Bacillus subtilis with high yield of glutaminase and application of bacillus subtilis

Abstract

The invention discloses bacillus subtilis with high yield of glutaminase and application of the bacillus subtilis, and belongs to the technical field of microorganisms. The bacillus subtilis B31 with high yield of glutaminase is screened through an ARTP mutagenesis technology, the glutaminase activity of the bacillus subtilis B31 is improved by 3 IU / mL compared with that of an original strain, and the bacillus subtilis B31 is resistant to high-concentration KCl and weak acid environments and suitable for natto fermentation. After the strain is used for natto fermentation, the content of amino acid nitrogen in a product reaches 8.59 g / 100g, and the product is rich in fragrance and taste and has an application prospect in the food industry.

Description

Technical Field

[0001] This invention relates to a high-glutaminase-producing Bacillus subtilis strain and its applications, belonging to the field of microbial technology. Background Technology

[0002] Glutaminase is a class of hydrolases that catalyze the deamidation of glutamine to produce glutamate and ammonia. Glutamate is an important flavor compound that imparts a significant umami taste to food and plays a crucial role in the flavor formation of fermented foods. Existing research indicates that glutaminase has broad application potential in the food industry, including fermented soy products, sauces, and condiments.

[0003] Currently, traditional processes for fermented soy products (such as fermented bean curd, fermented black beans, natto, and soy sauce) mainly rely on natural or artificially inoculated microbial communities. These microorganisms produce hydrolytic enzymes such as proteases and glutaminase during fermentation, thereby promoting the breakdown of soybean protein, releasing free amino acids, and creating unique flavors and textures. However, traditional natural fermentation processes suffer from long cycles, unstable enzyme activity levels, and significant flavor variations, making it difficult to standardize product quality. To improve fermentation results, existing technologies have attempted to directly add exogenous glutaminase preparations to increase the free glutamate content in fermented soy products. For example, US3912822A directly adds glutaminase to increase glutamate levels. While this method can enhance umami to some extent, the production cost of enzyme preparations is high, and exogenous enzymes exhibit poor stability in complex fermentation environments, thus limiting their large-scale application in traditional soy product fermentation.

[0004] Bacillus subtilis ( Bacillus subtilis Lactobacillus rhamnosus is an important fermentation strain widely used in soy products such as natto and fermented black beans. This strain can produce various extracellular hydrolases, such as proteases, amylases, and lipases, playing a crucial role in flavor formation during the fermentation process of soy products. Invention patent CN1553775A discloses a strain of Lactobacillus rhamnosus derived from accession number CNCM I-2433. Lactobacillus rhamnosus The study focuses on the development of salt- and heat-resistant glutaminase, which maintains high activity in high-salt fermentation systems such as soy sauce. However, most natural Bacillus subtilis strains have limited glutaminase secretion, making it difficult to effectively increase the level of free glutamate and umami intensity in fermented products. Therefore, screening for Bacillus subtilis strains that can efficiently secrete glutaminase and significantly enhance umami flavor in soybean fermentation systems has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a high-glutaminase-producing Bacillus subtilis strain and its application in fermenting natto to improve its quality. The purpose is to solve the problem of the lack of a high-glutaminase-producing Bacillus subtilis strain in the existing technology that can effectively improve the quality of fermented natto.

[0006] The first technical solution provided by this invention is a strain of Bacillus subtilis ( Bacillus subtilis B31 was deposited on October 27, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66985, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The second technical solution provided by the present invention is to provide a microbial preparation containing the Bacillus subtilis B31.

[0008] In one embodiment, the content of Bacillus subtilis B31 in the microbial preparation is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0009] The third technical solution provided by the present invention is a fermentation agent containing the aforementioned Bacillus subtilis B31.

[0010] In one embodiment, the method for preparing the fermenting agent includes: activating the Bacillus subtilis B31 strain, fermenting the activated strain in a culture medium, and preparing the fermenting agent.

[0011] In one embodiment, the activated strain is obtained by culturing Bacillus subtilis B31 in a culture medium.

[0012] In one embodiment, the culture temperature is 35°C. 39℃, incubation time is 20 30 hours, speed 120 250rpm; In one embodiment, the culture medium includes, but is not limited to, LB medium.

