Bacillus velezensis GS33 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus velezensis GS33 and application thereof. The fermentation supernatant of the bacillus laicisii GS33 has broad-spectrum antibacterial activity on common food-borne pathogenic bacteria and plant fungi, can generate a large number of bacteriostatic active substances after fermentation optimization, can be further developed as a probiotic agent, a growth promoting agent or an antibacterial agent, can promote animal growth or prevent diseases after being added into feed, and has a good application prospect. Therefore, the use of antibiotics can be reduced to a certain extent, and the application prospect is wide. According to the invention, the fermentation conditions of the bacillus velezensis GS33 for producing the bacteriostatic active substances are also optimized, and the bacteriostatic activity of the fermentation supernatant is improved by 30.14% through optimization of the fermentation conditions, so that the yield of the bacteriostatic active substances is effectively improved. A powerful tool is provided for solving the problems of drug resistance, drug residues, environmental pollution and the like caused by the application of antibiotics in livestock production.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Bacillus Velezii GS33 and application thereof. Background Art

[0002] At present, the massive increase in drug-resistant pathogenic strains and drug residues caused by the abuse of antibiotics have attracted great attention. The problems caused by the long-term and large-scale use of antibiotics are becoming more and more serious, posing a major threat to livestock and poultry products, food safety and human health. Therefore, it is urgent to find alternatives to antibiotics. The antibiotic alternatives that have been studied more include probiotic preparations, plant extracts, acidifiers and antimicrobial peptides.

[0003] In recent years, research on Bacillus Velez has mainly focused on the fields of feed, medicine, textiles, aquatic products, etc. As a new type of biocontrol microbial factor, Bacillus Velez has attracted much attention in plant disease prevention and control, promoting plant growth, etc. Bacillus Velez is a Gram-positive bacillus that can effectively inhibit a variety of plant pathogens. The antagonistic effect of Bacillus Velez on pathogenic microorganisms mainly works through the antibacterial active metabolites it secretes. The bacteria can secrete and produce a variety of bioactive substances, including cell wall degrading enzymes (glucanases and proteases), lipopeptide antibiotics (surfactants, iturin and fenbuconazole) and polyketide antibiotics. The antibacterial active substances produced by Bacillus have the advantages of strong antibacterial activity, good stability and no drug resistance. They can be used as green feed additives or veterinary drugs to replace antibiotics in animal production. The fermentation process of Bacillus is the basis for the production of antibacterial active substances. The environmental conditions of fermentation, such as temperature, fermentation time and composition of the culture medium, all affect the yield and activity of antibacterial substances.

[0004] Microbial fermentation is the process of using microorganisms to convert raw materials into products needed by humans through specific metabolic pathways under suitable conditions. Microbial fermentation depends not only on the genetic characteristics of the strain itself, but also requires suitable culture medium and fermentation conditions. Optimizing the fermentation process can give full play to the potential production capacity of the strain, improve production efficiency and reduce production costs. The fermentation yield of antibacterial active substances of this strain of Bacillus Velez is low, and the cost of the basic fermentation medium is relatively high. It is necessary to optimize the fermentation to reduce costs and increase the yield of antibacterial active substances.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a strain of Bacillus Velezciscous GS33 with broad-spectrum antibacterial activity and application thereof.

[0007] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a Bacillus Velezii ( Bacillus velezensis )GS33, its deposit number is GMCC NO.32069.

[0008] In a second aspect, the present invention provides a bacterial agent (probiotic agent) containing the Bacillus Velezii GS33.

[0009] In a third aspect, the present invention provides a fermentation product, which is obtained by fermenting the Bacillus Velezii GS33 or the bacterial agent.

[0010] In a fourth aspect, the present invention provides any of the following uses of the Bacillus Velezii GS33, the bacterial agent or the fermentation product: 1) Used for preparing antibacterial drugs; 2) Used as feed additive; 3) Used in animal production.

[0011] Preferably, the antibacterial drug has antibacterial effects on Gram-positive bacteria, Gram-negative bacteria and fungi.

