Preparation method of soy sauce koji with high germination rate and high spore number

Abstract

The application discloses a preparation method of soy sauce starter with high germination rate and high spore number, which comprises the following steps: mixing and rubbing a composite nutrition package A with dry non-moldy large bran, uniformly packing, sterilizing by heating, shaking the material while it is hot, and obtaining a starter culture medium after sterilization; during the first time of turning the starter, uniformly supplementing a composite nutrition package B into the starter culture medium through a sterile filter, continuing to culture until the surface of the starter culture medium is white and clumpy again, carrying out the second time of shaking bottle turning, culturing until the bottle is closed, and finally culturing until the starter culture matures, and obtaining the soy sauce starter. The application matches two kinds of nutrition packages of promoting growth and rough metabolism, and cooperates with shaking bottle turning, so that the activity of aspergillus oryzae is supplemented and intervened in stages, the spore germination rate of the starter culture is greater than or equal to 98%, the spore number is greater than or equal to 13 billion per gram, and the trehalose content of aspergillus oryzae in the starter culture is not higher than 4.50 mg / g.

Description

Technical Field

[0001] This invention belongs to the field of soy sauce preparation, specifically involving a method for preparing soy sauce starter with high germination rate and high spore number. Background Technology

[0002] Aspergillus oryzae is one of the most important microorganisms in soy sauce brewing, as it secretes a large number of enzymes to hydrolyze raw materials. Industrial production requires the preparation of starter culture to expand the cultivation of Aspergillus oryzae. The quality of the starter culture directly determines the quality of the soy sauce and is the core of soy sauce brewing.

[0003] However, the unstable quality of starter culture is a problem faced by most enterprises, which seriously affects the quality of soy sauce and causes unnecessary losses to the soy sauce industry.

[0004] Currently, the main characteristics of unstable or poor-quality koji seedlings include low spore germination rate, slow germination speed, insufficient spore number, and high bacterial content. Among these, low germination rate and insufficient spore number are the main reasons for poor koji seedling quality.

[0005] Therefore, there is an urgent need in this field for a method to prepare soy sauce starter with high germination rate and high spore number in order to improve the quality of soy sauce products. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing soy sauce starter with high germination rate and high spore number.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing soy sauce starter with high germination rate and high spore number, comprising,

[0010] Mix the compound nutrient pack A with dry, mold-free large pieces of wheat bran, knead them evenly, bottle them, heat them for sterilization, shake the material while it is still hot, and sterilize to obtain the seed culture medium.

[0011] Activated Aspergillus oryzae spores were scraped with a sterile inoculation loop under sterile conditions to prepare a dispersed spore suspension.

[0012] The spore suspension was inoculated into the koji culture medium under sterile conditions. After inoculation, the medium was shaken thoroughly and then incubated at a constant temperature until obvious clumping appeared. The first shake-flask inoculation was then performed.

[0013] During the first turning of the koji, the compound nutrient pack B is sterilized through a sterile filter and evenly added to the koji. The koji is then cultured until the surface turns white and clumps up again. The second turning of the koji is then performed, and the koji is cultured until it is covered with a bottle. Finally, the koji is cultured until it matures, and the koji is harvested to produce soy sauce koji.

[0014] The compound nutrient package A consists of glucose, zinc sulfate, calcium chloride, potassium dihydrogen phosphate and sterile water.

[0015] Compound Nutrition Pack B consists of ammonium sulfate, GDP-D-mannose, chitinase and phosphorylase, and sterile water.

[0016] In a preferred embodiment of the preparation method described in this invention, the mass ratio of the large pieces of wheat bran to the compound nutrient package A is 1:1 to 1.3.

[0017] As a preferred embodiment of the preparation method described in this invention, the composite nutrient package A comprises, by mass fraction of each raw material, 6 parts glucose, 2 parts zinc sulfate, 2 parts calcium chloride, 4 parts potassium dihydrogen phosphate, and 20-40 parts sterile water.

