Bacillus coagulans with high antibacterial activity as well as fermentation process optimization method and application thereof

Abstract

The invention discloses bacillus coagulans JBHBIO-BNJ13 with high antibacterial activity as well as a fermentation process optimization method and application of the bacillus coagulans JBHBIO-BNJ13, and relates to the technical field of microorganisms. The bacterial strain is obtained through NTG mutagenesis, UV mutagenesis and ARTP mutagenesis and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.32216. The concentration of lactic acid produced by the strain can reach 84.31 g / L, the inhibitory activity on Listeria monocytogenes is relatively strong, and the diameter of an inhibition zone reaches 13.68 mm. After the strain is cultured by using the optimization method disclosed by the invention, the spore concentration of the bacillus coagulans can reach 3.92 * 10 < 9 > CFU / mL after the strain is fermented for 42 hours in a 10L fermentation tank. The method is of great significance in lactic acid production, pathogenic bacteria inhibition, organism immunity improvement, organism digestion function enhancement, intestinal health promotion and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bacillus coagulans with high antibacterial activity, an optimization method for its fermentation process, and applications thereof. Background Art

[0002] Bacillus coagulans is an aerobic or facultative anaerobic spore-forming lactic acid bacterium. It has good heat tolerance, and its optimal growth temperature is 35 - 50 °C. On the one hand, Bacillus coagulans has the characteristic of lactic acid production of lactic acid bacteria, which can inhibit pathogenic bacteria, improve the body's immunity, enhance the body's digestive function, and promote intestinal health; on the other hand, it also has a rich enzyme system of Bacillus and biological characteristics such as strong stress resistance and easy storage.

[0003] Lactic acid is the main secondary metabolite of Bacillus coagulans, which plays an important role in the in vitro antibacterial activity of Bacillus coagulans and the in vivo regulation of intestinal flora. In addition, lactic acid can be used as a substrate for other intestinal microorganisms in the body, promoting the production of short-chain fatty acids (such as acetic acid, butyric acid, etc.). Short-chain fatty acids are beneficial to intestinal health, capable of providing energy, regulating intestinal flora, and inhibiting inflammation, etc. The ability to produce lactic acid is an important indicator for screening Bacillus coagulans. Chinese Patent CN116769637A discloses a preparation method and applications of Bacillus coagulans. The inventor carried out ultraviolet mutagenesis on the starting strain of Bacillus coagulans, and used a solid medium containing steam-exploded straw enzymatic hydrolysate for enrichment and domestication. After primary screening, secondary screening, and genetic stability investigation, a strain with a lactic acid production concentration of 48.03 g / L was selected. Chinese Patent CN116042441A obtained a Bacillus coagulans strain with high stress resistance and high acid production ability through ultraviolet mutagenesis breeding. After 48 h of fermentation culture, the acid production ability of this strain reached 18.35 g / L. The lactic acid production ability of the strains mentioned in the literature is just so-so, and it is necessary to screen Bacillus coagulans strains with stronger lactic acid production ability.

[0004] Listeria monocytogenes is a pathogenic bacterium that causes diseases in humans and livestock. It was listed as the fourth most important foodborne pathogenic bacterium by the World Health Organization in 2002. Listeria monocytogenes has stronger vitality than other pathogenic bacteria. It is not only resistant to drying, salt, but also more resistant to low temperature, and can survive at 0 - 45 °C. It is known as the "refrigerator killer", and livestock meat products, ready-to-eat foods, dairy products, and vegetables are very easily contaminated by Listeria monocytogenes. It has been found that lactic acid bacteria can inhibit Listeria monocytogenes in dairy products.

