A method for inhibiting miscellaneous bacteria of koji and improving protease activity by adjusting pH of solid-state fermentation soy sauce koji

Abstract

The application discloses a method for inhibiting miscellaneous bacteria in soy sauce Daqu and improving protease activity by adjusting pH of the solid-state fermentation soy sauce Daqu, which comprises the following steps: in a soy sauce Daqu preparation stage, spraying acetic acid solution on cooked beans, adjusting pH of the cooked beans to 6.0-6.5, then uniformly mixing the cooked beans with strain powder and wheat powder, and carrying out solid-state fermentation to prepare Daqu; when the Daqu is cultured for 14-16 hours, before turning the Daqu, adjusting pH of the Daqu to 6.8-7.2 by using edible alkali solution, and carrying out solid-state fermentation for 44-48 hours to obtain the Daqu. In different stages of the fermentation of the soy sauce Daqu, different pH is adjusted to meet the purpose of growth and reproduction of Aspergillus oryzae and enzyme production, so that the purpose of inhibiting miscellaneous bacteria in the Daqu and improving enzyme activity is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of condiment production, and particularly relates to a method for inhibiting miscellaneous bacteria of koji and improving protease activity by adjusting the pH of solid-state fermentation soy sauce koji. BACKGROUND

[0002] Soy sauce is one of Chinese traditional condiments, which is formed by decomposing and synthesizing soybean, wheat or wheat flour as main raw materials and enzymes secreted by Aspergillus oryzae as power. The soy sauce is a kind of condiment with full color, fragrance and taste. The common soy sauce brewing process in China mainly includes high-salt dilute-state fermentation process and low-salt solid-state fermentation process, and the koji-making process in the two processes is similar. Koji-making is a key process of brewing soy sauce, which mainly provides suitable conditions for cultivating Aspergillus oryzae and promoting the growth and reproduction of Aspergillus oryzae to secrete various enzymes required for brewing soy sauce.

[0003] In the production of soy sauce, more than 90% of enzymes are accumulated in the koji-making stage, and protease activity is an important indicator of koji-making. Protease can decompose the protein in soybean into polypeptide and amino acid, improve the content of amino acid nitrogen in soy sauce, and thus improve the flavor, color and taste of soy sauce. Therefore, improving protease activity is the key to improving the quality of soy sauce.

[0004] The main raw materials for brewing soy sauce have high content of protein and starch, and are good culture medium for bacteria. At present, the koji-making of soy sauce is mainly carried out in an open condition, and the hygiene of mechanical and air-sending equipment is not easy to be thoroughly managed, inoculation is not uniform, and temperature management is improper, which causes the koji-making process to be easily polluted by various bacteria, and makes the koji appear phenomena of flower koji, acid koji and smelly koji, which seriously affects the quality of koji and reduces the protein hydrolysis rate of raw materials, the taste and aroma of soy sauce.

[0005] Therefore, it is necessary to control and manage these microorganisms in the koji-making process. The most common method for controlling miscellaneous bacteria at present is low-temperature culture in the early stage of koji-making, which adjusts the culture temperature of koji to inhibit miscellaneous bacteria. The most suitable growth temperature of Aspergillus oryzae is 35℃, however, in order to inhibit the growth and reproduction of contaminating microorganisms such as Bacillus and acid-producing bacteria, the culture temperature in the early stage of koji-making is controlled below 30℃, which inhibits the growth of Aspergillus oryzae.

[0006] As the birthplace of soy sauce condiment, China has the largest production of soy sauce in the world, but most of the market share of high-quality soy sauce and condiments is occupied by Japanese and Korean enterprises. At the same time, with the improvement of people's living standards, people's demand and requirement for condiments are getting higher and higher. Therefore, it is urgent to improve the quality of soy sauce. SUMMARY

[0007] The application aims to provide a method for inhibiting miscellaneous bacteria in koji and improving protease activity by adjusting pH of koji for soy sauce solid fermentation, which adjusts different pH in different stages of koji fermentation for soy sauce to meet the purpose of growth and reproduction of Aspergillus oryzae and enzyme production, and further realizes the purpose of inhibiting miscellaneous bacteria in koji and improving enzyme activity.