[0018] In a preferred embodiment of the preparation method described in this invention, the activated Aspergillus oryzae spores include, wherein the activation process comprises,

[0019] Soak fresh soybeans in water for 8 hours, then filter them out. Add 5 to 7 times the mass of water and boil until the Baumé meter reading is 5. Filter out the soybean juice.

[0020] Dissolve 2g of glucose, 0.1g of potassium dihydrogen phosphate, 0.05g of ammonium sulfate, and 0.05g of magnesium sulfate in every 100mL of soy milk, then add 1.5% agar powder by mass and adjust the pH to 6.

[0021] The mixture was then placed into test tubes for sterilization to prepare test tube slant activation culture medium;

[0022] Use a sterile inoculation loop to pick up the spores to be activated and streak them in a Z-shape on the activation medium. Incubate at 30°C for 72 hours. Observe the Aspergillus oryzae until it turns green, which means it has been activated once. Repeat the steps for a total of two activations to obtain activated Aspergillus oryzae spores.

[0023] In a preferred embodiment of the preparation method described in this invention, the dispersed spore suspension obtained comprises,

[0024] Add 9g NaCl, 100μL Tween-80, and 1L water, stir well, sterilize, and use the resulting spore eluent to elute and mix.

[0025] Place a drop of spore suspension on a hemocytometer, observe and count the spores using an optical microscope, and adjust the number of spores per mL of spore suspension to 1 × 10⁻⁶ by adding spore elution buffer. 7 After that, a dispersed spore suspension was obtained.

[0026] As a preferred embodiment of the preparation method of the present invention, the spore suspension is inoculated into the seed culture medium under sterile conditions, wherein 0.5 to 1 mL of spore suspension is inoculated into every 10 g of seed culture medium.

[0027] In a preferred embodiment of the preparation method described in this invention, the mass ratio of the composite nutrient package B to the seed culture of the first fermentation is 0.1-0.5%wt.

[0028] As a preferred embodiment of the preparation method described in this invention, the composite nutrient package B comprises, by mass fraction of each raw material, 1 part ammonium sulfate, 0.4 parts GDP-D-mannose, 0.01 parts chitinase, 0.01 parts phosphorylase, and 1-2 parts sterile moisture.

[0029] In a preferred embodiment of the preparation method described in this invention, the interval between the first shaking and the second shaking is 12-14 hours, and the interval between the second shaking and the second shaking is 15.5-17.5 hours.

[0030] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing soy sauce starter with high germination rate and high spore number. The soy sauce starter has a spore germination rate of ≥98%, a spore number of ≥13 billion / g, and a trehalose content of Aspergillus oryzae in the starter not higher than 4.50 mg / g.

[0031] Beneficial effects of this invention:

[0032] This invention provides a method for preparing soy sauce starter with high germination rate and high spore count. A dispersed spore suspension is prepared to promote the uniformity of Aspergillus oryzae distribution in the starter, enhance the germination and enzyme production fermentation characteristics of Aspergillus oryzae, and prevent phenomena such as stale starter, burnt starter, and insufficient enzyme activity. This invention combines two nutrient packages that promote growth and crude metabolism, and uses shake-flask turning to supplement and intervene in the activity of Aspergillus oryzae in stages. The spore germination rate of the starter is ≥98%, the spore count is ≥13 billion / g, and the trehalose content of Aspergillus oryzae in the starter is not higher than 4.50 mg / g. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0034] Figure 1 The figures show the germination rate and survival status of spores in Examples 1 (top left), 2 (top right), and 3 (bottom middle) of the present invention, where spores are marked as inferior spores.

[0035] Figure 2 Examples 1 (right figure) and 5 (left figure) of the present invention illustrate the effect of dispersed spore suspension technology on the germination state of Aspergillus oryzae. Detailed Implementation

[0036] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0037] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0038] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0039] Materials used in this invention:

[0040] Glucose, zinc sulfate, calcium chloride, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, agar powder, GDP-D-mannose, Tween 80: Tianjin Jiangtian Chemical Technology Co., Ltd.