[0005] Mutation breeding is an effective way to improve the lactic acid production ability of microorganisms. Chemical mutagenesis (nitrosoguanidine NTG), ultraviolet mutagenesis, and ARTP mutagenesis are currently common methods of mutation breeding. Ultraviolet mutagenesis requires simple equipment, is easy to control during the operation process, and is relatively safe, making it a common mutagenesis method. Atmospheric and room temperature plasma mutagenesis (ARTP) is a technique that generates plasma at atmospheric pressure and room temperature to trigger the self-repair of cells, thereby causing gene mutations. ARTP is convenient, safe, and efficient, and is widely used in the field of biological science. Mutagenesis by these methods is expected to improve the lactic acid production level of Bacillus coagulans and its ability to inhibit Listeria monocytogenes.

[0006] A large amount of nutrients are consumed during the fermentation process of Bacillus coagulans, and the cost of the required nutrients is generally high. Therefore, it is necessary to select appropriate culture medium components and optimize the fermentation process parameters to reduce production costs. Although there have been some literature reports in this regard, the technology for controlling the production cost of Bacillus coagulans still needs to be improved, and research in this area has great practical value. Summary of the Invention

[0007] To solve the above technical problems, the present invention first obtained a strain of Bacillus coagulans with good antibacterial effect and strong lactic acid production ability through preliminary screening, then obtained 1 ideal strain after three rounds of mutagenesis, and then optimized the culture medium components and fermentation process to achieve high-density fermentation of the ideal strain.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] One object of the present invention is to provide a strain of Bacillus coagulans JBHBIO-BNJ13, which has a strong inhibitory effect on Listeria monocytogenes and a high lactic acid concentration. It was deposited at the China General Microbiological Culture Collection Center on October 15, 2024. The address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 32216.

[0010] The strain JBHBIO-BNJ13 was obtained by chemical (NTG) mutagenesis, ultraviolet (UV) mutagenesis, and atmospheric and room temperature plasma (ARTP) mutagenesis.

[0011] Further, the conditions for NTG mutagenesis are an NTG concentration of 2 mg / L and a treatment time of 0 - 60 s.

[0012] Further, the conditions for UV mutagenesis are a mutagenesis distance of 20 cm and a treatment time of 0 - 60 s.

[0013] Furthermore, the conditions for ARTP mutagenesis are high-purity helium gas, a power of 120 W, a working gas flow rate of 10 L / min, a mutagenesis distance of 2 mm, and a mutagenesis time of 0 - 400 s.

[0014] The second object of the present invention is to provide a microbial preparation, which contains the Bacillus coagulans JBHBIO - BNJ13 and / or its fermentation products.

[0015] The third object of the present invention is to provide a method for culturing the Bacillus coagulans JBHBIO - BNJ13. The medium formula used in the method includes: 10 - 20 g / L molasses, 5 - 15 g / L dried corn steep liquor, 5 - 10 g / L peptone, 0.5 - 5 g / L anhydrous dipotassium hydrogen phosphate, 0.5 - 2 g / L potassium chloride, 0.2 - 0.6 g / L magnesium sulfate, and 0.01 - 0.1 g / L manganese sulfate; the pH value is 6.0 - 8.0.

[0016] Furthermore, the shake flask culture conditions used in the method include: a fermentation culture period of 36 - 60 h, a temperature of 30 - 55 °C, an inoculum amount of 1% - 10% (v / v), a liquid loading amount of 10% - 50% (v / v), an initial pH value of 3 - 8, and a shaker speed of 100 - 200 r / min.

[0017] Furthermore, the culture conditions at the 10 L fermenter level in the method include: an inoculum amount of 2% - 8% (v / v), feeding in 1 - 3 times, and a culture period of 36 - 60 h.

[0018] Even further, the number of feeding times is 2 - 3 times. The first feeding contains 10 - 20 g / L molasses, and the second feeding is 1 - 2 times the whole medium.

[0019] Even more further, the whole medium contains 10 - 20 g / L molasses, 5 - 15 g / L dried corn steep liquor, 5 - 10 g / L peptone, 0.5 - 5 g / L anhydrous dipotassium hydrogen phosphate, 0.5 - 2 g / L potassium chloride, 0.2 - 0.6 g / L magnesium sulfate, and 0.01 - 0.1 g / L manganese sulfate.

