Bacillus pumilus T1-11 and application thereof

Abstract

The invention relates to the field of food microorganisms, and particularly discloses a bacillus pumilus T1-11 strain and an application of the bacillus pumilus T1-11 strain. The bacillus pumilus T1-11 is preserved in the China General Microbiological Culture Collection Center on January 10, 2025, and the preservation number is CGMCC NO.33025. The bacillus pumilus T1-11 has the characteristic of producing glutaminase, and can metabolize glutamine to generate glutamic acid; in addition, the strain T1-11 can also resist a salt environment, and the generated glutaminase can keep enzyme activity under the conditions of higher temperature and higher salinity. The bacillus pumilus T1-11 is further applied to soy sauce fermentation, the content of glutamic acid, isovaleraldehyde and amino nitrogen in soy sauce can be increased, and the delicate flavor of fermented food such as soy sauce can be improved. The bacillus pumilus T1-11 disclosed by the invention has an important industrial utilization value and a wide application prospect in the aspects of producing glutaminase and improving the delicate flavor of fermented foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of food microorganisms, and more specifically relates to a strain of Bacillus pumilus T1-11 and its application in fermented foods. Background Art

[0002] Glutaminases are important industrial enzymes that not only play key roles in metabolic regulation and energy balance within organisms but also serve as a vital raw material in food, pharmaceutical, and industrial production. They are widely distributed in a variety of microorganisms and animals, and are widely used in various industries, including food. Based on their microbial origin, glutaminases can be broadly divided into bacterial and fungal sources. Bacteria primarily include Bacillus, Escherichia, and Pseudomonas, while fungi primarily include Penicillium, Trichoderma, and Aspergillus. These microorganisms produce glutaminases, which participate in nitrogen metabolism within bacterial cells and are crucial for microbial growth and survival. They are also utilized in certain fermentation industries.

[0003] Glutaminase is an amidase that catalyzes the hydrolysis of glutamine to produce L-glutamate and ammonia. During the soy sauce fermentation process, glutaminase catalyzes the conversion of glutamine to glutamate, thereby enhancing the umami flavor of soy sauce. Glutamate is one of the main umami-contributing substances in soy sauce and has a significant impact on the umami flavor. However, the activity of glutaminase produced by the Aspergillus oryzae Shanghai Niang 3.042 strain, currently the main soy sauce fermentation strain on the market, is significantly inhibited at temperatures above 50°C and salt concentrations above 15%, which affects the flavor of the soy sauce to a certain extent. Therefore, there is a need to develop salt-tolerant strains that can produce high glutaminase to increase the glutamate content in soy sauce. Bacillus, as a major source of glutaminase in nature, is an important research and development direction for high-glutaminase-producing strains.

[0004] At present, Bacillus pumilus is mostly used for plant growth regulation and plant disease prevention and control. Its application in soy sauce fermentation is mostly to use Bacillus pumilus to increase the isoflavone content in soy sauce and shorten the soy sauce preparation time, or to use the aromatic hydrocarbon hydroxylase produced by Bacillus pumilus to reduce the bitterness of tangerine peel in sauces (patent CN1373206A, patent KR20170022516A, patent CN107455668B). There has been no report on its metabolization of glutamine to produce glutamate under higher salinity conditions, thereby increasing the content of glutamate, isovaleraldehyde, and amino nitrogen in soy sauce, thereby enhancing the umami flavor of soy sauce. Summary of the Invention

[0005] The present invention aims to overcome the defects and shortcomings of the aforementioned prior art by providing a strain of Bacillus pumilus T1-11 and its applications. The strain is salt-tolerant and produces glutaminase. The glutaminase produced by the strain can maintain its activity under high temperature and high salinity conditions, metabolizing glutamine to glutamate. Furthermore, application of Bacillus pumilus T1-11 in soy sauce fermentation can increase the content of glutamate, isovaleraldehyde, and amino nitrogen in soy sauce, thereby enhancing the flavor of fermented foods such as soy sauce.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a strain of Bacillus pumilus T1-11, which was deposited in the General Microbiology Center of the China Culture Collection Administration on January 10, 2025, with a deposit number of CGMCC NO.33025.

[0008] The present invention provides a preparation containing live bacteria of Bacillus pumilus T1-11.

[0009] The present invention provides a preparation, which contains a bacterial liquid obtained by activating and expanding the Bacillus pumilus T1-11. The nitrogen source of the activation and expansion culture medium is ammonium sulfate, the carbon source is glucose, the pH value is 4.5-6.0, and the culture medium further contains 0-1g / L of L-glutamine and 5-120g / L of sodium chloride.

[0010] Preferably, the formula of the activation and expansion culture medium is: ammonium sulfate 5-8g / L, glucose 3-7g / L, L-glutamine 0-1g / L, sodium chloride 5-120g / L, pH 4.5-6.0, and the rest is water.