[0041] Chitinase (Catalog No. C6137-50UN), Phosphorylase (Catalog No. P6635): Merck KGaA, Darmstadt, Germany.

[0042] Spore germination rate determination in this embodiment of the invention:

[0043] Take 1g of fresh spores and place them in a small Erlenmeyer flask containing 50mL of sterile water and 4-5 glass beads. Shake thoroughly for at least 5 minutes. Use a pipette to draw 1mL of the spore suspension and mix it with 9mL of sterile water in a test tube, then shake thoroughly.

[0044] Dip a sterile glass rod into a drop of spore dilution and place it on a clean, dry sterile coverslip. Quickly dip the glass rod into a drop of freshly heated and dissolved PCA medium and mix it thoroughly with the spore dilution on the coverslip in a clockwise circular motion. The diameter of the circle should not be too large, and should be less than 1 cm.

[0045] Place the coverslip on the groove on the back of the hemocytometer. The mixture will solidify under the action of agar within 30 seconds. Take 2 mL of sterile water and drop it into a sterile culture dish. Place two tubes to support the hemocytometer.

[0046] Cover the petri dish and place it in a 30℃ constant temperature incubator for incubation. Observe the germination of spores after 4 hours of incubation. Spores with a germination length greater than the spore diameter are defined as germinating spores.

[0047] Spore germination rate = number of germinating spores in the field of view ÷ total number of spores in the field of view. 3 to 5 fields of view need to be observed and calculated.

[0048] Plotting the standard curve of spore count in this embodiment of the invention:

[0049] Freshly prepared spore stock was used to prepare spore suspension. Spore eluent was added to adjust the concentration of the spore suspension. The concentration was counted and calculated using a hemocytometer. Seven suitable concentration gradients were selected so that the OD600 was approximately between 0.2 and 1.0. A standard curve of spore concentration from the spore stock was plotted.

[0050] Determination of spore count in this embodiment of the invention:

[0051] Take 5g of fresh koji and place it in a drying oven at 105℃. Dry until constant weight, then dilute with spore eluent to OD. 600 Set the temperature to 0.2–1.0, then shake well in a constant temperature shaking incubator for 20 minutes, and finally vortex for 5 minutes to fully mix the spores.

[0052] Based on the standard curve, the absorbance of the spore solution at 600 nm can be measured using an ultraviolet spectrophotometer to calculate the number of spores per gram of oven-dried spore culture.

[0053] Determination of trehalose content in spores in this invention:

[0054] Take 10g of seed culture, centrifuge and wash the spores at 4000r / min with a large amount of sterile spore elution buffer for 5min each time, and wash at least 3 times. Place the washed spore solution in a drying oven at 105℃ and dry it to constant weight. Weigh and measure the spore mass under the absolute dry condition.

[0055] Dry spores were dissolved in 4 mL of 0.4 mol / L TCA (trichloroacetic acid solution), extracted in an ice-water bath for 30 min, and then centrifuged to collect the extract. TCA was then added to further extract trehalose. Finally, the two extracts were combined. 100 μL of the extract was diluted to 2 mL with sterile water, and 5 mL of 0.2% anthrone-sulfuric acid reagent was added. The mixture was heated to boiling for 5 min, cooled to room temperature, and the absorbance at 630 nm was measured spectrophotometrically.

[0056] Based on the trehalose standard curve, the trehalose content per gram of oven-dried spores was measured and calculated.

[0057] Example 1

[0058] (1) By mass fraction, prepare compound nutrient pack A with 6 parts glucose, 2 parts zinc sulfate, 2 parts calcium chloride, 4 parts potassium dihydrogen phosphate, and 20 parts sterile water.

[0059] Mix equal mass ratios of compound nutrient pack A with dry, mold-free, large pieces of wheat bran, knead evenly, pack into Erlenmeyer flasks, heat to sterilize, shake to disperse the material while hot, and place in a sterile ultraviolet refrigeration unit for sterilization to obtain seed culture medium.