[0020] The fourth object of the present invention is to provide an application of the Bacillus coagulans JBHBIO - BNJ13, the microbial preparation, or any of the cultivation methods of the Bacillus coagulans JBHBIO - BNJ13 in the production of lactic acid.

[0021] The fifth object of the present invention is to provide an application of the Bacillus coagulans JBHBIO - BNJ13, the microbial preparation, or any of the cultivation methods of the Bacillus coagulans JBHBIO - BNJ13 in the preparation of products for inhibiting pathogenic bacteria, improving body immunity, enhancing body digestive function, and / or promoting intestinal health.

[0022] Furthermore, the pathogenic bacteria include Listeria monocytogenes.

[0023] Compared with the prior art, the present invention has the following technical effects:

[0024] Listeria monocytogenes seriously threatens human health and food safety. Through mutagenesis screening, the present invention obtained 1 strain of Bacillus coagulans with high lactic acid production and strong inhibitory activity against Listeria monocytogenes. The lactic acid concentration produced by this strain can reach 84.31 g / L, and it has strong inhibitory activity against Listeria monocytogenes, with the diameter of the inhibition zone reaching 13.68 mm. After optimizing the culture medium and fermentation conditions of this strain, the spore concentration of Bacillus coagulans reached 3.92×10 9 CFU / mL after 42 h of fermentation in a 10 L fermenter.

[0025] Explanation of the preservation certificate:

[0026] Preservation institution: China General Microbiological Culture Collection Center;

[0027] Preservation number: CGMCC No. 32216;

[0028] Preservation date: October 15, 2024;

[0029] Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0030] Taxonomic nomenclature: Bacillus coagulans. Description of the drawings

[0031] Figure 1 This is the result of the influence of the type of carbon source on the growth of Bacillus coagulans in Example 3 of the present invention;

[0032] Figure 2 This is the result of the influence of the carbon source concentration on the growth of Bacillus coagulans in Example 3 of the present invention;

[0033] Figure 3 This is the result of the influence of the addition amount of the complex nitrogen source on the growth of the strain in Example 3 of the present invention;

[0034] Figure 4 This is the result of the influence of the type of inorganic salt on the growth of Bacillus coagulans in Example 3 of the present invention;

[0035] Figure 5 This is the response result of the interactive effect of molasses, magnesium sulfate and manganese sulfate on the spore concentration in Example 3 of the present invention;

[0036] Figure 6 This is the result of the influence of the culture temperature on the growth of Bacillus coagulans in Example 3 of the present invention;

[0037] Figure 7 This is the result of the influence of the shaker speed on the growth of Bacillus coagulans in Example 3 of the present invention;

[0038] Figure 8 This is the result of the influence of the inoculum size on the growth of Bacillus coagulans in Example 3 of the present invention;

[0039] Figure 9 This is the result of the influence of the initial pH on the growth of Bacillus coagulans in Example 3 of the present invention;

[0040] Figure 10 This is the result of the influence of the liquid loading volume on the growth of Bacillus coagulans in Example 3 of the present invention;

[0041] Figure 11 This is the result of the influence of the culture time on the spore formation of the strain in Example 3 of the present invention;

[0042] Figure 12 This is the test result of the inoculation ratio in Example 3 of the present invention;

[0043] Figure 13 This is the optimization result of the fed-batch feeding time in Example 3 of the present invention;

[0044] Figure 14 This is the optimization result of the fed-batch feeding components and concentrations in Example 3 of the present invention;

[0045] Figure 15 This is the determination result of the fermentation culture time in Example 3 of the present invention. Detailed implementation manners

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention shall fall within the scope of the present invention. The reagents and instruments used in the following examples can be obtained commercially, and the methods used in the examples are the same as the commonly used methods unless otherwise specified.