[0011] More preferably, the formula of the activation and expansion culture medium is: ammonium sulfate 6.8g / L, glucose 5g / L, L-glutamine 1g / L, sodium chloride 5g / L, pH 5.5, and the rest is water.

[0012] The present invention provides the use of the Bacillus pumilus T1-11 or the above preparation in producing glutaminase.

[0013] The present invention provides application of the Bacillus pumilus T1-11 or the preparation in fermented food.

[0014] As a preferred embodiment of the application of the present invention, the fermented food is soy sauce, paste, MSG, chicken essence, condiments and other fermented foods that need to increase the content of glutamic acid, isovaleraldehyde and amino nitrogen.

[0015] Preferably, the fermented food is soy sauce.

[0016] Another object of the present invention is to provide a method for improving the umami taste of fermented food, which comprises using the above-mentioned Bacillus pumilus T1-11 strain or the above-mentioned preparation for fermentation to improve the umami taste of the fermented food.

[0017] As a preferred embodiment of the method for enhancing the umami flavor of fermented food according to the present invention, the method comprises the following steps:

[0018] (1) Activation and expansion culture: Activate and culture Bacillus pumilus T1-11 in culture medium until the logarithmic phase to obtain seed solution;

[0019] (2) Preparation of bacterial solution: The seed solution obtained in step (1) was subjected to refrigerated centrifugation, the supernatant was discarded, and the precipitated cells were resuspended in physiological saline to prepare a bacterial solution;

[0020] (3) Inoculation and fermentation: adding the bacterial solution prepared in step (2) to the soy sauce at the end of fermentation and mixing and fermenting.

[0021] Preferably, in the above-mentioned method, in step (1), the culture temperature is 37° C., and the activation culture is a shaking activation culture for 12 to 24 hours, followed by expansion culture for 24 to 48 hours.

[0022] Preferably, for the above-described method, in step (1), the culture medium is a culture medium that can achieve the growth and expansion of Bacillus pumilus T1-11, including but not limited to MRS, NB, YPD and LB culture media, and other improved culture media for achieving better growth or metabolism of Bacillus pumilus T1-11.

[0023] Preferably, for the above-described method, in step (1), the nitrogen source of the culture medium is ammonium sulfate, the carbon source is glucose, the pH value is 4.5-6.0, and the culture medium further contains 0-1 g / L of L-glutamine and 5-120 g / L of sodium chloride.

[0024] Preferably, for the above-described method, in step (1), the formula of the culture medium is: ammonium sulfate 5-8 g / L, glucose 3-7 g / L, L-glutamine 0-1 g / L, sodium chloride 5-120 g / L, pH 4.5-6.0, and the rest is water.

[0025] Preferably, the culture medium has a formula of: 6.8 g / L ammonium sulfate, 5 g / L glucose, 1 g / L L-glutamine, 5 g / L sodium chloride, a pH of 5.5, and the rest is water.

[0026] Preferably, for the above method, in step (2), the refrigerated centrifugation is carried out at 4°C and 5000 r / min for 10 to 20 minutes. Preferably, the refrigerated centrifugation is carried out at 4°C and 5000 r / min for 10 minutes.

[0027] Preferably, in the above method, in step (2), the concentration of the bacterial solution is 1 to 7 lg CFU / mL. Preferably, the concentration of the bacterial solution is 5 lg CFU / mL, 6 lg CFU / mL or 7 lg CFU / mL.

[0028] Preferably, in the above-mentioned method, in step (3), the bacterial liquid prepared in step (2) is added to the raw soy sauce or mash at the end of fermentation for mixed fermentation.

[0029] Preferably, for the above method, in step (3), the mixed fermentation time is 2 to 30 days, and the fermentation temperature is 30° C. to 40° C. Preferably, the mixed fermentation time is 2 to 14 days, and the fermentation temperature is 30° C.

[0030] The present invention provides a fermented food prepared by the above preparation method.

[0031] The present invention has the following beneficial effects:

[0032] The present invention provides a strain of Bacillus pumilus T1-11 and its application. The Bacillus pumilus T1-11 of the present invention is isolated from soybean mash and has the characteristics of salt tolerance and glutaminase production, so that it can produce glutaminase under conditions of higher salinity; the glutaminase produced by the Bacillus pumilus T1-11 of the present invention can maintain enzyme activity under conditions of higher temperature and higher salinity, metabolize glutamine to produce glutamate; further, the Bacillus pumilus T1-11 is applied to soy sauce fermentation, which can increase the content of glutamate, isovaleraldehyde, and amino nitrogen in soy sauce, and enhance the umami flavor of fermented foods such as soy sauce. The Bacillus pumilus T1-11 of the present invention has important industrial utilization value and broad application prospects in producing glutaminase and enhancing the umami flavor of fermented foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the evolutionary tree of Bacillus pumilus T1-11.