[0060] (2) Soak fresh soybeans in water for 8 hours, then filter them out. Add 7 times the mass of water and boil until the Baumé meter reading is 5. Filter out the soybean juice.

[0061] Dissolve 2g of glucose, 0.1g of potassium dihydrogen phosphate, 0.05g of ammonium sulfate, 0.05g of magnesium sulfate, and 1.5% agar powder in every 100ml of soy milk to adjust the pH to 6.

[0062] The mixture was then placed into test tubes for sterilization to prepare test tube slant activation culture medium;

[0063] Use a sterile inoculation loop to pick up the spores to be activated and streak them in a zigzag pattern on the activation medium. Incubate at 30°C for 72 hours. Observe the Aspergillus oryzae until it turns green, which means it has been activated once. Repeat the steps for a total of two activations.

[0064] (3) In a sterile operating table, scrape activated Aspergillus oryzae spores with a sterile inoculation loop, elute with a spore eluent prepared by mixing 9g NaCl, 100μL Tween-80, and 1L water, stirring thoroughly, and then sterilizing. Place a drop of the spore suspension on a hemocytometer, observe and count the spores using an optical microscope, and adjust the number of spores per ml of spore suspension to 1×10⁻⁶ by adding more spore eluent. 7 After that, a dispersed spore suspension was obtained.

[0065] (4) Inoculate the spore suspension into the koji culture medium under sterile conditions (inoculate 0.8 mL of spore suspension into every 10 g of koji culture medium). After inoculation, shake thoroughly and then incubate at a constant temperature of 30°C until obvious clumping occurs. Then perform the first shake-flask turning of the koji.

[0066] (5) By mass fraction, 1 part ammonium sulfate, 0.4 parts GDP-D-mannose, 0.01 parts chitin hydrolase, 0.01 parts phosphorylase, and 1 part sterile water are used to prepare compound nutrient pack B. The compound nutrient pack B is sterilized by a sterile filter and then evenly added to the seed koji (the mass ratio of compound nutrient pack B to the seed koji turned once is 0.4% wt).

[0067] Continue culturing until the surface of the starter turns white and clumps again, then perform a second shaking and turning of the starter, culturing for 48 hours and then covering the bottle, finally culturing for 72 hours until the starter matures, and then harvesting the starter to obtain soy sauce starter.

[0068] (6) The germination rate of the spores was 98.23%; the number of spores in the spores was 13 billion / g; and the trehalose content of the spores was 4.46 mg / g.

[0069] Example 2

[0070] (1) Mix equal mass ratio of water with dry, mold-free, large pieces of wheat bran and knead evenly. Pack the mixture into an Erlenmeyer flask, heat to sterilize, shake to disperse the material while hot, and place in a sterile ultraviolet hood for sterilization to obtain a starter culture medium.

[0071] (2) Soak fresh soybeans in water for 8 hours, then filter them out. Add 7 times the mass of water and boil until the Baumé meter reading is 5. Filter out the soybean juice.

[0072] Dissolve 2g of glucose, 0.1g of potassium dihydrogen phosphate, 0.05g of ammonium sulfate, 0.05g of magnesium sulfate, and 1.5% agar powder in every 100ml of soy milk to adjust the pH to 6.

[0073] The mixture was then placed into test tubes for sterilization to prepare test tube slant activation culture medium;

[0074] Use a sterile inoculation loop to pick up the spores to be activated and streak them in a zigzag pattern on the activation medium. Incubate at 30°C for 72 hours. Observe the Aspergillus oryzae until it turns green, which means it has been activated once. Repeat the steps for a total of two activations.

[0075] (3) In a sterile operating table, scrape activated Aspergillus oryzae spores with a sterile inoculation loop, elute with a spore eluent prepared by mixing 9g NaCl, 100μL Tween-80, and 1L water, stirring thoroughly, and then sterilizing. Place a drop of the spore suspension on a hemocytometer, observe and count the spores using an optical microscope, and adjust the number of spores per ml of spore suspension to 1×10⁻⁶ by adding more spore eluent. 7 After that, a dispersed spore suspension was obtained.