[0047] The culture medium formulation used in the present invention is as follows :

[0048] 1. Slant preservation medium: glucose 10 g / L, tryptone 10 g / L, yeast extract powder 5 g / L, agar powder 15 g / L, and the solvent is 1000 mL of distilled water.

[0049] 2. Seed medium (YPD): glucose 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, pH 6.8 - 7.2.

[0050] 3. Initial screening medium for plate separation: 10 g / L glucose, 10 g / L tryptone, 5 g / L yeast extract powder, 6 mL / L of 1 g / L bromocresol green indicator, 15 g / L agar.

[0051] 4. Nutrient broth solid medium: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar, pH 6.8 - 7.2.

[0052] The analysis method used in the present invention is as follows :

[0053] 1. Analysis of acid - producing ability: Spread the activated Bacillus coagulans seed liquid on the initial screening medium for plate separation, culture at 42 - 45 °C for 24 - 48 h, measure the diameter of the color - changing circle and the colony diameter of single colonies on the initial screening medium for plate separation, and calculate the ratio.

[0054] 2. Analysis of antibacterial effect: Spread the diluted pathogenic bacteria liquid evenly on the nutrient broth solid medium, place sterilized Oxford cups. Add the Bacillus coagulans fermentation broth into the Oxford cups, after diffusing and absorbing at 4 °C for 4 h, transfer to 42 °C and culture for 24 h, and measure the diameter of the antibacterial circle.

[0055] 3. Determination of lactic acid content: Centrifuge the Bacillus coagulans fermentation broth at 12000 r / min for 10 min, filter the obtained supernatant with a 0.22 - μm filter membrane, and analyze the lactic acid content in the fermentation broth by HPLC (chromatographic column: Gemini NX - C18 (250×4.6 mm, 5 μm), mobile phase is a system of 97% diammonium hydrogen phosphate and 3% methanol, flow rate 0.5 mL / min, ultraviolet detection wavelength 254 nm, column temperature 25 °C). Use the standard product concentration (ug / L) as the abscissa and the peak area as the ordinate to determine the acid standard curve. Substitute the sample peak area into the standard curve to calculate the acid content in the sample.

[0056] 4. Analysis of bacterial concentration: The viable bacteria concentration is determined by the dilution - plate counting method; the spore concentration is determined by diluting and spreading the cultured bacterial liquid on a plate after heating in a water bath at 80 °C for 10 min.

[0057] The technical solution of the present invention will be further elaborated in detail below in conjunction with the embodiments.

[0058] Example 1 Screening of the starting strain

[0059] 1. Strain activation: Seven strains of Bacillus coagulans preserved in the laboratory (numbered A, B, S, M, P, C, O respectively) were used as candidate strains. Pick single colonies from the slant medium and inoculate them into the sterile seed medium, then culture at 45 °C, 180 r / min with shaking for 24 h, and sub - culture 2 - 3 generations to obtain the seed liquid.

[0060] 2. Screening of acid production ability: The acid production ability of Bacillus coagulans was evaluated by the average diameter of the color-changing circle, and the experimental results are shown in Table 1.

[0061] Table 1 Primary screening of lactic acid-producing Bacillus coagulans

[0062]

[0063] 3. Screening of antibacterial effect: The antibacterial effects of seven strains of Bacillus coagulans against common pathogenic bacteria were evaluated by the average diameter of the antibacterial circle, and the experimental results are shown in Table 2.

[0064] Table 2 Primary screening of Bacillus coagulans with high antibacterial activity

[0065]

[0066] (Note: The outer diameter of the Oxford cup is 7.8 mm.)

[0067] Combined with Table 1 and Table 2, it can be seen that Bacillus coagulans A has the best acid production effect, and it has different degrees of inhibitory effects on pathogenic bacteria, especially the best inhibitory effect on Listeria monocytogenes. Therefore, Bacillus coagulans A was selected as the starting strain for mutagenesis.