[0008] The above-mentioned purpose of the application can be realized by the following technical scheme: a method for inhibiting miscellaneous bacteria in koji and improving protease activity by adjusting pH of koji for soy sauce solid fermentation, comprising the following steps: in a koji preparation stage for soy sauce, spraying acetic acid solution on cooled matured beans to adjust pH of the matured beans to 6.0-6.5, then mixing the matured beans with strain powder and wheat powder uniformly, and performing solid fermentation to prepare koji, and before turning the koji, adjusting pH of the koji to 6.8-7.2 by using edible alkali solution, and performing solid fermentation for 44-48 hours to obtain the koji.

[0009] The method adjusts pH of koji material in the koji preparation stage, adjusts pH to the most suitable pH for growth and reproduction of Aspergillus oryzae in the growth stage of Aspergillus oryzae, and adjusts pH to the most suitable pH for enzyme production in the enzyme production stage of Aspergillus oryzae, so that Aspergillus oryzae grows and reproduces rapidly, becomes dominant bacteria, and further inhibits growth and reproduction of other miscellaneous bacteria, and under suitable conditions, Aspergillus oryzae grows and reproduces fully, and secretes protease, amylase, and various enzymes such as oxidase, lipase, and cellulase to the maximum extent, and decomposes protein in raw materials in the next fermentation process, and improves raw material utilization rate of soy sauce.

[0010] Alternatively, the mass percentage content of the acetic acid solution is 18-22%, and the spraying speed is 1.8-2.2 mL / min.

[0011] More preferably, the mass percentage content of the acetic acid solution is 20%, the spraying speed is 2.0 mL / min, and the pH of the matured beans is adjusted to 6.3.

[0012] Alternatively, the mass percentage content of the edible alkali solution is 48-52%, the spraying speed is 98-102 mL / min, and the spraying time is 48-52 min.

[0013] More preferably, the mass percentage content of the edible alkali solution is 50%, the spraying speed is 100 mL / min, the spraying time is 50 min, and the pH of the koji is adjusted to 7.0 by using the edible alkali solution.

[0014] Alternatively, the edible alkali is sodium carbonate.

[0015] Optionally, during the koji-making process, the koji-cultivation temperature is controlled at 30-34℃ from 0 to 10 hours, 34-38℃ from 11 to 16 hours, 32-36℃ from 17 to 26 hours, 30-34℃ from 27 to 32 hours, and 28-32℃ from 33 hours to the koji release. The koji is turned twice, at 14-16 hours and 25-26 hours respectively.

[0016] Optionally, the inoculum powder is Aspergillus oryzae inoculum powder, and its addition amount is 8-12‰ of the total mass of cooked soybeans and wheat flour.

[0017] More preferably, the inoculum powder is Aspergillus oryzae inoculum powder, and its addition amount is 10‰ of the total mass of cooked soybeans and wheat flour.

[0018] Optionally, the amount of wheat flour used is 0.5 to 0.7 times the total mass of cooked beans.

[0019] More preferably, the amount of wheat flour used is 0.6 times the total mass of cooked beans.

[0020] Therefore, this invention aims to alter the pH of the starter culture at different stages during the fermentation process of soy sauce koji. Specifically, when the cooked soybeans are cooled, acetic acid is sprayed to bring the pH to 6.0–6.5. This mixture is then combined with inoculum powder and wheat flour to create a blend. This pH is suitable for the growth and reproduction of Aspergillus oryzae, allowing it to grow rapidly and become the dominant microorganism in the starter culture, inhibiting the growth and reproduction of other miscellaneous bacteria (Bacillus, Micrococcus, etc.). After approximately 15 hours of fermentation, just before turning the starter culture, edible alkali liquid is sprayed, followed by turning, bringing the pH to approximately 7.0. This pH is conducive to the secretion of proteases by Aspergillus oryzae, especially neutral protease activity. This process not only inhibits the growth and reproduction of miscellaneous bacteria in the starter culture but also increases the activity of neutral protease secreted by Aspergillus, thereby improving the quality of the starter culture. This, in turn, increases the utilization rate of raw materials and the amino acid nitrogen production rate. With fewer miscellaneous bacteria, secondary precipitation in the soy sauce is also reduced, ultimately improving the quality of the soy sauce.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) In the method of this invention, acetic acid is sprayed onto the cooked soybeans, reducing their pH from approximately 7.0 to 6.0–6.5, which is the optimal pH for Aspergillus oryzae. This allows Aspergillus oryzae to grow and multiply rapidly in the koji material, inhibiting the growth and reproduction of other microorganisms. Consequently, the total bacterial count and spore count in the mature koji material are reduced by an order of magnitude compared to existing processes, with the total bacterial count decreasing from 10... 7 Reduced to 10 6 The number of spores increased from 10 5 Reduced to 10 4The reduction of the number of miscellaneous bacteria in the Daqu directly influences secondary precipitation of the finished soy sauce, and the secondary precipitation of the soy sauce is reduced by about 60% after the overall application of the technology.