[0076] (4) Inoculate the spore suspension into the koji culture medium under sterile conditions (inoculate 0.8 mL of spore suspension into every 10 g of koji culture medium). After inoculation, shake thoroughly and then incubate at a constant temperature of 30°C until obvious clumping occurs. Then perform the first shake-flask turning of the koji.

[0077] (5) By mass fraction, 1 part ammonium sulfate, 0.4 parts GDP-D-mannose, 0.01 parts chitin hydrolase, 0.01 parts phosphorylase, and 1 part sterile water are used to prepare compound nutrient pack B. The compound nutrient pack B is sterilized by a sterile filter and then evenly added to the seed koji (the mass ratio of compound nutrient pack B to the seed koji turned once is 0.4% wt).

[0078] Continue culturing until the surface of the starter turns white and clumps again, then perform a second shaking and turning of the starter, culturing for 48 hours and then covering the bottle, finally culturing for 72 hours until the starter matures, and then harvesting the starter to obtain soy sauce starter.

[0079] (6) The germination rate of the spores was 87.49%; the number of spores in the spores was 7.9 billion / g; and the trehalose content of the spores was 8.76 mg / g.

[0080] A comparison of Examples 1 and 2 shows that Compound Nutrient Pack A has a significant impact on the quality of Aspergillus oryzae.

[0081] Compound nutrient pack A is rich in fast-acting and homeostatic components (glucose, zinc sulfate, calcium chloride, and potassium dihydrogen phosphate) essential for the growth and development of Aspergillus oryzae. After Aspergillus oryzae is inoculated into the culture medium, it can use these components to rapidly multiply into a dominant bacterial population, rather than slowly decomposing the nutrients in the culture medium. This prevents Aspergillus oryzae from becoming contaminated and burning due to resource competition and slow metabolism.

[0082] Example 3

[0083] (1) By mass fraction, prepare compound nutrient pack A with 6 parts glucose, 2 parts zinc sulfate, 2 parts calcium chloride, 4 parts potassium dihydrogen phosphate, and 20 parts sterile water.

[0084] Mix equal mass ratios of compound nutrient pack A with dry, mold-free, large pieces of wheat bran, knead evenly, pack into Erlenmeyer flasks, heat to sterilize, shake to disperse the material while hot, and place in a sterile ultraviolet refrigeration unit for sterilization to obtain seed culture medium.

[0085] (2) Soak fresh soybeans in water for 8 hours, then filter them out. Add 7 times the mass of water and boil until the Baumé meter reading is 5. Filter out the soybean juice.

[0086] Dissolve 2g of glucose, 0.1g of potassium dihydrogen phosphate, 0.05g of ammonium sulfate, 0.05g of magnesium sulfate, and 1.5% agar powder in every 100ml of soy milk to adjust the pH to 6.

[0087] The mixture was then placed into test tubes for sterilization to prepare test tube slant activation culture medium;

[0088] Use a sterile inoculation loop to pick up the spores to be activated and streak them in a zigzag pattern on the activation medium. Incubate at 30°C for 72 hours. Observe the Aspergillus oryzae until it turns green, which means it has been activated once. Repeat the steps for a total of two activations.

[0089] (3) In a sterile operating table, scrape activated Aspergillus oryzae spores with a sterile inoculation loop, elute with a spore eluent prepared by mixing 9g NaCl, 100μL Tween-80, and 1L water, stirring thoroughly, and then sterilizing. Place a drop of the spore suspension on a hemocytometer, observe and count the spores using an optical microscope, and adjust the number of spores per ml of spore suspension to 1×10⁻⁶ by adding more spore eluent. 7 After that, a dispersed spore suspension was obtained.