[0068] Example 2 Strain mutagenesis

[0069] Bacillus coagulans A was subjected to three rounds of mutagenesis, namely nitrosoguanidine (NTG) mutagenesis, ultraviolet (UV) mutagenesis, and atmospheric and room temperature plasma (ARTP) mutagenesis.

[0070] 1. NTG mutagenesis

[0071] After treatment with 2 mg / mL NTG for 30 s, the lethality was 90% (the highest mutation rate was reported in the literature). After treatment under this condition, 17 strains of bacteria were isolated. The lactic acid concentration of these strains was 24.12 - 50.83 g / L; the antibacterial circle diameter against Listeria monocytogenes was 8.0 - 10.42 mm. Among them, strain A4 (lactic acid concentration 50.83 g / L, antibacterial circle diameter 10.24 mm) was the best, and it was used as the starting strain for ultraviolet mutagenesis.

[0072] 2. UV mutagenesis

[0073] The best lethality was obtained by irradiating with an ultraviolet lamp at 20 cm for 30 s. After treatment under this condition, 11 strains of bacteria were isolated. Their lactic acid concentration was 50.63 - 68.02 g / L, and the antibacterial circle diameter against Listeria monocytogenes was 8.85 - 11.65 mm. Among them, strain A4-10 (lactic acid concentration 64.30 g / L; antibacterial circle diameter 11.65 mm) was the best, and it was used as the starting strain for ARTP mutagenesis.

[0074] 3. ARTP mutagenesis

[0075] The optimal lethal rate was obtained after 280 s of ARTP mutagenesis treatment. After treatment under this condition, 21 strains were isolated. The lactic acid concentration produced by them was 37.21 - 84.31 g / L, and the inhibition zone diameter against Listeria monocytogenes was 8.74 - 13.68 mm. Among them, strain W20 (lactic acid concentration 84.31 g / L; inhibition zone diameter against Listeria monocytogenes 13.68 mm) was the best, and its culture medium and liquid fermentation process were optimized subsequently.

[0076] Example 3 Optimization of liquid fermentation process

[0077] The initial culture medium formula was: molasses 10 g / L, yeast extract powder 20 g / L, sodium chloride 10 g / L, anhydrous dipotassium hydrogen phosphate 5 g / L, manganese sulfate 0.01 g / L, pH 6.8 - 7.2. (Initial viable cell concentration 5.93×10 8 CFU / mL, spore concentration 2.31×10 8 CFU / mL.)

[0078] 1. Optimization of culture medium components

[0079] (1) Single - factor optimization

[0080] The types and concentrations of carbon sources, nitrogen sources and inorganic salts were optimized in turn. Each time, one parameter was optimized, and the optimal result obtained was used for the optimization of the next parameter. The selected carbon sources were sucrose, maltose, glucose, lactose, bran, molasses, glycerol and soluble starch, and the carbon source addition amount (g / L) was 5, 10, 20, 30, 40, 50; the selected nitrogen sources were yeast extract, soybean meal, tryptophan, peptone, yeast extract, corn starch, fish meal, beef extract, urea, and the nitrogen source addition amount (g / L) was 5, 10, 15, 20, 25, 30. Then, the three single nitrogen sources with the best effects were selected and compounded pairwise in a certain proportion; the inorganic salts were anhydrous dipotassium hydrogen phosphate 5 g / L, sodium chloride 5 g / L, calcium carbonate 2 g / L, potassium chloride 2 g / L, calcium chloride 1 g / L, magnesium sulfate 0.3 g / L and manganese sulfate 0.01 g / L. The specific results are shown in Figures 1 to 4 and Tables 3 - 4.

[0081] Table 3 Effects of nitrogen source types on the growth of Bacillus coagulans

[0082]

[0083] Table 4 Effects of compound nitrogen source types and ratios on the growth of strains

[0084]

[0085] Based on the experimental results, considering factors such as the spore concentration and production cost, the finally determined optimal medium formulation is: molasses 20 g / L, nitrogen source (corn steep liquor powder: peptone = 1:2) 20 g / L, dipotassium hydrogen phosphate anhydrous 5 g / L, potassium chloride 2 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.01 g / L.