[0023] (2) The Daqu is cultured for about 15 hours, before the first turning of the Daqu, edible alkali liquid is sprayed to the Daqu material, and then the Daqu is turned again to make it uniform, so as to adjust the pH of the Daqu material to about 7.0, which is beneficial to the secretion of more neutral protease by Aspergillus, compared with the commonly used process, the neutral enzyme activity is increased by about 20%, the amino acid nitrogen content of the raw oil fermented by the process is increased by about 7% compared with the commonly used process, and the total acid is reduced by about 8%. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be described in detail below with specific examples, so that the technical solutions of the present application can be better understood and implemented by the skilled in the art. The following examples are only used for illustrative purposes, and should not be understood as limiting the present application. Unless otherwise specified, the reagents or materials used in the examples are obtained from commercial channels. Unless otherwise specified, the experimental instruments used are conventional laboratory instruments.

[0025] In order to explain the present application in more detail, the following examples are carried out. It should be emphasized that the following examples are only used to explain the present application, and should not be used to limit the essential scope or content of the present application.

[0026] Example 1 Determination of the best process of spraying vinegar acid on cooked beans

[0027] (1) Preparation of acid liquid

[0028] According to different proportions, the acid liquid is prepared, and the prepared proportions of the acid liquid are 10%, 15%, 20%, 25% and 30% (mass percentage) of vinegar acid, which are sprayed on the cooked beans (after cooling);

[0029] (2) Determination of spraying speed

[0030] The spraying speed is 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min and 5 mL / min, respectively, which is sprayed on the surface of the cooked beans, and the pH of the cooked beans is used as the determination index to determine the best concentration of vinegar acid and spraying speed.

[0031] Table 1 pH of cooked beans corresponding to different spraying concentrations and spraying speeds

[0032]

[0033] From Table 1, it can be seen that the pH of cooked beans is 6.3 when the concentration of acetic acid is 20% and the spraying speed is 2 mL / min, which is most suitable for the growth and reproduction of Aspergillus oryzae.

[0034] The pH value of cooked beans was determined by using an automatic potentiometric titrator: 10.00 g of cooked beans was ground and added to 90 mL of tertiary water to prepare a solution with a dilution of 10 g / 100 mL in a 100 mL beaker. The stirring rod and electrode were washed with tertiary water and placed in the stirring rod. The electrode was immersed in the sample solution. The pH measurement program was selected and the "start" key was pressed to start the detection. When the program automatically stopped, the reading was the pH value of the sample.

[0035] The effect of spraying acetic acid on cooked beans was verified in a pilot-scale test in Example 2

[0036] The best concentration of acetic acid for spraying on cooked beans was determined to be 20% in the small-scale test, and the best spraying speed of acetic acid was 2 mL / min. In the pilot-scale test, the prepared acetic acid solution was sprayed on cooked beans, and the pH of 10 batches of cooked beans was recorded (see Table 2 below). Cooked beans, strains, and wheat flour were mixed (the strain was Aspergillus oryzae, and the addition amount was 10‰ of the total mass of cooked beans and wheat flour, and the addition amount of wheat flour was 0.6 times the total mass of cooked beans). The mixture was pumped to the fermentation tank through a conveyor belt pump for koji making. The koji was cultured for 42-44 h to obtain matured Daqu. The neutral protease activity and acid protease activity of the matured Daqu, the total number of bacteria and the total number of spores of the Daqu were detected (see Table 3 below). The matured Daqu was mixed with 20 Baume's brine to prepare a mash, which was subjected to open-air fermentation for 180 d to produce raw soy sauce. The total acid, amino acid nitrogen, salt content, and total nitrogen of the raw soy sauce were detected.

