Bacillus subtilis, leavening agent, and preparation method and application thereof

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to a Bacillus subtilis, a fermentation agent, a preparation method and applications thereof, fermented feed and a preparation method. The Bacillus subtilis is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 21166. The Bacillus subtilis provided in the present application has strong acid and bile salt resistance, can stably colonize in the intestinal tract, and can produce various amylases, cellulases and proteases in the animal digestive tract, thereby making up the vacancy of endogenous enzymes in the body, reducing anti-nutritional factors in feed, and having the effects of antibiosis, disease prevention and disease resistance. The fermented feed using the Bacillus subtilis of the present application has improved bioavailability and metabolic rate.

Description

Technical Field

[0001] This invention relates to the field of fermentation, specifically disclosing a strain of Bacillus subtilis, a fermenting agent, a method for preparing the fermenting agent, and the application of the fermenting agent prepared according to the method, the Bacillus subtilis or the fermenting agent in fermented feed, and also disclosing a method for preparing fermented feed and a fermented feed. Background Technology

[0002] Bacillus subtilis, a representative species of the genus Bacillus, is a Gram-positive bacterium that widely survives in various environments. It secretes a variety of enzyme systems, including amylase, lipase, protease, and cellulase, and possesses multiple functions such as improving livestock and poultry growth performance, improving intestinal flora balance, and enhancing immunity. It plays a positive role in promoting animal growth and development and maintaining animal health. Bacillus subtilis can break down starch in the intestine into monosaccharides, which are then synthesized into lactic acid by intestinal bacteria. This effectively lowers the pH of the livestock and poultry intestine, enhancing the activity of endogenous enzymes in an acidic environment and improving the body's absorption and utilization of nutrients. After colonizing the animal intestine, it releases a large number of active substances, inhibiting cell apoptosis, protecting intestinal epithelial cells, maintaining intestinal morphology, and providing an anaerobic environment for the growth of lactic acid bacteria. This plays an important role in the animal's nutrient utilization, growth, and disease prevention. Studies have shown that Bacillus subtilis can secrete a large number of antibacterial substances, such as bacteriocins, bacteriocinoids, and organic acids, exhibiting broad-spectrum antibacterial activity.

[0003] Barley is the main raw material for beer production, and malt root, a byproduct of malting, accounts for about 3% of the total feed weight. my country produces approximately 4 million tons of malt annually, generating over 100,000 tons of malt root, a considerable quantity. Based on total malt root mass, it contains 25%-34% protein (of which enzymes account for 2%-3%), 1.6%-2.2% fat, 6%-10% crude fiber, 35%-44% non-nitrogenous extract, <7.0% moisture, as well as vitamins and minerals. While malt root is rich in nutrients, its low cost due to its status as a byproduct of malt production has historically led to its underutilization. It has been processed into inexpensive livestock feed in both domestic and international markets, resulting in low economic returns.

[0004] Therefore, a strain of Bacillus subtilis with good enzyme production and antibacterial ability, as well as the ability to improve the balance of intestinal flora in livestock and poultry and enhance the body's immunity, is needed. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a strain of Bacillus subtilis, a fermenting agent, a method for preparing the fermenting agent, and the application of the fermenting agent prepared according to the method, the Bacillus subtilis, or the fermenting agent in fermented feed. Furthermore, a method for preparing fermented feed and a fermented feed are disclosed. This Bacillus subtilis can produce various amylases, cellulases, and proteases in the animal digestive tract and can also reduce anti-nutritional factors in feed. Having antibacterial and disease-preventing effects, the application of Bacillus subtilis in the preparation of fermented feed can improve the bioavailability of the fermented feed.

[0006] To achieve the above objectives, the present invention provides a strain of Bacillus subtilis, the preservation number of which is CGMCC No. 21166.

[0007] A second aspect of the present invention provides a fermentation agent comprising the above-described Bacillus subtilis.

[0008] A third aspect of the present invention provides a method for preparing a fermenting agent, the method comprising fermenting the above-mentioned Bacillus subtilis in a fermentation medium.

[0009] A fourth aspect of the present invention provides a fermentation agent prepared by the above method.

[0010] The fifth aspect of the present invention provides the application of the above-mentioned Bacillus subtilis or the above-mentioned fermenting agent in fermented feed.

[0011] The sixth aspect of the present invention provides a method for preparing fermented feed, the method comprising: contacting the above-mentioned Bacillus subtilis or the above-mentioned fermenting agent with a fermentation substrate for fermentation.

[0012] A seventh aspect of the present invention provides a fermented feed, which is prepared by the above-described preparation method.

[0013] The beneficial effects obtained by the present invention through the above technical solution are as follows:

[0014] 1. Safe, healthy, and low-cost: The Bacillus subtilis CGMCC No. 21166 of this invention is a safe strain that can be used in food and has been screened from traditional dough leavening agents. The method of this invention has no chemical additives, is green and natural, and is nutritious and healthy.

