Bacillus subtilis with high glutamate decarboxylase activity and application thereof in fermented bean products

Abstract

The invention discloses bacillus subtilis with high glutamate decarboxylase activity and application of the bacillus subtilis in fermented bean products, and belongs to the technical field of fermentation engineering. The bacillus subtilis SNBA-1 with high glutamate decarboxylase activity is obtained through screening and is preserved in the China General Microbiological Culture Collection Center on November 7, 2022, the preservation address is No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 26053. According to the present invention, the Bacillus subtilis B.subtilis SNBA-1 has good glutamic acid decarboxylase production capacity, and can be directly used for fermentation production of bean products, and the GABA content in the fermented soya beans is substantially improved by adding the Bacillus subtilis B.subtilis SNBA-1 fermentation agent during the fermentation process of the fermented soya beans so as to improve the pressure relieving and depression improving functions of the fermented soya beans, such that the fermented soya beans have the good application prospects in the field of fermented soya bean production. Meanwhile, the comprehensive quality of the fermented soya beans is obviously improved according to sensory evaluation results.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering, and relates to a Bacillus subtilis with high glutamate decarboxylase activity and application thereof in fermented bean products, and specifically to a Bacillus subtilis with high glutamate decarboxylase activity and application thereof in fermented fermented black bean products suitable for relieving stress and depression for modern people. Background Art

[0002] As a new resource food, studies have shown that γ-aminobutyric acid (GABA) exhibits certain anti-depressant ability and is believed to help improve sleep and relieve stress to a certain extent, which is why it has attracted much attention.

[0003] Light black beans are the only fermented Chinese medicine with both medicinal and edible properties included in all editions of the Chinese Pharmacopoeia. They have the effects of relieving exterior symptoms, relieving restlessness, and relieving pent-up heat. Researchers have found that GABA is also present in light black beans, and it is speculated that this may be the effective ingredient in light black beans that can relieve depression. At the same time, ingredient studies have shown that black beans, the raw material of light black beans, contain almost no GABA, which means that GABA is mostly accumulated by microorganisms during the fermentation process. Therefore, the strains screened from light black beans have a strong advantage as GABA fermentation strains.

[0004] Bacillus subtilis belongs to the genus Bacillus, and is an aerobic or facultative anaerobic Gram-positive rod-shaped bacterium with excellent stability, acid resistance, alkali resistance, high temperature resistance, etc., and the survival rate is still about 80% in a 90℃ water bath for 10 minutes. As a dominant strain in light fermented black beans, Bacillus subtilis has a strong ability to secrete glutamate decarboxylase related to GABA accumulation, and has certain advantages as a GABA-rich soy product fermentation strain.

[0005] In summary, therefore, screening and obtaining Bacillus subtilis with high GABA production and fermenting to prepare a fermented soy product rich in GABA can not only give play to the advantages of microbial resources of traditional Chinese fermented foods, but also serve as a functional food to improve stress, depression and other related conditions in modern people. Summary of the invention

[0006] In order to solve the technical problems existing in the background technology, the purpose of the present invention is to provide a Bacillus subtilis with high glutamate decarboxylase activity and the application of the same in fermenting bean products.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides a strain of Bacillus subtilis SNBA-1 with high glutamate decarboxylase activity, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on November 7, 2022, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26053.

[0009] In a second aspect, the present invention provides a high-efficiency fermentation agent, which is prepared by culturing the above-mentioned Bacillus subtilis SNBA-1.

[0010] Based on the above technical solution, further, the preparation process of the high-efficiency starter comprises the following steps: inoculating the Bacillus subtilis SNBA-1 in a fermentation medium at an inoculation rate of 1-5%, and culturing for 12-72 hours at 25-45°C and pH 4-10.

[0011] Based on the above technical solution, further, the fermentation medium is LB medium.

[0012] In a third aspect, the present invention provides the use of the above-mentioned Bacillus subtilis SNBA-1 with high glutamate decarboxylase activity and high-efficiency starter culture in fermenting bean products.

[0013] Based on the above technical solution, further, the soy product includes fermented black beans.

[0014] Based on the above technical solution, further, the fermented black beans contain a high content of GABA, which has a certain anti-depression and sleep-promoting effect.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The Bacillus subtilis SNBA-1 provided by the present invention has good glutamate decarboxylase production ability, can be directly applied to the fermentation production of bean products, and by adding the Bacillus subtilis SNBA-1 starter in the fermentation process of fermented black beans, the GABA content in the fermented black beans is greatly improved, thereby improving the function of the fermented black beans in relieving stress and improving depression, and according to the sensory evaluation result, it is found that the comprehensive quality of the fermented black beans is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The colony morphology and bacterial morphology of Bacillus subtilis SNBA-1 of the present invention are shown in FIG.

[0018] Figure 2 It is a growth curve diagram of Bacillus subtilis SNBA-1 of the present invention.

[0019] Figure 3These are the test results of the levels of inflammatory factors IL-6 and TNF-α in the blood and the content of neurotransmitter GABA in the brain tissue of each group of mice in Example 4. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0021] The Bacillus subtilis SNBA-1 described in the embodiment of the present invention has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 7, 2022, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26053.