[0037] During the koji making process, the culture temperature was controlled as follows: 0-10 h, 30-34℃; 11-16 h, 34-38℃; 17-26 h, 32-36℃; 27-32 h, 30-34℃; 33 h to the end of the koji making process, 28-32℃. The koji was turned twice at the 16th hour and the 26th hour.

[0038] In the existing process, soybeans were soaked, cooked, and then mixed with wheat flour and strain powder. The mixture was pumped to the fermentation tank through a conveyor belt pump for solid-state fermentation. The fermentation time was 44-48 h, and the matured Daqu was mixed with 20 Baume's brine to prepare a mash, which was subjected to open-air fermentation for 180 d to produce raw soy sauce.

[0039] Table 2 pH of different batches of cooked beans

[0040] Batch 1 2 3 4 5 6 7 8 9 10 pH 6.3 6.4 6.5 6.4 6.6 6.3 6.5 6.2 6.3 6.5

[0041] Table 3 Neutral protease activity and acid protease activity of different batches of Daqu

[0042]

[0043]

[0044] Using the existing process to make Daqu, 10 batches of Daqu were extracted for physicochemical index detection, as control samples, the indexes are shown in Table 4:

[0045] Table 4 Physicochemical indexes of 10 batches of Daqu of the existing process

[0046] Batch 1 2 3 4 5 Neutral protease activity 2032 2101 2020 2205 2103 Acid protease activity 254 232 245 210 247 Spore number 2.32E+05 2.63E+05 6.27E+05 5.21E+05 4.87E+05 Total bacterial count 4.91E+107 4.52E+107 8.54E+107 9.97E+107 5.97E+107 Batch 6 7 8 9 10 Neutral protease activity 2105 2015 2153 2231 2054 Acid protease activity 235 234 221 254 237 Spore number 5.61E+05 5.42E+05 2.03E+05 3.54E+05 5.47E+05 Total bacterial count 3.54E+107 3.22E+107 6.56E+107 6.35E+107 5.88E+107

[0047] From Table 3 and Table 4, it can be seen that the concentration of acetic acid is 20%, the spraying speed is 2 mL / min, and the pH of cooked beans is 6.2-6.5, which is most suitable for the growth and reproduction of Aspergillus oryzae. From Table 2, it can be seen that adjusting the pH of cooked beans to about 6.3 is beneficial to the rapid reproduction of Aspergillus oryzae and inhibits the growth of other bacteria. The number of spores and the total number of bacteria in Daqu are both reduced by one order of magnitude compared with the control. The neutral and acid enzyme activities increase slightly, by 8.6% and 14%, respectively.

[0048] Example 3 Determination of the best process for spraying edible alkali when Daqu is cultured for about 15 h (based on the experiment of spraying acetic acid on cooked beans)

[0049] (1) Different starch raw materials were used to make Daqu on a laboratory scale, and the protease activity of the resulting Daqu was compared

[0050] ①Preparation of edible alkali (sodium carbonate) liquid

[0051] According to different mass percentage ratios, different concentrations of edible alkali (sodium carbonate) liquid were prepared. The preparation ratios were 20%, 30%, 40%, 45%, and 50% acetic acid for spraying onto cooked beans.

[0052] ②Spraying time

[0053] Using a fermentation tank as a unit, the spraying speed was 100 mL / min, and the spraying time was 10 min, 20 min, 30 min, 40 min, and 50 min, respectively, until the pH of the semi-mature Daqu was 7.0. See Table 5 below to determine the best spraying time. After spraying, the material was turned to ensure uniform pH.

[0054] Table 5 pH of mature koji corresponding to different spraying concentrations and spraying times

[0055] Spray concentration 20％ 30％ 40％ 45％ 50％ 10 min 6.3 6.3 6.4 6.4 6.5 20 min 6.3 6.4 6.4 6.5 6.5 30 min 6.5 6.5 6.6 6.6 6.7 40 min 6.6 6.6 6.7 6.7 6.8 50 min 6.6 6.7 6.8 6.8 7.0

[0056] As shown in Table 5, the edible alkali with concentrations of 20%, 30%, 40%, 45% and 50% is sprayed onto the surface of the koji at a spraying speed of 100 mL / min, and the spraying time is 10 min, 20 min, 30 min, 40 min and 50 min respectively, and the pH of the cooked beans is detected. As shown in Table 5, when the concentration of the edible alkali is 50% and the spraying time is 50 min, the pH of the cooked beans is 7.0 (determined), which is most suitable for the neutral protease activity of Aspergillus oryzae. The optimal concentration of the edible alkali and the spraying time are determined for the first time in this round of pilot test, and the pH of the koji is 7.0 when the koji is sprayed according to the process for the first time, which is most suitable for the neutral protease activity of Aspergillus oryzae.

