Preparation method of finished koji for soy sauce and preparation method of soy sauce

Abstract

The invention relates to the field of soy sauce brewing, and discloses a preparation method of finished koji for soy sauce and a preparation method of the soy sauce. The preparation method of finished koji comprises the following steps: carrying out first mixing on raw materials and mold koji, and carrying out first culture; mixing the material with bacillus subtilis for the second time, and culturing for the second time; by adopting the method provided by the invention to prepare the finished koji for the soy sauce, the obtained finished koji is richer in protease system and better in enzyme activity, and is more beneficial to release of substances such as amino acids and flavor peptides. In cooperation with the preparation method of the soy sauce, the faint scent type soy sauce can be obtained, it is further guaranteed that the soy sauce has rich flavor substances and organic acid, and the rancidity and the content of aflatoxin B1 can be better controlled.

Description

Technical Field

[0001] The invention relates to the field of soy sauce brewing, and in particular to a method for preparing koji for soy sauce and a method for preparing soy sauce. Background Art

[0002] Soy sauce is a traditional condiment in my country. Brewing soy sauce offers a high-quality platform for the comprehensive utilization of various protein and starch byproducts, enabling the reuse of these byproducts. The soy sauce brewing process generally requires the preparation of koji (koji). However, existing koji preparation processes often suffer from low koji protease activity and a simple enzyme system, which hinders the generation and release of flavor compounds during the soy sauce preparation process, resulting in a simple flavor profile.

[0003] Furthermore, peanuts, a major oilseed and cash crop in my country, have seen their production approach 20 million tons in recent years, ranking first in the world. More than half of these peanuts are used to produce edible oil, resulting in an average of 9 million tons of peanut meal annually. Currently, hot-pressing is the primary method for producing peanut oil in my country, resulting in a significant portion of the resulting peanut meal being hot-pressed. Hot-pressed peanut meal often uses very high temperatures during processing, which severely denatures the peanut protein, impacting its nutritional value and functional properties to varying degrees. Furthermore, peanut meal has a higher oil content than soybean meal. Fermenting soy sauce with peanut meal can easily produce a rancid flavor due to oil oxidation, and the resulting mash is highly acidic. Furthermore, peanuts can be contaminated by molds such as Aspergillus flavus during growth, harvesting, storage, and transportation, resulting in the contamination of aflatoxins, such as aflatoxin B1. Consequently, peanut meal is mostly used as fertilizer or animal feed, resulting in a significant waste of peanut protein resources. Using peanut meal in soy sauce production could significantly improve resource utilization. However, as mentioned above, the production of soy sauce using peanut meal as a raw material presents numerous challenges, at least for these reasons. Summary of the Invention

[0004] The present invention aims to overcome the above-mentioned problems existing in the prior art and provides a method for preparing a koji for soy sauce and a method for preparing soy sauce. When the method provided by the present invention is used to prepare the koji for soy sauce, the protease system of the koji is richer and the enzyme activity is higher, which is more conducive to fully utilizing the raw materials and promoting the release of substances such as amino acids and flavor peptides. In addition, the koji obtained in this way is used in combination with the method for preparing soy sauce provided by the present invention to obtain peanut sauce richer in flavor substances and organic acids, and at the same time, the rancid smell and aflatoxin B1 content of the soy sauce can be better controlled.

[0005] To achieve the above object, the present invention provides a method for preparing koji for soy sauce, the method comprising: first mixing raw materials with seed koji and performing a first culture; then second mixing the materials with Bacillus subtilis and performing a second culture;

[0006] The seed koji includes Aspergillus oryzae and Aspergillus niger; wherein the first mixing is such that the initial content of the seed koji in the material after the first mixing is 5×10 6 -5×10 7 cfu / g; the second mixing is such that the initial content of Bacillus subtilis in the material after the second mixing is 1×10 2 -5×10 3 cfu / g.

[0007] A second aspect of the present invention provides a method for preparing soy sauce, comprising: mixing brine and soy sauce prepared by the method according to the first aspect with koji, and fermenting the mixture.

[0008] The third aspect of the present invention provides soy sauce prepared by the method described above.

