Application of bacillus subtilis in increasing content of free soy isoflavone in water fermented soya beans
By fermenting soybeans from traditional Sichuan vinegar and vinegar mash, Bacillus subtilis isolated from Sichuan's traditional vinegar and vinegar mash, combined with the control of the strain addition amount and fermentation temperature, the problem of low isoflavones content in water fermented black beans was solved, and the product quality was improved and the antioxidant capacity was enhanced.
Patent Information
- Application Number
- CN202510041910.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-10
- Publication Date
- 2025-05-16
AI Technical Summary
In the existing water fermented black bean preparation process, free soybean isoflavone content is low, and the product quality is unstable, which is easily affected by the climate and fermentation environment.
Bacillus subtilis with high protease activity was isolated from traditional Sichuan vinegar and vinegar mash. Combined with the control of the strain addition amount and fermentation temperature, it was used to ferment soybeans to prepare water fermentation black beans.
It significantly improves the free soy isoflavone content in water fermented black beans, improves the antioxidant ability of the product, and does not require the addition of food additives.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to application of Bacillus subtilis in increasing the content of free soybean isoflavones in fermented black beans. Background Art
[0002] Douchi is a traditional specialty fermented soybean product. According to the different types of fermented microorganisms, it can be divided into four categories: Mucor type Douchi, Rhizopus type Douchi, Aspergillus type Douchi and bacterial type Douchi. Water Douchi is a representative of bacterial Douchi. It is a product with unique flavor and nutritional value made from soybeans as the main raw material and using bacterial fermentation to decompose soybean protein. Soybeans have more bioactive substances after bacterial fermentation, such as soy isoflavones, soy saponins, Douchi fibrinolytic enzymes, melanoidins, etc. Among them, soy isoflavones are the main bioactive substances, which exist in two forms: free and glycoside. Free soy isoflavones have stronger biological activity than glycoside soy isoflavones. At present, the preparation of water Douchi mostly uses traditional natural fermentation technology. During the natural fermentation process, it is easily affected by climate, fermentation environment, etc., and is also easily affected by gas-producing bacteria. The product quality is extremely unstable, and the content of free soy isoflavones is low. In view of the above situation, the existing technology uses a single strain of Bacillus Velez to ferment soybeans to produce water-fermented black beans, so as to improve the quality of water-fermented black beans products, but the effect of increasing the content of free soybean isoflavones is not obvious. Summary of the invention
[0003] Based on the above reasons, the purpose of the present invention is to provide an application of Bacillus subtilis in increasing the content of free soybean isoflavones in water-fermented soy bean curd. The Bacillus subtilis is isolated from Sichuan traditional sun-dried vinegar mash and used in the preparation of water-fermented soy bean curd. Combined with the control of the strain addition amount and the fermentation temperature, the content of free soybean isoflavones in the prepared water-fermented soy bean curd can be significantly increased.
[0004] In order to achieve the above-mentioned purpose, the present invention discloses an application of Bacillus subtilis in increasing the content of free soybean isoflavones in water-fermented black beans. The Bacillus subtilis is isolated from Sichuan traditional sun-dried vinegar mash and was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 17, 2024, with a deposit number of GDMCC No: 65652.
[0005] Preferably, the method for preparing the water fermented black beans is: after steaming and cooling the soybeans, inoculating Bacillus subtilis to carry out constant temperature fermentation.
[0006] Preferably, the inoculation amount of Bacillus subtilis is 2%-2.5% of the mass of the soybeans after cooking and cooling.
[0007] Preferably, the fermentation temperature is 30-33°C and the fermentation time is 90-96 h.
[0008] Preferably, the cooking time is 50-60 min.
[0009] Preferably, the cooking further includes soaking treatment, and the soaking time is 14-16 hours.
[0010] Preferably, after the fermentation is completed, the process further includes adding auxiliary materials for mixing, filling and sterilizing.
[0011] Preferably, the amount of the auxiliary material added is 20%-25% of the mass of the cooled soybeans.