[0013] The present invention also provides a method for improving the quality of natto, wherein the method involves using the Bacillus subtilis B31 or the fermenting agent for natto fermentation.

[0014] In one embodiment, the improvement of fermented natto quality includes, but is not limited to: increasing the content of flavor substances such as umami amino acids and sweet amino acids, and improving the complexity and intensity of the aroma.

[0015] In one embodiment, the umami amino acids include, but are not limited to, glutamic acid, and the sweet amino acids include, but are not limited to, serine and proline.

[0016] The present invention also provides a method for fermenting natto, wherein the Bacillus subtilis B31 or the fermenting agent is inoculated into washed and soaked soybeans, and fermented at 35-40°C for a period of time and then cooled to 0-4°C for post-ripening.

[0017] In one embodiment, the inoculation is performed at a rate of 1 × 10⁻⁶. 8 The *Bacillus subtilis* B31 was inoculated at a concentration of CFU / g soybean.

[0018] In one embodiment, the method specifically involves: washing soybeans thoroughly, soaking them in three times their volume of deionized water at room temperature for 12 hours, and then adding 1×10... 8 The *Bacillus subtilis* was inoculated at a concentration of CFU / g soybean.

[0019] In one embodiment, the fermentation is carried out at 37 °C for 20-28 hours, followed by post-fermentation at 4 °C for 20-28 hours to obtain the finished product.

[0020] The present invention also provides the application of the aforementioned Bacillus subtilis B31 in the food industry.

[0021] Beneficial effects: 1. This invention utilizes ARTP mutagenesis technology to treat existing Bacillus subtilis strains, resulting in the screening of a high-glutaminase-producing Bacillus subtilis strain B31, which effectively improves the quality of fermented natto. The glutaminase activity of this strain is 3 IU / mL higher than the original strain, representing an increase of approximately 60%. This strain also exhibits tolerance to KCl concentrations of 100 g / L and below, and pH levels of 4.4 and above, making it suitable for fermented natto production and showing promising application prospects in the food industry.

[0022] 2. The Bacillus subtilis B31 obtained by screening is used to ferment natto, and the amino acid nitrogen content reaches 8.59 g / 100 g, with a rich overall aroma and flavor. Attached Figure Description

[0023] Figure 1 The results show the glutaminase activity of the bacterial strains before and after mutagenesis; where the original strain represents the original strain Bacillus subtilis B4, and B31 represents Bacillus subtilis B31.

[0024] Figure 2 The results show the KCl and pH tolerance of the mutant Bacillus subtilis B31.

[0025] Figure 3The glutaminase activity of fermented natto is represented by: Blank represents the blank fermented natto group, B4 represents the original strain Bacillus subtilis B4 fermented natto group, and B31 represents the Bacillus subtilis B31 fermented natto group.

[0026] Figure 4 The content of amino acid nitrogen in fermented natto; where Blank represents the blank fermented natto group, B4 represents the original strain Bacillus subtilis B4 fermented natto group, and B31 represents the Bacillus subtilis B31 fermented natto group.

[0027] Figure 5 The results are the electronic tongue assay results for fermented natto; where Blank represents the blank fermented natto group, B4 represents the original strain Bacillus subtilis B4 fermented natto group, and B31 represents the Bacillus subtilis B31 fermented natto group.

[0028] Figure 6 The results show the determination of free amino acids in fermented natto; where Blank represents the blank fermented natto group, B4 represents the original strain Bacillus subtilis B4 fermented natto group, and B31 represents the Bacillus subtilis B31 fermented natto group.

[0029] Figure 7 The results are obtained by gas chromatography-ion mobility spectrometry (GC-IMS) for fermented natto; where Blank represents the blank fermented natto group, B4 represents the original strain Bacillus subtilis B4 fermented natto group, and B31 represents the Bacillus subtilis B31 fermented natto group.

[0030] Preservation of biological materials Bacillus subtilis ( Bacillus subtilis B31, categorized and named Bacillus subtilis It was deposited on October 27, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66985, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0031] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0032] Test method: Glutaminase activity is defined as the amount of enzyme required to hydrolyze the substrate Z-Gln-Gly (benzyloxycarboxyl-glutamine-glycine) to produce 1 μmol of glutamate in 30 min at 37°C and pH 6.5. It is defined as one unit of glutaminase activity, expressed as IU.