[0090] (4) Inoculate the spore suspension into the koji culture medium under sterile conditions (inoculate 0.8 mL of spore suspension into every 10 g of koji culture medium). After inoculation, shake thoroughly and then incubate at a constant temperature of 30°C until obvious clumping occurs. Then perform the first shake-flask turning of the koji.

[0091] (5) Continue to cultivate until the surface of the starter turns white and clumps again, then shake the bottle to turn the starter over for the second time, cultivate for 48 hours and then cover the bottle, and finally cultivate for 72 hours until the starter matures, and then harvest the starter to obtain soy sauce starter.

[0092] The spore germination rate was 79.57%; the number of spores in the spore germination rate was 7.1 billion / g; and the trehalose content in the spores was 9.01 mg / g.

[0093] Comparing Examples 1, 2, and 3, it can be seen that Compound Nutrient Pack B is more important for the quality of the inoculum than A. Compound Nutrient Pack B mainly contains nutrients and enzymes (including ammonium sulfate, GDP-D-mannose, chitin hydrolase, and phosphorylase) that optimize the metabolic activity and growth and reproductive physiological characteristics of Aspergillus oryzae.

[0094] like Figure 1 As shown, compound nutrient packs A and B significantly improved the condition of Aspergillus oryzae spores, resulting in a substantial decrease in the defect rate.

[0095] Example 4

[0096] (1) By mass fraction, prepare compound nutrient pack A with 6 parts glucose, 2 parts zinc sulfate, 2 parts calcium chloride, 4 parts potassium dihydrogen phosphate, and 20 parts sterile water.

[0097] Mix equal mass ratios of compound nutrient pack A with dry, mold-free, large pieces of wheat bran, knead evenly, pack into Erlenmeyer flasks, heat to sterilize, shake to disperse the material while hot, and place in a sterile ultraviolet refrigeration unit for sterilization to obtain seed culture medium.

[0098] (2) Soak fresh soybeans in water for 8 hours, then filter them out. Add 7 times the mass of water and boil until the Baumé meter reading is 5. Filter out the soybean juice.

[0099] Dissolve 2g of glucose, 0.1g of potassium dihydrogen phosphate, 0.05g of ammonium sulfate, 0.05g of magnesium sulfate, and 1.5% agar powder in every 100ml of soy milk to adjust the pH to 6.

[0100] The mixture was then placed into test tubes for sterilization to prepare test tube slant activation culture medium;

[0101] Use a sterile inoculation loop to pick up the spores to be activated and streak them in a zigzag pattern on the activation medium. Incubate at 30°C for 72 hours. Observe the Aspergillus oryzae until it turns green, which means it has been activated once. Repeat the steps for a total of two activations.

[0102] (3) In a sterile operating table, scrape activated Aspergillus oryzae spores with a sterile inoculation loop, elute with a spore eluent prepared by mixing 9g NaCl, 100μL Tween-80, and 1L water, stirring thoroughly, and then sterilizing. Place a drop of the spore suspension on a hemocytometer, observe and count the spores using an optical microscope, and adjust the number of spores per ml of spore suspension to 1×10⁻⁶ by adding more spore eluent. 7 After that, a dispersed spore suspension was obtained.

[0103] (4) Inoculate the spore suspension into the koji culture medium under sterile conditions (inoculate 0.8 mL of spore suspension into every 10 g of koji culture medium). After inoculation, shake thoroughly and then incubate at a constant temperature of 30°C until obvious clumping occurs. Then perform the first shake-flask turning of the koji.

[0104] (5) By mass fraction, 0.4 parts of GDP-D-mannose, 0.01 parts of chitin hydrolase, 0.01 parts of phosphorylase, and 1 part of sterile water are used to prepare compound nutrient pack B, which is then sterilized by a sterile filter and evenly added to the seed koji (the mass ratio of compound nutrient pack B to the seed koji turned once is 0.4%wt).

