Bacterial strain Bacillus subtilis subsp.subtilis CEGF012 and culture method thereof

Abstract

The invention relates to a bacillus subtilis subsp.subtilis CEGF012 and a culture method thereof, the preservation number of the bacillus subtilis subsp.subtilis CEGF012 is CCTCC (China Center For Type Culture Collection) NO: M20241702, the preservation date is July 31, 2024, and the preservation number of the bacillus subtilis subsp.subtilis CEGF012 is CCTCC NO: M20241702, and the preservation number of the bacillus subtilis subsp.subtilis CEGF012 is CCTCC NO: M20241702, and the preservation number of the bacillus subtilis subsp.subtilis CEGF012 is CCTCC NO: M20241702. The culture method comprises the following steps: inoculating a seed solution of the Bacillus subtilis subsp.subtilis CEGF012 into a solid culture medium, and culturing the seed solution of the Bacillus subtilis subsp.subtilis CEGF012 in the solid culture medium; a nitrogen source in the solid culture medium comprises plant protein with the protein content being 15% or above. The activity of the screened bacillus subtilis subsp.subtilis CEGF012 strain is as high as 9.16 * 10 < 9 > CFU / g, the strain can grow well in a culture medium taking plant degradation-resistant protein with the protein content of 15%-30% as a nitrogen source, and the mass ratio of a carbon source to the nitrogen source is (50-83): (17-50), so that the cost of culturing by taking peptone as the nitrogen source is reduced, and the industrial large-scale production of the strain is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and more specifically to a strain Bacillus subtilissubsp.subtilis CEGF012 and a culture method thereof. Background Art

[0002] Bacillus subtilis is a species of Bacillus, which is widely used in the fields of food, feed additives, seed protection, biological control, biological preservation, animal breeding, crop pest control and biofertilizer production. Currently, the production methods of Bacillus subtilis include solid-state fermentation and liquid fermentation.

[0003] However, the commonly used typical culture medium usually uses starch, glucose, etc. as carbon source, and beef extract, peptone, etc. as nitrogen source, which is difficult to promote and apply in industry due to its high cost. In order to reduce the cost, industrial large-scale production generally uses industrial culture medium for cultivation, which easily leads to the strains screened out by the typical culture medium being difficult to achieve the expected effect on the industrial culture medium. Therefore, screening Bacillus subtilis that can grow on industrial culture medium and has high activity is conducive to promoting its application in industry.

[0004] In addition, Bacillus subtilis strains are diverse, and different Bacillus subtilis strains have different suitable culture media and fermentation conditions. For example, conventional fermentation technology cannot achieve the expected effect of high-density fermentation for fermentation production of Bacillus subtilis YXSY-01. Therefore, it is necessary to study the appropriate culture methods for different Bacillus subtilis to promote their industrial large-scale production. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a strain Bacillus subtilis subsp. subtilis CEGF012 and a cultivation method thereof, the purpose of which is to separate and purify microorganisms from the roots of Populus euphratica in the Taklimakan Desert in Xinjiang, screen out a highly active strain, which is identified as a new strain of Bacillus subtilis, named Bacillus subtilis subsp. subtilis CEGF012, with a preservation number of CCTCC NO: M 20241702. The strain can be cultured with low-cost plant-based refractory protein as a nitrogen source, thereby solving the technical problem that it is difficult to achieve the expected effect when the strains screened by typical culture medium are expanded in industrial culture medium.

[0006] To achieve the above objectives, according to one aspect of the present invention, a highly active strain Bacillus subtilis subsp. Subtilis CEGF012 is provided, whose preservation number is CCTCC NO: M20241702 and the preservation date is July 31, 2024. The strain can be cultured using industrial culture medium and has high activity.

[0007] According to another aspect of the present invention, a bacterial agent is provided, comprising the Bacillus subtilis subsp. Subtilis CEGF012 as claimed in claim 1.

[0008] According to another aspect of the present invention, a method for culturing Bacillus subtilis subsp.subtilisCEGF012 is provided, comprising the steps of inoculating seed solution of Bacillus subtilissubsp.subtilis CEGF012 with a preservation number of CCTCC NO:M 20241702 into a solid culture medium for culturing; the nitrogen source in the solid culture medium comprises plant protein with a protein content of more than 15%.