[0033] Raw materials used in the examples: 1. Bacillus subtilisB4 is selected from naturally fermented salted beans.

[0034] 2. Bacillus subtilis B31 by Bacillus subtilis It was obtained through ARTP induction.

[0035] 3. The LB agar medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 20 g / L agar.

[0036] 4. LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0037] Example 1: Screening of Bacillus subtilis Using Bacillus subtilis B4, screened from naturally fermented salted beans, as the starting strain, 5 mL of the original strain Bacillus subtilis B4, cultured to the logarithmic growth phase and exhibiting vigorous activity, was taken. B4 was screened from naturally fermented salted beans and identified as Bacillus subtilis. After centrifugation, the bacterial cells were washed and resuspended with sterile physiological saline to prepare a bacterial suspension, maintaining a bacterial concentration of 10%. 8 CFU / mL, 10 μL of the diluted bacterial suspension was dropped into the center of a sterilized metal slide, and the slide was then placed in an ARTP plasma mutagenesis instrument for radiation mutagenesis. The parameters were set as follows: power 120 W, aeration rate 10 SLM, and treatment time 4 s. After treatment, the slide containing the bacterial suspension was placed in 1 mL of sterile physiological saline and thoroughly shaken to wash off the bacteria. The washed bacterial suspension was serially diluted, and 100 μL was plated onto LB agar plates. Single colonies were picked from the plates and inoculated into LB liquid medium for incubation at 37 ℃, 200 rpm, and for 24 h.

[0038] After ten subcultures, the glutaminase activity of the mutant bacteria was measured. A genetically stable mutant B31 with high glutaminase production was selected. Its glutaminase activity after ten subcultures was 9.94 IU / mL, which was about 5 IU / mL higher than that of the original strain (4.93 IU / mL), and it had good subculture stability (Table 1).

[0039] Table 1. Glutaminase activity after different passage numbers

[0040] After identification, strain B31 was determined to belong to Bacillus subtilis, and therefore it was renamed Bacillus subtilis B31. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 66985.

[0041] Example 2: Characteristic Analysis of Bacillus subtilis The characteristics of Bacillus subtilis B31 prepared in Example 1 were analyzed. The specific steps were as follows: (1) Salt tolerance: Take a bacterial concentration of 1×10 8 Bacillus subtilis B31 bacterial suspensions at CFU / mL were inoculated into 100 mL LB liquid medium with KCl concentrations of 0, 20, 40, 60, 80, 100, 120, 140, 160, and 180 g / L, respectively. The culture temperature was 37℃, the rotation speed was 200 rpm, and the culture time was 24 h. After the culture, the absorbance (OD) of the medium at 600 nm was measured. 600nm ).

[0042] (2) Acid resistance: Take 10 8 Bacillus subtilis B31 bacterial suspension at CFU / mL was inoculated into 100 mL LB liquid medium at pH 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, and 3.0, respectively. The culture temperature was 37 °C, the rotation speed was 200 rpm, and the culture time was 24 h. After the culture, the absorbance (OD) of the medium at 600 nm was measured. 600nm ).

[0043] The results showed that the upper limit of salt tolerance for Bacillus subtilis B31 was about 140-160 g / L, and its growth was significantly inhibited after 80 g / L; the suitable pH was 5.0-6.5, and it could tolerate a minimum pH of 4.0.

[0044] Example 3: Bacillus subtilis fermentation of natto Using soybeans as a substrate, Bacillus subtilis B31 prepared in Example 1 was used for fermentation. The specific steps are as follows: Wash the soybeans thoroughly and soak them in three times their volume of deionized water at room temperature for 12 hours. Bottle 50g of soybeans and sterilize at 121°C for 15 minutes.

[0045] Bacillus subtilis ( Bacillus subtilisB31 was stored in 25% glycerol / LB medium (v / v) before use. LB medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The culture was inoculated at a 5% (v / v) rate into 50 mL Erlenmeyer flasks containing 40 mL of LB medium. The culture was incubated at 37 °C, 200 rpm, and for 24 h to obtain the first activated bacterial suspension. This second inoculation was repeated in 50 mL Erlenmeyer flasks containing 40 mL of LB medium, yielding the activated bacterial suspension. The bacterial suspension was washed three times with 0.85% sterile physiological saline. The washed strain was then inoculated at a 4% (w / w) rate with 10... 8 A co-inoculation of CFU / mL was applied to a 500 mL sterile, sealed fermenter containing 100 g of sterile soybeans. Fermentation was carried out at 37 °C for 24 hours, followed by post-fermentation at 4 °C for 24 hours. Natto samples were collected after fermentation was completed.

