 0.05-0.1%, there are no active yeast or lactic acid bacteria. Free fatty acid comes from the lipid in soybean, which is hydrolyzed by lipases from starter mold. Some fatty acids form lipo-ester components contributing to miso flavor. Therefore, the determination of fatty acids or lipo-esters becomes the index of aging.
For quality consistence, blending is needed after aging. Granular texture-type miso can be used for blending directly, but fine texture-type miso needs chopping through a 1-2 mm sieve. A too-small-diameter sieve or a slow-speed chopper may cause sticky product. When preservative is needed, we can add sorbic acid (less than 0.1%) or K-sorbate (0.05%). Alcohol (2-3%) can also be used as a substitute preservative. The level of residual alcohol in miso can be determined to estimate the amount of alcohol needed for addition. It is very important to blend the alcohol thoroughly. Sterilizing the aged product in hot water can also preserve the miso. The yeasts in raw miso cause continuous fermentation after bottling. In small operation, miso is packed in small packs, sealed, and sterilized at 60°C for 10 min or 70 °C for 5 min. For continuous sterilization and packaging, miso is heated and cooled in pumping pipes followed by packaging. However, this method is not done under strictly aseptic conditions, and additional care is needed to prevent contamination.
Single polyethylene bags were first used for small packs of miso. Now, laminated films such as cellophane and polyethylene coated with vinyliden chloride or laminate poly-cellophane are available. In order to maintain quality, film with low gas permeability, which can slow down the browning of product, is recommended. Plastic containers are also available.
There are manual and automatic filler packaging machines. Before sealing the package, we should remove the air to prevent rapid browning. Product stored under room temperature will undergo color and flavor deterioration. If miso is stored at high temperature, severe browning occurs, and miso pH continues to drop with increases in titratable acidity, resulting in a browning and off-flavor. Low temperature storage (lower than 15°C) can prohibit such deterioration.
Good-quality miso can be described as having (1) unique aroma without off-flavor, (2) light sweetness and no odd-odor or sour taste, (3) even mycelial growth on each rice kernel without undesired microorganisms, and (4) soft but elastic texture with proper adhesiveness of rice kernels. A good miso product must be free from off-flavor (sour flavor) or alcohol flavor, mold infection, and discoloration (3).
By Republic of China National Standard (10), miso should meet the following criteria: (1) possesses proper miso aroma, (2) total nitrogen is higher than 1.51, (3) nonsalt solid content is higher than 40.80%, (4) pH ranges from 4.85 to 5.85, (5) packaging material should be clean and waterproof.
VI. DOU-PAN CHIANG (OR TOU-PAN CHIANG) A. Introduction
Dou-pan chiang is a traditional Chinese food. It is manufactured in various places in China. Traditionally, it was made mostly from Vicia faba (faba beans, horse beans, or wing beans). However, with the easy access to soybeans, it is now also commonly manufactured using soybeans, such as is done in Taiwan, using similar procedures.
Besides soybeans, dou-pan-chiang also uses wheat, brine, and starter as the major ingredients. Various seasonings such as chili pepper, pepper, fennel, and anise seeds can be added to modify the flavor to meet consumer demands. Its nutritional composition is similar to miso. This section discusses the handling of raw materials, manufacturing process, and changes during fermentation in the making of dou-pan-chiang.
B. Manufacturing of Dou-Pan-Chiang
The flowchart of dou-pan-chiang manufacturing is presented in Fig. 17.
1. Raw Material Handling a. Soybeans Handling of the soybeans for making dou-pan chiang follows the same procedure for making soy sauce (see Chapter 29). The cooked soybeans are mixed with roasted (or pan-fried) wheat at a ratio of 10:2 (w/w) for the koji starter preparation. The wheat is in a wheat flour form instead of broken pieces (grits). Other steps are the same as in the making of soy sauce koji starter.
b. Wheat Flour The wheat flour is browned in a frying pan or roasted to light brownish yellow color.
c. Brine Commercial crude salt is used to prepare a brine of 20-21° Baume21.18-22.24% salt) at 15°C.
2. Preparation of Seed Koji (Starter)
In the past, the seed koji (inoculum) came from the leftover Dou-pan-chiang from the previous making. It was mixed with the medium to prepare the starter. With the availability of pure cultures, this traditional method is no longer used. The current method is to use slightly (1-2%) polished rice as the raw material. It is washed, soaked, steamed, and cooled to 40° C or lower, followed by mixing with 2% potash made from
Figure 17 Flowchart on Dou-pan chiang manufacturing.