[0057] Example 4 Verification of the effect of spraying vinegar on cooked beans and spraying edible alkali on koji in pilot scale

[0058] The cooked bean spraying process and the edible alkali spraying process have been determined in the small scale test, and the pilot scale test is carried out according to the process. The pH of 10 batches of cooked beans is recorded (see Table 6 below); the cooked beans, the strain and the wheat flour are mixed (the strain is Aspergillus oryzae strain, and the addition amount is 10 ‰ of the total mass of the cooked beans and the wheat flour, and the addition amount of the wheat flour is 0.6 times the total mass of the cooked beans), and the mixed material is pumped to the fermentation tank through the transmission belt pump to carry out koji making. The edible alkali is sprayed during the koji making process, and the koji is cultured in the fermentation tank for 42-44 h to obtain mature koji. The neutral protease activity and the acid protease activity of the mature koji, the total number of bacteria and the total number of spores of the mature koji are detected (see Table 7); the mature koji is mixed with 20 Baume brine to prepare soy sauce mash, and the mash is subjected to sunning fermentation. The mash is fermented for 180 d, and then oil is added to prepare raw soy sauce. The total acid, the amino acid nitrogen, the salt content, the total nitrogen and other indicators of the raw soy sauce fermented for 180 d are detected (see Table 8).

[0059] The existing process is used to ferment raw soy sauce.

[0060] During the koji making process, the koji culture temperature control and the koji turning are the same as those in Example 2.

[0061] Table 6 pH of cooked beans prepared in different batches according to the technology of the present application

[0062] Batch 1 2 3 4 5 6 7 8 9 10 pH 6.2 6.4 6.5 6.4 6.5 6.3 6.5 6.2 6.3 6.5

[0063] Table 7 Neutral protease activity and acid protease activity of koji prepared in different batches according to the technology of the present application

[0064] Batch 1 2 3 4 5 Neutral protease activity 2430 2475 2364 2258 2431 Acid protease activity 305 321 219 298 278 Spore number 1.23E+04 2.41E+04 2.34E+04 2.54E+04 2.51E+04 Total bacterial count 4.23E+106 4.97E+106 5.21E+106 4.63E+106 3.25E+106 Batch 6 7 8 9 10 Neutral protease activity 2364 2541 2532 2500 2587 Acid protease activity 302 312 298 278 294 Spore number 2.48E+04 2.53E+04 2.63E+04 2.53E+04 2.23E+04 Total bacterial count 2.58E+106 7.45E+106 6.34E+106 3.97E+106 4.56E+106

[0065] Table 8 Physico-chemical indexes of different batches of soy sauce brewed by the present technology

[0066] Batch 1 2 3 4 5 Total acid 1.94 1.93 1.92 1.90 1.89 Amino acid nitrogen 1.10 1.09 1.08 1.05 1.04 Salt content 16.32 16.26 16.28 16.86 16.47 Total nitrogen 1.67 1.64 1.65 1.67 1.69 Batch 6 7 8 9 10 Total acid 1.94 1.96 1.92 1.93 1.89 Amino acid nitrogen 1.06 1.08 1.12 1.08 1.04 Salt content 16.42 16.21 16.84 16.31 16.71 Total nitrogen 1.68 1.67 1.63 1.67 1.64

[0067] The existing technology was used to produce Daqu and ferment soy sauce. Ten batches of Daqu and corresponding soy sauce were extracted as control tanks. The physico-chemical indexes of the ten batches of Daqu produced by the existing technology are shown in Table 9, and the physico-chemical indexes of the natural oil fermented by the Daqu produced by the existing technology are shown in Table 10.