[0009] The method provided by the present invention is used to prepare koji for soy sauce. The resulting koji has a richer enzyme system and better enzyme activity, which is more conducive to fully hydrolyzing the raw materials. Using such koji to prepare soy sauce is more conducive to the release of substances such as amino acids and flavor peptides, and the resulting soy sauce is richer in flavor substances and organic acids. In addition, the method provided by the present invention can produce a fragrant soy sauce, further ensuring that the soy sauce is rich in flavor substances and organic acids. The resulting soy sauce has a more fragrant and delicious taste, and can also better control the rancidity and aflatoxin B1 content of the soy sauce, which is conducive to the full resource utilization of peanut meal. DETAILED DESCRIPTION

[0010] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0011] In a first aspect, the present invention provides a method for preparing koji for soy sauce, the method comprising: first mixing raw materials with seed koji and performing a first culture; then second mixing the materials with Bacillus subtilis and performing a second culture;

[0012] The seed koji includes Aspergillus oryzae and Aspergillus niger; wherein the first mixing is such that the initial content of the seed koji in the material after the first mixing is 5×10 6 -5×10 7 cfu / g; the second mixing is such that the initial content of Bacillus subtilis in the material after the second mixing is 1×10 2 -5×10 3 cfu / g.

[0013] The inventors of the present invention have found in their research that the koji obtained by the method described above has a richer protease system and higher enzyme activity. When such koji is used to prepare soy sauce, the rich protease can hydrolyze the raw materials more thoroughly, which is beneficial to the release of substances such as amino acids and flavor peptides. The resulting soy sauce will be richer in flavor substances and organic acids.

[0014] The present invention has no particular limitation on the type and amount of raw materials, which may be conventional in the art. However, preferably, the raw materials include a protein raw material and a starch raw material, and the mass ratio of the protein raw material to the starch raw material on a dry basis is 1:(0.7-1.3).

[0015] Preferably, the protein raw material comprises peanut meal and / or soybean meal.

[0016] Preferably, the starchy raw material comprises oat flakes (such as raw oat flakes).

[0017] Preferably, on a dry basis, the raw materials are peanut meal, soybean meal and oatmeal in a mass ratio of 1:(0.8-1.2):(1.8-2.2).

[0018] Preferably, before the first mixing, the method further comprises: steaming the raw materials.

[0019] Preferably, before cooking, the method further comprises: performing a third mixing of peanut meal, soybean meal, and water, and then performing a fourth mixing of the third mixed material and oatmeal; in the third mixing, the mass amount of water used is 0.9-1.1 times the total mass of the peanut meal, soybean meal, and oatmeal, and the third mixing time is 20-40 minutes. During the third mixing, the peanut meal and soybean meal are fully moistened with water. The present invention has no particular limitations on the cooking operation, and it can also be a conventional choice in the art. For example, the cooking temperature can be 100-130° C., the cooking time can be 0.3-0.5 h, and the pressure can be 0.05-0.15 MPa.

[0020] According to the present invention, preferably, the temperature of the first mixing is 38-42° C. (for example, 38, 39, 40, 41, 42, and a range formed by any two of the above values, and a value within the range)° C. The steamed raw materials can be cooled to the above temperature before being mixed with the seed koji.

[0021] According to the present invention, preferably, in the first mixing, the inoculation amounts of Aspergillus oryzae and Aspergillus niger are such that the ratio of their initial viable counts in the material after the first mixing is 1:(0.8-1.2).

[0022] The present invention is not particularly limited to the specific species of Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis, and can be selected from conventional sources in the art. For example, Aspergillus oryzae Shanghai Niang 3.042 purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd. can be used; Aspergillus niger can be ATCC16404; and Bacillus subtilis can be ATCC6633.

[0023] According to the present invention, preferably, the conditions for the first culture include: a temperature of 32-35 (for example, 32, 33, 34, 35, and a range formed by any two of the above values, and values ​​within the range) ° C, and a time of 6-8 h.

[0024] According to the present invention, preferably, the second culture method includes: first culturing at 28-32°C (for example, 28, 29, 30, 31, or 32°C) for 5-8 hours, then performing a first turning over, then culturing at 28-30°C (for example, 28, 29, or 30°C) for 6-8 hours after flattening, then performing a second turning over, and then continuing to culture at 28-30°C (for example, 27, 28, or 29°C) for 8-10 hours. After the second culture is completed, mature Daqu (i.e., finished qu) can be obtained.