Figure 17 Flowchart on Dou-pan chiang manufacturing.
wide-leaf wood and 1-2% (w/w) dou-pan chiang koji starter (seed koji) or 1-2 x 108/g spores of the starter (Aspergillus oryzae or A. sojae).
The purpose of adding the potash is to
1. Provide inorganic nutrients for the formation of the spores
2. Provide an alkaline environment to inhibit undesirable microorganisms
3. Adjust the moisture content of the mixture so that the mixture will not stick together and has better aeration
The koji starter is incubated at 25-30 °C for 5-6 days in the incubator room, taken out from the incubator room, and sun-dried or mechanically dried to contain 5-8% moisture. It is then weighed and packed for marketing or can be used directly for the making of dou-pan-chiang. Commercially, the dou-pan-chiang koji starter either can be granules of the koji starter or, if has been sieved, contains only the starter powder of the spores.
Preparation of the koji starter is basically the same as the preparation of koji starter for soy sauce making (see Chapter 29). In the preparation of dou-pan-chiang koji starter, there are other microorganisms such as bacteria and yeasts from the environment besides the inoculated A. oryzae. In each gram of the koji starter, the total bacterial count, mold (spore) count, yeast count, and lacto-bacteria count are about 107-109, 107-108, 104-105, and 104-105, respectively. The environmental requirements determine the microbial counts and changes in the kinds of microorganisms. If the foreign microbial counts are too high, they will affect the growth of the koji mold, and directly influence the utilization rate of raw materials, product quality, and product stability. Therefore, it is important that the environment be kept sanitary to avoid unnecessary contamination.
In the preparation of Dou-pan chiang koji starter, with incubation of A. oryzae at 30 °C for 72 hr, the activity of neutral protease (related to the hydrolysis of nitrogenous compounds) reaches 2397-3688 units/g dry matter. The activity of neutral protease is highest after 48-hr incubation. At 60-hr incubation, the acid protease activity is highest. The a-amylase activity increases gradually with incubation time. The activity of h-amylase is highest at 23-60 hr. The activities of a-galactosidase, cellulase, and lipase reach their peak at 30-60 hr. The pH value as well as amino nitrogen, ammonia nitrogen, and free fatty acids contents increase gradually with incubation time. Moisture and crude fat contents decrease gradually. However, no significant change in total nitrogen was observed. Total and reducing sugars increase at the beginning and then decrease afterwards.
The Dou-pan-chiang koji starter is mixed with 20-21 °Baume (21.18-22.24% salt) brine at a ratio of 1:1 (w/w) in tubs. These tubs are left outdoors for solar heating and stirred with a wooden paddle once every day. They are covered at night or when it is raining. In the winter, supplemental heat insulation may be considered. Depending on the intermediate product temperature, the fermentation may take 2-5 months.
When addition of seasoning such as chili pepper or pepper is desired, it can be added at the end of fermentation in the form of powder or crushed seasoning (Fig. 18). Chili pepper can also be added in form of chili pepper oil made by heating seedless chili pepper in peanut oil. Addition of 20-40% cooked soybean at the time of packing into jars will
increase the flavor. Dou-pan chiang is usually packed in ceramic or glass jars (Figs. 19, 20). The jars have to be heat-sterilized in boiling water for about 20 min and are then capped and sealed.
C. Changes During Fermentation (13)
1. Changes in Microbial Population During Fermentation
At the initial stage of fermentation, the mold count decreases rapidly. More salt-tolerant lactic acid bacteria and fewer yeast increase accordingly. As fermentation progresses, lactic
acid bacteria and yeasts continue to increase, and eventually there are more yeasts than lactic acid bacteria. The lactic acid bacteria include Pediococcus, Streptococcus, and Tetracoccus sp. Yeasts include Candida, Cryptococcus, Kluyveromyces, Rhodotorula, Saccharomyces, and Torulopsis. They all contribute to the flavor of Dou-pan-chiang.
2. Changes in Enzymatic Activities During Fermentation
At the beginning of fermentation, acid protease has higher activity. Neutral protease has higher activity at the end—about 40%, compared to acid protease activity decrease to only about 20%.
