Bacillus velezensis and preparation method and application of preparation thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses bacillus velezensis and a preparation method and application of a preparation of the bacillus velezensis, the bacillus velezensis is bacillus velezensis MS-3, the bacillus velezensis MS-3 is preserved in China General Microbiological Culture Collection Center on January 16, 2025, the preservation number is CGMCC No.33440, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bacillus velezensis MS-3 disclosed by the invention has the effects of promoting the growth of the micropterus salmoides, improving the digestive ability and the antioxidant ability and improving the immunity of the micropterus salmoides.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a Bacillus velezensis and a preparation method and application thereof. Background Art

[0002] In recent years, while the aquaculture industry has developed rapidly, various diseases have also broken out, causing significant losses. According to statistics, there are currently more than 100 kinds of aquaculture animal diseases that can cause economic losses in China, directly resulting in economic losses of tens of billions of yuan. The culture of Micropterus salmoides and Channa argus is frequently affected by diseases, and the pathogen types are complex and diverse, including bacterial diseases such as septicemia and skin ulcer caused by Aeromonas hydrophila, ascites disease caused by Citrobacter freundii, and nodular disease caused by Nocardia seriolae, viral diseases such as iridovirus and rhabdovirus, and parasitic diseases caused by Trichodina and Ichthyophthirius multifiliis. Probiotics are currently considered an effective and environmentally friendly alternative to various reagents and antibiotics in the prevention of fish diseases due to their unique physiological functions, lack of drug resistance, and safety. In recent years, probiotics have been widely used in aquaculture. Finding a new, safe, and green antibiotic alternative has become one of the hotspots in current aquaculture research. Among them, Bacillus has been widely used in aquaculture because it can produce spores to resist extreme environments and is easy to store and transport during industrial production.

[0003] Microecological preparations achieve the purpose of preventing and controlling diseases of aquaculture animals by "using bacteria to control bacteria". On the one hand, beneficial microorganisms produce metabolites and physiologically active substances to inhibit or kill pathogenic bacteria. At the same time, they compete with pathogenic bacteria for nutrients in the aquaculture water body and the intestines of aquaculture animals, occupy ecological niches, and form dominant flora to achieve the purpose of preventing and controlling diseases. On the other hand, beneficial microorganisms produce a variety of digestive enzymes to help with the metabolism of proteins, carbohydrates, and fats, improve the utilization rate and conversion rate of feed, and achieve the purpose of reducing resource consumption and increasing production.

[0004] Chinese Patent Publication No. CN102586144A discloses a Bacillus pumilus, a probiotic preparation, and its preparation method and application. Bacillus pumilus LV149 was deposited with the China Center for Type Culture Collection (CCTCC) on November 23, 2011, with the deposit number: CCTCC NO: M2011411. The Bacillus pumilus LV149 of this invention has strong extracellular protease, lipase, and amylase activities, has a wide range of inhibitory effects on Vibrio, and does not have hemolytic activity. Using this bacterium as a fermentation strain, through solid-state fermentation, drying, and pulverization, a Bacillus pumilus probiotic preparation with Bacillus pumilus LV149 as the active ingredient is obtained. Adding it to the feed of penaeid shrimp and feeding the shrimp can not only inhibit the growth of pathogenic Vibrio in the shrimp intestine, reduce the occurrence of Vibrio disease in shrimp, but also promote the growth of shrimp, reduce the feed coefficient, improve the commercial quality, shorten the breeding cycle, thereby reducing breeding risks and breeding costs, and having high biological safety, and has a wide application prospect in aquaculture.

[0005] However, at present, no microorganisms and preparations that can promote the growth of Micropterus salmoides and improve the disease resistance of Micropterus salmoides have been found. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a Bacillus velezensis and its preparation method and application.

[0007] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0008] The first technical solution of the present invention provides a Bacillus velezensis, which is Bacillus velezensis MS-3, deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number: CGMCC No. 33440, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The second technical solution of the present invention provides a Bacillus velezensis preparation, which contains the Bacillus velezensis MS-3 as described in claim 1, and the viable count of the Bacillus velezensis preparation is 1×10 9 CFU / mL~1×10 10 CFU / mL.

[0010] The third technical solution of the present invention provides a preparation method of a Bacillus velezensis preparation, including the following steps:

[0011] Bacillus velezensis MS-3 was subjected to liquid fermentation to obtain a Bacillus velezensis fermentation broth; the fermentation medium used for liquid fermentation consisted of the following components: glucose 2% W / V, soybean meal 1% W / V, K2HPO4 0.1% W / V, NaCl 0.5% W / V, MgSO4 0.08% W / V.