[0015] 2. The Bacillus subtilis CGMCC No. 21166 of the present invention can effectively improve the bioavailability of feed: The present invention uses Bacillus subtilis CGMCC No. 21166 as a fermentation agent, which can effectively improve the bioavailability of feed and improve its nutritional value;

[0016] 3. The Bacillus subtilis CGMCC No. 21166 of the present invention can effectively exert the probiotic function: As a fermenting agent, Bacillus subtilis CGMCC No. 21166 of the present invention has strong acid and bile salt resistance, can stably colonize in the intestine, and is resistant to a variety of pathogenic bacteria, which can improve the balance of intestinal flora in livestock and poultry; the fermented feed prepared has high dry matter digestibility, energy digestibility and crude protein digestibility in simulated pig digestion tests, and the fermented feed has good quality;

[0017] 3. The Bacillus subtilis CGMCC No. 21166 of the present invention is easy to cultivate and prepare as a starter culture, and the concentration of live bacteria in the prepared starter culture is high.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description section.

[0019] Biological Preservation

[0020] The Bacillus subtilis provided by this invention was deposited on November 11, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21166. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (CGMCC). Attached Figure Description

[0021] Figure 1 This invention describes the colony morphology of Bacillus subtilis on a culture medium.

[0022] Figure 2 The cell morphology of Bacillus subtilis in this invention is shown. Detailed Implementation

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] The first aspect of the present invention provides a strain of Bacillus subtilis, wherein the preservation number of Bacillus subtilis is CGMCC No. 21166.

[0025] The Bacillus subtilis CGMCC No.21166 described in this invention was screened from traditional dough leavening agents.

[0026] The Bacillus subtilis CGMCC No. 21166 described above has the following properties:

[0027] (1) Morphological characteristics: On LB medium, the colonies are milky white with neat edges, dry surface and opaque appearance. Under a microscope, the cells are rod-shaped and Gram-positive.

[0028] (2) In vivo tolerance: Bacillus subtilis had a survival rate of 91% after 1 hour of acid treatment (pH 2.0) and 65% after 2 hours; the strain also had strong tolerance to bile salts, with a survival rate of 85% after 1 hour of bile salt treatment (0.3% bile salt concentration) and 60% after 2 hours.

[0029] (3) Antibacterial effect: This strain has significant resistance to Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Salmonella, Shigella flexneri, Salmonella choleraesuis and Salmonella paratyphi B.

[0030] (4) The Bacillus subtilis of the present invention can significantly increase the crude protein content of grain and oil by-products after fermentation, and significantly improve the crude protein digestibility, dry matter digestibility and energy digestibility of the prepared feed by animals.

[0031] The Bacillus subtilis provided by this invention can produce a large number of live Bacillus subtilis cells through liquid culture. The culture method is not particularly demanding, as long as it enables the Bacillus subtilis to proliferate. For example, a 103 ratio can be used. 6 -10 8 The live Bacillus subtilis cells are inoculated into a Bacillus subtilis culture medium at an inoculum volume of CFU / mL, and cultured under anaerobic or aerobic conditions at 15-40°C for 8-72 hours to obtain a culture solution. The culture medium for Bacillus subtilis can be any culture medium known in the art suitable for culturing Bacillus subtilis, such as LB medium.

[0032] This invention can further isolate live Bacillus subtilis cells from the above-mentioned culture medium. The method of separation is not particularly limited, as long as it can enrich the cells from the culture medium. For example, it can be achieved by centrifugation and / or filtration. The conditions for centrifugation and filtration can be known conditions, which will not be elaborated here.

[0033] A second aspect of the present invention provides a fermentation agent comprising the aforementioned Bacillus subtilis.

[0034] In some embodiments of the present invention, the number of viable bacteria in the fermentation agent is 10. 8 cfu / mL or higher.

[0035] In this invention, the fermenting agent can be a liquid fermenting agent, a semi-liquid fermenting agent, a concentrated fermenting agent, or a solid fermenting agent.

[0036] In this invention, the liquid fermentation agent refers to the fermentation broth obtained by fermenting and culturing Bacillus subtilis; the semi-liquid fermentation agent refers to the fermentation agent prepared by mixing the fermentation broth and the fermentation substrate; the concentrated fermentation agent refers to the fermentation agent prepared by concentrating the fermentation broth; and the solid fermentation agent refers to the fermentation agent obtained by concentrating and drying the fermentation broth or by directly drying the fermentation broth.

[0037] In this invention, the fermentation agent can be in various forms commonly used in the art, including but not limited to capsules, tablets, oral liquids, and powders.

[0038] In some embodiments of the present invention, the fermentation agent may also contain a freeze-drying protectant. In the present invention, the freeze-drying protectant may be any of the various cryoprotectants conventional in the art, such as at least one selected from skim milk powder, maltodextrin, trehalose, dextran, and glycerol.