[0012] The beneficial effects of the present invention are: The invention separates a Bacillus subtilis with high protease activity from Sichuan traditional sun-dried vinegar mash, utilizes the strain to ferment soybeans to prepare water-fermented black beans, and controls the added amount of the strain and the fermentation temperature. Without using food additives, the content of free soybean isoflavones in the water-fermented black beans can be significantly increased, and the antioxidant capacity of the product is also improved. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 This is the colony morphology of Bacillus subtilis isolated from Sichuan traditional sun-dried vinegar mash. DETAILED DESCRIPTION
[0014] In order to make the purpose, technical scheme and advantages of the present invention clearer, the scheme of the present invention is further described in detail below in conjunction with embodiment.It will be appreciated by those skilled in the art that the following examples are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, those not indicating specific conditions in the examples are carried out according to the conditions of normal conditions or manufacturer's recommendations.Reagents used or instruments not indicating manufacturers are conventional products that can be obtained by commercial purchase.
[0015] The embodiment of the present invention provides an application of Bacillus subtilis in increasing the content of free soybean isoflavones in fermented black beans. The Bacillus subtilis is isolated from Sichuan traditional sun-dried vinegar fermented grains and is classified and named Bacillus subtilis ( Bacillus subtilis ), deposited in Guangdong Provincial Microbiological Culture Collection Center on December 17, 2024, with the deposit number GDMCC No: 65652.
[0016] In this embodiment, the method for preparing water fermented black beans is to steam and cool soybeans, and then inoculate Bacillus subtilis to perform constant temperature fermentation.
[0017] It should be noted that, during the fermentation process described in the present embodiment, the enzyme secreted by Bacillus subtilis will act on the soybean isoflavone glycosides in soybeans, hydrolyzing them into free isoflavones, thereby obtaining water fermented soybeans with certain biological activity. However, the existing Bacillus subtilis has a weak enzyme production ability during the soybean fermentation process, and the obtained water fermented soybeans have a low content of free isoflavones and poor biological activity. In order to improve the biological activity of water fermented soybeans, the prior art generally adds enzyme preparations or optimizes the fermentation substrate soybeans first to increase the enzyme content in the fermentation system, and achieves the purpose of improving the biological activity of water fermented soybeans by increasing the total content of soybean isoflavones in water fermented soybeans, but the effect of improving the content of free soybean isoflavones is not obvious. The Bacillus subtilis isolated from Sichuan traditional sun-dried vinegar fermented grains in this embodiment has a high protease activity and can be directly used for fermentation of soybeans. During the fermentation process, more glucosides can be hydrolyzed, and the total content of soybean isoflavones in the product can be increased. On the other hand, the conversion of glycoside-type soybean isoflavones to free soybean isoflavones can be increased, thereby significantly increasing the content of free soybean isoflavones in water-fermented soybeans. In addition, the Bacillus subtilis in this embodiment can also produce soybean antioxidant peptides by hydrolyzing soybean protein in soybeans, thereby improving the antioxidant capacity of the product.
[0018] It should be further explained that the fermentation temperature of the existing Bacillus subtilis is generally 25°C-37°C. When it is used to ferment soybeans, the fermentation temperature is usually controlled at 35°C or above. The Bacillus subtilis of the present embodiment is used to ferment soybeans, and the fermentation temperature needs to be controlled at 30-33°C. At the same time, the inoculation amount of the strain needs to be 2%-2.5% of the mass of soybeans to ensure that the enzyme production capacity of the strain is optimal, and the free soybean isoflavone content in the obtained water fermented soybeans is significantly improved. Raising or lowering the fermentation temperature, the free soybean isoflavone content in the obtained water fermented soybeans is low. Reducing the inoculation amount of Bacillus subtilis, the fermentation and maturation cycle of water fermented soybeans is extended, and the effect of improving the content of free soybean isoflavones is not good. If the inoculation amount of Bacillus subtilis is increased, the acid-base imbalance of the fermentation system will be caused, and the growth and reproduction of the strain will be easily affected. The content of free soybean isoflavones in water fermented soybeans is still low, and the aroma will be weakened.
[0019] Under the above fermentation conditions, the soybeans can be completely fermented after 90-96 hours of fermentation. Mucus will appear between the fermented soybeans, and they will become silky when picked up with chopsticks.
[0020] In this embodiment, the soybeans are steamed at 50-60° C., and soaked for 14-16 h before being steamed. The soybeans are naturally cooled to room temperature after being steamed.