[0034] Figure 2 This is the Gram staining result of Bacillus pumilus T1-11.

[0035] Figure 3 This is the growth curve of Bacillus pumilus T1-11 after inoculation into LB medium without additional NaCl.

[0036] Figure 4 This is the growth curve of Bacillus pumilus T1-11 after inoculation into LB medium supplemented with 6% NaCl.

[0037] Figure 5 is the relative enzyme activity of Bacillus pumilus T1-11 glutaminase under different temperature conditions.

[0038] Figure 6 Thermostability analysis of glutaminase from Bacillus pumilus T1-11.

[0039] Figure 7 Figure 3 shows the changes in amino nitrogen content within 2 to 14 days after Bacillus pumilus T1-11 was inoculated into raw soy sauce for fermentation. ES-5: strain T1-11 was inoculated into raw soy sauce for fermentation at 5 lg CFU / mL; ES-6: strain T1-11 was inoculated into raw soy sauce for fermentation at 6 lg CFU / mL; ES-7: strain T1-11 was inoculated into raw soy sauce for fermentation at 7 lg CFU / mL.

[0040] Figure 8 The changes in amino nitrogen content within 2 to 14 days after inoculation of Bacillus pumilus T1-11 into soybean mash fermentation (MS-5: strain T1-11 inoculated into soybean mash fermentation at 5 lg CFU / mL; MS-6: strain T1-11 inoculated into soybean mash fermentation at 6 lg CFU / mL; MS-7: strain T1-11 inoculated into soybean mash fermentation at 7 lg CFU / mL) DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0043] The culture medium formula used in the embodiment of the present invention is as follows:

[0044] MRS solid medium: 10 g peptone, 8 g beef extract powder, 4 g yeast extract powder, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 14 g agar, 1 g Tween 80. Adjust the pH to 6.3-6.7 and add distilled water to 1 L.

[0045] NB solid medium: 3 g beef extract, 5 g peptone, 15 g agar, adjust the pH to 6.8-7.0, and add distilled water to 1 L.

[0046] YPD solid medium: 10 g yeast extract, 20 g peptone, 20 g glucose, 15 g agar, and distilled water to 1 L.

[0047] LB solid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, adjust the pH to 7.0-7.2, and add distilled water to 1 L.

[0048] Primary screening culture medium: 0.5 g / L KCl, 0.5 g / L MgSO4, 1 g / L KH2PO4, 0.1 g / L ZnSO4, 5 g / L L-glutamine, 60 g / L NaCl, 0.04 g / L bromocresol purple, 0.05 g / L pyridoxal phosphate, dissolved in distilled water and adjusted to pH 5.5.

[0049] NB liquid medium: 3 g beef extract, 5 g peptone, adjust the pH to 6.8-7.0, and add distilled water to 1 L.

[0050] LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, adjust the pH to 7.0-7.2, and add distilled water to 1 L.

[0051] Example 1 Isolation and Identification of Bacillus pumilus T1-11

[0052] Bacillus pumilus T1-11 was isolated from soybean paste and deposited in the General Microbiology Center of China Culture Collection Administration on January 10, 2025, with the deposit number CGMCCNO.33025.

[0053] The specific separation and identification process is as follows:

[0054] 1. Isolation of strain T1-11

[0055] The sauce mash collected in the present invention comes from Guangdong Chubang Food Co., Ltd., adopts the five-point sampling method, is fully mixed and placed in a sterile glycerol EP tube and stored in a -80°C refrigerator.

[0056] The collected mash was diluted with sterile saline (0.75%, w / v) at a mass ratio of 1:2. 5 , 10 6 , 10 7 After gradient dilution of cfu / mL, the cells were spread on MRS, NB, YPD and LB solid culture media respectively.

[0057] After spreading, incubate the LB and NB plates at 37°C, the YPD plates at 30°C, and the MRS plates at 28°C until distinct single colonies appear. Pick a single colony from the MRS solid medium and repeatedly streak it on MRS solid medium for purification, pick a single colony from the NB solid medium and repeatedly streak it on NB solid medium for purification, pick a single colony from the YPD solid medium and repeatedly streak it on YPD solid medium for purification, pick a single colony from the LB solid medium and repeatedly streak it on LB solid medium for purification, and finally obtain strains that have been repeatedly streaked and purified on MRS, NB, YPD, and LB solid media, respectively.