[0009] Preferably, in the culture method, the nitrogen source in the solid culture medium includes a plant-derived non-degradable protein having a protein content of 15% to 30%, wherein the mass ratio of the carbon source to the nitrogen source is 50 to 83:17 to 50.

[0010] Preferably, in the culture method, the mass ratio of the carbon source to the nitrogen source in the solid culture medium is 50-75:25-50.

[0011] Preferably, in the culture method, the Bacillus subtilis subsp. subtilis CEGF012 seed solution is prepared according to the following method:

[0012] Bacillus subtilis subsp. subtilis CEGF012 was inoculated into LB liquid culture medium at a rate of 2%, and after recovery and propagation for 3 generations, the seed solution of Bacillus subtilis subsp. subtilis CEGF012 was obtained.

[0013] Preferably, the culture method determines the inoculation amount, culture temperature and culture time according to the preset biomass of Bacillus subtilis subsp. subtilis CEGF012 according to the following prediction model:

[0014] Y=675.64+61.79X1-154.94X2-2.89X3-39.36X1X2-6.03X1X3+19.72X2X3-

[0015] 227.78X1 2 -133.14X2 2 -399.64X3 2

[0016] Where: Y is the preset biomass, based on the number of colonies 10 7 CFU / g; X1 is the inoculum size, in %; X2 is the culture temperature, in °C; X3 is the culture time, in h.

[0017] Preferably, in the culture method, the culture conditions are as follows: the inoculation amount of Bacillus subtilissubsp.subtilis CEGF012 seed solution is 4% to 10%, the culture temperature is 35 to 40° C., and the culture time is 24 to 36 hours.

[0018] Preferably, in the culture method, the culture conditions are as follows: the inoculation amount of Bacillus subtilissubsp.subtilis CEGF012 seed solution is 6% to 7%, the culture temperature is 36 to 37° C., and the culture time is 28 to 29 hours.

[0019] Preferably, in the culture method, the mass ratio of the carbon source to the nitrogen source in the culture medium is 1:1.

[0020] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0021] The strain Bacillus subtilis subsp.subtilis CEGF012 provided by the present invention is separated and purified from the roots of Populus euphratica forest in the Taklimakan Desert in Xinjiang, and screened with a specific screening medium prepared from organic matter with a cellulose content of more than 10% and a protein content of less than 10%, and a strain with high activity is screened out, and the activity is as high as 9.16×10 9 CFU / g. It was identified as a new strain of Bacillus subtilis, named Bacillus subtilis subsp.subtilisCEGF012. Compared with other Bacillus subtilis, this strain can grow well on the culture medium with plant-based refractory protein as the nitrogen source.

[0022] In addition, the cultivation method provided by the present invention can simulate the situation of large-scale cultivation using industrial culture medium in actual applications. Adding plant protein with a protein content of more than 15% as a nitrogen source in the culture medium can not only reduce the cost of large-scale cultivation, but also it is found that using plant-based difficult-to-degrade protein with a protein content of 15% to 30% as a nitrogen source can also significantly improve the activity of Bacillus subtilis subsp.subtilis CEGF012, thereby realizing efficient and low-cost cultivation of Bacillus subtilis subsp.subtilis CEGF012, which is conducive to promoting its large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the effect of different nitrogen sources on the activity of Bacillus subtilis subsp.subtilis CEGF012;

[0024] Figure 2 is the effect of the ratio of chickpea to carbon source on the activity of Bacillus subtilis subsp.subtilis CEGF012;

[0025] Figure 3 is the response surface analysis of inoculum size and incubation time;

[0026] Figure 4 is the response surface analysis of inoculum size and culture temperature;

[0027] Figure 5 It is the response surface analysis of culture temperature and culture time. DETAILED DESCRIPTION

[0028] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0029] The inventors isolated and purified strains from the roots of Populus euphratica in the Taklimakan Desert in Xinjiang, and screened out a strain with high activity and good activity consistency from the isolated strains. The bacterium was identified as a new strain of Bacillus subtilis subsp. subtilis CEGF012 by 16S rRNA, and was named Bacillus subtilis subsp. subtilis CEGF012. It was deposited in the China Center for Type Culture Collection at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province on July 31, 2024 (Wuhan University Collection Center), and its preservation number is CCTCC NO: M 20241702, and the preservation date is July 31, 2024.