[0012] More preferably, the antibacterial drug is effective against Escherichia coli ( Escherichia coli ),salmonella( Salmonella ) and Staphylococcus aureus ( Staphylococcus aureus ) has antibacterial effect.

[0013] In a fifth aspect, the present invention provides a pharmaceutical composition, wherein the main active ingredient of the pharmaceutical composition is the Bacillus Velezii GS33, the bacterial agent or the fermentation product.

[0014] In a sixth aspect, the present invention provides a feed additive, wherein the main active ingredient of the feed additive is the Bacillus Velez subtilis GS33, the bacterial agent or the fermentation product.

[0015] In the seventh aspect, the present invention provides a fermentation method of the Bacillus Velez GS33, which has special requirements on the components of the fermentation medium, specifically: the carbon source of the fermentation medium is yeast culture, the nitrogen source is cottonseed meal, and the mass ratio of the carbon source to the nitrogen source is 1~3:2~4, preferably 2:3.

[0016] Preferably, 2% to 4% MnSO4 is added to the fermentation medium, more preferably 3% MnSO4 is added.

[0017] Preferably, the age of the seed liquid is 16±2h, the fermentation temperature is 37±2°C, and the fermentation time is 24±3h.

[0018] Beneficial effects: The present invention provides a Velez subtilis GS33 and its application. The fermentation supernatant of the Velez subtilis GS33 has broad-spectrum antibacterial activity against common foodborne pathogens and plant fungi, and can produce a large amount of antibacterial active substances after fermentation optimization. It can be further developed as a probiotic, growth promoter or antibacterial drug. Adding it to feed can promote animal growth or prevent diseases, thereby reducing the use of antibiotics to a certain extent, and has broad application prospects. The present invention also optimizes the fermentation conditions for the production of antibacterial active substances by Velez subtilis GS33. After the fermentation conditions are optimized, the antibacterial activity of the fermentation supernatant is increased by 30.14%, effectively increasing the yield of antibacterial active substances. It provides a powerful tool for overcoming the problems of drug resistance, drug residues and environmental pollution caused by the use of antibiotics in animal husbandry production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0020] Figure 1 These are some results of the antibacterial test of Bacillus velezensis GS33 in the examples of the present invention.

[0021] Figure 2 The figure shows the effect of the carbon source type on the diameter of the inhibition zone of the GS33 fermentation broth in the examples of the present invention.

[0022] Figure 3 The figure shows the effect of the type of nitrogen source on the diameter of the inhibition zone of GS33 fermentation broth in the examples of the present invention.

[0023] Figure 4 The figure shows the effect of the total amount and ratio of carbon and nitrogen sources on the diameter of the inhibition zone of GS33 fermentation broth in the embodiment of the present invention.

[0024] Figure 5 The figure shows the effect of the types of inorganic salts on the diameter of the inhibition zone of the GS33 fermentation broth in the examples of the present invention.

[0025] Figure 6 The figure shows the effect of manganese sulfate concentration on the diameter of the inhibition zone of GS33 fermentation broth in the embodiment of the present invention.

[0026] Figure 7 The figure shows the effect of fermentation time on the diameter of the inhibition zone of GS33 fermentation liquid in the embodiment of the present invention.

[0027] Figure 8 The figure shows the effect of inoculum size on the diameter of the inhibition zone of GS33 fermentation broth in the examples of the present invention.

[0028] Fig. 9 The figure shows the effect of the bacterial age of the seed solution on the diameter of the inhibition zone of the GS33 fermentation solution in the embodiment of the present invention.

[0029] Fig.10 The figure shows the effect of fermentation temperature on the diameter of the inhibition zone of GS33 fermentation liquid in the embodiment of the present invention.

[0030] Fig.11 The figure shows the effect of the initial pH value on the diameter of the inhibition zone of the GS33 fermentation liquid in the embodiment of the present invention.

[0031] Fig.12 Comparison of the antibacterial activity of GS33 fermentation broth before and after optimization in the examples of the present invention. DETAILED DESCRIPTION

[0032] The present invention separates and purifies a strain of bacteria GS33 from Tibetan pig feces, which has an inhibitory effect on a variety of pathogenic bacteria. After identification, the strain GS33 is Bacillus Velezii ( Bacillus velezensis ), which has been deposited in the China General Microbiological Culture Collection Center (CGMCC) on September 25, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, with the deposit number CGMCC No. 32069.