[0058] The above strains were inoculated into the primary screening culture medium with L-glutamine as the sole carbon and nitrogen source and bromocresol purple as the indicator. After culturing at 37°C for 5 days, the color change was observed and the OD was measured. 600 A total of 10 strains were obtained with color changes to purple-red and OD 600 500 μL of bacterial suspension of the above 10 strains were added to 9.5 ml NB liquid medium supplemented with 6% NaCl and 9.5 ml mash, respectively, and cultured at 30°C for 6 h.

[0059] After 6 hours of incubation, the cells were centrifuged at 5000 rpm and 4°C for 10 minutes. The pelleted cells were resuspended in 0.85% saline and ultrasonically disrupted. The supernatant was then centrifuged and the intracellular glutaminase activity of the 10 strains was measured using an enzyme-linked immunosorbent assay kit from Shanghai Yuanju Biotechnology Center. Finally, a strain with the highest intracellular glutaminase activity was identified, designated T1-11, and used for subsequent fermentation.

[0060] The preservation method of strain T1-11 is as follows: pick a single colony from the plate and place it in NB liquid medium. After culturing for 12 hours, add an equal volume of 60% glycerol and store it at -80°C.

[0061] 2. Identification of strain T1-11

[0062] The bacterial 16S rDNA universal primers 27F and 1492R (see Table 1 for details) were used as primers to PCR amplify the 16S rDNA nucleotide sequence of strain T1-11 using the PCR amplification system and program in Table 2, and the product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA nucleotide sequence of strain T1-11 was compared with the existing sequence in NCBI. The similarity between the sequence and the 16S rDNA sequence of Bacillus was 99%, and the bacterium was determined to be Bacillus. The 16S rDNA nucleotide sequence of strain T1-11 is shown in SEQ ID NO.1. At the same time, a phylogenetic tree was constructed based on the 16S rDNA gene sequence of Bacillus obtained from NCBI (see Figure 1 ),from Figure 1 It was found that strain T1-11 was most closely related to Bacillus pumilus XD-PI, and strain T1-11 was identified as Bacillus pumilus and named Bacillus pumilus T1-11.

[0063] At the same time, Gram staining was used to identify strain T1-11. The Gram staining result of Bacillus pumilus T1-11 was positive (for details, see Figure 2 ).

[0064] Table 1 Primer pairs used for identification of strain T1-11

[0065]

[0066] Table 2 PCR amplification system

[0067]

[0068] PCR program: pre-denaturation at 94°C for 10 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min; final extension at 72°C for 5 min; and insulation at 4°C.

[0069] Example 2 Salt tolerance test of Bacillus pumilus T1-11

[0070] Bacillus pumilus T1-11 was inoculated into 10 mL of NB liquid medium and cultured on a shaker at 37°C and 160 rpm for 12 to 16 hours until the strain reached the logarithmic phase. The cells were centrifuged at 5000 rpm for 10 minutes, and the precipitated cells were washed with 0.75% saline, resuspended, and 1% of the bacterial suspension was inoculated into 100 mL of LB liquid medium without or with 6% NaCl, respectively. The cells were cultured at 37°C and 160 rpm. The LB liquid medium without NaCl was used as the control group. Samples were taken every 2 hours, and the OD value of the bacterial suspension was measured using a microplate reader. 600 The values ​​were recorded and plotted for Bacillus pumilus T1-11 without the addition of NaCl (see Figure 3 ) and an additional 6% NaCl (see Figure 4 ) environment.

[0071] Results: As Figure 3 and Figure 4 As shown in the results, NaC1 had a certain inhibitory effect on the early growth of Bacillus pumilus T1-11. As the culture time prolonged, when the culture time exceeded 10 h, the inhibitory effect of NaC1 on the growth of Bacillus pumilus T1-11 showed a decreasing trend. After the culture time reached 20 h, the growth curve of Bacillus pumilus T1-11 was similar to that of the control group, showing strong adaptability and salt tolerance to NaC1.

[0072] Example 3 Optimization of culture medium enzyme production conditions and enzyme activity determination of Bacillus pumilus T1-11

[0073] To determine the effects of the nitrogen source, carbon source, L-glutamine concentration, sodium chloride concentration, and pH in the culture medium on glutaminase production by Bacillus pumilus T1-11, the present invention optimized the culture medium conditions for enzyme production by Bacillus pumilus T1-11. The gene sequence of the glutaminase from Bacillus pumilus T1-11 is shown in SEQ ID NO. 4.

[0074] The experiment was divided into fifteen groups. The culture medium formulas for experimental groups 1 to 15 are detailed in Table 3. The experiment used group 11 (detailed in Table 3) as the basal medium (tryptone 10 g / L, sodium chloride 5 g / L, glucose 5 g / L, pyridoxal phosphate 0.05 g / L, yeast extract 2 g / L, glutamine 1 g / L, pH 5.5) to optimize the nitrogen source, carbon source, L-glutamine concentration, sodium chloride concentration, and pH value of the culture medium.