[0039] In some embodiments of the present invention, the fermenting agent further contains auxiliary fermentation bacteria. In the present invention, the auxiliary fermentation bacteria can be commonly used strains in the art for preparing fermented feed, such as lactic acid bacteria, yeast, acetic acid bacteria, etc.

[0040] In this invention, the fermentation agent can be in various forms commonly used in the art, including but not limited to capsules, tablets, oral liquids, and powders.

[0041] In some preferred embodiments of the present invention, the fermenting agent is a bacterial powder, and the viable count of Bacillus subtilis in the bacterial powder is 10. 8 More than one per gram.

[0042] A third aspect of the present invention provides a method for preparing a fermenting agent, the method comprising fermenting the above-mentioned Bacillus subtilis in a fermentation medium.

[0043] In some embodiments of the present invention, the fermenting agent exists in the form of a liquid fermenting agent, a semi-liquid fermenting agent, a concentrated fermenting agent, or a solid fermenting agent.

[0044] In some embodiments of the present invention, the method includes: fermenting the above-mentioned Bacillus subtilis in a fermentation medium to achieve a viable cell count of 102. 8 When the concentration of cfu / mL is above a certain level, a fermentation broth is obtained, which is fermentation agent A.

[0045] In this invention, the fermentation medium can be any of the conventional fermentation media for fermenting Bacillus subtilis in the art, such as the LB medium as described above, or any of the various media optimized based on the LB medium that are suitable for fermenting Bacillus subtilis.

[0046] In this invention, the fermentation conditions can be those commonly known in the art for the fermentation culture of Bacillus subtilis, for example, the fermentation temperature can be 30-40℃.

[0047] In some embodiments of the present invention, the method further includes further processing the above-mentioned fermentation broth to obtain a fermenting agent.

[0048] In some embodiments of the present invention, the processing method is selected from one of the following methods:

[0049] (1) The obtained fermentation broth is mixed with the fermentation substrate to obtain fermentation agent B;

[0050] (2) The obtained fermentation broth was concentrated to obtain fermentation agent C;

[0051] (3) The obtained fermentation broth is concentrated and dried to obtain fermentation agent D;

[0052] (4) The obtained fermentation broth is dried to obtain fermentation agent E.

[0053] In this invention, fermenting agent A is a liquid fermenting agent, fermenting agent B is a semi-liquid fermenting agent, fermenting agent C is a concentrated fermenting agent, and fermenting agents D and E are solid fermenting agents.

[0054] In this invention, in method (1), the fermentation substrate can vary depending on the intended use of the fermenting agent. For example, when the fermenting agent is intended for use with malt roots, the fermentation substrate is malt roots. The fermentation substrate includes, but is not limited to, malt roots, wheat bran, rice bran, wheat husk, and soybean meal.

[0055] The amount of substrate used is not particularly limited, as long as it can convert the liquid fermentation agent obtained in step (1) into a semi-liquid fermentation agent.

[0056] In this invention, in methods (2) and (3), the concentration method can be a commonly used technique in the field, as long as it can achieve the concentration of the fermentation broth, such as filtration, centrifugation, membrane filtration, etc., as long as it does not significantly affect the activity of Bacillus subtilis.

[0057] In this invention, the centrifugation conditions can be those commonly used in the field, such as centrifugation at 5000-12000 rpm for 5-20 min in a refrigerated centrifuge.

[0058] In some preferred embodiments of the present invention, the fermentation agent C is prepared by centrifuging the fermentation broth in a refrigerated centrifuge at a speed of 5000-12000 rpm for 5-20 min to obtain a cell precipitate, thereby obtaining the fermentation agent C.

[0059] In this invention, in methods (3) and (4), the drying method can be a commonly used technique in the art, including but not limited to vacuum freeze-drying, spray drying, etc. Preferably, the drying method is vacuum freeze-drying.

[0060] In some embodiments of the present invention, method (3) further includes mixing the concentrated material with a freeze-drying protectant before drying.

[0061] In some embodiments of the present invention, method (4) further includes mixing the fermentation broth with a freeze-drying protectant before drying.

[0062] In this invention, the freeze-drying protectant can be any of the conventional freeze protectants in the art, such as at least one selected from skim milk powder, maltodextrin, trehalose, dextran, and glycerol. Those skilled in the art can select and adjust the amount of protectant as needed.

[0063] In some embodiments of the present invention, the method further includes mixing the above-mentioned fermenting agent with auxiliary fermentation bacteria.

[0064] In some preferred embodiments of the present invention, the method for preparing the fermenting agent includes: culturing Bacillus subtilis CGMCC No. 21166 in a fermentation medium to achieve a viable count of 10⁻⁶. 8 The fermentation broth was obtained by adding a cfu / mL concentration of 1 or higher, mixing it evenly, and then performing vacuum freeze-drying to obtain the fermentation agent.