[0033] The present invention further provides a bacterial agent containing the Bacillus Velez subtilis GS33, and a medicine or composition containing the Bacillus Velez subtilis GS33 and / or its fermentation product.

[0034] The invention also provides a fermentation method of the Bacillus Velezii GS33.

[0035] Preferably, the fermentation method comprises: the liquid volume is 30 ml / 100 L, the inoculation volume is 2.5% v / v, the seed liquid age is 16 hours, the fermentation temperature is 37° C., and the fermentation time is 24 hours. Fermentation medium: the carbon source is yeast culture, the nitrogen source is cottonseed meal, the total amount of carbon and nitrogen sources is 5%, the mass ratio of carbon and nitrogen sources is 2:3, MnSO4 is 3%, and pH is 7.0.

[0036] Cottonseed meal has attracted wide attention as a good protein resource, but it contains a variety of anti-nutritional factors such as free cottonyphenol, glucosinolates, phytic acid, etc., which not only affect the digestion and absorption of nutrients by animals, but also may cause nutritional metabolic diseases, limiting its wide application in livestock and poultry feed. The present invention can change the physicochemical structure of nutrients in cottonseed meal through microbial fermentation, degrade macromolecules into small molecules that are easy to digest and absorb, and improve feed conversion rate. The present invention uses cottonseed meal as a culture medium component and performs microbial liquid fermentation to produce antibacterial active substances, thereby reducing fermentation costs and improving the utilization efficiency of cottonseed meal.

[0037] The present invention also provides the use of the Velez subtilis GS33 and / or its fermentation product for preparing an antibacterial drug or composition. The bacteria include but are not limited to Gram-positive bacteria, Gram-negative bacteria and fungi. Preferably, the bacteria are selected from Escherichia coli ( Escherichia coli ),salmonella( Salmonella ) and Staphylococcus aureus ( Staphylococcus aureus ).

[0038] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The endpoints and any values ​​of the ranges disclosed in this specification 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0040] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0041] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0042] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0043] The enzymatically hydrolyzed soybean meal used in the following examples was purchased from Liaoning Compulide Biotechnology Co., Ltd.

[0044] Example 1

[0045] This example illustrates the screening and identification process of Bacillus Velezii GS33.

[0046] Tibetan pig feces were collected and further isolated and purified to obtain strain GS33 after antibacterial test. The 16S rDNA sequence of strain GS33 is:

[0047] Based on the results of microbiological and molecular biological identification, strain GS33 was identified as Bacillus velezinoffii ( Bacillus velezensis ).

[0048] Example 2

[0049] In this example, the antibacterial activity of Bacillus velez GS33 strain was tested.

[0050] (1) Activation of Bacillus velez GS33: Perform sterile operation in a clean bench, pick Bacillus Velez GS33 with an inoculation loop, streak on LB agar medium, and culture overnight in a 37°C incubator. Pick a loop of GS33 with an inoculation loop and inoculate it into 30mL / 100mL LB liquid medium, culture it at 37°C in a shaker at 220rpm for 12h and set aside.

[0051] (2) Preparation of indicator bacteria suspension: When the indicator bacteria are the following bacteria, Escherichia coli K88, O157:H7, 987P and 1515, Staphylococcus aureus CVCC1882 and ATCC4330, Salmonella CVCC1791, pick the test bacteria with an inoculation loop under a sterile environment, streak on LB agar medium, and culture in a 37°C incubator overnight. Pick a loop of bacteria with an inoculation loop and inoculate it in 10mL / 50mL LB liquid medium, culture it in a 37°C shaker at 220rpm overnight, count the live bacteria, and dilute it with physiological saline to a concentration of 10 6 cfu / mL.