[0030] Further, the effects of different nitrogen sources, inoculation amounts, culture temperatures and culture times on the activity of Bacillus subtilissubsp.subtilis CEGF012 were studied. The results showed that the strain can grow well on a culture medium with a plant-based non-degradable protein with a protein content of 15% to 30% as a nitrogen source, and can be used to replace peptone in a typical culture medium as a nitrogen source, such as chickpeas. Wherein preparing a solid culture medium according to a mass ratio of carbon source to nitrogen source of 50 to 83:17 to 50 for cultivation can significantly promote the growth of Bacillus subtilis subsp.subtilis CEGF012, preferably preparing a solid culture medium according to a mass ratio of carbon source to nitrogen source of 50 to 75:25 to 50 for cultivation.

[0031] Based on this, the present invention provides a highly active strain Bacillus subtilis subsp.SubtilisCEGF012, whose preservation number is CCTCC NO:M 20241702 and the preservation date is July 31, 2024. The strain can grow well on a culture medium with high-fiber and low-protein organic matter as a carbon source and plant protein with a protein content of more than 15% as a nitrogen source. The high-fiber and low-protein organic matter is an organic matter with a cellulose content of more than 10% and a protein content of less than 10%. In some embodiments, plant-based refractory protein can be selected as a nitrogen source, and the plant-based refractory protein is a plant protein with a fiber content of 5% to 20% and a protein content of more than 15%, such as chickpeas.

[0032] In addition, the present invention also provides a bacterial agent, which includes the Bacillus subtilissubsp.Subtilis CEGF012 described in the present invention.

[0033] In addition, the present invention also provides a method for efficiently culturing Bacillus subtilis subsp.subtilisCEGF012, which comprises the steps of inoculating the Bacillus subtilis subsp.subtilisCEGF012 seed solution of the present invention into a solid culture medium for culturing;

[0034] The nitrogen source in the solid culture medium includes plant protein with a protein content of more than 15%.

[0035] Preferably, the nitrogen source in the solid culture medium includes plant-derived non-degradable protein with a protein content of 15% to 30%, wherein the mass ratio of the carbon source to the nitrogen source is 50 to 83:17 to 50; more preferably, the mass ratio of the carbon source to the nitrogen source is 50 to 75:25 to 50.

[0036] In some embodiments, the Bacillus subtilis subsp. subtilis CEGF012 seed solution is prepared according to the following method:

[0037] The purified Bacillus subtilis subsp. subtilis CEGF012 was inoculated into LB liquid culture medium at a rate of 2% for activation, and cultured in a shaking incubator at 37° C. for 3 generations to obtain the seed liquid of Bacillus subtilis subsp. subtilis CEGF012.

[0038] Preparation of LB liquid medium (1 L): add 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl to 950 mL of ddH2O. Adjust the pH to 7.0 with 1 M NaOH, make up to 1 L, sterilize by autoclaving at 121°C for 20 min, and store at 4°C.

[0039] Furthermore, the appropriate inoculation amount, culture temperature and culture time can be calculated and determined according to the following prediction model based on the preset biomass of Bacillus subtilis subsp. subtilisCEGF012:

[0040] Y=675.64+61.79X1-154.94X2-2.89X3-39.36X1X2-6.03X1X3+19.72X2X3-

[0041] 227.78X1 2 -133.14X2 2 -399.64X3 2

[0042] Where: Y is the preset biomass, based on the number of colonies 10 7 CFU / g; X1 is the inoculum size, in %, X2 is the culture temperature, in °C, and X3 is the culture time, in h.

[0043] In some embodiments, the culture conditions are that the inoculation amount of Bacillus subtilis subsp.subtilisCEGF012 seed solution is 4% to 10%, the culture temperature is 35 to 40°C, and the culture time is 24 to 36 hours; preferably, the inoculation amount is 6% to 7%, the culture temperature is 36 to 37°C, and the culture time is 28 to 29 hours; more preferably, the mass ratio of the carbon source to the nitrogen source in the solid culture medium is 1:1, and the nitrogen source is selected from chickpea. In the present invention, the carbon source of the culture medium may also include organic matter with a cellulose content of more than 10% and a protein content of less than 10%.

[0044] According to the culture method, the inoculation amount of Bacillus subtilis subsp. subtilis CEGF012 seed solution is 6% to 7%, the culture temperature is 36 to 37°C, and the culture time is 28 to 29 hours. The colony number of Bacillus subtilissubsp. subtilis CEGF012 can reach 8×10 9 CFU / g and above.