[0068] Table 9 Physico-chemical indexes of ten batches of Daqu produced by the existing technology

[0069] Batch 1 2 3 4 5 Neutral protease activity 2103 2143 2015 2045 2064 Acid protease activity 284 254 251 273 230 Spore number 3.24E+05 2.53E+05 5.28E+05 6.32E+05 4.87E+05 Total bacterial count 2.58E+107 3.69E+107 4.32E+107 2.39E+107 4.36E+107 Batch 6 7 8 9 10 Neutral protease activity 2053 2203 2156 2143 2035 Acid protease activity 262 253 245 293 260 Spore number 2.36E+05 3.78E+05 3.03E+05 3.74E+05 6.47E+05 Total bacterial count 4.31E+107 3.22E+107 7.15E+107 3.65E+107 5.43E+107

[0070] Table 10 Physico-chemical indexes of natural oil fermented by Daqu produced by the existing technology (unit: g / 100 mL)

[0071] Batch 1 2 3 4 5 Total acid 2.12 2.05 2.14 2.06 2.04 Amino acid nitrogen 0.96 1.03 1.01 0.98 1.02 Salt content 16.37 16.25 16.61 16.52 15.92 Total nitrogen 1.62 1.57 1.64 1.67 1.59 Batch 6 7 8 9 10 Total acid 1.97 1.99 2.12 1.16 2.13 Amino acid nitrogen 1.03 1.0 0.97 1.02 1.02 Salt content 16.43 16.35 16.54 16.09 16.14 Total nitrogen 1.60 1.63 1.57 1.61 1.69

[0072] The results of the pilot-scale verification test using the new technology show that the total number of bacteria and the number of spores of mature Daqu each decrease by one order of magnitude. The total number of bacteria decreases from 10 7 to 10 6 , and the number of spores decreases from 10 5 to 10 4 . The neutral protease activity increases by 16.8% compared with the control, and the acid enzyme activity increases by 11.5%. The total acid content of the fermented crude oil decreases by about 8%, and the amino acid nitrogen content increases by 7%.

[0073] Example 5: Effect of production-scale verification of the present technology

[0074] Soy sauce fermented by the new technology: The steamed soybeans were sprayed with a 20% acetic acid liquid at a spraying speed of 2 mL / min, and the pH of the soybeans was adjusted to 6.3. The soybeans, strain powder, and wheat powder (the strain powder was Aspergillus oryzae strain powder, and the amount added was 10‰ of the total mass of the soybeans and wheat powder, and the amount of wheat powder was 0.6 times the total mass of the soybeans) were mixed uniformly, and then pumped to the fermentation tank through a conveyor belt for solid fermentation. The Daqu was cultured for about 15 h, and then a 50% edible alkali solution was sprayed on the Daqu at a spraying time of 50 min and a spraying speed of 100 mL / min, and the pH of the Daqu was adjusted to about 7.0. The solid fermentation was performed for 44-48 h, and then the Daqu was discharged. The neutral protease activity and acid protease activity of the Daqu, and the total number of bacteria and the total number of spores of the Daqu were detected (see Table 11). The mature Daqu was mixed with 20 Baume's brine to make wort, and then exposed to the sun for 180 d for fermentation to produce soy sauce. The total acid, amino acid nitrogen, salt content, total nitrogen, and other indexes of the soy sauce fermented for 180 d were detected (see Table 12).

[0075] Soy sauce fermented by the existing technology was the same as in Example 2.

[0076] In the koji-making process, the koji culture temperature control and the koji turning are the same as in Example 2.

[0077] Table 11 Neutral protease activity and acid protease activity of different batches of koji made by the present technology

[0078] Batch 1 2 3 4 5 Neutral protease activity 2345 2256 2456 2458 2322 Acid protease activity 324 245 305 256 298 Spore number 3.25E+04 2.56E+04 6.75E+04 5.52E+04 6.21E+04 Total bacterial count 2.31E+106 3.58E+106 4.36E+106 6.37E+106 5.47E+106 Batch 6 7 8 9 10 Neutral protease activity 2368 2453 2451 2389 2458 Acid protease activity 256 278 245 278 251 Spore number 3.20E+04 2.57E+04 3.56E+04 4.31E+04 5.26E+04 Total bacterial count 6.34E+106 5.21E+106 7.21E+106 3.45E+106 2.10E+106

[0079] Table 12 Physicochemical indexes of different batches of raw soy sauce brewed by the present technology