[0025] The inventors of the present invention have discovered through research that by using the method provided by the present invention, adding Aspergillus oryzae, Aspergillus niger and Bacillus subtilis in steps, and combining them with specific dosage ratios and culture methods, a koji with a richer protease system and higher enzyme activity can be obtained, which is more conducive to the preparation of soy sauce richer in flavor substances and organic acids.

[0026] In a second aspect, the present invention provides a method for preparing soy sauce, comprising: mixing brine and soy sauce prepared by the method according to the first aspect with koji, and fermenting the mixture.

[0027] As described above, the soy sauce koji obtained by the first aspect has a richer protease system and higher enzyme activity. Therefore, when such koji is used in soy sauce preparation, peanut butter with richer flavor substances and organic acids can also be obtained.

[0028] According to the present invention, preferably, the amount of brine used is 2-3 kg relative to 1 kg of soy sauce koji.

[0029] Preferably, the mass concentration of salt in the brine is 18-25 wt% (for example, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, and any range formed by any two of the above values, and values ​​within the range). It is understood that in soy sauce fermentation, the salt in the brine refers to sodium chloride.

[0030] According to the present invention, preferably, the fermentation includes temperature control during the fermentation process, and the temperature control method includes: first performing temperature-raising fermentation, and when the temperature is raised to a constant temperature, performing constant temperature fermentation until the fermentation ends.

[0031] Preferably, the starting temperature of the fermentation is 14-16°C.

[0032] Preferably, the heating rate of the temperature-raising fermentation is 0.8-1.2°C / day.

[0033] Preferably, the constant temperature is 29-31°C (for example, 29°C, 30°C, or 31°C).

[0034] Preferably, the total fermentation time is 90-190 days, more preferably 160-180 days.

[0035] The inventors of the present invention also found in their research that by gradually increasing the fermentation temperature from a lower temperature as described above, the proteases in the finished koji can better function, including increasing the activity of alcohol and aldehyde dehydrogenases, and promoting the conversion of rancid substances such as nonanal.

[0036] According to the present invention, preferably, the fermentation comprises: inoculating lactic acid bacteria into the fermentation system on the 14th to 16th day of fermentation; when the pH of the fermentation system drops to 4.8-5.2 (generally around the 30th day of fermentation), inoculating yeast into the fermentation system;

[0037] Wherein, the lactic acid bacteria include halophilic Tetragenococcus and Lactobacillus plantarum;

[0038] The yeasts include Saccharomyces rouxii, Saccharomyces cerevisiae and Saccharomyces fulvicinii.

[0039] Through the above method, the various strains can cooperate better with each other, further ensuring that the protease in the finished koji can play a better role, and can better activate the alcohol and aldehyde dehydrogenases. While ensuring that the flavor substances and organic acids of the soy sauce are richer, it can also significantly reduce the rancid smell and aflatoxin B1 content of the soy sauce.

[0040] According to the present invention, preferably, the inoculation amount of lactic acid bacteria is such that the initial content of lactic acid bacteria in the fermentation system is 1×10 6 -1×10 7 CFU / g.

[0041] According to the present invention, preferably, the inoculation amounts of Tetragenococcus halophilus and Lactobacillus plantarum are such that the ratio of their initial viable bacterial counts in the fermentation system is 1:(0.8-1.2).

[0042] According to the present invention, preferably, the inoculation amount of yeast is such that the initial content of yeast in the fermentation system is 1×10 6 -1×10 7 CFU / g.

[0043] According to the present invention, preferably, the inoculation amounts of Saccharomyces rouxii, Saccharomyces cerevisiae and Saccharomyces fulvidraco are such that the ratio of their initial viable counts in the fermentation system is 1:(0.8-1.2):(0.8-1.2).

[0044] By adopting the soy sauce preparation method provided by the present invention and the above-mentioned dosage ratio, it is possible to further ensure that the flavor substances and organic acids are more abundant, especially to better control the rancid smell and aflatoxin B1 content of the soy sauce, and to enhance the caramel aroma, floral aroma, sweet aroma, etc. of the soy sauce.