[0075] The nitrogen sources in the fifteen experimental culture media were ammonium sulfate, urea, arginine, lysine, and tryptone; the carbon sources were glucose, soluble starch, and sucrose; the L-glutamine concentrations were 0g / L, 0.5g / L, and 1g / L (w / v), respectively; the sodium chloride concentrations were 5g / L, 60g / L, and 120g / L (w / v), respectively; and the pH values ​​were 4.5, 5.0, 5.5, and 6.0, respectively. To optimize the nitrogen and carbon sources in the culture media, the amounts of nitrogen sources (ammonium sulfate, urea, arginine, lysine, and tryptone) and carbon sources (glucose, soluble starch, and sucrose) added to each experimental group were varied to ensure the same molar ratios of nitrogen and carbon in the culture media.

[0076] Specifically, experimental groups 1 to 4 and group 11 were for the optimization of the nitrogen source conditions of the culture medium. Groups 1 to 4 and group 11 used ammonium sulfate, urea, arginine, lysine, and tryptone as nitrogen sources in the culture medium, respectively, and other components of the culture medium remained unchanged; experimental groups 5, 6, and group 11 were for the optimization of the carbon source conditions of the culture medium. In the culture medium with tryptone as the nitrogen source, sucrose, soluble starch, and glucose were used as carbon sources, respectively, and other components of the culture medium remained unchanged; experimental groups 7, 8, and group 11 were for the optimization of the L-glutamine concentration in the culture medium. In the culture medium with tryptone as the nitrogen source, L- The glutamine concentrations were 0 g / L, 0.5 g / L, and 1 g / L, respectively, and the other components of the culture medium remained unchanged; experimental groups 9, 10, and 11 were for optimizing the sodium chloride concentration in the culture medium. In the culture medium with tryptone as the nitrogen source, the sodium chloride concentrations were set to 60 g / L, 120 g / L, and 5 g / L, respectively, and the other components of the culture medium remained unchanged; experimental groups 12, 13, 14, and 15 were for optimizing the pH value in the culture medium. In the culture medium with ammonium sulfate as the nitrogen source, the pH values ​​of the culture medium were set to 4.5, 5.0, 5.5, and 6.0, respectively, and the other components of the culture medium remained unchanged.

[0077] Table 3 Culture medium formulas for experimental groups 1 to 15

[0078]

[0079]

[0080] The specific experimental steps are as follows: (1) Activation and expansion: 100 μL of bacterial solution was drawn from the glycerol tube containing Bacillus pumilus T1-11 and inoculated into 10 mL The activated bacterial liquid was cultured in NB liquid culture medium at 37°C and 160 rpm for 12 to 16 hours until the strain entered the logarithmic phase. The activated bacterial liquid was inoculated into 100 mL of NB liquid culture medium at a ratio of 1% for expansion culture. The culture was cultured at 37°C and 160 rpm for 24 hours to obtain the culture liquid. (2) Enzyme production: 500 μL of the culture liquid was transferred to 9.5 mL of the above-mentioned fifteen groups of culture medium (see Table 3) and cultured statically for 6 hours. (3) Enzyme activity determination: After the culture was completed, the bacterial liquid was centrifuged at 5000 r / min at 4°C for 10 minutes, the supernatant was discarded, the precipitated cells were resuspended in 0.85% physiological saline, and ultrasonically disrupted. After ultrasonic disruption, the supernatant was centrifuged and the intracellular glutaminase activity of each experimental group was determined using the enzyme-linked immunosorbent assay kit of Shanghai Yuanju Biotechnology Center. The glutaminase activity of experimental groups 1 to 15 is shown in Table 4.

[0081] Results: As shown in Table 4, in the optimization of nitrogen source conditions of the culture medium (experimental groups 1 to 4 and group 11), the enzyme activity of glutaminase in the culture medium with ammonium sulfate as the nitrogen source was the highest, which was 3850.33±347.91U / g; in the optimization of carbon source conditions of the culture medium (experimental groups 5, 6 and group 11), the enzyme activity of glutaminase in the culture medium with glucose as the carbon source was the highest, which was 1503.99±276.89U / g; in the optimization of L-glutamyl In the optimization of amine concentration (experimental groups 7, 8 and 11), when glutamine was 1 g / L, the enzyme activity of Bacillus pumilus T1-11 glutaminase was the highest, which was 1503.99±276.89 U / g; in the optimization of sodium chloride concentration in the culture medium (experimental groups 9, 10 and 11), when sodium chloride was 5 g / L, the enzyme activity of Bacillus pumilus T1-11 glutaminase was the highest, which was 1503.99±276.89 U / g. Sodium chloride has a certain inhibitory effect on the enzyme activity of Bacillus pumilus T1-11 glutaminase, but when the sodium chloride concentration is 120g / L, the enzyme activity of Bacillus pumilus T1-11 glutaminase is still 770.25±245.35U / g, showing strong salt tolerance; in the optimization of the pH value in the culture medium (experimental groups 12, 13, 14 and 15), when the pH value is 5.5, the enzyme activity of Bacillus pumilus T1-11 glutaminase is the highest, which is 5716.95±168.14U / g.