[0052] (3) Preparation of fermentation supernatant of Bacillus velezinii: The activated Bacillus Velez GS33 was inoculated into 30mL / 100mL LB liquid medium at 1 / 1000 inoculation amount, and cultured at 37°C shaker 200rpm for 12h, and then inoculated into 100mL / 500mL basic fermentation medium (1% glucose, 1% maltose, 1% peptone, 1% tryptone and 1% yeast extract) at 1% inoculation amount, and cultured at 37°C shaker 200rpm for 24h. The bacteria were removed by centrifugation at 4°C, 4,000rpm for 10min in a high-speed refrigerated centrifuge, and the supernatant was filtered with a 0.22μm filter membrane to obtain the GS33 fermentation supernatant.

[0053] (4) Determination of antibacterial activity of GS33 fermentation supernatant: When the indicator bacteria are bacteria, under sterile conditions, use a spreading rod to spread the prepared 100 μL test strain suspension on the LB agar culture dish to obtain a bacteria-containing plate. Use a sterile hole puncher to punch holes on the bacteria-containing plate, remove the agar in the holes, punch two holes on each plate, and each group of three parallels, and add 150 μL of GS33 fermentation supernatant or sterile saline to each hole. Place the bacteria-containing plate at room temperature for 30 minutes to allow the solution to fully diffuse, and then place all bacteria-containing plates at the optimal growth temperature of the indicator bacteria overnight. Use a vernier caliper to measure the diameter of the inhibition zone using the cross method, and use the diameter of the inhibition zone (mm) as an indicator of the strength of the antibacterial activity.

[0054] The antibacterial spectrum of Bacillus Velezii is shown in Table 1. It is found that it has good antibacterial effects on a variety of pathogens, including animal pathogens and plant pathogens ( Figure 1 ).

[0055]

[0056] Example 3

[0057] In this example, the fermentation conditions for producing antibacterial active substances by Bacillus Velezii GS33 were optimized.

[0058] With Escherichia coli K88 as the indicator bacteria and the diameter of the inhibition zone of the fermentation supernatant as the indicator of antibacterial activity, a single-factor experimental design was used to optimize the optimal fermentation conditions for the production of antibacterial active substances by Bacillus velez GS33.

[0059] 1. Optimization of fermentation medium composition

[0060] Using basal culture medium (1% glucose, 1% maltose, 1% peptone, 1% tryptone, and 1% yeast extract) as the basal fermentation medium, the carbon source, nitrogen source, carbon-nitrogen ratio, and inorganic salt types of the culture medium for GS33 fermentation to produce antimicrobial peptides were optimized and screened.

[0061] 1.1 Screening of carbon source types: Based on the basic fermentation medium of Bacillus Velez GS33, 2% maltose, sucrose, glucose, soluble starch, yeast culture, soybean molasses, cane molasses and beet molasses were used to replace the glucose and maltose in the basic fermentation medium to prepare 30 mL of medium, which was placed in a 100 mL conical flask, inoculated with 1% seed liquid, and placed in a 37°C shaker at 220 rpm for 24 hours. The bacteria were removed by centrifugation at 4°C, 4,000 rpm for 10 minutes in a high-speed refrigerated centrifuge, and the supernatant was filtered with a 0.22 μm filter membrane to obtain the fermentation supernatant. The antibacterial activity of the fermentation supernatant of GS33 was determined according to the method of Example 2 to determine the optimal carbon source.

[0062] The results showed that the order of influence of different carbon sources on antibacterial activity was: yeast culture > glucose > maltose > soluble starch = sucrose > soybean molasses > beet molasses > sugarcane molasses. Therefore, the optimal carbon source was yeast culture, and the diameter of the inhibition zone was 18.0 mm. The results are shown in Figure 2 .