[0045] The specific strains of the above bacteria are not particularly limited. For example, Tetragenococcus halophilus may be ATCC13623, Lactobacillus plantarum may be ATCC8014, R. rouxii may be ATCC10383, Saccharomyces cerevisiae may be ATCC4098, and Saccharomyces fulvidraco may be ATCC36309.

[0046] According to the present invention, preferably, during the fermentation process, the method further comprises: aerating and stirring the fermented product 2-4 times daily, each time for 20-40 minutes, after inoculating the yeast. The inventors of the present invention have further discovered that this method can further promote the growth of fungi (including yeast), further inhibit possible anaerobic bacteria, further ensure healthy microbial homeostasis in the fermentation system, further inhibit aflatoxin B1, and further promote the production of flavor substances and organic acids.

[0047] It is understood that after the fermentation is completed, the mash can be filtered and then kept at 75-85° C. for 20-40 minutes for pasteurization, and the precipitate can be removed to obtain the finished soy sauce product.

[0048] In a third aspect, the present invention provides soy sauce prepared by the method described above.

[0049] As described above, the soy sauce prepared according to the above method has a fresh and fragrant taste, is rich in flavor substances and organic acids, and has significantly suppressed rancidity and aflatoxin B1 content.

[0050] The present invention will be described in detail below through examples.

[0051] In the following examples, the cooking conditions include: temperature of 110°C, time of 0.4 h, and pressure of 0.1 MPa;

[0052] Unless otherwise specified, Aspergillus oryzae was Aspergillus oryzae Shanghai Niang 3.042 purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd.; Aspergillus niger was ATCC16404; Bacillus subtilis was ATCC6633; Tetragenococcus halophilus was ATCC13623; Lactobacillus plantarum was ATCC8014; Saccharomyces rouxii was ATCC10383, Saccharomyces cerevisiae was ATCC4098, and Saccharomyces cerevisiae was ATCC36309.

[0053] Example 1

[0054] Used to illustrate the preparation method of soy sauce koji and soy sauce provided by the present invention

[0055] (1) Raw material cooking: The mass ratio of peanut meal, soybean meal and raw oat flakes is 1:1:2. Water (1 times the total mass of the raw materials) is added to the peanut meal and soybean meal. After 30 minutes, the raw oat flakes are mixed in and the mixture is evenly mixed before cooking.

[0056] (2) When the raw material after cooking is cooled to 40°C, Aspergillus oryzae and Aspergillus niger are mixed into the raw material so that the initial total content of Aspergillus oryzae and Aspergillus niger in the raw material is 8×10 6 cfu / g, and the initial viable count ratio of Aspergillus oryzae to Aspergillus niger was 1:1.

[0057] After culturing at 35℃ for 6h, Bacillus subtilis was inoculated. The initial content of Bacillus subtilis in the material was 1×10 3 cfu / g; continue to incubate at 29°C for 6 hours and then turn the koji over once. After spreading, continue incubating at 28°C for 7 hours and then turn the koji over again. Lower the temperature to 28°C and incubate for 9 hours to obtain mature koji.

[0058] (3) Fermentation: The mature Daqu obtained as above was mixed with 20 wt% brine at a ratio of 1:2.5 by mass and fermented. The fermentation temperature was 15°C on the first day and then increased to 30°C at a rate of 1°C / day.

[0059] On the 15th day of fermentation, the fermentation system was inoculated with halophilic Tetragenococcus and Lactobacillus plantarum so that the ratio of the initial viable bacteria in the fermentation system was 1:1 and the initial total content of the two in the fermentation system was 1×10 6 CFU / g.

[0060] When the pH of the mash dropped to about 5 (about the 30th day of fermentation), Saccharomyces cerevisiae, Saccharomyces cerevisiae and Saccharomyces glutinosus were inoculated. The ratio of the initial viable bacteria count of the three in the fermentation system was 1:1:1, and the initial total content of the three in the fermentation system was 1×10 7 CFU / g. Aerate and stir three times a day from the time of yeast inoculation, each time for 30 minutes.