[0021] In this embodiment, after the fermentation is completed, the process further includes adding auxiliary materials for mixing, filling, and sterilization. The amount of the auxiliary materials added is 20%-25% of the mass of the cooled soybeans.
[0022] It is understood that the components and proportions of the auxiliary materials can be conventionally adjusted by those skilled in the art according to the taste of the final product, and the present invention does not specifically limit them, but only provides some preferred embodiments. For example, the auxiliary material is prepared by mixing pepper powder, soy sauce, minced garlic, and minced ginger in a mass ratio of (5-7): (3-5): (6-8): (6-8).
[0023] Specific embodiments for implementing the present invention will be disclosed below, as well as corresponding comparative examples to demonstrate the relevant technical effects of the present invention.
[0024] Example 1 Isolation and screening of strains The strain of the present invention is isolated from Sichuan traditional sun-dried vinegar fermented mash, which is obtained from Zigong Qiantian Baiwei Food Co., Ltd. It is made of rice and bran as the main raw materials, supplemented by Chinese herbal medicine for making koji, and is produced through fermented mash making, fermented mash turning and sun-dried mash processes. Under sterile conditions, 25 g of Sichuan traditional sun-dried vinegar fermented mash sample is taken and placed in a sterile homogenizing bag containing 225 mL of physiological saline, and then beaten with a slapping homogenizer for 2 minutes and then diluted 10 times in a gradient, and 10 3 -10 6 The samples with multiple dilutions were spread on BHI agar medium and inverted for culture at 37°C for 24 h; then typical colonies of Bacillus were selected and isolated and cultured using BHI agar medium.
[0025] Example 2 Identification of strains (1) Preliminary identification of strains The strains isolated and cultured above were subjected to colony morphology observation and physiological and biochemical identification. The colony characteristics included colony size, color, shape, edge, gloss, transparency, etc.
[0026] The colony morphology of the strain is as follows Figure 1 As shown, A is the colony morphology on the BHI plate; B is the colony morphology under a stereo microscope. Figure 1 It can be seen that the colonies of the strain are light yellow and flat, with irregular colony edges, a rough, wrinkled and opaque surface; under a stereomicroscope, it can be seen that the colonies have branches inside and are hard tuberculosis-like.
[0027] The physiological and biochemical identification results of the strain are as follows: the aerobic growth, propionate utilization and hemolytic reaction tests of the strain are all negative, and the VP, nitrate reduction, starch hydrolysis, gelatin liquefaction, citrate, D-xylose, L-arabinose, D-mannitol, 7% sodium chloride growth, and pH 5.7 growth tests are all positive.
[0028] The strain was preliminarily identified as Bacillus subtilis.
[0029] (2) Molecular identification of strains The strain DNA was extracted and PCR amplified using the following system: 12.5 μL 2×Mix, 1.0 μL upstream and downstream primers, 8.5 μL ddH2O, and 2 μL DNA template; Amplification conditions were as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 15 s, 35 cycles, and extension at 72°C for 5 min.
[0030] The PCR amplification products were subjected to agarose gel electrophoresis and then sequenced. The sequencing results were compared for nucleic acid sequence similarity using Blast, and a phylogenetic tree was constructed using Mega 6. The species with the closest genetic distance was selected for identification. The strain was identified as Bacillus subtilis and was named Bacillus subtilis ( Bacillus subtilis ), and was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 17, 2024, with the deposit number GDMCC No: 65652.
[0031] Example 3 Determination of protease activity of strains The protease activity of the strain isolated above was measured by the following method: (1) Drawing of standard curve Prepare L-tyrosine standard solution with water to the following values: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL; Take 1.00 mL of each of the above solutions, add 5.00 mL of sodium carbonate solution and 1.00 mL of Folin reagent solution to each, shake evenly, place in a 40°C water bath for color development for 20 min, take out, and use a spectrophotometer to measure its absorbance at a wavelength of 680 nm, using tube 0 without tyrosine as the blank, and then draw a standard curve with absorbance A as the ordinate and tyrosine concentration c as the abscissa.