[0105] Continue culturing until the surface of the starter turns white and clumps again, then perform a second shaking and turning of the starter, culturing for 48 hours and then covering the bottle, finally culturing for 72 hours until the starter matures, and then harvesting the starter to obtain soy sauce starter.

[0106] (6) The germination rate of the spores was 91%; the number of spores in the spores was 11.4 billion / g; and the trehalose content of the spores was 5.41 mg / g.

[0107] Comparing Examples 3 and 4, it is evident that ammonium sulfate is a key nutrient stimulating the metabolic activity of *Aspergillus oryzae*. Samples from Examples 3 and 4 were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for transcriptome analysis. It was found that ammonium sulfate intervention upregulated cell wall synthesis, spore convolution, and the mycelial anti-gene pathway in *Aspergillus oryzae*. Figure 2).

[0108] Example 5

[0109] (1) By mass fraction, prepare compound nutrient pack A with 6 parts glucose, 2 parts zinc sulfate, 2 parts calcium chloride, 4 parts potassium dihydrogen phosphate, and 20 parts sterile water.

[0110] Mix equal mass ratios of compound nutrient pack A with dry, mold-free, large pieces of wheat bran, knead evenly, pack into Erlenmeyer flasks, heat to sterilize, shake to disperse the material while hot, and place in a sterile ultraviolet refrigeration unit for sterilization to obtain seed culture medium.

[0111] (2) Soak fresh soybeans in water for 8 hours, then filter them out. Add 7 times the mass of water and boil until the Baumé meter reading is 5. Filter out the soybean juice.

[0112] Dissolve 2g of glucose, 0.1g of potassium dihydrogen phosphate, 0.05g of ammonium sulfate, 0.05g of magnesium sulfate, and 1.5% agar powder in every 100ml of soy milk to adjust the pH to 6.

[0113] The mixture was then placed into test tubes for sterilization to prepare test tube slant activation culture medium;

[0114] Use a sterile inoculation loop to pick up the spores to be activated and streak them in a zigzag pattern on the activation medium. Incubate at 30°C for 72 hours. Observe the Aspergillus oryzae until it turns green, which means it has been activated once. Repeat the steps for a total of two activations.

[0115] (3) In a sterile operating table, use a sterile inoculation loop to scrape the activated Aspergillus oryzae spores and inoculate them into the seed culture medium under sterile conditions (inoculate 0.8 mL of spore suspension for every 10 g of seed culture medium). Repeat three times. After inoculation, shake thoroughly and then incubate at 30°C until obvious clumping occurs. Then perform the first shake-flask turning of the koji.

[0116] (4) By mass fraction, 1 part ammonium sulfate, 0.4 parts GDP-D-mannose, 0.01 parts chitin hydrolase, 0.01 parts phosphorylase, and 1 part sterile water are used to prepare compound nutrient pack B. The compound nutrient pack B is sterilized by a sterile filter and then evenly added to the seed koji (the mass ratio of compound nutrient pack B to the seed koji turned once is 0.4% wt).

[0117] Continue culturing until the surface of the starter turns white and clumps again, then perform a second shaking and turning of the starter, culturing for 48 hours and then covering the bottle, finally culturing for 72 hours until the starter matures, and then harvesting the starter to obtain soy sauce starter.

[0118] The spore germination rate was 84.19%; the number of spores in the spore germination rate was 8.9 billion / g; and the trehalose content in the spores was 7.96 mg / g.

[0119] Comparing Examples 1 and 5, it can be seen that preparing a dispersed spore suspension is beneficial to the physiological morphology and growth state of the koji, because dispersed inoculation is conducive to the uniform distribution of Aspergillus oryzae in the culture medium, avoiding competition from accumulation, contamination by other microorganisms, and koji burning. Using the dispersed spore suspension technology, the germination rate of Aspergillus oryzae is significantly improved.