[0012] The fourth technical solution of the present invention provides an application of a Bacillus velezensis preparation in the preparation of a feed additive for Micropterus salmoides.

[0013] Compared with the prior art, Bacillus velezensis MS-3 of the present invention has a significant effect in promoting the growth of Micropterus salmoides. Bacillus velezensis MS-3 was subjected to liquid fermentation and added to the feed at a concentration of 10 6 cfu / g (the viable bacteria content per gram of feed) to feed Micropterus salmoides. It was found that after 30 days of feeding, compared with the control group without adding probiotics, the average body weight growth rate of Micropterus salmoides in the experimental group fed with the feed added with Bacillus velezensis MS-3 increased by 15.3%. At the same time, the specific growth rate was significantly increased and the feed conversion rate was significantly decreased (P < 0.05). The intestinal trypsin, lipase and amylase of Micropterus salmoides in the probiotic MS-3 experimental group were all significantly higher than those in the control group (P < 0.05). It has a significant effect in promoting the growth of Micropterus salmoides and improving the digestive ability of Micropterus salmoides. In addition, compared with the control group without adding probiotics, the superoxide dismutase and catalase in the liver of Micropterus salmoides in the Bacillus velezensis MS-3 experimental group were significantly higher than those in the control group, and the content of malondialdehyde in the liver was significantly decreased (P < 0.05). At the same time, the activities of serum acid phosphatase, serum alkaline phosphatase, serum nitric oxide, serum immunoglobulin M and serum lysozyme were also significantly increased. Detailed implementation mode

[0014] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the following examples are used to further elaborate on the present invention in detail. The specific examples described herein are only used to explain the present invention and are not used to limit the invention.

[0015] Example 1: Isolation and screening of Bacillus velezensis MS-3

[0016] Bacillus velezensis MS-3 was isolated from the intestine of Micropterus salmoides. The specific isolation and screening process is as follows:

[0017] The juvenile Micropterus salmoides and Channa argus were fished out and put into a bucket mixed with eugenol (1:10000) for anesthesia. After anesthesia, they were taken out and placed in a laminar flow hood for subsequent steps. The surface of the experimental fish was disinfected with 75% alcohol cotton balls. The fish intestine was taken out in the laminar flow hood. After washing the surface fat and digestive substances in the intestine with PBS for multiple times, the intestine was cut into pieces and put into a centrifuge tube, and 1 mL of PBS was added. After the intestinal tissue was broken, the homogenate was spread on LB, YPD, and MRS plates at different dilution multiples for screening. After culturing for 24 h, single colonies were picked and inoculated into the corresponding LB, YPD, and MRS liquid media for amplification. Through multiple streak purifications, purified single colonies were obtained.

[0018] The bacterial DNA was extracted by the boiling method and the 16S rRNA gene was amplified. The PCR reaction system (25 μL): 2×Mix ExTaq 12.5 μL, 27F and 1492R each 1 μL, bacterial DNA 1 μL, ddH2O 9.5 μL. The reaction conditions were 96°C for 5 min; 96°C for 30 s, 55°C for 45 s, 72°C for 90 s, for 33 cycles; 72°C for 10 min. The amplification quality was detected by 1% agarose gel electrophoresis. The successfully amplified PCR products were sent for sequencing, and the sequencing results were:

[0019]

[0020] The sequencing results were compared, and a phylogenetic tree (Neighbor-Joining method) was constructed using MEGA 7 software with the bacterial sequences of different species having the highest homology among them; a biochemical identification kit was used to conduct biochemical identification of the samples, and the morphology of the isolated bacteria was observed by Gram staining. The biochemical characteristics of MS-3 were consistent with the descriptions in Bergey's Manual of Systematic Bacteriology; the Gram staining observation was positive; MS-3 had high homology with Bacillus velezensis.