[0063] 1.2 Screening of nitrogen source types: On the basis of determining the optimal carbon source, 2% yeast culture was used as the nitrogen source, and 3% ordinary soybean meal (provided by Hebei Chengde Jiuyun Agriculture and Animal Husbandry Co., Ltd., the specific components of soybean meal are as follows: dry matter 91.19%, crude protein 45.22%, crude fat 2.13%, crude ash 6.61%), enzymatically hydrolyzed soybean meal and cottonseed meal were used to replace the peptone, trypsin and yeast extract in the basic fermentation medium to prepare the culture medium. The fermentation supernatant of GS33 was prepared according to the method of 1.1 in Example 3, and the antibacterial activity of the fermentation supernatant of GS33 was determined according to the method of Example 2 to determine the optimal nitrogen source. The results showed that on the basis of the optimal carbon source of 2% yeast culture, the order of influence of different nitrogen sources on the antibacterial activity was: cottonseed meal > enzymatically hydrolyzed soybean meal > ordinary soybean meal. Therefore, the optimal carbon source was cottonseed meal, and the diameter of the inhibition zone was 16.8 mm. The results are shown in Table 2. Figure 3 .

[0064] 1.3 Screening of different total amounts and ratios of carbon and nitrogen sources: On the basis of determining the most suitable carbon and nitrogen sources, the selected yeast culture was used as the carbon source and cottonseed meal as the nitrogen source, and different total amounts and ratios of carbon and nitrogen sources were used to replace the carbon source and nitrogen source in the basic fermentation medium to prepare the medium, wherein the total amounts of carbon and nitrogen sources were set to three gradients of 2%, 4% and 5%, and the mass ratios of carbon and nitrogen sources under different total amounts of carbon and nitrogen sources were set to four gradients of 1:1, 1:2, 2:1 and 2:3. The fermentation supernatant of GS33 was prepared according to the method of 1.1 in Example 3, and the antibacterial activity of the fermentation supernatant of GS33 was determined according to the method of Example 2 to determine the most suitable total amounts and ratios of carbon and nitrogen sources. The results showed that, based on the optimum carbon and nitrogen sources of 2% yeast culture and 3% cottonseed meal, the total amount and ratio of carbon and nitrogen sources in the culture medium were changed for fermentation. It was found that when the total amount of carbon and nitrogen sources was 5% and the mass ratio of carbon and nitrogen sources was 2:3, the antibacterial activity was the highest, and the diameter of the inhibition zone was 16.7 mm. Figure 4 .

[0065] 1.4 Screening of inorganic salt types: Based on the above experiments, yeast culture was used as the carbon source, cottonseed meal was used as the nitrogen source, the total amount of carbon and nitrogen sources was 5%, and the ratio of carbon to nitrogen sources was 2:3. 1% of MnSO4, MgSO4, KH2PO4 and NaCl were added respectively to prepare the culture medium. The fermentation supernatant of GS33 was prepared according to the method in 1.1 of Example 3. The antibacterial activity of the fermentation supernatant of GS33 was determined according to the method in Example 2 to determine the most suitable inorganic salt types. The results showed that on the basis of yeast culture as the carbon source, cottonseed meal as the nitrogen source, the total amount of carbon source and nitrogen source was 5%, and the mass ratio of carbon source and nitrogen source was 2:3, 1% of different types of inorganic salts were added. The order of influence of different types of inorganic salts on antibacterial activity was MnSO4>MgSO4>NaCl>KH2PO4. Therefore, the most suitable inorganic salt type was MnSO4. The results are shown in Table 2. Figure 5 .

[0066] 1.5 Screening of inorganic salt concentration: After determining that the best carbon source is yeast culture, the best nitrogen source is cottonseed meal, the total amount of carbon and nitrogen sources is 5%, the mass ratio of carbon and nitrogen sources is 2:3, and the best inorganic salt is MnSO4, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, and 4% of MnSO4 are added to prepare the culture medium, and the fermentation supernatant of GS33 is prepared according to the method of 1.1 in Example 3. The antibacterial activity of the fermentation supernatant of GS33 is determined according to the method of Example 2 to determine the optimal inorganic salt addition concentration. The results show that on the basis of yeast culture as the carbon source, cottonseed meal as the nitrogen source, the total amount of carbon source and nitrogen source is 5%, and the mass ratio of carbon source and nitrogen source is 2:3, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% and 4% of MnSO4 are added to conduct experiments and select the optimal inorganic salt addition amount. The results showed that the antibacterial activity was the highest when the addition amount of MnSO4 was 2.5%, and the diameter of the inhibition zone was 17.5 mm. Figure 6 .