[0061] (4) Pasteurization: After fermentation for 180 days, the mash is filtered and then kept at 80°C for 30 minutes to remove the sediment to obtain the finished soy sauce.

[0062] Example 2

[0063] Used to illustrate the preparation method of soy sauce koji and soy sauce provided by the present invention

[0064] (1) Raw material cooking: The mass ratio of peanut meal, soybean meal and raw oat flakes is 1:0.8:2.2. Water 0.9 times the total mass of the raw materials is added to the peanut meal and soybean meal. After 25 minutes, the raw oat flakes are mixed in and the mixture is evenly mixed before cooking.

[0065] (2) When the cooked raw material is cooled to 39°C, Aspergillus oryzae and Aspergillus niger are mixed into the raw material so that the initial total content of Aspergillus oryzae and Aspergillus niger in the raw material is 5×10 7 cfu / g, and the initial viable count ratio of Aspergillus oryzae to Aspergillus niger was 1:0.8.

[0066] After culturing at 33°C for 6 h, Bacillus subtilis was inoculated. The initial content of Bacillus subtilis in the material was 5×10 2 cfu / g; continue to incubate at 30℃ for 7 hours and then turn the koji over once. After spreading, continue to incubate at 30℃ for 6.5 hours and then turn the koji over again. Lower the temperature to 28℃ and incubate for 10 hours to obtain mature koji.

[0067] (3) Fermentation: The mature Daqu obtained as above was mixed with saline solution having a mass concentration of 19 wt % at a ratio of 1:2.7 and then fermented. The fermentation temperature was 15° C. on the first day and then increased to 30° C. at a rate of 1° C. / day.

[0068] On the 15th day of fermentation, the fermentation system was inoculated with halophilic Tetragenococcus and Lactobacillus plantarum so that the ratio of the initial viable bacteria in the fermentation system was 1:0.8, and the initial total content of the two in the fermentation system was 1×10 7 CFU / g.

[0069] When the pH of the mash dropped to 4.9, Saccharomyces cerevisiae, Saccharomyces cerevisiae and Saccharomyces glutinosus were inoculated. The ratio of the initial viable bacteria counts of the three in the fermentation system was 1:0.8:1.2, and the initial total content of the three in the fermentation system was 8×10 6 CFU / g. Aerate and stir three times a day from the time of yeast inoculation, each time for 25 minutes.

[0070] (4) Pasteurization: After fermentation for 160 days, the mash is filtered and then kept at 80°C for 30 minutes to remove the sediment to obtain the finished soy sauce.

[0071] Example 3

[0072] Used to illustrate the preparation method of soy sauce koji and soy sauce provided by the present invention

[0073] (1) Raw material cooking: The mass ratio of peanut meal, soybean meal and raw oat flakes is 1:1.2:1.8. Water (1.1 times the total mass of the raw materials) is added to the peanut meal and soybean meal. After 35 minutes, the raw oat flakes are mixed in and the mixture is evenly mixed before cooking.

[0074] (2) When the raw material after cooking is cooled to 38°C, Aspergillus oryzae and Aspergillus niger are mixed into the raw material so that the initial total content of Aspergillus oryzae and Aspergillus niger in the raw material is 5×10 6 cfu / g, and the initial viable count ratio of Aspergillus oryzae to Aspergillus niger was 1:1.2.

[0075] After culturing at 32°C for 8 h, Bacillus subtilis was inoculated. The initial content of Bacillus subtilis in the material was 1×10 3 cfu / g; continue to incubate at 30℃ for 7.5 hours, then turn the koji once. After spreading, continue incubating at 30℃ for 7.5 hours, turn the koji twice, and lower the temperature to 28℃ and incubate for 8 hours to obtain mature koji.

[0076] (3) Fermentation: The mature Daqu obtained as above was mixed with saline solution having a mass concentration of 23 wt% at a ratio of 1:2.2 and then fermented. The fermentation temperature was 15°C on the first day and then increased to 30°C at a rate of 1°C / day.

[0077] On the 15th day of fermentation, the fermentation system was inoculated with halophilic Tetragenococcus and Lactobacillus plantarum so that the ratio of the initial viable bacteria in the fermentation system was 1:1.2, and the initial total content of the two in the fermentation system was 5×10 6 CFU / g.