[0082] Conclusion: In summary, the optimal nitrogen source for glutaminase production by Bacillus pumilus T1-11 is ammonium sulfate, the optimal carbon source is glucose, the optimal L-glutamine concentration is 1 g / L, the optimal sodium chloride concentration is 5 g / L, and the optimal pH is 5.5. The optimal medium formula for glutaminase production by Bacillus pumilus T1-11 is: ammonium sulfate 6.8 g / L, glucose 5 g / L, L-glutamine 1 g / L, sodium chloride 5 g / L, pH 5.5, dissolved in distilled water.

[0083] At the same time, sodium chloride had a certain inhibitory effect on the activity of glutaminase of Bacillus pumilus T1-11, but when the concentration of sodium chloride was 120 g / L, the enzyme activity of glutaminase of Bacillus pumilus T1-11 was still 770.25±245.35 U / g, showing strong salt tolerance.

[0084] Table 4 Intracellular glutaminase activity of experimental groups 1 to 15

[0085]

[0086]

[0087] Example 4 Test of the High Temperature Resistance of Bacillus pumilus T1-11 Glutaminase

[0088] (1) Preparation of glutaminase

[0089] The optimal culture medium for glutaminase production by Bacillus pumilus T1-11 was used instead of the NB liquid culture medium. Bacillus pumilus T1-11 was activated and expanded according to the steps of Example 3 to obtain a culture solution. The culture solution was transferred to the optimal culture medium for glutaminase production by Bacillus pumilus T1-11 at a ratio of 1:100. After culturing on a shaker at 37°C and 160 rpm for 12 hours, the culture solution was centrifuged at 5000 r / min at 4°C for 10 minutes, the supernatant was discarded, the precipitated cells were resuspended in 0.85% physiological saline, and ultrasonically disrupted. After ultrasonic disruption, the supernatant was centrifuged and crudely purified by dialysis to obtain a glutaminase enzyme solution.

[0090] (2) Test of high temperature resistance of glutaminase

[0091] Take 1 mL of a 50 mmol / L PBS buffer solution containing L-glutamine, place it in a 30°C water bath to preheat for 5 minutes, add 50 μL of glutaminase solution, and then react in a constant temperature water bath at 30°C, 40°C, 50°C, 60°C, and 70°C for 5 minutes, and in a 100°C water bath for 10 minutes to terminate the reaction. The enzyme activity of glutaminase at 30°C, 40°C, 50°C, 60°C, and 70°C was determined using the method of Example 3, and the relative enzyme activity of glutaminase at each temperature was calculated (see Figure 5).

[0092] (3) Study on the thermal stability of glutaminase

[0093] The purified glutaminase enzyme solution was placed at 30°C, 40°C, 50°C, 60°C, and 70°C, respectively, and kept warm for 24 hours. 1 mL of a 50 mmol / L PBS buffer solution containing L-glutamine was added as substrate. After the reaction was terminated, the enzyme activity of glutaminase was determined using the method of Example 3, and the relative enzyme activity was calculated (see Figure 6 ).

[0094] Results: From Figure 5 It can be seen that the glutaminase activity of Bacillus pumilus T1-11 strain is the highest at 50℃, and then decreases with the increase of temperature, and loses its activity at 70℃. Figure 6 It can be seen that after being treated at 60°C for 24 hours, the glutaminase of the Bacillus pumilus T1-11 strain still maintained an activity of more than 60%.

[0095] Example 5 Application of Bacillus pumilus T1-11 in raw soy sauce

[0096] (1) Activation and expansion culture: Bacillus pumilus T1-11 was inoculated into the optimal culture medium for glutaminase production of Bacillus pumilus T1-11, and activated and cultured at 37°C, 160 rpm, on a shaking platform for 12 to 24 hours until the strain reached the logarithmic phase to obtain seed solution;

[0097] (2) Preparation of bacterial suspension: The seed solution was cultured in a shaker at 37°C and 160 rpm for 24–48 h. The supernatant was discarded after centrifugation at 4°C and 5000 rpm for 10 min. The precipitated cells were resuspended in 0.85% saline to prepare high-density bacterial suspensions of 5 lg CFU / mL, 6 lg CFU / mL, and 7 lg CFU / mL.