[0022] The Bacillus subtilis SNBA-1 is separated from naturally fermented light fermented black beans, and has a good protein conversion rate. At the same time, it has a certain glutamate decarboxylase ability. The produced black beans accumulate a certain amount of GABA, have better stress-relieving and depression-relieving abilities, and have broad application prospects.

[0023] The present invention will be further described below in conjunction with specific embodiments.

[0024] Example 1: Fermentation strain screening

[0025] A total of 13 strains of microorganisms were selected from the previous strain library. After culturing for 24 hours, they were centrifuged at 1000×g for 20 minutes, 10 μL of the supernatant was removed, diluted to 50 μL with distilled water, and tested with a glutamate decarboxylase kit (enzyme-linked immunosorbent assay) to screen out strains with higher glutamate decarboxylase activity. The results of measuring the glutamate decarboxylase activity of the strains are shown in Table 1. The strain with the strongest glutamate decarboxylase activity was SNBA-1.

[0026] Table 1 Enzyme production capacity of different strains

[0027] Strain number Glutamate decarboxylase (U / mL) SNBA-1 251.45±0.34 15-1 63.73±0.42 15-2 113.29±0.26 15-3 161.10±0.37 15-4 104.08±0.32 15-6 122.06±0.39 10-2 104.96±0.28 P10-1 141.80±0.40 5-2 161.54±0.35 5-4 179.52±0.38 20-1 158.90±0.31 20-2 152.32±0.34 LKX15-2 144.87±0.47

[0028] Combined with the colony morphology of SNBA-1 strain ( Figure 1 ), physiological and biochemical characteristics and molecular biological identification results showed that the SNBA-1 strain was a new strain of Bacillus subtilis, and it was named Bacillus subtilis SNBA-1.

[0029] The applicant has deposited the sample with the General Microbiology Center of China Culture Collection Administration on November 7, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26053.

[0030] Example 2: Effect of Bacillus subtilis SNBA-1 on GABA content in fermented black beans

[0031] Select black beans with full grains of basically uniform size and no diseases, wash and remove impurities, soak in clean water at 25℃ for 24h; drain, cook at 120℃, 2.0MPa for 30min, and make koji when the center temperature of black beans cools to below 35℃. The whole process of making koji takes 72h. The natural koji control group is directly placed in an open incubator, and the bean temperature in the first 24h is kept at 28-39℃. The soybeans on the koji bed are turned over once every 24h-48h to make the bean temperature even and fully fermented and avoid sticking and agglomeration; the bean temperature is kept at 30-35℃ for 24h-48h; the bean temperature is below 30℃ during 48h-72h, and the koji room is kept dry throughout the process to prevent water drops from dripping into the fermentation tank. After that, wash the beans and put them in a closed environment and ferment them at 35℃ for 15 days. Pure strain fermentation uses LB medium to activate Bacillus subtilis SNBA-1, and after culturing for 12 hours, it is inoculated into cooled black beans and placed in an incubator for fermentation at 35°C for 15 days. Pay attention to keeping the overall environment sterile, and finally dry to obtain the finished product.

[0032] Weigh 200 mg of each sample, add 10 mL of 0.1 mol / mL hydrochloric acid, mix well, ultrasonicate for 40 min, centrifuge at 8 000 r / min, take 1 mL of the supernatant and filter with a 0.45 μm filter membrane, and retain the filtrate.

[0033] The HPLC derivatization reagent was prepared by mixing o-phthalaldehyde (OPA) dissolved in methanol, borate buffer (0.5 M, pH 9.9) and 2-MCE (40:10:1, v / v / v). 114 μL of the derivatization reagent was mixed with 36 μL of the extract / standard and 10 μL of the internal standard (IS, 250 mg / L L-norvaline aqueous solution) to derivatize the free amino acids in the extract / standard. The derivatization reaction was carried out at 22°C for 4 minutes. Then, 20 μL of the derivatized extract was injected into an HPLC column (Nucleodur 100-5C18ec column, 5 μm; 150 mm×4.6 mm), and the mobile phase A was an aqueous solution prepared by V (Waters AccQ·TagEluent A): V (ultrapure water) = 1:10, the mobile phase B was acetonitrile, and the mobile phase C was ultrapure water; the elution conditions are shown in Table 2. A standard curve was prepared using GABA / Glu concentrations ranging from 1 mg / L to 100 mg / L.

[0034] Table 2 Elution conditions

[0035]

[0036]

[0037] The GABA content in fermented black beans was detected by pre-column derivatization-ultra-performance liquid chromatography. The results are shown in Table 3. It was found that the GABA content in fermented black beans produced by pure Bacillus subtilis SNBA-1 was significantly higher than that produced by natural fermentation.