[0086] (2) Plackeet - Burman experimental design

[0087] On the basis of the above single - factor experiments, using the Plackett - Burman experimental design, the above - mentioned influencing factors were screened. Each factor was taken at two levels, low (-1) and high (+1), with a total of 12 experimental combinations. The experimental design is shown in Table 5, the experimental results are shown in Table 6, and the main effects and variance analysis of the experimental results are shown in Table 7.

[0088] Table 5 Plackett - Burman test factor and level design

[0089]

[0090] Table 6 Plackett - Burman test design and results

[0091]

[0092] Table 7 Plackett - Burman test variance and main effect analysis

[0093]

[0094] From the P - values of each factor in Table 7, it can be obtained that the addition amounts of molasses, magnesium sulfate, and manganese sulfate have a significant impact on the growth of Bacillus coagulans, among which the P of the addition amount of manganese sulfate < 0.01 is extremely significant. An equation was fitted with the spore concentration of the strain as the response value: spore concentration Y = 9.69 - 1.45A - 0.3418B + 0.5052C + 0.5807D + 0.3005E + 1.40F + 2.04G. By comparing the P - values of each factor, it can be obtained that the order of the influence degree of each factor on the spore concentration from low to high is potassium chloride, corn steep liquor powder, peptone, dipotassium hydrogen phosphate anhydrous, magnesium sulfate, molasses, and manganese sulfate.

[0095] (3) Experiment on the minimum addition amount of insignificant factors

[0096] The minimum addition amounts of the insignificant factors affecting the spore concentration of the strain were explored. Considering the spore concentration and production cost comprehensively, finally, dipotassium hydrogen phosphate anhydrous 1 g / L, potassium chloride 0.5 g / L, peptone 8 g / L, and corn steep liquor powder 10 g / L were selected as the minimum addition amounts.

[0097] (4) Steepest Climb Experiment of Significant Factors

[0098] The steepest climb test was carried out on the factors that had the most significant influence on the spore concentration (molasses, magnesium sulfate, and manganese sulfate). The climbing direction and change step length were determined by the coefficients of each factor obtained from the Plackett-Burman test. The design and results of the steepest climb test are shown in Table 8. It can be seen from the table that when the molasses was 16 g / L, magnesium sulfate was 0.4 g / L, and manganese sulfate was 0.07 g / L, the spore concentration was the highest, reaching 1.4×10 9 CFU / mL.

[0099] Table 8 Design and Results of the Steepest Climb Experiment

[0100]

[0101] (5) Response Surface Optimization

[0102] Using the central composite design principle of Box-Behnken, three levels were taken for each of the three significant influencing factors of molasses, magnesium sulfate, and manganese sulfate with the center point obtained from the steepest climb test. The response surface analysis design is shown in Table 9, and the test results are shown in Table 10.

[0103] Table 9 Response Surface Analysis Design

[0104]

[0105] Table 10 Results of Response Surface Analysis Design

[0106]

[0107] Using Design-Expert software for regression analysis, the regression equation with the spore concentration (Y) as the response value was obtained as: Y = 13.40 - 0.3750A + 0.6738B + 0.7363C + 0.1975AB - 0.2775AC + 0.1000BC - 2.28A 2 - 1.60B 2 - 2.05C 2 . The regression analysis of the regression model was carried out, and the results are shown in Table 11. The P value of this regression model was < 0.01, which was extremely significant, and the P value of the lack-of-fit term was 0.6795 > 0.05, which was not significant. This indicated that the regression model fitted well with the test results and could be used for subsequent spore concentration prediction. R 2 (Prediction) = 0.8916, which indicated that only 10.84% of the cases could not be explained by this regression model. The three linear terms and three quadratic terms of the fitted equation all had a significant influence on the formation of spores of the strain, and the interaction terms had no significant influence on the spore concentration.