[0098] (3) Inoculation and fermentation: The prepared high-density bacterial solution was added to the raw soy sauce at the end of fermentation and mixed and fermented at 30°C for 14 days. The raw soy sauce without the addition of Bacillus pumilus T1-11 bacterial solution was used as the control group. Samples were taken 2 days, 7 days, and 14 days after the high-density bacterial solution of 5lg CFU / mL, 6lg CFU / mL, and 7lg CFU / mL was inoculated into the raw soy sauce. The content of amino nitrogen and isovaleraldehyde in the experimental group was measured; the content of glutamic acid in the experimental group was measured 2 days, 7 days, and 14 days after the high-density bacterial solution of 5lg CFU / mL and 7lg CFU / mL was inoculated into the raw soy sauce. The content of amino nitrogen, glutamic acid, and isovaleraldehyde in the control group was measured 0 days after the experimental group was inoculated into the raw soy sauce.

[0099] The glutamic acid content was determined according to the method in GB 5009.124-2016; the amino nitrogen content was determined according to the method in GB 5009.235-2016; and the isovaleraldehyde content was determined according to the method in the article “Study on the Change Pattern and Formation Mechanism of Key Aroma Substances in High-Salt Dilute Soy Sauce”.

[0100] Results: (1) Amino nitrogen content: Figure 7 As shown, the amino nitrogen content in the raw soy sauce from the experimental group fermented with Bacillus pumilus T1-11 was significantly higher than that in the control group. The amino nitrogen content in the raw soy sauce from the experimental group increased with the number of days of culture, and the higher the bacterial concentration, the higher the content. On day 14 of fermentation, the amino nitrogen content reached a maximum of 1.33±0.01g / 100mL, produced at a bacterial concentration of 71g CFU / mL. In the raw soy sauce from the control group without Bacillus pumilus, the amino nitrogen content was only 0.99±0.01g / 100mL.

[0101] (2) Glutamic acid content: As shown in Table 5, the glutamate content in the raw soy sauce of the experimental group increased compared with that of the control group within 2 days, 7 days, and 14 days after the high-density bacterial liquid of 5 lg CFU / mL and 7 lg CFU / mL was inoculated into the raw soy sauce. Among them, the glutamate content was the highest at 44.72±12.85mmol / L when the bacterial liquid of 5 lg CFU / mL was added to the raw soy sauce on the 14th day of cultivation, while the glutamate content in the control group without adding Bacillus pumilus was only 29.85±4.34mmol / L.

[0102] Table 5 Glutamic acid content of raw soy sauce fermented with Bacillus pumilus T1-11 for 14 days

[0103]

[0104] (Note: 5T-2d means strain T1-11 was inoculated into raw soy sauce at 5 lg CFU / mL and fermented for 2 days, and so on)

[0105] (3) Isovaleraldehyde Content: As shown in Table 6, the isovaleraldehyde content in the raw soy sauce of the experimental group fermented with Bacillus pumilus T1-11 was significantly higher than that of the control group. When the raw soy sauce was inoculated with 7 lg CFU / mL for 7 days, the isovaleraldehyde content reached a maximum of 32.83±1.52 μg / L, while the isovaleraldehyde content in the control group without Bacillus pumilus was only 0.24±0.00 μg / L.

[0106] Table 6 Relative content of isovaleraldehyde in raw soy sauce fermented with Bacillus pumilus T1-11 for 14 days

[0107]

[0108] (Note: 5T-2d means strain T1-11 was inoculated into raw soy sauce at 5 lg CFU / mL and fermented for 2 days, and so on)

[0109] Conclusion: In summary, the contents of glutamate, amino nitrogen and isovaleraldehyde in raw soy sauce fermented with Bacillus pumilus T1-11 were significantly increased compared with those in raw soy sauce fermented without Bacillus pumilus T1-11.

[0110] Example 6 Application of Bacillus pumilus T1-11 in mash

[0111] Bacillus pumilus T1-11 was activated and expanded, and a bacterial solution was prepared according to the steps in Example 5 to obtain high-density bacterial solutions of 51 CFU / mL, 61 CFU / mL, and 71 CFU / mL. The prepared high-density bacterial solutions were added to the mash at the end of fermentation and mixed and fermented at 30°C for 14 days. The mash without the addition of Bacillus pumilus T1-11 bacterial solution served as the control group. Samples were taken from the experimental groups 2, 7, and 14 days after the addition of the 51 CFU / mL, 61 CFU / mL, and 71 CFU / mL high-density bacterial solutions to the mash to determine the content of amino nitrogen and isovaleraldehyde. Samples were taken from the experimental groups 2 and 14 days after the addition of the 51 CFU / mL and 61 CFU / mL high-density bacterial solutions to the mash to determine the content of glutamate. Samples were taken from the experimental groups 0 day after the addition of the experimental groups to the mash to determine the content of amino nitrogen, glutamate, and isovaleraldehyde in the control group.