[0078] When the pH of the mash dropped to 5.1, Saccharomyces cerevisiae, Saccharomyces cerevisiae and Saccharomyces glutinosus were inoculated. The ratio of the initial viable bacteria counts of the three in the fermentation system was 1:1.2:0.8, and the initial total content of the three in the fermentation system was 1×10 7 CFU / g. Aerate and stir three times a day from the time of yeast inoculation, each time for 45 minutes.

[0079] (4) Pasteurization: After fermentation for 180 days, the mash is filtered and then kept at 80°C for 30 minutes to remove the sediment to obtain the finished soy sauce.

[0080] Example 4

[0081] The method of Example 1 was followed until step (2) was completed, except that Bacillus subtilis was not inoculated.

[0082] Example 5

[0083] The method of Example 1 was followed, except that no aeration was performed during the fermentation process.

[0084] Example 6

[0085] The method of Example 1 was followed, except that during the fermentation process, lactic acid bacteria and yeast were not inoculated, and aeration was not performed.

[0086] Example 7

[0087] The method of Example 1 was followed, except that Aspergillus niger was not inoculated, but the initial content of Aspergillus oryzae was the same as the initial total content of Aspergillus oryzae and Aspergillus niger in Example 1.

[0088] Comparative Example 1

[0089] The method of Example 1 was followed, except that in step (2), when the raw material after cooking was cooled to 40° C., only Aspergillus oryzae was inoculated, so that the initial content of Aspergillus oryzae in the raw material was 8×10 7 cfu / g, and cultured until a finished koji was obtained in the manner of step (2) of Example 1, except that Aspergillus niger and Bacillus subtilis were not inoculated during this process. Then, steps (3)-(4) of Example 1 were followed, except that in step (3), lactic acid bacteria and yeast were not inoculated, and aeration was not performed.

[0090] Test Example 1

[0091] The protease activity of the finished koji prepared in the above examples was determined according to the "SB / T 10317-1999 Protease Activity Assay Method". The results are shown in Table 1.

[0092] Table 1 Comparison of enzyme activity of Daqu in different fermentation methods

[0093] Acid protease activity (U / g) Neutral protease activity (U / g) Alkaline protease activity (U / g) Example 1 1231.10 2074.29 1471.22 Example 2 1360.26 2137.78 1545.39 Example 3 1196.22 2042.35 1468.64 Example 4 847.36 1643.24 1256.85 Example 7 1003.66 1578.54 1235.38 Comparative Example 1 549.74 1482.38 928.56

[0094] As can be seen from the results of Table 1, the protease activity of the obtained Daqu is greatly improved by adopting the method for preparing soy sauce with koji of the present invention. During the fermentation of mash, the acidity of peanut meal soy sauce is higher than that of soybean meal soy sauce, and the alkaline protease activity in Aspergillus oryzae is higher, while the acidic protease activity is lower, which is not conducive to fermentation. When the method of the present invention is adopted to simultaneously adopt Aspergillus oryzae, Aspergillus niger and Bacillus subtilis to mix and make koji, the protease system is more abundant, and the raw material utilization is more thorough, which contributes to the accumulation of substances such as amino acids and short peptides.

[0095] Test Example 2

[0096] The soy sauce prepared in the above example was evaluated as follows:

[0097] The activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) was evaluated as follows:

[0098] To a 10 mL colorimetric tube, add 0.5 mL of McIlvaine buffer (pH 4.0), 0.1 mL of 1.0 mol / L ethanol (acetaldehyde) solution, 0.1 mL of soy sauce sample, and 0.1 mL of 6% Triton X-100. Incubate at 25°C for 5 minutes, then add 0.2 mL of 0.1 mol / L potassium ferricyanide solution. Allow to react at 25°C for 5 minutes (simultaneously perform a blank control). Terminate the reaction by adding 0.5 mL of ferric sulfate Dupanol solution. Incubate at 25°C for 20 minutes, then add 35 mL of distilled water and mix. The optical density at 660 nm is measured using a UV-visible spectrophotometer. At 25°C and pH 4.0, 1 unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of ethanol in 1 minute; an optical density of 4.0 corresponds to 1 μmol of ethanol oxidized. Enzyme activity (U / mL) = A660 × 1 / 4 × 1 / 5 × 1 / sample (mL) × dilution factor. (A660 is the absorbance value at a wavelength of 660nm)

[0099] Sensory evaluation, the method is as follows:

[0100] The panel consisted of 14 participants (7 men and 7 women, aged 22 to 45 years), all of whom were familiar with five taste qualities (umami, saltiness, bitterness, sweetness, and sourness) and had experience in sensory evaluation (at least one year of training).