[0021] Morphological characteristics: MS-3 was rod-shaped, with a size of (1.0 - 1.5) μm × (1.0 - 1.5) μm. The colonies were milky white and round, with a smooth surface, slightly convex, viscous texture, and large oval shape. It was named Bacillus velezensis MS-3 and was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025. The deposit number was: CGMCC No. 33440, and the deposit address was No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0022] Example 2. Bioassay of the growth performance and related enzyme activities of Micropterus salmoides by Bacillus velezensis MS-3

[0023] Cultivation of Bacillus velezensis: Bacillus velezensis MS-3 was cultivated in the order of solid plate, liquid seed, and liquid fermentation. The solid and seed media were conventional nutrient media or broth media; the formula of the fermentation medium was: glucose 2% W / V, soybean meal 1% W / V, K2HPO4 0.1% W / V, NaCl 0.5% W / V, MgSO4 0.08% W / V. After the obtained Bacillus velezensis fermentation broth was serially diluted 10-fold with physiological saline, 100 μl was taken and spread on an LB plate and cultured at 37 °C for 24 h, and the viable cell count was calculated to be 1×10 8 cfu / mL.

[0024] The largemouth bass used in the experiment was purchased from Guangdong Yangyang Fisheries Co., Ltd., with a body weight of about 12 g. It was temporarily raised in the breeding base for one week for domestication, and the formal experiment was carried out when the body weight reached about 14 g. The volume of the fish tank was 80 L, and 60 L of water was put into it. 100 largemouth bass were stocked in each fish tank. The experiment was divided into a control group and an experimental group, with three parallels in each group. The experimental group was fed the commercial largemouth bass feed (Shangshang Yijian breeding treasure feed) added with Bacillus velezensis MS-3 every day, and the control group was only fed the above commercial largemouth bass feed without bacteria; Preparation method of the largemouth bass feed in the experimental group: Take 80 mL of the above Bacillus velezensis fermentation broth, appropriately dilute it with sterile normal saline and evenly spray it on the surface of 1000 g of feed, stir evenly to ensure that the viable bacteria concentration in the feed is 8×10 6 CFU / g. The experiment lasted for 30 days, and the feed was fed 2 times a day, and 30% of the water was changed every day. After 30 days, the average body weight increase of the largemouth bass was counted, and antioxidant enzymes, digestive enzymes and immune-related enzymes were measured; The measurement results are shown in Table 1 - Table 4.

[0025] Table 1 Effects of MS-3 on the growth performance of largemouth bass

[0026]

[0027] Table 2 Effects of MS-3 on the intestinal digestive enzyme activities of largemouth bass

[0028]

[0029] Table 3 Effects of MS-3 on the liver antioxidant enzyme activities of largemouth bass

[0030]

[0031] Table 4 Effects of MS-3 on the serum non-specific immune enzyme activities of largemouth bass

[0032]

[0033] As can be seen from Table 1 and Table 2, after 30 days of feeding, Bacillus velezensis MS-3 significantly increased the average weight gain of Micropterus salmoides, which was 15.3% higher than that of the control group. In addition, the activities of trypsin, lipase and amylase in the intestine of Micropterus salmoides in the MS-3 probiotic treatment group were significantly higher than those in the control group (P<0.05). Furthermore, as can be seen from Table 3, the activities of superoxide dismutase and catalase in the liver of Micropterus salmoides in the Bacillus velezensis MS-3 experimental group were significantly higher than those in the control group, and the malondialdehyde content in the liver showed a significant decrease (P<0.05). Finally, as can be seen from Table 4, the activities of acid phosphatase, alkaline phosphatase, nitric oxide, immunoglobulin M and lysozyme in the serum of Micropterus salmoides in the Bacillus velezensis MS-3 experimental group were significantly increased compared with those in the control group (P<0.05).

[0034] In summary, Bacillus velezensis MS-3 can promote the growth of Micropterus salmoides, improve its digestive ability and antioxidant capacity, and enhance its immunity.

[0035] The technical solution of the present invention is not limited to the above specific embodiments, and any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A Bacillus velezensis, characterized in that, It is Bacillus velezensis MS-3, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number: CGMCC No. 33440, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A Bacillus velezensis preparation, characterized in that, Containing Bacillus velezensis MS-3 as described in claim 1, the viable count of the Bacillus velezensis preparation is 1×10 9 CFU / mL~1×10 10 CFU / mL.

3. A preparation method of the Bacillus velezensis preparation according to claim 2, characterized in that, It includes the following steps: Perform liquid fermentation on Bacillus velezensis MS-3 to obtain a Bacillus velezensis fermentation broth; the fermentation medium used for liquid fermentation consists of the following components: glucose 2% W / V, soybean meal 1% W / V, K2HPO4 0.1% W / V, NaCl 0.5% W / V, MgSO4 0.08% W / V.

4. Use of a Bacillus velezensis preparation as described in claim 2 in the preparation of a feed additive for Micropterus salmoides.

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