[0032] (2) Protease activity assay The isolated Bacillus subtilis strain was inoculated into a broth medium, and after culturing for 24 h, it was inoculated into the broth medium again at a ratio of 2%, and cultured in a shaking incubator at 37°C and 180 rpm for 24 h, and then the cultured broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken to obtain an enzyme solution for subsequent protease activity determination; First, preheat the casein solution in a constant temperature water bath at 40°C for 5 min, take 1 mL of the above enzyme solution, add 1.00 mL of the preheated casein solution, mix well, preheat in a constant temperature water bath at 40°C for 10 min, then add 2.00 mL of trichloroacetic acid, mix well, let stand for 10 min, and then filter to obtain the filtrate; Take 1.00 mL of the above filtrate, add 5.0 mL of sodium carbonate solution and 1.00 mL of Folin reagent solution, mix well, place in a water bath at 40°C for 20 min, take out, and use a spectrophotometer to measure its absorbance at a wavelength of 680 nm.
[0033] The result of protease activity test of the strain was: 35.88 U / mL.
[0034] Example 4 The water-fermented fermented bean curd sample was prepared using the Bacillus subtilis isolated and cultured in Example 1 above, and the preparation method is as follows: Sample 1: Weigh 50 kg of soybeans with full grains and no foreign matter, wash them with clean water, add 150 kg of pure water and soak them for 16 h, then drain the water, put them in a pot and steam for 60 min, then naturally cool them to room temperature to obtain the steamed material; The fermentation material was placed in a fermentation tank, inoculated with 2% of the mass of the cooking material, stirred evenly, sealed, and fermented at a constant temperature of 32°C for 94 hours to obtain a fermented material; Add auxiliary materials to the fermented material, stir evenly, then fill, sterilize, and preserve at low temperature. The amount of the auxiliary materials added is 20% of the mass of the soybeans. The auxiliary materials are prepared by mixing pepper powder, soy sauce, minced garlic, and minced ginger in a mass ratio of 5:3:6:6.
[0035] Sample 2: Weigh 50 kg of soybeans with full grains and no foreign matter, wash them with clean water, add 150 kg of pure water and soak them for 14 h, then drain the water, put them in a pot and steam them for 50 min, then naturally cool them to room temperature to obtain the steamed material; The cooking material is placed in a fermentation tank, inoculated with 2.5% of the mass of the cooking material, Bacillus subtilis, stirred evenly, sealed, and fermented at a constant temperature of 30° C. for 90 hours to obtain a fermented material; Add auxiliary materials to the fermented material, stir evenly, then fill, sterilize, and preserve at low temperature. The amount of the auxiliary materials added is 20% of the mass of the soybeans. The auxiliary materials are prepared by mixing pepper powder, soy sauce, minced garlic, and minced ginger in a mass ratio of 5:3:6:6.
[0036] Sample 3: Weigh 50 kg of soybeans with full grains and no foreign matter, wash them with clean water, add 150 kg of pure water and soak them for 15 h, then drain the water, put them in a pot and steam for 55 min, then naturally cool them to room temperature to obtain the steamed material; The cooking material is placed in a fermentation tank, inoculated with 2% of the mass of the cooking material, Bacillus subtilis, stirred evenly, sealed, and fermented at a constant temperature of 33° C. for 96 h to obtain a fermented material; Add auxiliary materials to the fermented material, stir evenly, then fill, sterilize, and preserve at low temperature. The amount of the auxiliary materials added is 20% of the mass of the soybeans. The auxiliary materials are prepared by mixing pepper powder, soy sauce, minced garlic, and minced ginger in a mass ratio of 7:5:8:8.
[0037] Sample 4: Weigh 50 kg of soybeans with full grains and no foreign matter, wash them with clean water, add 150 kg of pure water and soak them for 16 h, then drain the water, put them in a pot and steam for 60 min, then naturally cool them to room temperature to obtain the steamed material; The cooking material is placed in a fermentation tank, inoculated with 2.3% of the weight of the cooking material, Bacillus subtilis, stirred evenly, sealed, and fermented at a constant temperature of 30° C. for 94 h to obtain a fermented material; Add auxiliary materials to the fermented material, stir evenly, then fill, sterilize, and preserve at low temperature. The amount of the auxiliary materials added is 20% of the mass of the soybeans. The auxiliary materials are prepared by mixing pepper powder, soy sauce, minced garlic, and minced ginger in a mass ratio of 7:5:8:8.