[0045] The following are examples

[0046] Example 1 Isolation, purification and screening of strains

[0047] 1. Isolation and Purification of Strains

[0048] Sample source: Roots of Populus euphratica forest in the Taklimakan Desert, Xinjiang

[0049] Separation material: soil

[0050] Separation method: 500 g of soil sample from the root of Populus euphratica forest in the Taklimakan Desert, Xinjiang was taken, 10 g was taken by quartering method and put into 90 mL of phosphate buffered saline (PBS) containing glass beads, after shaking on a shaker for 10 min, 1 mL was taken from each sample and diluted to 10 -4 , 10 -5 , 10 -6 , 10 -7 , draw 0.1mL of 10 -4 , 10 -5 , 10 -6 , 10 -7 The dilution was spread on LB medium, with 3 replicates for each gradient, and inverted culture was performed at 30°C. The count was observed every 3 days, and the culture was stopped after 6 days. The colonies were classified and counted according to their morphology, and colonies of different morphologies in the sample were selected and numbered, and stored in a -80°C refrigerator after purification.

[0051] 2. Screening of highly active strains

[0052] Conventional microbial culture generally uses a typical culture medium (such as containing glucose, beef extract, peptone and other ingredients) for culture. In this embodiment, a typical culture medium is first used for strain culture, separation and purification, and then a specific screening culture medium is used for screening. The screening culture medium uses high-fiber and low-protein organic matter as a carbon source to simulate a culture medium containing high-fiber and low-protein microorganisms that is difficult to use, so as to screen strains with high activity in practical applications.

[0053] In this embodiment, the organic matter rich in dietary fiber can be selected from Cistanche deserticola, in which the cellulose content is 10% to 30%, the polysaccharide content is 5% to 20%, and the protein content is 2% to 8%; the screening of high-activity strains is as follows:

[0054] (1) Preparation of screening medium: Soak dry Cistanche deserticola until saturated, filter out excess water, weigh 50 g of wet Cistanche deserticola and crush it into soybean size, add water to the medium until the water content is 20%, and adjust the medium according to (V 水 :m 湿肉苁蓉 )*100%, high temperature sterilization at 121℃ for 20min.

[0055] (2) Cultivation: Each strain isolated in Example 1 was taken out from a -80°C refrigerator, inoculated with LB liquid culture medium for activation, and inoculated at an inoculum size of 2%. After culturing for three generations in a shaking incubator at 37°C and 200 rpm, fresh fermentation broth was inoculated into the screening culture medium at an inoculum size of 6% of the wet weight of the Cistanche screening culture medium, stirred evenly, and placed in a 37°C incubator for static culture for 24 h.

[0056] (3) Strain screening: The plate colony counting method was used to screen strains with high activity. Specifically, 0.1 g of the fermentation product after culturing for 24 h in step (2) was taken and placed in 9 mL of PBS containing steel beads. The product was shaken on a vortex for 1 min. 1 mL was taken and vortexed for 10 min. -4 , 10 -5 , 10 -6 , 10 -7 Gradient dilution. Take a certain amount of dilution liquid and apply it to the LB plate. After culturing in a 37℃ incubator for 1 day, count the number of colonies on the plate. According to the dilution degree and the amount of liquid applied to the plate, calculate the number of live bacteria in the original bacterial solution. The results are as follows:

[0057] Table 1 Comparison of the activities of different strains

[0058] NO. serial number Bacteria Bacteria CFU(10^7CFU / g) 1 NA2 Bacillus subtilis subsp.subtilis Bacillus subtilis 200 2 NA3 Bacillus subtilis subsp.subtilis Bacillus subtilis 40 3 NA4 Bacillus subtilis subsp.subtilis Bacillus subtilis 916 4 NA5 Bacillus tropicus Bacillus tropicalis 0.5 5 CE30CL1 Aerococcus urinaeequi Urinary Marneffei 205 6 CE30CL2 Aerococcus urinaeequi Urinary marneffei 0.01 7 CE30CL3 Aerococcus urinaeequi Urinary marneffei 115 8 CE30CL4 Bacillus tequilensis Bacillus tequila 124 9 CE31SL1 Bacillus subtilis Bacillus subtilis 40 10 CE31SL2 Bacillus tequilensis Bacillus tequila 50 11 CE31SL3 Bacillus subtilis Bacillus subtilis 10 12 CE31SL4 Bacillus subtilis Bacillus subtilis 70 13 CE31SL5 Bacillus vallismortis Bacillus death valley 80 14 CE31SL6 Bacillus tequilensis Bacillus tequila 100 15 CE31SL7 Bacillus tequilensis Bacillus tequila 110 16 CE31SL8 Bacillus tequilensis Bacillus tequila 200 17 CE31SL9 Lysinibacillus macroides Lysinibacillus elongatus 90 18 CE31SL10 Bacillus tequilensis Bacillus tequila 13 19 CE31SL11 Bacillus tequilensis Bacillus tequila 110 20 CE31SL12 Bacillus tequilensis Bacillus tequila 50 21 CE31SL13 Bacillus tequilensis Bacillus tequila 38 22 CE31SL15 Bacillus tequilensis Bacillus tequila 200 23 Natto Heyndrickxia coagulans Bacillus coagulans 100 24 CE32L1 Bacillus subtilis Bacillus subtilis 4 25 CE32L2 Bacillus subtilis Bacillus subtilis 78 26 CE32L3 Bacillus subtilis Bacillus subtilis 529 27 CE32L4 Bacillus subtilis Bacillus subtilis 705

[0059] The strain with the highest activity screened out was Bacillus subtilis subsp.subtilis corresponding to number NA4. The strain was identified by 16S rRNA as a new strain of Bacillus subtilis, named Bacillus subtilissubsp.subtilis CEGF012, and the purified and isolated new strain was sent to the China Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province for preservation (Wuhan University Collection Center), with a preservation number of CCTCC NO: M20241702 and a preservation date of July 31, 2024.

[0060] Example 2 Effect of fermentation conditions on the activity of Bacillus subtilis subsp. subtilis CEGF012

[0061] In a typical culture medium, beef extract, peptone and other ingredients are often used as nitrogen sources, which are costly in actual large-scale culture. In this embodiment, plant protein with lower cost is used as a nitrogen source to study the effects of culture medium and culture conditions on the activity of Bacillus subtilissubsp.subtilis CEGF012. The plant protein is selected from soybean meal, black beans, chickpeas, etc., wherein the soybean meal has a cellulose content of 15% to 20% and a protein content of 30% to 50%, the black beans have a dietary fiber content of 10 to 15% and a protein content of 30% to 36%, and the chickpeas have a cellulose content of 6% to 10% and a protein content of 19% to 22%.

[0062] (1) Effect of nitrogen source on the activity of Bacillus subtilis subsp. subtilis CEGF012

[0063] Preparation of culture medium: Soaked soybean meal, black beans, chickpeas, potassium nitrate, and ammonium nitrate were added to the culture medium at 1% of the carbon source weight, and water was added to the medium until the water content of the matrix was 25% ((v 水 :m 湿培养基 )×100%), and sterilize under high pressure at 121°C and 0.1 MPa for 20 min to obtain culture medium.

[0064] Inoculation culture: Inoculate the activated Bacillus subtilissubsp.subtilis CEGF012 seed solution at 6% of the wet weight of the culture medium, and culture it at 37℃ for 24h to study the effect of nitrogen source on the activity of the strain. The results are as follows Figure 1 Wherein the seed solution of Bacillus subtilis subsp.subtilis CEGF012 is prepared according to the following method:

[0065] The purified Bacillus subtilis subsp. subtilis CEGF012 stored at -80°C was taken out, inoculated into LB liquid medium at a 2% inoculation rate for activation, and the fermentation liquid after culturing for 3 generations at 37°C and 200 rpm in a shaking incubator was the seed liquid.

[0066] Preparation of LB liquid medium (1 L): add 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl to 950 mL of ddH2O. Adjust the pH to 7.0 with 1 M NaOH, make up to 1 L, sterilize by autoclaving at 121°C for 20 min, and store at 4°C.

[0067] Depend on Figure 1 The results show that there is no significant difference in the activity of the strain when soybean meal, black bean or potassium nitrate is used as the nitrogen source. Compared with soybean meal, black bean, potassium nitrate or ammonium nitrate as the nitrogen source, chickpea with a lower protein content can significantly increase the colony count of Bacillus subtilis subsp. subtilis CEGF012 and significantly promote its growth, so it is preferred as a nitrogen source for the cultivation of Bacillus subtilis subsp. subtilis CEGF012.