At the initial stage of fermentation, both a- and h-amylase activities increase gradually. With progression of fermentation, they decrease gradually. At the end of the fermentation, a-amylase retains about 40% activity, whereas h-amylase retains 60% activity. The amylase demonstrated higher salt-tolerance than protease.
Cellulase stays at about 35% activity at the beginning and at the end of the fermentation. Its salt tolerance is somewhat between that of amylase and protease.
The activity of a-galactosidase increases slightly at the beginning and then decreases afterwards, with only about 5% activity at the end of fermentation. It is not very salttolerant.
Lipase activity increases slightly at the beginning followed by a rapid decrease, and then again increases. This kind of enzymatic change shows that the lipase from the koji starter is not salt-tolerant, and the lipase from yeasts is more salt-tolerant.
3. Changes in pH, Acidity, and Alcohol Content
During the maturation period of Dou-pan-chiang fermentation, the salt-tolerant microorganisms produce organic acids. The koji enzymes hydrolyze the substrates into free fatty acids and amino acids. The autolysis of the microbial cells produces the nucleotides. With all the acids produced, the pH gradually drops to about 5.4, and the total acidity gradually increases. The production of alcohol is irregular at the beginning, and in the mid-fermentation period increases to about 0.45% due to an increase in the yeast population.
At the end of fermentation, alcohol decreases due to reactions with free fatty acids and organic acid to form the volatile flavoring compounds.
The color of Dou-pan-chiang changes with the progression of fermentation. At the initial stage of fermentation, the color brightness increases slightly, and then decreases with the progression of fermentation. The color changes from slightly green to slightly red with an increase in yellowness, then to a dull brown color. These color changes are caused by factors such as the temperature, pH value, iron ions, amino nitrogen and reducing sugar contents, time, polyphenol oxidase activity, heating, and enzymatic and nonenzymatic browning reactions.
The soluble solids content in the fermentation mixture increases during the maturation period. This is due to the breakdown of the large molecules to small molecules during fermentation.
5. Changes in Selected Chemical Composition of the Fermentation Mixture
Total nitrogen content of the fermentation mixture increases at the initial stage of fermentation, followed by gradual decrease with progression of fermentation. The amino nitrogen and ammonia nitrogen contents increase gradually during fermentation with a more stable situation at the later period. Proteins in the raw materials are first hydrolyzed by the endoprotease into peptides, followed by the exoprotease hydrolysis to amino acids. The deaminase and decarboxylase then hydrolyze the amino acids to ammonia and amines.
The reducing sugars of the fermentation mixture increase at the initial stage and then gradually decrease.
Crude fat and free fatty acids in the fermentation mixture increase slightly at the initial stage of fermentation, followed by a gradual decrease as fermentation progresses. This is because free fatty acids, produced in the initial stage of fermentation, react with the alcohol produced to form esters.
Organic acids such as acetic acid, lactic acid, oxalic acid, and succinic acid increase with fermentation. This is due to activities of the lactic acid bacteria and yeasts.
Nucleotides such as 5'-UMP, 5'-IMP and 5'-GMP are produced during dou-pan-chiang fermentation. Both 5'-IMP and 5'-GMP showed contents above their taste threshold levels (0.012% for 5'-IMP and 0.0035% for 5'-GMP), with 5 IMP at 0.1 mg/g dry matter and 5'-GMP at 2.1 mg/g dry matter after 96 days. The production of these nucleotides contributes partially to the umami taste of Dou-pan-chiang.
The essential amino acid leucine increases threefold during dou-pan-chiang fermentation. Glutamic acid increases fourfold during the fermentation.
Dou-pan-chiang contains various easily absorbable amino acids, reducing sugars, fatty acids, organic acids, and nucleotides that contribute to its characteristic flavor. The flavor of this fermented soy product is well accepted by the consumers. It is usually available in sterilized ceramic or glass jars.
According to the Republic of China National Standards (CNS 611) (14), dou-pan-chiang should meet the following requirements:
1. Outer appearance: easily recognized bean cotyledons
2. Color: characteristic brownish yellow color
3. Odor: characteristic fermented dou-pan-chiang odor, absence of caramel odor, furfural odor, and other undesirable odors
4. Water: total water and volatile content less than 70% (wt. basis)
6. Crude protein at least 8% (wt. basis)
7. Ash: not more than 19%, including salt
8. Foreign matter: contaminants and foreign matter not permitted
9. Labeling: must meet CNS 3192 food packaging requirements
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