[0038] Table 3 GABA content of fermented sour soybean milk with different strains

[0039] Strain number GABA content (μmol / L) SNBA-1 785.85 Natural fermentation 445.16

[0040] Example 3: Effect of Bacillus subtilis SNBA-1 on the free amino acid content of fermented black beans

[0041] The free amino acids in the fermented bean curd samples were detected by pre-column derivatization-ultra-performance liquid chromatography. Weigh 200 mg of fermented bean curd samples (naturally fermented samples and pure strain Bacillus subtilis SNBA-1 fermented samples) and add 6 mL of ultrapure water, ultrasonically extract and filter for 40 min, wash the filter residue with 10 mL of ultrapure water, repeat the ultrasonic once, combine the filtrate, centrifuge at 6000 r / min for 10 min, filter the supernatant through a 0.22 μm filter membrane, and derive the liquid phase method as in Example 2. The standard curve uses 200 mg of each amino acid sample, adds 10 mL of 0.1 mol / mL of hydrochloric acid, mixes, ultrasonically centrifuges for 40 min, and centrifuges at 8000 r / min. Take 1 mL of the supernatant and filter it with a 0.45 μm filter membrane to derive the liquid phase. The results are shown in Table 4. The samples of natural fermentation are compared with the samples of pure strain fermentation. It is detected that the total amino acid content of the pure strain Bacillus subtilis SNBA-1 fermented sample is significantly greater than the total amino acid content of the natural fermentation.

[0042] Table 4 Comparison of free amino acid content (μmol / L) of SNBA-1 fermentation samples and natural fermentation samples

[0043]

[0044] Example 4: In vitro antidepressant experiment of fermented black beans

[0045] 20 57bl / 6 male mice were divided into 4 groups, namely blank group, model group, fluoxetine group (20mg / kg), sample group (0.97g / kg), mice were adaptively raised for 7d, and from the 8th day, continuous gavage for 13d, fluoxetine group gavage 20mg / kg fluoxetine, sample group gavage 0.97g / kg fermented black beans after Bacillus subtilis SNBA-1 in embodiment 2, blank group and model group were given corresponding volume of normal saline. On the 10th day, model group, fluoxetine group and sample group were administered after 1.5mg / kg intraperitoneal injection of LPS. On the 13th day (24h after the last intraperitoneal injection), after 1h of administration, mouse behavior test was started. Open field test, tail suspension test and forced swimming test were carried out respectively. After the behavioral experiment, the mice were sacrificed after blood was collected from their orbits, and their brains were quickly removed on ice. The levels of inflammatory factors IL-6 and TNF-α in the blood and the level of neurotransmitter GABA in the brain tissue were detected by enzyme-linked immunosorbent assay (ELISA) according to the instructions. Figure 3 As shown, the results showed that fermented black beans played a certain role in optimizing the inflammatory factors in the blood of depressed mice, and at the same time increased the GABA content in the mouse brain. Behavioral experiments also showed that fermented black beans played a certain role in improving the depressive-like behavior of mice.

[0046] Example 5: Sensory Evaluation

[0047] Twenty experienced sensory evaluators were selected, including 10 boys and 10 girls, aged between 25 and 30 years old, with normal sensory function and experience. After systematic training, they referred to the food sensory evaluation standard "GB / T15682-2009 Inspection of grain and oil, sensory evaluation method for steamed and cooked rice" to conduct a comprehensive evaluation and scoring of the color, aroma, taste and body shape of the fermented black beans. The detailed scoring rules are shown in Table 5.

[0048] Table 5 Sensory evaluation standards for fermented black beans

[0049]

[0050] Table 6 Sensory evaluation results of pure strain Bacillus subtilis SNBA-1 fermentation samples and natural fermentation samples

[0051] project Naturally fermented black beans Pure fermented black beans form 22 19.5 Color 19 20.75 aroma 20.75 23 taste 20.5 22.75 Total score 82.25 86

[0052] The results are shown in Table 6. The results show that the color, taste and morphology of the fermented black beans inoculated with Bacillus subtilis SNBA-1 are improved, which is similar to the results of the previous experiments, indicating that adding Bacillus subtilis fermentation agent to fermented black beans has the effect of improving the flavor quality of fermented black beans.

[0053] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bacillus subtilis SNBA-1 having high glutamate decarboxylase activity, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 7, 2022, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.26053.

2. A highly efficient fermentation agent, characterized in that: The method is prepared by culturing the Bacillus subtilis SNBA-1 according to claim 1.

3. The efficient leavening agent according to claim 2, characterized in that: The preparation process of the high-efficiency starter comprises the following steps: inoculating the Bacillus subtilis SNBA-1 in a fermentation medium at an inoculation rate of 1-5%, and culturing for 12-72 hours at 25-45°C and pH 4-10 to obtain the starter.

4. The high-efficiency leavening agent according to claim 3, characterized in that: The fermentation medium is LB medium.

5. Use of the Bacillus subtilis SNBA-1 with high glutamate decarboxylase activity according to claim 1 and the high-efficiency starter according to any one of claims 2 to 4 in fermenting soy products.

6. The use according to claim 5, characterized in that: The bean product includes fermented black beans.

7. The use according to claim 6, characterized in that: Fermented black beans contain high levels of GABA, which has certain anti-depression and sleep-promoting effects.

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