[0068] (2) Effect of nitrogen source and carbon source ratio on the activity of Bacillus subtilis subsp. subtilis CEGF012

[0069] Chickpea was used as nitrogen source, and the carbon source and nitrogen source were mixed according to the mass ratio of 100:0, 99:1, 90:10, 83:17, 75:25, 50:50, and 0:100, respectively, so that the percentage of carbon source in the total mass of the two was 100%, 99%, 90%, 83%, 75%, 50%, and 0%, respectively, and water was added to make the water content of the matrix 25% ((v 水 :m 湿培养基 )×100%), sterilized at 121℃, 0.1MPa for 20min, prepared culture medium, inoculated with 6% of the wet weight of culture medium, seed solution of Bacillus subtilis subsp.subtilis CEGF012 after 3 generations of recovery and propagation, and cultured at 37℃ for 24h to study the effect of nitrogen source and carbon source ratio on strain activity. The results are as follows Figure 2 shown.

[0070] Depend on Figure 2 It can be seen that compared with chickpea cultivation alone, the number of viable bacteria of Bacillus subtilis subsp. subtilis CEGF012 was significantly improved when the culture medium was prepared according to the mass ratio of carbon source to chickpea at 50-83:17-50, indicating that the two can synergistically promote the growth of Bacillus subtilis subsp. subtilisCEGF012 within this ratio range, and the culture medium is preferably prepared according to the mass ratio of carbon source to chickpea at 50-75:25-50 for cultivation.

[0071] Example 3 Optimization of the culture process of Bacillus subtilis subsp. subtilis CEGF012

[0072] Based on the single-factor preliminary experiment, a response surface optimization experiment was carried out. The three factors with greater influence, namely inoculation size (factor A), culture temperature (factor B), and culture time (factor C), were selected. The biomass of strain Bacillus subtilissubsp.subtilis CEGF012 was used as the evaluation index. The DesignExpert 8.0.6 software was used to carry out a 3-factor 3-level Box-Behnken response surface optimization experimental design, as shown in the table below.

[0073] Table 2 Box-Behnken test factor levels

[0074]

[0075] Based on the optimization results of the single-factor experiment, a 3-factor 3-level Box-Behnken response surface test plan was carried out according to the Design-Expert Version 8.0.6 experimental design. The results are shown in the table below.

[0076] Table 3 Box-Behnken test plan and results

[0077]

[0078]

[0079] There are 17 test points in the table above, among which the central point (6% inoculation, 37°C fermentation for 36h) was repeated 5 times to detect the test error. Then the standard polynomial regression method was used for analysis and fitting, and the response values ​​obtained from the above 17 test points were analyzed by response surface design. The results are as follows Figure 3 , Figure 4 and Figure 5 The Box-Behnken test variance analysis table and the quadratic polynomial of the relationship between the response value and the independent variable were obtained, and the results are as follows.

[0080] Table 4 Analysis of variance in Box-Behnken test

[0081]

[0082]

[0083] Note: The difference is extremely significant (P<0.01); the difference is significant (P<0.05).

[0084] Get a quadratic polynomial that relates the response to the independent variable:

[0085] Y=675.64+61.79X1-154.94X2-2.89X3-39.36X1X2-6.03X1X3+19.72X2X3-

[0086] 227.78X1 2 -133.14X2 2 -399.64X3 2

[0087] Where: Y is the predicted response value, i.e. biomass, and the number of colonies is 10 7 CFU / g; X1 is the inoculation amount (%), X2 is the culture temperature (°C), and X3 is the culture time (h).

[0088] From the above table, we can see that the model R 2 =0.9147>0.9, indicating that the model has a good fit, the linear relationship between the independent variable and the response value reaches a significant level, and the optimal fermentation parameters of Bacillus subtilis subsp. subtilis CEGF012 can be determined using the regression equation.

[0089] according to Figure 3 , Figure 4 and Figure 5 The response surface and contour lines show that:

[0090] When the culture temperature remains constant, the biomass of Bacillus subtilis subsp. subtilis CEGF012 will increase first and then decrease according to the increase of inoculation amount and time;

[0091] When the culture time remained unchanged, the biomass of Bacillus subtilis subsp. subtilis CEGF012 showed a trend of increasing and then decreasing with the increase of temperature and inoculum size;

[0092] When the inoculum size remained unchanged, the biomass of Bacillus subtilis subsp. subtilis CEGF012 increased first and then decreased with the increase of temperature and time.