[0067] It can be seen that by optimizing the fermentation medium formula, the medium formula that can improve the antibacterial activity of Bacillus Velez GS33 is obtained as follows: when the liquid volume is 30mL / 100mL, the carbon source is yeast culture, the nitrogen source is cottonseed meal, the total amount of carbon and nitrogen sources is 5%, the mass ratio of carbon to nitrogen sources is 2:3, and MnSO4 is 2.5%.

[0068] 2. Optimization of fermentation conditions

[0069] The culture medium was prepared based on the determination of the optimal types, total amounts and proportions of carbon and nitrogen sources, the optimal inorganic salts and their addition ratios, and the fermentation conditions for the production of antibacterial active substances by Bacillus Velezii GS33 were optimized using a single-factor experimental scheme.

[0070] 2.1 Optimization of fermentation time: GS33 with an inoculation age of 12 hours was inoculated into the optimized culture medium at a 1% inoculation amount, and the liquid volume was 30mL / 100mL. It was placed in a shaker at a temperature of 37°C and a speed of 220rpm for constant temperature shaking culture for 0, 12, 24, 36, 48, 60 and 72 hours to prepare the fermentation supernatant of GS33. The antibacterial activity of the fermentation supernatant of GS33 was determined according to the method of Example 2 to determine the optimal fermentation time. The results showed that when the fermentation time was 24 hours, the antibacterial activity was the highest, and the diameter of the antibacterial zone was 18.5mm. The results are shown in Figure 7 .

[0071] 2.2 Optimization of inoculation size: Bacillus Velezii GS33 with an inoculation age of 12h was inoculated in the optimized culture medium with inoculation sizes of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0% and 4.0%, respectively. The liquid volume was 30mL / 100mL and the culture was carried out in a shaker at 37°C and 220rpm for constant temperature shaking culture. The fermentation time was 12h. The fermentation supernatant of GS33 was prepared, and the antibacterial activity of the fermentation supernatant of GS33 was determined according to the method of Example 2 to determine the optimal inoculation size. The results showed that when the inoculation size was 2.5%, the antibacterial activity was the highest, and the diameter of the inhibition zone was 17.7mm. The results are shown in Table 2. Figure 8 .

[0072] 2.3 Optimization of seed liquid age: Seed liquids with ages of 8, 12, 16, 20, 24 and 28 hours were inoculated into the optimized culture medium at a 2% inoculation rate, with a liquid volume of 30mL / 100mL, and placed in a shaker at a temperature of 37°C and a speed of 220rpm for constant temperature shaking culture for 24 hours to prepare the fermentation supernatant of GS33. The antibacterial activity of the fermentation supernatant of GS33 was determined according to the method of Example 2 to determine the optimal seed liquid age. The results showed that when the seed liquid age was 16 hours, the antibacterial activity was the highest, and the diameter of the inhibition zone was 19.6mm. See the results in Fig. 9 .

[0073] 2.4 Optimization of fermentation temperature: The seed liquid was inoculated into the optimized culture medium with a liquid volume of 30 mL / 100 mL, and placed in a shaker at 25, 28, 31, 34, 37 and 40 ° C and a speed of 220 rpm for constant temperature shaking culture for 12 hours to prepare the fermentation supernatant of GS33. The antibacterial activity of the fermentation supernatant of GS33 was determined according to the method of Example 2 to determine the optimal fermentation temperature. The results showed that when the fermentation temperature was 37 ° C, the antibacterial activity was the highest, and the diameter of the antibacterial zone was 18.5 mm. Results are shown in Fig.10 .

[0074] 2.5 Optimization of initial pH value: The seed liquid with a bacterial age of 20 hours was inoculated at an inoculation rate of 2% into the optimized culture medium with initial pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, respectively. The liquid volume was 30mL / 100mL, and the culture was carried out at a constant temperature of 37°C and a rotation speed of 220rpm in a shaker for 12 hours to prepare the fermentation supernatant of GS33. The antibacterial activity of the fermentation supernatant of GS33 was determined according to the method of Example 2 to determine the optimal initial pH value. The results showed that when the initial pH value was 7.0, the antibacterial activity was the highest, and the diameter of the antibacterial zone was 18.9mm. See the results. Fig.11 .