[0080] Batch 1 2 3 4 5 Total acid 1.89 1.96 1.95 1.90 1.92 Amino acid nitrogen 1.09 1.11 1.12 1.07 1.03 Salt content 16.42 16.35 16.54 16.68 16.74 Total nitrogen 1.70 1.68 1.65 1.67 1.63 Batch 6 7 8 9 10 Total acid 1.93 1.95 1.92 1.93 1.90 Amino acid nitrogen 1.04 1.08 1.07 1.06 1.04 Salt content 16.35 16.56 16.83 16.74 16.32 Total nitrogen 1.66 1.62 1.65 1.68 1.63

[0081] Koji and raw soy sauce were made by the existing technology, and 10 batches of koji and corresponding raw soy sauce were extracted as control tanks. The physicochemical indexes are shown in Tables 13 and 14:

[0082] Table 13 Physicochemical indexes of 10 batches of koji made by the existing technology

[0083] Batch 1 2 3 4 5 Neutral protease activity 2031 2056 2057 2041 2131 Acid protease activity 276 254 259 268 220 Spore number 2.54E+05 3.21E+05 4.23E+05 5.54E+05 6.34E+05 Total bacterial count 6.35E+107 5.21E+107 4.32E+107 3.96E+107 2.58E+107 Batch 6 7 8 9 10 Neutral protease activity 2045 2067 2098 2034 2056 Acid protease activity 230 245 248 265 248 Spore number 5.34E+05 4.51E+05 6.30E+05 3.65E+05 2.17E+05 Total bacterial count 1.98E+107 2.78E+107 3.64E+107 4.32E+107 5.47E+107

[0084] Table 14 Physicochemical indexes of raw soy sauce fermented by koji made by the existing technology (unit: g / 100mL)

[0085] Batch 1 2 3 4 5 Total acid 2.05 2.14 2.06 2.12 1.98 Amino acid nitrogen 1.02 0.97 1.03 0.99 1.00 Salt content 16.45 16.58 16.32 16.50 15.98 Total nitrogen 1.67 1.62 1.65 1.63 1.67 Batch 6 7 8 9 10 Total acid 2.15 1.99 1.97 2.12 2.17 Amino acid nitrogen 1.02 1.01 0.98 1.04 1.03 Salt content 16.21 16.36 16.74 16.85 16.63 Total nitrogen Batch Neutral protease activity Acid protease activity Spore number Total bacterial count Batch Neutral protease activity Acid protease activity Spore number Total bacterial count Batch Total acid Amino acid nitrogen Salt content Total nitrogen Batch Total acid Amino acid nitrogen Salt content Total nitrogen 1.63 1.60 1.57 1.63 1.61

[0086] The production test scale was used to verify the process effect of the present technology. The results showed that the total number of bacteria and the number of spores of mature koji each decreased by one order of magnitude. The total number of bacteria decreased from 10 7 to 10 6 , and the number of spores decreased from 10 5 to 10 4 . The neutral protease activity increased by 16.8% compared with the control, and the acid enzyme activity increased by 11.5%. The total acid content of the fermented crude oil decreased by about 8%, and the amino acid nitrogen content increased by 7%. The present technology was used to brew raw soy sauce, which achieved the increase of amino acid nitrogen in raw soy sauce, reduced the production cost of soy sauce, and improved the quality of soy sauce.

[0087] Example 6 Comparison test of secondary precipitation of soy sauce brewed by the present technology and soy sauce brewed by the existing technology

[0088] New process fermentation of raw soy sauce: soybean is steamed, and 20% acetic acid liquid is sprayed on the steamed soybean at a spraying speed of 2 mL / min, and the pH of the steamed soybean is adjusted to 6.3; the steamed soybean, strain powder and wheat powder (the amount is the same as in Example 5) are uniformly mixed, and are pumped to a fermentation tank through a conveying belt for solid fermentation; the koji is cultured for about 15 h, 50% edible alkali solution is sprayed on the koji at a spraying speed of 100 mL / min for 50 min, and the pH of the koji is adjusted to about 7.0; the solid fermentation is performed for 44-48 h, and the koji is discharged; the matured koji is mixed with 20 Baume's brine to make a mash, and is exposed to the sun for 180 d for fermentation to obtain raw soy sauce; after the raw soy sauce is blended, diatomite filtered, sterilized and packaged, a finished soy sauce is obtained, and the secondary precipitate in the sample is weighed after the sample is left at room temperature for 60 d.