[0065] In some embodiments of the present invention, the method further includes washing the concentrated material or fermentation broth with a buffer solution before adding the lyophilization protectant to the concentrated material or fermentation broth. The buffer solution may be a conventional buffer solution for rinsing bacterial cells, such as physiological saline or PBS buffer.

[0066] In some preferred embodiments of the present invention, the method for preparing the fermenting agent includes:

[0067] (1) Bacillus subtilis CGMCC No.21166 was fermented in a fermentation medium until the viable count reached 10⁻⁶. 8 A fermentation broth was obtained with a cfu / mL concentration of above 100 cfu / mL.

[0068] (2) Centrifuge the fermentation broth obtained in step (1), rinse with buffer solution, add freeze-drying protectant, and adjust the viable cell concentration to 10. 10 After mixing thoroughly with cfu / mL or higher, the mixture is freeze-dried under vacuum to obtain the starter culture.

[0069] In this invention, the fermentation agent can be in various forms commonly used in the art, including but not limited to capsules, tablets, oral liquids, and powders.

[0070] A fourth aspect of the present invention provides a fermentation agent prepared by the above method.

[0071] The fifth aspect of the present invention provides the application of the above-mentioned Bacillus subtilis or the above-mentioned fermenting agent in fermented feed.

[0072] In some embodiments of the present invention, the application is in the fermentation of malt roots. Preferably, the application involves inoculating malt roots with the aforementioned Bacillus subtilis and fermenting or allowing the Bacillus subtilis to survive under temperature and pressure conditions conducive to its reproduction. In this invention, the inoculation method can be a commonly used method in the art, and will not be elaborated further here. By inoculating malt roots with Bacillus subtilis for fermentation, its metabolites impart certain excellent characteristics such as acidity and aroma to the malt roots, improving their nutritional value and flavor characteristics, and enhancing their bioavailability.

[0073] The sixth aspect of this invention provides a method for preparing fermented feed, the method comprising: contacting the aforementioned Bacillus subtilis or the aforementioned fermenting agent with a fermentation substrate for fermentation. The inoculum amount of Bacillus subtilis may be 1-10g per 100g of fermentation substrate, and the fermentation conditions include: a temperature of 20-40℃ and a time of 3-7 days.

[0074] In some embodiments of the present invention, the fermentation substrate is a by-product of grain and oil processing. Preferably, the grain and oil processing by-product is at least one selected from malt root, wheat bran, rice bran, wheat husk, and soybean meal. The water content of the fermentation substrate is 20-70 wt%. More preferably, the fermentation substrate is selected from barley malt root and / or wheat bran. When the fermentation substrate is barley malt root and wheat bran, the weight ratio of barley malt root to wheat bran is a commonly used weight ratio in the art, for example, barley malt root: wheat bran (weight ratio) = 2:4-6.

[0075] In this invention, the preparation method of the fermented feed is a conventional preparation method in the art, and will not be described in detail here.

[0076] A seventh aspect of the present invention provides a fermented feed, which is prepared by the above-described preparation method.

[0077] The present invention will be described in detail below through embodiments.

[0078] In the following examples, unless otherwise specified, all reagents and raw materials used are commercially available.

[0079] In the following embodiments, unless otherwise specified, all operations are conventional in the art.

[0080] LB solid medium (Luria-Bertani): 5g yeast extract, 10g tryptone, 10g sodium chloride, 15g agar, add water to 1000mL, adjust pH to 7.0 with NaOH, autoclave at 121℃ for 20min.

[0081] LB liquid medium (Luria-Bertani): 5g yeast extract, 10g tryptone, 10g sodium chloride, add water to 1000mL, adjust pH to 7.0 with NaOH, autoclave at 121℃ for 20min.

[0082] The Bacillus subtilis strain used as the control was purchased from the China Industrial Microbial Culture Collection Center, strain number CICC10164.

[0083] Example 1

[0084] This example illustrates the isolation and purification of Bacillus subtilis CGMCC No. 21166.

[0085] Collect traditional dough leavening agent and inoculate it into 9 mL of physiological saline solution for 10 days. -1 Dilute by multiples, and then dilute to 10. -2 10 -3 10 -4 10 -5 10 -6 Dilute the colonies multiple times, and then plate each dilution onto LB agar plates sequentially, incubating at 36±1℃ for 72 h. Use an inoculation needle to select colonies with different morphologies and streak them onto LB agar plates until uniform single colonies of consistent size and morphology appear.

[0086] like Figure 1 As shown, the colony surface is rough and wrinkled, opaque, and dirty white. The colonies are round, crater-shaped, and Gram-positive. The spores are elliptical to columnar, located in the center or slightly off-center of the bacterial cell. After spore formation, the bacterial cell does not swell, initially suggesting a Bacillus genus strain. The strain tentatively identified as Bacillus was activated for three generations in LB liquid medium for physiological, biochemical, and molecular biological identification. The resistance of Bacillus subtilis to gastrointestinal acidic and bile salt environments, as well as its antibacterial activity, were investigated. After multiple rounds of research and validation, a single Bacillus subtilis strain was finally screened from numerous wild lactic acid bacteria.