[0108] Table 11 Variance Analysis of the Response Surface Regression Model

[0109]

[0110] According to the regression equation and the variance analysis of the experimental results, the Design-Expert V11.0.1 software was used to draw the three-dimensional surface diagram of the two-factor response surface and the corresponding contour diagram, as Figure 5 shown. The results show that within a certain range, the concentration of spores formed by the strain increases non-linearly with the increase in the addition amounts of molasses, magnesium sulfate, and manganese sulfate; when their addition amounts reach a certain value, the spore concentration of the strain reaches the maximum value. At this time, if the addition amounts of the three culture components are further increased, the spore concentration will decrease. The optimal addition amounts of the three factors finally obtained are 14.64 g / L of molasses, 0.4848 g / L of magnesium sulfate, and 0.0833 g / L of manganese sulfate, and the theoretically predicted maximum value of the spore concentration is 1.36×10 9 CFU / mL.

[0111] The optimal culture medium composition obtained according to the above experimental results is: 14.64 g / L of molasses, 10 g / L of corn steep liquor powder, 8 g / L of peptone, 1 g / L of anhydrous dipotassium hydrogen phosphate, 0.5 g / L of potassium chloride, 0.4848 g / L of magnesium sulfate, 0.0833 g / L of manganese sulfate, and pH 6.0.

[0112] 2. Optimization of culture conditions

[0113] The initial fermentation conditions were: fermentation time 46 h; fermentation temperature 45 °C; pH 6.8; inoculum size 4%; liquid filling volume 20%; rotation speed 180 r / min. The selected fermentation times were 24 h, 28 h, 32 h, 36 h, 38 h, 42 h, 46 h, 48 h, 57 h; the fermentation temperatures were 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C; the initial pH was 3.0, 4.0, 5.0, 6.0, 7.0, 8.0; the inoculum sizes were 1%, 2%, 4%, 6%, 8%, 10%; the liquid filling volumes were 10%, 20%, 30%, 40%, 50%; the rotation speeds were 100 r / min, 120 r / min, 140 r / min, 160 r / min, 180 r / min, 200 r / min. The experimental results are shown in Figures 6 to 11 .

[0114] According to the experimental results, considering factors such as the spore concentration of the strain and production cost, the finally determined optimized culture conditions were: inoculum size 4%, culture temperature 40 °C, rotation speed 140 r / min, fermentation time 46 h, and liquid filling volume 30%. Under these optimized conditions, three shake flask verification tests were carried out, and the average spore concentration was 1.45×10 9 CFU / mL, which is 6.3 times that before optimization (2.31×10 8 CFU / mL).

[0115] Optimization of Fermentation Process in 3.10L Fermenter

[0116] (1) Initial fermentation parameters: liquid filling volume 50%, initial pH 6.0, fermentation temperature 40°C, rotation speed 400 r / min, aeration rate 1:1 vvm; tank pressure 0.035 Mpa.

[0117] (2) Optimization of inoculum size: Five inoculum sizes of 2%, 3%, 4%, 5%, and 6% were selected for experiments. From Figure 12 the results, it can be seen that the optimal inoculation ratio is 4%.

[0118] (3) Optimization of fed-batch time: According to the change of pH value of the fermentation broth, an optimization experiment of fed-batch time was designed (Table 12).

[0119] Table 12 Optimization Table of Fed-batch Time

[0120]

[0121] From Figure 13 the experimental results shown, the optimal condition of fed-batch time is No. 3 (i.e., the first uniform fed-batch takes 5 h and the second uniform fed-batch takes 3 h).

[0122] (4) Optimization of the concentration of the components in two fed-batches: On the basis of determining the feeding time, the optimization of the concentration of the components in the uniform fed-batch was carried out, and the experimental scheme is shown in Table 13.