[0089] Existing process fermentation of raw soy sauce: soybean is soaked, steamed and cooked, and is uniformly mixed with wheat powder and strain powder, and is pumped to a fermentation tank through a conveying belt for solid fermentation for 44-48 h; the koji is discharged, and the matured koji is mixed with 20 Baume's brine to make a mash, and is exposed to the sun for 180 d for fermentation to obtain raw soy sauce; after the raw soy sauce is blended, diatomite filtered, sterilized and packaged, a finished soy sauce is obtained, and the secondary precipitate in the sample is weighed after the sample is left at room temperature for 60 d, as shown in Table 15.

[0090] In the koji-making process, the koji culture temperature is controlled and the koji is turned over, which is the same as in Example 2.

[0091] Method for measuring the secondary precipitate in a finished soy sauce: after the soy sauce product is left at room temperature for 60 d, centrifugation is performed at 0°C and 10,000 r / min for 10 min, the supernatant is removed by siphoning, and the precipitate is collected; the collected precipitate is dried at 105°C to constant weight, and the amount of the secondary precipitate in the soy sauce is calculated, as shown in Table 15.

[0092] Table 15: Secondary precipitate content in soy sauce produced by the existing process and the process of the present application

[0093]

[0094] As shown in Table 15, the amount of the secondary precipitate in the soy sauce produced by the process of the present application is much less than that produced by the existing process, and the amount of the secondary precipitate in the soy sauce is reduced by 60% after the application of the process of the present application as a whole.

[0095] This example is only an explanation and description of the concept of the present application, and is not a limitation thereof, that is, equivalent changes and modifications made by those skilled in the art without departing from the concept of the present application also belong to the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A method for inhibiting miscellaneous bacteria of koji and improving protease activity by adjusting pH of solid-state fermentation soy sauce Daqu, characterized in that The method comprises the following steps: In the soy sauce Daqu preparation stage, acetic acid solution is sprayed on cooked beans to adjust the pH value of the cooked beans to 6.0-6.5, and then the cooked beans are mixed with strain powder and wheat powder to perform solid fermentation to prepare Daqu; when the Daqu is cultured for 14-16 hours, the pH value of the Daqu is adjusted to 6.8-7.2 by using edible alkali solution before turning the Daqu, and the solid fermentation is performed for 44-48 hours to obtain the Daqu; The acetic acid solution has a mass percentage of 18-22%, and the spraying speed is 1.8-2.2 mL / min; The edible alkali is sodium carbonate; The edible alkali solution has a mass percentage of 48-52%, the spraying speed is 98-102 mL / min, and the spraying time is 48-52 min; During the Daqu preparation, the Daqu culture temperature is controlled as follows: 30-34 ℃ for 0-10 hours, 34-38 ℃ for 11-16 hours, 32-36 ℃ for 17-26 hours, 30-34 ℃ for 27-32 hours, and 28-32 ℃ for 33 hours to the end of the Daqu preparation; the Daqu is turned twice at the 14th-16th hour and the 25th-26th hour; The strain powder is Aspergillus oryzae strain powder, and the addition amount of the strain powder is 8-12 ‰ of the total mass of the cooked beans and the wheat powder; the amount of the wheat powder is 0.5-0.7 times the total mass of the cooked beans.

2. The method for inhibiting miscellaneous bacteria of koji and improving protease activity by adjusting pH of solid-state fermentation soy sauce Daqu according to claim 1, characterized in that: The acetic acid solution has a mass percentage of 20%, the spraying speed is 2.0 mL / min, and the pH value of the cooked beans is adjusted to 6.

3.

3. The method for inhibiting miscellaneous bacteria and improving protease activity by adjusting pH of solid-state fermentation soy sauce Daqu according to claim 1, characterized in that: The edible alkali solution has a mass percentage of 50%, the spraying speed is 100 mL / min, the spraying time is 50 min, and the pH value of the Daqu is adjusted to 7.0 by using the edible alkali solution.

4. The method for inhibiting miscellaneous bacteria and improving protease activity by adjusting pH of solid-state fermentation soy sauce Daqu according to claim 1, characterized in that: The addition amount of the strain powder is 10 ‰ of the total mass of the cooked beans and the wheat powder, and the amount of the wheat powder is 0.6 times the total mass of the cooked beans.

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