[0038] The following comparative samples were set up to demonstrate the synergistic effect of the Bacillus subtilis and fermentation conditions of the present invention: Comparative sample 1 The preparation method is the same as that of Example 2, except that Bacillus subtilis is replaced by commercially available Bacillus subtilis (purchased from Beijing Biobo Biotechnology Co., Ltd.), and the fermentation temperature is set to the optimal fermentation temperature of 35°C for the commercially available Bacillus subtilis.
[0039] Comparative sample 2 The preparation method is the same as that of Example 2, except that Bacillus subtilis is replaced by commercially available Bacillus Velez (purchased from Beijing Biobo Biotechnology Co., Ltd.), and the fermentation temperature is set to the optimal fermentation temperature of 30°C for the commercially available Bacillus Velez.
[0040] Comparative sample 3 The preparation method is the same as that of Example 2, except that the inoculation amount of Bacillus subtilis is 1.8%.
[0041] Comparative sample 4 The preparation method is the same as that of Example 2, except that the inoculation amount of Bacillus subtilis is 2.8%.
[0042] Comparative sample 5 The preparation method is the same as that of Example 2, except that the fermentation temperature is 28°C.
[0043] Comparative sample 6 The preparation method is the same as that of Example 2, except that the fermentation temperature is 35°C.
[0044] Comparative sample 7 The preparation method is the same as that of Example 2, except that Bacillus subtilis is not inoculated and natural fermentation is used for fermentation.
[0045] According to the method specified in the national standard "GB / T 26625-2011 Grain and Oil Inspection - Determination of Soybean Isoflavones by High Performance Liquid Chromatography", the soy isoflavones in the above samples and comparison samples were tested, including the six soy isoflavones specified in the above national standard, namely, daidzein, genistin, glycitein, daidzein, genistein and glycitein, and the total content of free soy isoflavones (including daidzein, genistein and glycitein) with strong physiological activity was calculated. The test results are shown in Table 1.
[0046] Table 1 shows the test results of the contents of 6 kinds of soybean isoflavones in each group of samples. It can be seen from the table that the total content of soybean isoflavones and the content of free soybean isoflavones in samples 1-4 are relatively high, especially the total content of free soybean isoflavones (daidzein, genistein, glycitein) is significantly higher than that of each comparative example, indicating that the Bacillus subtilis isolated from the traditional Sichuan sun-dried vinegar mash of the present invention can significantly increase the content of free soybean isoflavones in water fermented black beans and enhance the physiological activity of the product.
[0047] Table 1 Determination results of soybean isoflavones content in samples of each group .
[0048] The total antioxidant capacity and free radical cation scavenging capacity of the above samples and the comparative samples were measured. The total antioxidant capacity was measured using a FRAP kit, and the free radical cation scavenging capacity was measured using an ABST kit and a DPPH kit. The measurement results are shown in Table 2.
[0049] According to the results in Table 2, the overall antioxidant capacity of samples 1-4 is higher than that of the comparative examples, indicating that the Bacillus subtilis isolated from the traditional Sichuan sun-dried vinegar mash of the present invention can effectively improve the antioxidant capacity of water-fermented black beans.
[0050] Table 2 Antioxidant capacity test results of samples in different groups .
[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. The application of Bacillus subtilis in increasing the content of free soybean isoflavones in fermented black beans is characterized in that: The Bacillus subtilis was isolated from Sichuan's traditional sun-dried vinegar mash and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 17, 2024, with a deposit number of GDMCC No: 65652.
2. The use according to claim 1, characterized in that The preparation method of the water fermented black beans comprises: steaming and cooling soybeans, and then inoculating Bacillus subtilis to perform constant temperature fermentation.
3. The use according to claim 2, characterized in that The inoculation amount of the Bacillus subtilis is 2%-2.5% of the mass of the soybeans after cooking and cooling.
4. The use according to claim 2, characterized in that The fermentation temperature is 30-33°C and the fermentation time is 90-96 h.
5. The use according to claim 2, characterized in that The cooking time is 50-60 min.
6. The use according to claim 2 or 5, characterized in that The cooking process also includes soaking for 14-16 hours.
7. The use according to claim 2, characterized in that After the fermentation is completed, the steps include adding auxiliary materials for mixing, filling and sterilization.
8. The use according to claim 2, characterized in that The amount of the auxiliary material added is 20%-25% of the mass of the cooled soybeans.