[0075] It can be seen that through the above single factor experiment, the optimal fermentation conditions for Bacillus Velezii GS33 to produce antibacterial active substances are as follows: fermentation time is 24h, inoculation size is 2.5%, seed liquid age is 16h, fermentation temperature is 37℃, initial pH is 7.0, and liquid volume is 30mL / 100mL.

[0076] In summary, the optimal fermentation conditions for Bacillus Velez GS33 to produce antibacterial active substances are as follows: when the liquid volume is 30mL / 100mL, the carbon source is yeast culture, the nitrogen source is cottonseed meal, the total amount of carbon and nitrogen sources is 5%, the mass ratio of carbon and nitrogen sources is 2:3, MnSO4 is 2.5%, the fermentation time is 24h, the inoculation size is 2.5%, the seed liquid age is 16h, the fermentation temperature is 37℃, and the initial pH is 7.0.

[0077] 3. Optimization and verification of fermentation conditions

[0078] 3. Optimization and verification of fermentation conditions of Bacillus velez GS33

[0079] The basic culture medium (1% glucose, 1% maltose, 1% peptone, 1% tryptone, 1% yeast extract), fermentation time of 24h, inoculation size of 1%, seed liquid age of 24h, fermentation temperature of 37℃, and liquid volume of 30mL / 100mL were used as the control (before optimization of fermentation process). The optimized fermentation conditions: when the liquid volume was 30mL / 100mL, the carbon source was yeast culture, the nitrogen source was cottonseed meal, the total amount of carbon and nitrogen sources was 5%, the ratio of carbon and nitrogen sources was 2:3, MnSO4 was 2.5%, the fermentation time was 24h, the inoculation size was 2.5%, the seed liquid age was 16h, the fermentation temperature was 37℃ and the initial pH value was 7.0 for verification (after optimization of fermentation process). The results showed that the diameter of the inhibition zone was 14.8mm before optimization and 19.4mm after optimization, which was 30.14% higher than the original conditions. The results are shown in Table 1. Fig.12 .

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Bacillus Velez Bacillus velezensis )GS33, its deposit number is GMCC NO.32069.

2. A bacterial agent containing the Bacillus Velez subtilis GS33 according to claim 1.

3. A fermentation product, characterized in that It is prepared by fermenting the Bacillus Velez GS33 described in claim 1 or the bacterial agent described in claim 2.

4. Any of the following uses of the Bacillus Velez GS33 of claim 1, the bacterial agent of claim 2, or the fermentation product of claim 3: 1) Used for preparing antibacterial drugs; 2) Used as feed additive; 3) Used in animal production.

5. The use according to claim 4, characterized in that: The antibacterial drug has antibacterial effects on Gram-positive bacteria, Gram-negative bacteria and fungi; Preferably, the antibacterial drug is effective against Escherichia coli ( Escherichia coli ),salmonella( Salmonella ) and Staphylococcus aureus ( Staphylococcus aureus ) has antibacterial effects.

6. A pharmaceutical composition, characterized in that The main active ingredient of the pharmaceutical composition is the Bacillus Velezii GS33 described in claim 1, the bacterial agent described in claim 2 or the fermentation product described in claim 3.

7. A feed additive, characterized in that The main active ingredient of the feed additive is the Bacillus Velez GS33 described in claim 1, the bacterial agent described in claim 2 or the fermentation product described in claim 3.

8. The fermentation method of Bacillus Velez GS33 according to claim 1, characterized in that: The carbon source of the fermentation medium is yeast culture, and the nitrogen source is cottonseed meal. The mass ratio of the carbon source to the nitrogen source is 1~3:2~4.

9. The fermentation method according to claim 8, characterized in that: 2%~4% MnSO4 was added to the fermentation medium.

10. The fermentation method according to claim 8 or 9, characterized in that: The seed liquid age is 16±2h, the fermentation temperature is 37±2℃, and the fermentation time is 24±3h.

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