[0120] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing a soy sauce starter with high germination rate and high spore number, characterized in that: include, Mix the compound nutrient pack A with dry, mold-free large pieces of wheat bran, knead them evenly, bottle them, heat them for sterilization, shake the material while it is still hot, and sterilize to obtain the seed culture medium. Activated Aspergillus oryzae spores were scraped with a sterile inoculation loop under sterile conditions to prepare a dispersed spore suspension. The spore suspension was inoculated into the koji culture medium under sterile conditions. After inoculation, the medium was shaken thoroughly and then incubated at a constant temperature until obvious clumping appeared. The first shake-flask inoculation was then performed. During the first turning of the starter culture, the compound nutrient pack B is sterilized through a sterile filter and evenly added to the starter culture. The culture continues until the surface of the starter culture turns white and clumps up again. The second turning of the starter culture is then performed, and the culture continues until the bottle is covered. Finally, the starter culture is cultivated until it matures, and the starter culture is harvested to obtain soy sauce starter culture. The compound nutrient pack A is composed of glucose, zinc sulfate, calcium chloride, potassium dihydrogen phosphate and sterile water. The proportions of each ingredient by mass are as follows: glucose 6 parts, zinc sulfate 2 parts, calcium chloride 2 parts, potassium dihydrogen phosphate 4 parts, and sterile water 20-40 parts. Compound Nutrition Pack B consists of ammonium sulfate, GDP-D-mannose, chitinase, phosphorylase, and sterile water. The proportions of each ingredient by weight are as follows: ammonium sulfate 1 part, GDP-D-mannose 0.4 parts, chitinase 0.01 parts, phosphorylase 0.01 parts, and sterile water 1-2 parts. The chitinase is purchased from Merck Group, catalog number C6137-50UN; the phosphorylase is purchased from Merck Group, catalog number P6635.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the large pieces of wheat bran to compound nutrient pack A is 1:1 to 1.

3.

3. The preparation method according to claim 1, characterized in that: The activated Aspergillus oryzae spores, wherein the activation process includes, Soak fresh soybeans in water for 8 hours, then filter them out. Add 5 to 7 times the mass of water and boil until the Baumé meter reading is 5. Filter out the soybean juice. Dissolve 2 g of glucose, 0.1 g of potassium dihydrogen phosphate, 0.05 g of ammonium sulfate, and 0.05 g of magnesium sulfate in every 100 mL of soy milk, then add 1.5% agar powder by mass and adjust the pH to 6. The mixture was then placed into test tubes for sterilization to prepare test tube slant activation medium. Use a sterile inoculation loop to pick up the spores to be activated and streak them in a Z-shape on the activation medium. Incubate at 30°C for 72 h. Observe that Aspergillus oryzae grows green, which means that the activation has been completed once. Repeat the steps for a total of two activations to obtain activated Aspergillus oryzae spores.

4. The preparation method according to claim 1, characterized in that: The prepared dispersed spore suspension includes, Add 9 g NaCl, 100 μL Tween-80, and 1 L of water, stir well, sterilize, and use the resulting spore eluent to elute and mix. Place a drop of spore suspension on a hemocytometer, observe and count the spores using an optical microscope, and adjust the number of spores per mL of spore suspension to 1 × 10⁻⁶ by adding spore elution buffer. 7 After that, a dispersed spore suspension was obtained.

5. The preparation method according to claim 1 or 4, characterized in that: The spore suspension is inoculated into the koji culture medium under sterile conditions, wherein 0.5-1 mL of spore suspension is added to every 10 g of koji culture medium.

6. The preparation method according to claim 1, characterized in that: The mass ratio of the compound nutrient pack B to the seed culture of the first fermentation is 0.1~0.5%wt.

7. The preparation method according to claim 1, characterized in that: The interval between the first shake and the second shake is 12–14 h, and the interval between the second shake and the second shake is 15.5–17.5 h.

8. The soy sauce starter culture prepared by any one of claims 1 to 7, characterized in that: The germination rate of the soy sauce starter spores is ≥98%, the number of spores is ≥13 billion / g, and the trehalose content of Aspergillus oryzae in the starter spores is not higher than 4.50 mg / g.

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