[0087] 1. Morphological identification

[0088] The selected Bacillus subtilis was cultured in LB solid medium at 36±1℃ for 72 h, as follows Figure 1 As shown, on LB solid medium, the colonies are milky white, with neat edges, dry surfaces, and are opaque. Figure 2 As shown, Bacillus subtilis cells under a microscope are rod-shaped and Gram-positive.

[0089] 2. Molecular identification

[0090] The 16S rDNA of the isolated strain was cloned and sequenced. The nucleotide sequence of its 16S rDNA gene is shown in SEQ ID NO: 1. The 16S rDNA sequence of the strain of the present invention was compared with the sequence of Bacillus subtilis from NCBI. The 16S rDNA sequence of the strain of the present invention showed 99% similarity to the sequence of Bacillus subtilis CICC10164 (purchased from China Industrial Microbial Culture Collection Center).

[0091] SEQ ID NO: 1:

[0092]

[0093] The isolated strain was identified as Bacillus subtilis and deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 21166 and deposit date of November 11, 2020.

[0094] Example 2

[0095] This embodiment illustrates the preparation of Bacillus subtilis control bacteria and the seed culture and fermentation broth of Bacillus subtilis CGMCC No. 21166 of this invention.

[0096] Seed culture and fermentation broth of Bacillus subtilis CGMCC No. 21166 and control strain of the present invention were prepared according to the following methods:

[0097] One loopful of bacterial culture was inoculated from a glycerol storage tube onto an LB slant medium and incubated at 37°C for 24 h to obtain a single colony of Bacillus subtilis. One loopful of this single colony was then inoculated into 5 mL of sterile LB liquid medium and incubated at 37°C and 180 rpm for 24 h to obtain the seed culture (OD). 600 =2). Inoculate 5% (v / v) into a 100 mL Erlenmeyer flask containing 20 mL of fermentation medium (LB liquid medium) and incubate at 37 °C and 180 r / min for 48 h to obtain the fermentation broth (OD). 600 =2).

[0098] Example 3

[0099] This example illustrates the acid resistance test of Bacillus subtilis CGMCC No. 21166.

[0100] Acid treatment: The pH value in the animal stomach will fluctuate between 2.0 and 5.8. The acidic environment of the stomach was simulated by adding HCl to LB liquid medium to adjust the pH value to 2.0. Then, the Bacillus subtilis seed liquid and control bacterial seed liquid prepared according to Example 2 were inoculated at a rate of 0.5% (v / v). The culture conditions were 37°C and 180 r / min.

[0101] The above seed culture was inoculated into LB liquid medium without HCl treatment at a rate of 0.5% (v / v) and cultured under the same conditions as a control group.

[0102] Bacterial cultures treated with acid for 1 hour and 2 hours, as well as the control group, were plate counted to calculate the survival rate.

[0103] The survival rate of Bacillus subtilis is calculated using the formula: S(%) = S1 / S0 × 100%, where S1 is the average number of viable bacteria in the bacterial solution after acid treatment for different times; and S0 is the average number of viable bacteria in the bacterial solution without acid treatment at the corresponding concentration and time.

[0104] Conclusion: As shown in Table 1, the survival rate of Bacillus subtilis CGMCC No.21166 of the present invention was 91% after 1 hour of acid treatment (pH 2.0) and 65% after 2 hours of acid treatment, showing better acid resistance compared with the control bacteria.

[0105] Table 1

[0106]

[0107] Example 4

[0108] This example illustrates the bile salt tolerance test of Bacillus subtilis CGMCC No. 21166.

[0109] Bile salt treatment: Bile salts are sodium or potassium salts formed by the combination of bile acids secreted by hepatocytes and glycine or taurine. They are the main components in bile involved in fat digestion and absorption and have bactericidal effects. Porcine bile salts were added to LB liquid medium to a concentration of 0.3% to simulate the duodenal environment of animals. The Bacillus subtilis seed culture and control seed culture prepared according to Example 2 were inoculated at a rate of 0.5% (v / v). The culture conditions were 37°C and 180 rpm.

[0110] The above seed culture was inoculated into LB liquid medium without porcine bile salts at a rate of 0.5% (v / v) and cultured under the same conditions as a control group.

[0111] Bacterial suspensions treated with bile salts for 1 hour and 2 hours, as well as the control group, were used for plate colony counting to calculate the survival rate.

[0112] The survival rate of Bacillus subtilis is calculated using the formula: S(%) = S1 / S0 × 100%, where S1 is the average number of viable bacteria in the bacterial solution treated with bile salts for different times, and S0 is the average number of viable bacteria in the bacterial solution without bile salt treatment at the corresponding concentration and time.