[0046] Physicochemical properties of fermented natto were analyzed: (1) Quantitative methods for amino acid nitrogen: Take 1 g of natto and add 20 mL of deionized water to homogenize. Centrifuge at 10000 g for 15 min. Take 10 mL of the supernatant and dilute to volume in a 50 mL volumetric flask. Pour the diluted solution into a 100 mL beaker. Take 5 mL of the diluted solution and add it to a 100 mL beaker. Add 30 mL of deionized water and 10 mL of formaldehyde solution. Start a magnetic stirrer and titrate with NaOH standard solution until the pH reaches 9.2. Record the volume of NaOH standard solution used after adding formaldehyde. Perform a reagent blank test simultaneously.

[0047] Formula for calculating amino acid nitrogen content: X%=(V-V0)×N×0.014 / ((W×5 / 200))×100; Wherein, X - amino acid nitrogen content; V - volume of sodium hydroxide consumed in sample titration; V0 - volume of sodium hydroxide consumed in blank sample titration; N - concentration of NaOH standard solution; W - sample weight, g; 0.014 - milligram equivalents of nitrogen; (2) Method for determining the electronic tongue: The taste characteristics of natto were analyzed using an electronic tongue taste analysis system. Before measurement, an internal solution (saturated AgCl + 3.3 mol / L KCl) was added to the sensor, followed by activation with a standard solution (30 mmol KCl + 0.3 mmol / L tartaric acid) for 24 h. Simultaneously, the electrode was added to the same internal solution and activated with 3.3 mol / L KCl solution for 24 h. A self-test was then performed. After passing the test, 40 mL of filtered and de-oiled natto extract was measured in a measuring cup. A total of four tests were conducted, and the results from the last three tests were analyzed.

[0048] (3) Methods for determining free amino acids: Take 1 g of natto and add 9 mL of deionized water to homogenize for 30 min. After standing at 4 °C for 24 h, centrifuge at 8000 rpm for 15 min. Take 2 mL of the supernatant of the natto extract and filter it through a 0.22 μm microporous aqueous filter membrane. Mix the filtrate with acetone at a ratio of 1:3, shake to mix, and centrifuge (12000 r / min, 10 min). Take the supernatant and blow it with nitrogen to ensure complete evaporation of acetone and water. Redissolve the natto in 1 mL of 0.02 mol / L hydrochloric acid, filter it through a 0.22 μm microporous aqueous filter membrane, and then analyze the solution. Free amino acid content was determined using an automated amino acid analyzer. The chromatographic column was a Hitachi 3 μm cation exchange resin column (4.6 mm × 60 mm); the injection volume was 20 μL; the flow rate of four sodium citrate buffers + regeneration buffer was 0.4 mL / min, and the column temperature was 57 °C; the flow rate of ninhydrin solution was 0.35 mL / min, and the column temperature during the reaction was 135 °C; a UV detector was used (proline was detected at 440 nm, and other amino acids were detected at 570 nm). The prepared samples were placed sequentially into the injection tray, and the detection program was set.

[0049] (4) GC-IMS determination method: Take 3 g of natto sample and place it in a 20 mL headspace vial. Run at 500 rpm for 10 minutes before injection at 60 °C. GC-IMS detection was performed under the following conditions: analysis time 30 min; MXT-WAX column (30 mm × 0.53 mm × 1 μm), column temperature 60 °C, N2 as carrier / drift gas, IMS temperature 45 °C, automated headspace injection (1 mL volume), and syringe temperature 85 °C. The gas chromatographic conditions were as follows: for the first 2 minutes, the drift gas was set to 150 mL / min and the carrier gas to 2 mL / min. From 2 to 10 minutes, the drift gas was kept constant at 150 mL / min, while the carrier gas was gradually increased from 2 mL / min to 10 mL / min. Between 10 and 20 minutes, the drift gas was maintained at 150 mL / min, while the carrier gas was increased from 10 mL / min to 100 mL / min. Finally, from 20 to 30 minutes, the drift gas was maintained at 150 mL / min, while the carrier gas was maintained at 100 mL / min.