[0101] The sensory evaluation was performed using the following concentration solutions as standard solutions: umami: 0.3wt% MSG solution (containing 0.024wt% MSG and 0.06wt% sodium chloride), saltiness: 0.5wt% sodium chloride solution, sweetness: 1.6wt% sucrose solution, sourness: 0.1wt% citric acid solution, and bitterness: 0.05wt% caffeine solution.

[0102] The flavor of soy sauce was evaluated using an 11-point categorical scale, with 0-2 being very weak, 2-4 being weak, 4-6 being moderate, 6-8 being strong, and 8-10 being very strong.

[0103] Aroma is assessed by training sensory panelists on different concentrations of standard samples of typical flavor substances in soy sauce. Finally, a sensory evaluation is conducted based on a concentration of 10 times the threshold of the flavor substances. The scoring scale is 0-2 for very weak, 2-4 for weak, 4-6 for medium, 6-8 for strong, and 8-10 for very strong.

[0104] Evaluation of aflatoxin content: The aflatoxin content in each soy sauce was determined using a total aflatoxin detection kit (Nanjing Herbal Source Biotechnology Co., Ltd.).

[0105] Total acid content of soy sauce was determined according to GB 18186-2000 Brewing Soy Sauce. The results are shown in Table 2-4.

[0106] Table 2

[0107]

[0108]

[0109] By adopting the preferred soy sauce preparation method provided by the present invention, the activities of alcohol and aldehyde dehydrogenases are significantly higher, and the rancid smell is significantly controlled.

[0110] Table 3

[0111] Caramel aroma fruity Floral scent malt aroma sweet fragrance Smoky incense Creamy aroma mellow aroma Example 1 8.7 5 7 6 7 4 5.5 7 Example 2 8.9 6.3 7.4 6.6 6.8 4.6 5.3 6.4 Example 3 8.2 5.7 6.6 6.4 7.4 3.6 5.2 6.7 Example 5 7.8 5 6.3 6 6 6.3 5.5 6 Example 6 5.8 4 4 7 3 6.5 2 5 Example 7 7.3 4 4 7 4 6.5 3 5.5 Comparative Example 1 7 4.7 4 7 2.8 7 3 4

[0112] As can be seen, the mixed koji production using Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis, followed by fermentation with multiple strains of lactic acid bacteria and yeast, allows for more complete raw material utilization and enriches the umami and flavor components. The peanut butter oil prepared using the preferred method of the present invention exhibits reduced smoky and malty notes, while significantly enhancing caramel, floral, and sweet aromas. Peanut butter oil produced using only Aspergillus oryzae koji production exhibits more pronounced smoky and malty notes, while other flavors are less pronounced.

[0113] Table 4

[0114] Total acid (g / 100mL) Aflatoxin (μg / kg) Example 1 2.74 1.32 Example 2 2.53 1.18 Example 3 2.69 1.26 Example 5 2.38 1.78 Example 6 1.54 7.53 Example 7 1.63 3.36 Comparative Example 1 1.39 7.88

[0115] It can be seen that the soy sauce prepared by the method for preparing soy sauce provided by the present invention has a high total acid content and a significantly lower aflatoxin content (significantly lower than the national limit of 5 μg / kg).

[0116] The above results show that the soy sauce prepared by the multi-strain collaborative fermentation method provided by the present invention has a fresh fragrance, improves the stability of the fermentation system, and significantly improves the sensory quality of the soy sauce.