[0113] Conclusion: As shown in Table 2, after treatment with 0.3% bile salts (at a mass concentration of 0.3% bile salts) for 1 hour, the survival rate of Bacillus subtilis CGMCC No. 21166 of the present invention was 85%, and after treatment for 2 hours, the survival rate of Bacillus subtilis was 60%, showing better bile salt tolerance compared with the control bacteria.

[0114] Table 2

[0115]

[0116] Example 5

[0117] This example illustrates the antibacterial activity of Bacillus subtilis CGMCC No. 21166.

[0118] Bacterial suspensions of *Escherichia coli*, *Enterococcus faecalis*, *Staphylococcus aureus*, *Salmonella*, *Shigella flexneri*, *Salmonella choleraesuis*, and *Salmonella typhimurium* were obtained using the following methods: A loopful of bacterial suspension was inoculated from a glycerol storage tube onto an LB agar slant and incubated at 37°C for 24 hours to obtain single colonies; a loopful of each single colony was then inoculated into 5 mL of sterile LB liquid medium and incubated at 37°C and 180 rpm for 12 hours to obtain bacterial suspensions. The bacterial suspension concentration (OD) was then adjusted. 600 It is version 2.0.

[0119] LB solid medium was melted by heating and cooled to 45°C. The LB solid medium was divided into 7 portions, and each portion was inoculated with 1 μL of bacterial suspension (Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Salmonella, Shigella flexneri, Salmonella choleraesuis, and Salmonella paratyphi B) at a rate of 1 μL of bacterial suspension per 1 mL of medium. The mixture was shaken to mix thoroughly, poured into sterile plates, and allowed to solidify horizontally. Two Oxford cups were placed on each plate, spaced equidistantly. 200 μL of the Bacillus subtilis test suspension (i.e., the seed culture prepared according to Example 2) and the control bacterial suspension (i.e., the seed culture prepared according to Example 2) were added to each Oxford cup. The plates were capped and placed in a 37°C incubator, upright, for static incubation. After 24 hours of incubation, the plates were opened, the Oxford cups were removed, and the diameter of the inhibition zone (i.e., the inhibition ability) was measured using calipers. The results are shown in Table 3.

[0120] Table 3

[0121]

[0122] This strain exhibits significant resistance to Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Salmonella, Shigella flexneri, Salmonella choleraesuis, and Salmonella typhimurium, with superior antibacterial effects compared to the control strain.

[0123] Example 6

[0124] This example illustrates the enzyme production test of Bacillus subtilis CGMCC No. 21166.

[0125] The fermentation broths of Bacillus subtilis CGMCC No. 21166 and the control bacteria prepared according to Example 2 were centrifuged at 8000 r / min and 4℃ for 15 min to obtain fermentation supernatant (i.e. crude enzyme solution). The fermentation supernatant was then used to determine the corresponding enzyme activity.

[0126] 1. Determination of protease activity

[0127] Add 0.5 mL of the fermentation supernatant to 1.5 mL of 1% casein (pH 7.0). Incubate at 37°C for 10 min, then add 2 mL of 0.4 M trichloroacetic acid (TCA) to terminate the reaction. Filter the solution using medium-speed qualitative filter paper to obtain the supernatant. Then, take 1 mL of the supernatant and react it with 5 mL of 0.4 M Na₂CO₃ and 1 mL of Folin-Ciocalteu reagent at 37°C for 20 min to obtain the corresponding solution. Measure the absorbance of the solution at 660 nm. Tyrosine was used as the standard (0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, and 80 μg / mL tyrosine standard solutions); the blank was 1.5 mL of 1% casein (pH 7.0).

[0128] Enzyme activity is defined as the amount of enzyme that hydrolyzes a 1% casein solution to produce 1 μg of tyrosine within 1 minute in 1 mL of fermentation supernatant under reaction conditions of pH 7 and 37℃. One unit of enzyme activity (U) is defined as the amount of enzyme that hydrolyzes a 1% casein solution to produce 1 μg of tyrosine within 1 minute in 1 mL of fermentation supernatant.

[0129] 2. Determination of amylase activity

[0130] A reaction mixture was prepared by mixing 1.25 mL of 1% soluble starch solution, 0.5 mL of 0.1 M acetate buffer (pH 5.0), and 0.25 mL of crude enzyme solution (fermentation supernatant). After incubation at 50 °C for 10 min, the reducing sugar (maltose equivalent) was determined by the dinitrosalicylic acid (DNS) method (the absorbance of the solution was measured at 540 nm; maltose was used as a standard (0 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, and 80 mg / mL maltose standard solutions); the blank was a mixture of 0.75 mL of 0.1 M acetate buffer (pH 5.0) and 1.25 mL of 1% soluble starch solution.