[0050] All experimental procedures were repeated three times, and the results are expressed as mean ± standard deviation. Data were statistically analyzed using SPSS 27.0 and graphically analyzed using Origin 2024.

[0051] The results showed that the Bacillus subtilis B31 fermentation group had the highest amino nitrogen content, increasing 5.7 times compared to the control group and 55% higher than the Bacillus subtilis B4 fermentation group. The Bacillus subtilis B31 fermentation group had the strongest umami flavor (20% higher than the control group), followed by the Bacillus subtilis B4 fermentation group. The astringency and aftertaste of the B31 fermentation group were slightly lower than those of the B4 fermentation group, but still better than the control group. Overall, the natto from the B31 fermentation group was fresher, richer, and had a more harmonious flavor. The total amino acid content of the B31 fermentation group was about 18% higher than that of the B4 fermentation group and nearly 3 times higher than that of the control group. The B31 fermentation group showed a more significant increase in umami and sweet amino acids. Specifically, the glutamic acid (20.94 μg / g), serine (33.09 μg / g), and proline (140.61 μg / g) content of the B31 fermentation group was higher than that of the B4 fermentation group (7.95 μg / g and 29.64 μg / g). The levels of acetone (μg / g) and proline (101.81 μg / g) showed significant increases; changes in volatile flavor compounds were also observed in the B31 fermentation group, with increased levels of diacetone (with creamy and buttery aroma), linalool oxide (with woody aroma), and 2-ethyl-3-methylpyrazine (with nutty aroma).

[0052] Comparative Example 1: The specific implementation method is the same as in Example 2, except that the starting strain is Bacillus subtilis (… Bacillus subtle B4 was used as the fermentation strain for natto fermentation, and the results were compared with those of Bacillus subtilis B31 used in Example 3.

[0053] The soybean pretreatment and fermentation methods were as described in Example 3. The physicochemical properties of the fermented natto were analyzed. The results showed that the Bacillus subtilis B31 fermentation group had the highest amino nitrogen content, increasing by 55% compared to the Bacillus subtilis B4 fermentation group. The Bacillus subtilis B31 fermentation group had the strongest umami flavor, followed by the Bacillus subtilis B4 fermentation group. The astringency and aftertaste of the B31 fermentation group were slightly lower than those of the B4 fermentation group. Overall, the natto from the B31 fermentation group was fresher, richer, and had a more harmonious flavor. The total amino acid content of the B31 fermentation group was about 18% higher than that of the B4 fermentation group, with a more significant increase in umami and sweet amino acids. Changes in the intensity of volatile flavor compounds also showed that the aroma complexity and intensity of the B31 fermentation group were significantly improved.

[0054] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Bacillus subtilis ( Bacillus subtilis B31 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 27, 2025, with accession number GDMCC No: 66985.

2. A microbial preparation containing Bacillus subtilis B31 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The content of Bacillus subtilis B31 in the microbial preparation is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

4. A fermentation agent containing Bacillus subtilis B31 as described in claim 1.

5. The fermenting agent according to claim 4, characterized in that, The method for preparing the fermenting agent includes: activating the Bacillus subtilis B31 strain, culturing the activated strain in a culture medium, and preparing the fermenting agent.

6. The fermenting agent according to claim 5, characterized in that, The culture temperature is 35°C. 39℃, incubation time is 20 30 hours, speed 120 250rpm.

7. A method for improving the quality of natto, characterized in that, The Bacillus subtilis B31 of claim 1 or any of the fermenting agents of claims 4 to 6 are used for natto fermentation.

8. A method for fermenting natto, characterized in that, The Bacillus subtilis B31 of claim 1 or the fermentation agent of any one of claims 4 to 6 is inoculated into washed and soaked soybeans, and fermented at 35 to 40°C for a period of time, and then cooled to 0 to 4°C for post-ripening.

9. The method according to claim 8, characterized in that, The vaccination is administered at a dose of 1×10 8 The *Bacillus subtilis* B31 was inoculated at a concentration of CFU / g soybean.

10. The application of Bacillus subtilis B31 as described in claim 1 in the food industry.

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