[0112] Results: (1) Amino nitrogen content: Figure 8 The amino nitrogen content in the mash of the experimental group fermented with Bacillus pumilus T1-11 was significantly higher than that in the control group. The amino nitrogen content in the mash of the experimental group increased with the number of days of culture, and the higher the bacterial concentration, the higher the content. On day 14 of fermentation, the amino nitrogen content reached a maximum of 1.01±0.01g / 100mL at a bacterial concentration of 71g CFU / mL. In the mash of the control group without Bacillus pumilus, the amino nitrogen content was only 0.83±0.01g / 100mL.

[0113] (2) Glutamic acid content: As shown in Table 7, the glutamate content in the sauce mash of the experimental group increased compared with that of the control group within 2 days and 14 days after the high-density bacterial solution of 5lg CFU / mL and 6lg CFU / mL was inoculated into the sauce mash. Among them, the glutamate content was the highest at 41.59±6.64mmol / L when the bacterial solution with a concentration of 6lg CFU / mL was added to the sauce mash and cultured for 14 days, while the glutamate content in the control group without adding Bacillus pumilus was only 25.52±5.71mmol / L.

[0114] Table 7 Glutamic acid content of Bacillus pumilus T1-11 inoculated into mash fermentation within 14 days

[0115]

[0116] (Note: 5J-2d means strain T1-11 was inoculated into mash at 5 lg CFU / mL for 2 days, and so on)

[0117] (3) Isovaleraldehyde content: As shown in Table 8, the isovaleraldehyde content in the fermented mash of the experimental group with Bacillus pumilus T1-11 was significantly higher than that in the control group. When 6 lg CFU / mL was inoculated into the mash for 2 days, the isovaleraldehyde content reached a maximum of 141.97±102.59 μg / L, while the isovaleraldehyde content in the control group without Bacillus pumilus was only 1.87±0.25 μg / L.

[0118] Table 8 Isovaleraldehyde content after inoculation of Bacillus pumilus T1-11 into mash fermentation

[0119]

[0120] (Note: 5J-2d means the strain was inoculated into mash at 5lg CFU / mL for 2 days, and so on)

[0121] Conclusion: In summary, the contents of glutamate, amino nitrogen and isovaleraldehyde in the mash fermented with Bacillus pumilus T1-11 were higher than those in the mash fermented without Bacillus pumilus T1-11.

[0122] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Bacillus pumilus T1-11, characterized in that: It was deposited in the General Microbiology Center of China Culture Collection Administration on January 10, 2025, with the deposit number CGMCC NO.33025.

2. A preparation, characterized in that Containing the live bacteria of Bacillus pumilus T1-11 according to claim 1.

3. A preparation, characterized in that The invention relates to a bacterial liquid obtained by activating and expanding the Bacillus pumilus T1-11 according to claim 1; the nitrogen source of the culture medium used for the activation and expansion is ammonium sulfate, the carbon source is glucose, the pH value is 4.5-6.0, and the culture medium also contains 0-1g / L L-glutamine and 5-120g / L sodium chloride.

4. Use of the Bacillus pumilus T1-11 according to claim 1 or the preparation according to claim 2 or 3 in the production of glutaminase.

5. Use of the Bacillus pumilus T1-11 according to claim 1 or the preparation according to claim 2 or 3 in the production of fermented foods.

6. The use according to claim 5, wherein the fermented food is soy sauce.

7. A method for improving the flavor of fermented food, characterized in that: The fermentation is carried out using the Bacillus pumilus T1-11 strain according to claim 1 or the preparation according to claim 2 or 3.

8. A method for enhancing the flavor of fermented food or producing fermented food, characterized in that: The method comprises the following steps: (1) Activation and expansion culture: activating and culturing the Bacillus pumilus T1-11 according to claim 1 in a culture medium until the logarithmic phase to obtain a seed solution; (2) Preparation of bacterial solution: The seed solution obtained in step (1) was subjected to refrigerated centrifugation, the supernatant was discarded, and the precipitated cells were resuspended in physiological saline to prepare a bacterial solution; (3) Inoculation and fermentation: adding the bacterial solution prepared in step (2) to the soy sauce at the end of fermentation and mixing and fermenting.

9. The method according to claim 8, characterized in that In step (1), the nitrogen source of the culture medium is ammonium sulfate, the carbon source is glucose, the pH value is 4.5-6.0, and the culture medium also contains 0-1 g / L of L-glutamine and 5-120 g / L of sodium chloride.

10. The fermented food prepared by the method according to any one of claims 7 to 9.

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