[0131] Enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 μmol / L of maltose from a 1% soluble starch solution within 1 minute under reaction conditions of pH 5.0 and 50℃. One unit of enzyme activity (U) is defined as the amount of enzyme that catalyzes the production of 1 μmol / L of maltose from 1 mL of fermentation supernatant within 1 minute.

[0132] 3. Determination of cellulase activity

[0133] Add 0.5 mL of crude enzyme solution (fermentation supernatant) and 1.5 mL of 1% CMC-Na (carboxymethyl cellulose sodium) substrate (prepared with citrate-disodium hydrogen phosphate buffer at pH 4.8) to a test tube and react at 50 °C for 30 min. Then add 1.5 mL of DNS reagent, mix well, and boil for 5 min. Transfer to a cold water bath to cool, and make up to 10 mL. Measure the absorbance at 540 nm. Glucose was used as the standard (0 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, and 8 mg / mL glucose standard solutions); the blank was 2.0 mL of 1% CMC-Na (prepared with citrate-disodium hydrogen phosphate buffer at pH 4.8).

[0134] Enzyme activity is defined as the ability of 1 mL of fermentation supernatant to catalyze the production of 1 μmol of glucose from CMC-Na substrate within 1 min at 50°C. One unit of enzyme activity (U) is defined as this.

[0135] Conclusion: As shown in Table 4, the Bacillus subtilis strain of the present invention has a strong ability to produce protease, amylase and cellulase, all of which are superior to the control strain.

[0136] Table 4

[0137] Protease activity (U / mL) Amylase activity (U / mL) Cellulase activity (U / mL) CGMCC NO.21166 50.35 61.58 20.94 CICC10164 (control strain) 35.67 46.71 15.00

[0138] Example 7

[0139] This example illustrates the application of Bacillus subtilis CGMCC No. 21166 in the production of starter cultures.

[0140] Bacillus subtilis CGMCC No. 21166 was inoculated into LB liquid medium at an inoculum rate of 2-4% (v / v) and cultured at 37-42℃ for 20-24 h at 150 rpm until the viable count of Bacillus subtilis CGMCC No. 21166 reached 10⁻⁶. 8 CFU / mL or higher, centrifuge (4000 rpm, 10 min), wash the precipitate twice with PBS buffer (pH 6.5-7.5), add skim milk and trehalose as lyophilization protectants, and adjust the cell concentration to 10-1. 9 The mixture was concentrated at cfu / mL and then freeze-dried under vacuum to obtain the starter culture. 8 -10 10 The starter culture has a concentration of cfu / mL. It can be added directly to fermented products for fermentation, or it can be formulated into a dosage form acceptable for the food industry or clinical use by adding appropriate excipients according to conventional methods. The dosage forms include various dosage forms such as capsules, microcapsules, tablets, and powders.

[0141] Fermentation agents made from the Bacillus subtilis strains selected above also include products that maintain the activity of the strains through domestication and other technical means.

[0142] Example 8

[0143] This embodiment demonstrates the preparation of fermented feed using Bacillus subtilis of the present invention.

[0144] The *Bacillus subtilis* strain of the present invention and the control strain were activated with LB liquid medium and cultured at 36±1℃ and 150 rpm for 72 h to the logarithmic phase, respectively, to obtain the liquid fermentation primordial (10) of *Bacillus subtilis* CGMCC No. 21166 of the present invention. 8 CFU / mL) and the liquid fermentation broth of the control strain (10 8 CFU / mL). Barley malt roots and wheat bran were weighed into a beating bag according to the proportions in Table 5 to prepare the raw materials. The moisture content was adjusted to 50 wt%. The liquid fermentation agent of Bacillus subtilis CGMCC No. 21166 of this invention (10...) was added. 8 The liquid fermentation agents of Bacillus subtilis (CFU / mL) and control bacteria were inoculated at an inoculation rate of 4% (w / w), mixed well, and the bag openings were tied tightly with rubber bands. The mixtures were then incubated at 32°C for 5 days to prepare fermented feeds 1-2, 2-2, and 3-2 of Bacillus subtilis CGMCC No. 21166 of this invention, and fermented feeds 1-1, 2-1, and 3-1 of control bacteria. Raw material 1-3, which was not inoculated with fermentation agent, was used as the control feed. The number of Bacillus subtilis in the feeds was detected, and the results are shown in Table 6.

[0145] Table 5

[0146]

[0147] Table 6

[0148]

[0149]

[0150] Example 9

[0151] This embodiment is used to illustrate the results of simulated pig digestion tests on feed samples prepared by Bacillus subtilis and control bacteria according to the present invention.