[0123] Table 13 Optimization Test Table of Fed-batch Components and Concentrations

[0124]

[0125] (Note: The composition of the complete medium is molasses 14.64 g / L, corn steep liquor dry powder 10 g / L, peptone 8 g / L, anhydrous dipotassium hydrogen phosphate 1 g / L, potassium chloride 0.5 g / L, magnesium sulfate 0.4848 g / L, manganese sulfate 0.0833 g / L)

[0126] From Figure 14 the experimental results, it can be seen that the best effect is achieved when the molasses in the first feeding is 15 g / L and the second feeding is 1.5 times the complete medium.

[0127] (5) Screening of fermentation time: Within 48 h of fermentation culture, fermentation broth samples were taken every 4 h to detect the spore concentration to judge the fermentation end point. From Figure 15 the results, it can be seen that 42 h is the fermentation end point.

[0128] Based on the above results, the optimal fermentation process in a 10 L fermenter is as follows: the inoculation amount is 4%; the first continuous feeding (15 g / L molasses) starts at 4 h for 5 h, and then the second feeding (1.5 times the full medium) is carried out for 3 h; the fermentation is cultured until 42 h ends; the spore concentration obtained under these conditions is 3.92×10 9 CFU / mL, which is 2.7 times that of the optimized result at the shaker level (1.45×10 9 CFU / mL).

[0129] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Bacillus coagulans JBHBIO-BNJ13, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.32216.

2. A microbial preparation, characterized in that: It contains the Bacillus coagulans JBHBIO-BNJ13 and / or its fermentation product according to claim 1.

3. A method for culturing the Bacillus coagulans JBHBIO-BNJ13 of claim 1, characterized in that: The culture medium formula used includes: 10-20 g / L molasses, 5-15 g / L corn steep liquor dry powder, 5-10 g / L peptone, 0.5-5 g / L anhydrous dipotassium hydrogen phosphate, 0.5-2 g / L potassium chloride, 0.2-0.6 g / L magnesium sulfate and 0.01-0.1 g / L manganese sulfate; the pH value is 6.0-8.

0.

4. The method according to claim 3, characterized in that The shaking flask culture conditions used include: fermentation culture cycle 36-60h, temperature 30-55°C, inoculation amount 1%-10% (v / v), liquid volume 10%-50% (v / v), initial pH value 3-8, and shaking table speed 100-200r / min.

5. The method according to claim 3, characterized in that The culture conditions at the level of a 10L fermentation tank include: an inoculation amount of 2% to 8% (v / v), 1 to 3 feedings, and a culture period of 36 to 60 hours.

6. The method according to claim 5, characterized in that The feeding times are 2 to 3 times, wherein the first feeding contains 10 to 20 g / L molasses, and the second feeding contains 1 to 2 times the full culture medium.

7. The method according to claim 6, characterized in that The complete culture medium contains 10-20 g / L molasses, 5-15 g / L corn steep liquor dry powder, 5-10 g / L peptone, 0.5-5 g / L anhydrous dipotassium hydrogen phosphate, 0.5-2 g / L potassium chloride, 0.2-0.6 g / L magnesium sulfate and 0.01-0.1 g / L manganese sulfate.

8. Use of the Bacillus coagulans JBHBIO-BNJ13 according to claim 1, the microbial preparation according to claim 2, or the cultivation method of Bacillus coagulans JBHBIO-BNJ13 according to any one of claims 3 to 7 in the production of lactic acid.

9. Use of the Bacillus coagulans JBHBIO-BNJ13 according to claim 1, the microbial preparation according to claim 2, or the culture method of Bacillus coagulans JBHBIO-BNJ13 according to any one of claims 3 to 7 in the preparation of products for inhibiting pathogens, improving immunity, enhancing digestive function, and / or promoting intestinal health.

10. The use according to claim 9, characterized in that: The pathogenic bacteria include Listeria monocytogenes.

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