[0117] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for preparing koji for soy sauce, characterized in that: The method comprises: first mixing the raw material with the seed koji and performing a first culture; then second mixing the material with Bacillus subtilis and performing a second culture; The seed koji includes Aspergillus oryzae and Aspergillus niger; wherein the first mixing is such that the initial content of the seed koji in the material after the first mixing is 5×10 6 -5×10 7 cfu / g; the second mixing is such that the initial content of Bacillus subtilis in the material after the second mixing is 1×10 2 -5×10 3 cfu / g.

2. The method according to claim 1, wherein The raw materials include protein raw materials and starch raw materials, and the mass ratio of the protein raw materials to the starch raw materials is 1: (0.7-1.3) on a dry basis; Preferably, the protein raw material includes peanut meal and / or soybean meal; Preferably, the starchy raw material comprises oatmeal; Preferably, on a dry basis, the raw materials are peanut meal, soybean meal and oatmeal in a mass ratio of 1:(0.8-1.2):(1.8-2.2); Preferably, before the first mixing, the method further comprises: steaming the raw materials; Preferably, before cooking, the method further comprises: performing a third mixing of peanut meal, soybean meal and water, and then performing a fourth mixing of the third mixed material and oat flakes; in the third mixing, the mass amount of water used is 0.9-1.1 times the total mass of the peanut meal, soybean meal and oat flakes, and the time of the third mixing is 20-40 minutes.

3. The method according to claim 1 or 2, wherein: The temperature of the first mixing is 38-42°C; And / or, in the first mixing, the inoculation amounts of Aspergillus oryzae and Aspergillus niger are such that the ratio of their initial viable bacterial counts in the material after the first mixing is 1:(0.8-1.2).

4. The method according to claim 1, wherein The conditions for the first culture include: a temperature of 32-35°C and a time of 6-8 hours; And / or, the second culturing method includes: first culturing at 28-32°C for 5-8 hours and then turning over for the first time, then culturing at 28-30°C for 6-8 hours after spreading and then turning over for the second time, and then continuing to culture at 28-30°C for 8-10 hours.

5. A method for preparing soy sauce, characterized in that: The method comprises: mixing salt water and soy sauce prepared by the method according to any one of claims 1 to 4 with koji, and fermenting the mixture.

6. The method according to claim 5, wherein: For every 1kg of soy sauce, 2-3kg of brine is needed. and / or, the mass concentration of salt in the brine is 18-25 wt%; And / or, the fermentation includes temperature control during the fermentation process, wherein the temperature control method includes: first performing temperature-raising fermentation, and when the temperature is raised to a constant temperature, performing constant temperature fermentation until the fermentation is completed; Preferably, the starting temperature of fermentation is 14-16°C; Preferably, the heating rate of the temperature-raising fermentation is 0.8-1.2°C / day; Preferably, the constant temperature is 29-31°C; And / or, the total fermentation time is 90-190 days, more preferably 160-180 days.

7. The method according to claim 5 or 6, wherein: The fermentation comprises: inoculating lactic acid bacteria into the fermentation system on the 14th to 16th day of fermentation; and inoculating yeast into the fermentation system when the pH of the fermentation system drops to 4.8-5.2; Wherein, the lactic acid bacteria include halophilic Tetragenococcus and Lactobacillus plantarum; The yeasts include Saccharomyces rouxii, Saccharomyces cerevisiae and Saccharomyces fulvicinii.

8. The method according to claim 7, wherein: The inoculation amount of lactic acid bacteria was such that the initial content of lactic acid bacteria in the fermentation system was 1×10 6 -1×10 7 CFU / g; and / or, the inoculation amounts of Tetragenococcus halophilus and Lactobacillus plantarum are such that the ratio of their initial viable bacterial counts in the fermentation system is 1:(0.8-1.2); And / or, the inoculation amount of yeast is such that the initial content of yeast in the fermentation system is 1×10 6 -1×10 7 CFU / g; And / or, the inoculation amounts of Saccharomyces rouxii, Saccharomyces cerevisiae and Saccharomyces fulvidraco are such that the ratio of their initial viable bacterial counts in the fermentation system is 1:(0.8-1.2):(0.8-1.2).

9. The method according to claim 7, wherein: During the fermentation process, the method further comprises: after inoculating the yeast, aerating and stirring 2-4 times a day, each time for 20-40 minutes.

10. Soy sauce prepared by the method according to any one of claims 5 to 9.

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