[0152] This experiment was conducted according to the operating manual of the monogastric animal biomimetic digestive system of the State Key Laboratory of Animal Nutrition, using the SDS3 monogastric animal biomimetic digestive system. The biomimetic digestion results (dry matter-based) of fermented feeds 1-2, 2-2, and 3-2 prepared with Bacillus subtilis CGMCC No. 21166 (prepared in Example 8), fermented feeds 1-2, 2-2, and 3-2 prepared with control bacteria, and control feed 1-3 were tested. This was to illustrate the dry matter digestibility, enzyme hydrolysate energy value, and crude protein digestibility of the feed samples prepared with Bacillus subtilis of this invention, and to compare them with the dry matter digestibility, enzyme hydrolysate energy value, and crude protein digestibility of the feed samples prepared with control bacteria. The samples were tested using a pig energy digestion biomimetic program, with 5 replicates per sample, to determine the enzyme hydrolysate energy value; the samples were also tested using a pig crude protein biomimetic digestion program, with 5 replicates per sample, to determine the crude protein digestibility.

[0153] The results are shown in Table 7.

[0154] Table 7

[0155]

[0156]

[0157] As can be seen from Table 7, compared with the control feed, the fermented feed prepared by Bacillus subtilis CGMCC NO.21166 of the present invention has improved dry matter digestibility, energy digestibility, crude protein content and crude protein digestibility; and the quality of the fermented feed prepared by Bacillus subtilis of the present invention is better than that of the fermented feed prepared by the control bacteria.

[0158] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention. SEQUENCE LISTING <110> COFCO Group Limited COFCO Nutrition and Health Research Institute Co., Ltd. <120> Bacillus subtilis, fermentation agent, preparation method and their application <130> I71345COF <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1294 <212> DNA <213> Bacillus subtilis <400> 1 gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta 60 gcgattccag cttcacgcag tcgagttgca gactgcgatc cgaactgaga acagatttgt 120 gggattggct taacctcgcg gtttcgctgc cctttgttct gtccattgta gcacgtgtgt 180 agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttctc cggtttgtca 240 ccggcagtca ccttagaggtg cccaactgaa tgctggcaac taagatcaag ggttgcgctc 300 gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 360 cactctgccc ccgaagggga cgtcctatct ctaggattgt cagaggatgt caagacctgg 420 taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 480 tcaattcctt tgagtttcag tcttgcgacc gtactcccca ggcggagtgc ttaatgcgtt 540 agctgcagca ctaaggggcg gaaaccccct aacacttagc actcatcgtt tacggcgtgg 600 actaccaggg tatctaatcc tgttcgctcc ccacgctttc gctcctcagc gtcagttaca 660 gaccagagag tcgccttcgc cactggtgtt cctccacatc tctacgcatt tcaccgctac 720 acgtggaatt ccactctcct cttctgcact caagttcccc agtttccaat gaccctcccc 780 ggttgagccg ggggctttca catcagactt aagaaaccgc ctgcgagccc tttacgccca 840 ataattccgg acaacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 900 gtggctttct ggttaggtac cgtcaaggta ccgccctatt cgaacggtac ttgttcttcc 960 ctaacaacag agctttacga tccgaaaacc ttcatcactc acgcggcgtt gctccgtcag 1020 actttcgtcc attgcggaag attccctact gctgcctccc gtaggagtct gggccgtgtc 1080 tcagtcccag tgtggccgat caccctctca ggtcggctac gcatcgttgc cttggtgagc 1140 cgttacctca ccaactagct aatgcgccgc gggtccatct gtaagtggta gccgaagcca 1200 ccttttatgt ttgaaccatg cggttcaaac aaccatccgg tattagcccc ggtttcccgg 1260 agttatccca gtcttacagg caggttaccc acgt 1294

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis) Its characteristics are, The Bacillus subtilis has the accession number CGMCC No. 21166.

2. A fermenting agent, characterized in that, The fermenting agent comprises Bacillus subtilis as described in claim 1.

3. The fermenting agent according to claim 2, wherein, The number of viable bacteria in the fermentation agent is 10. 8 cfu / mL or higher.

4. The fermenting agent according to claim 2 or 3, wherein, The fermenting agent is a liquid fermenting agent, a semi-liquid fermenting agent, a concentrated fermenting agent, or a solid fermenting agent.

5. A method for preparing a fermenting agent, characterized in that, The method includes fermenting the Bacillus subtilis of claim 1 in a fermentation medium.

6. The fermenting agent prepared by the preparation method according to claim 5.

7. The use of Bacillus subtilis as described in claim 1 or the fermenting agent as described in any one of claims 2-4 and 6 in fermented feed.

8. A method for preparing fermented feed, characterized in that, The method includes: contacting the Bacillus subtilis of claim 1 or the fermenting agent of any one of claims 2-4 and 6 with the fermentation substrate to carry out fermentation.

9. The method according to claim 8, wherein, The fermentation substrate is a by-product of grain and oil processing.

10. The method according to claim 9, wherein, The fermentation substrate is at least one of malt root, wheat bran, rice bran, wheat husk, and soybean meal.

11. A fermented feed, characterized in that, The fermented feed is prepared by the preparation method described in any one of claims 8-10.

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