[0093] The first-order partial derivatives of the variables (X1, X2, X3) were set to 0, and the derivatives of the fitted three-variable linear equations were taken. The optimal point predicted by the model was solved as follows: inoculation amount 6.76%, culture time 28.66h, and culture temperature 36.86℃. Substituting into the regression equation, the theoretical biomass of Bacillus subtilis subsp. subtilis CEGF012 was obtained as Y=728.859×10 7 CFU / g.

[0094] Example 4 Model Verification

[0095] The optimal fermentation conditions (inoculation amount 6.76%, culture time 28.66h, culture temperature 36.86°C) obtained by the prediction model in Example 3 were used for cultivation. After the fermentation, the biomass of Bacillus subtilis subsp. subtilisCEGF012 was measured to verify the reliability of the model prediction. The results are shown in the following table:

[0096]

[0097] It can be seen from the above table that when the optimal fermentation conditions determined by the model are used for cultivation, the biomass of Bacillus subtilissubsp.subtilis CEGF012 is consistent with the model prediction value by 91.7%, indicating that the prediction results of the model are accurate and reliable, and can be used to determine the appropriate inoculation size, culture temperature and culture time according to the preset biomass, or to predict the biomass of Bacillus subtilissubsp.subtilis CEGF012 after the cultivation is completed according to the preset inoculation size, culture temperature and culture time.

[0098] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A strain Bacillus subtilis subsp.subtilis CEGF012, characterized in that: Its deposit number is CCTCC NO:M 20241702, and the deposit date is July 31, 2024.

2. A bacterial agent, characterized in that The method comprises the Bacillus subtilissubsp. Subtilis CEGF012 as claimed in claim 1.

3. A method for culturing Bacillus subtilis subsp. subtilis CEGF012, characterized in that: The method comprises the steps of inoculating the seed solution of Bacillus subtilis subsp. subtilis CEGF012 according to claim 1 into a solid culture medium for culturing; The nitrogen source in the solid culture medium includes plant protein with a protein content of more than 15%.

4. The culture method according to claim 3, characterized in that The nitrogen source in the solid culture medium comprises plant-derived difficult-to-degrade protein with a protein content of 15% to 30%, wherein the mass ratio of the carbon source to the nitrogen source is 50 to 83:17 to 50.

5. The culture method according to claim 4, characterized in that The mass ratio of the carbon source to the nitrogen source in the solid culture medium is 50-75:25-50.

6. The culture method according to any one of claims 3 to 5, characterized in that: The Bacillus subtilissubsp.subtilis CEGF012 seed solution is prepared according to the following method: The purified Bacillus subtilis subsp. subtilis CEGF012 was inoculated into LB liquid culture medium at a rate of 2%, and revived and propagated for 3 generations to obtain the seed solution of Bacillus subtilis subsp. subtilis CEGF012.

7. The culture method according to claim 6, characterized in that Based on the preset biomass of Bacillus subtilissubsp.subtilis CEGF012, the inoculation amount, culture temperature and culture time were determined according to the following prediction model: Y=675.64+61.79X1-154.94X2-2.89X3-39.36X1X2-6.03X1X3+19.72X2X3- 227.78X1 2 -133.14X2 2 -399.64X3 2 Where: Y is the preset biomass, based on the number of colonies 10 7 CFU / g; X1 is the inoculum size, in %; X2 is the culture temperature, in °C; X3 is the culture time, in h.

8. The culture method according to claim 7, characterized in that The culture conditions are as follows: the inoculation amount of Bacillus subtilissubsp.subtilis CEGF012 seed solution is 4% to 10%, the culture temperature is 35 to 40° C., and the culture time is 24 to 36 hours.

9. The culture method according to claim 8, characterized in that The culture conditions are as follows: the inoculation amount of Bacillus subtilissubsp.subtilis CEGF012 seed solution is 6% to 7%, the culture temperature is 36 to 37° C., and the culture time is 28 to 29 hours.

10. The culture method according to claim 9, characterized in that: The mass ratio of the carbon source to the nitrogen source in the culture medium is 1:1.

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