An immune enhancer for aquatic animals and its use

Abstract

The application discloses an immune enhancer suitable for aquatic animals and application thereof, and the active component of the immune enhancer is bacillus velezensis BSG-2 and / or a fermentation product thereof, and the preservation number is CCTCC NO: M 20222096; the strain is isolated from the intestinal tract of healthy Micropterus salmoides, has good stress resistance, and has excellent probiotic property; when the strain is used in aquatic breeding, the strain can effectively improve the utilization of nutrition of the body, provides immune protection for the body when infected by bacterial and viral pathogens, reduces the mortality rate of infection, and has a good application prospect in fish breeding.

Description

Technical Field

[0001] This invention belongs to the field of microbial immunology technology, specifically relating to an immune enhancer suitable for aquatic animals. The active ingredient of the immune enhancer is Bacillus belyssus BSG-2 and / or its fermentation products. This immune enhancer can effectively promote fish growth, improve disease resistance, and reduce the likelihood of damage to aquatic animals. Background Technology

[0002] Fish and other aquatic products are important sources of protein. However, due to the increasing pursuit of intensive and high-density farming and the frequent use of drugs in aquaculture, the types and severity of diseases in aquaculture are becoming increasingly serious, severely hindering the healthy and sustainable development of the aquaculture industry. At the same time, insufficient feed utilization is common in aquaculture, leading to feed waste, environmental pollution, and increased farming costs, which greatly dampens farmers' enthusiasm. Therefore, improving nutrient utilization and safely and effectively preventing and controlling diseases are urgent problems that need to be solved in the development of aquaculture.

[0003] Probiotics are currently one of the most commonly used feed additives. They can help the body absorb nutrients by producing digestive enzymes. They can also be used in fermentation processes to transform large protein molecules in raw materials into easily absorbed smaller nutrient molecules. However, due to the scarcity of fish-derived probiotics, most companies adding probiotics to improve fish feed fermentation processes simply copy the livestock and poultry model, using engineered bacteria derived from livestock and poultry. Because these engineered bacteria cannot adapt well to and colonize the digestive tract of aquatic animals, the effects are often unsatisfactory. Research on the role of exogenous probiotics in regulating other fish species has been reported; however, some potential problems, such as the "uncomfortable" characteristics of probiotics from non-homologous species in the body, have not been assessed. For example, how well do probiotics from non-homologous species colonize the body, and whether the virulence genes of the probiotics themselves pose a safety threat to different fish species?

[0004] Other studies have shown that probiotics regulate nutrient utilization in the body and also participate in the body's mucosal immune response, thereby improving immunity and disease resistance. However, there are many types of probiotics, and their probiotic capabilities vary. Currently, Bacillus spp. are widely used probiotics due to their strong resistance and significant effects, and are therefore widely applied in aquaculture. Therefore, seeking high-performance fish-derived probiotic Bacillus spp. is urgently needed to improve nutrient utilization in fish and to safely and effectively control fish diseases, which has significant practical implications for fish farming. Summary of the Invention

[0005] In view of this, the present invention aims to provide a microbial immune enhancer suitable for aquatic animals. This microorganism can not only improve the nutritional utilization of fish, but also be used for the prevention and treatment of common harmful bacterial and viral pathogens in fish, so as to promote the healthy development of aquaculture.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] The first aspect of this invention provides an immune enhancer suitable for aquatic animals, the active ingredient of which is Bacillus velezensis BSG-2 and / or its fermentation products; the whole genome of this strain was sequenced to identify it as Bacillus velezensis, with the Latin scientific name Bacillus velezensis BSG-2, and this strain was deposited at the China Center for Type Culture Collection on December 29, 2022, with accession number CCTCC NO: M 20222096, and the deposit address is Wuhan University, Wuhan, China.

[0008] This strain was isolated from the intestine of California bass (Micropterus salmoides). As a symbiotic Bacillus derived from a homologous species, it can greatly reduce the repulsion between species, cause less damage to the homologous species, and facilitate its colonization in the intestine. Therefore, this strain is suitable for aquatic animals, especially California bass.

[0009] Among the aforementioned immune enhancers, Bacillus vesiculus BSG-2 can be in the form of lyophilized bacterial powder or bacterial suspension, and the fermentation product of Bacillus vesiculus BSG-2 can be in the form of fermentation broth, fermentation broth extract, or lyophilized powder of fermentation extract.

[0010] Furthermore, the aforementioned immune enhancers may also include carriers, protectants, adjuvants, and other substances that do not affect the activity of Bacillus belyssus or its fermentation products.

[0011] A second aspect of the present invention provides the application of this immune enhancer as a feed additive in the preparation of fish feed.

[0012] When using this immune enhancer to prepare fish feed, the following method can be referenced: Inoculate Bacillus vesiculosus BSG-2 into liquid culture medium, centrifuge and discard the supernatant, wash with PBS and resuspend the bacterial precipitate, spray the obtained bacterial suspension into the feed, and then feed it.

[0013] When microbial materials are used as feed additives, they must overcome the challenges of the gastrointestinal tract and the high-temperature processing of feed. Furthermore, long-term colonization of the body and its beneficial effects on health are prerequisites for evaluating the efficacy of probiotics. Therefore, probiotics need to possess good stress resistance to ensure, to a certain extent, their effective arrival and colonization in the intestines as additives. This invention conducted in vitro stress resistance experiments on Bacillus belyceca var. salina (BSG-2), including acid and heat resistance tests. The results showed that BSG-2 has good resistance to adverse environments, suggesting that it can effectively reach and colonize the intestines.

[0014] In addition, through analysis of the enzyme production capacity of Bacillus belyssus BSG-2, this invention found that Bacillus belyssus BSG-2 has a stable ability to produce digestive enzymes compared to other Bacillus species. Therefore, when it successfully colonizes the intestine, it has the potential to promote the body's nutrient utilization. Furthermore, in vivo regression experiments showed that feeding this strain can significantly reduce the feed conversion ratio and significantly increase the weight gain rate and specific growth rate.

[0015] A third aspect of the present invention provides the use of the immune enhancer in the preparation of medicaments for the prevention and / or treatment of bacterial and / or viral diseases in aquaculture.

[0016] In vitro antibacterial and antiviral experiments, as well as feeding and pathogen infection experiments with Bacillus vesiculosus, show that the Bacillus vesiculosus BSG-2 provided by this invention can provide immune protection for the body under bacterial and viral infections and reduce the mortality rate of pathogen infection. It can be seen that this strain can be used to prevent and treat bacterial and viral infectious diseases in both in vivo and in vitro environments.

[0017] In the above applications, the pathogens of the bacterial diseases include, but are not limited to, *Aeromonas hydrophila*, *Nocardia amberjack*, and *Edwards tarda*. In vitro experiments have demonstrated that *Bacillus belye* BSG-2 exhibits good antibacterial activity against *Aeromonas hydrophila*, *Nocardia amberjack*, and *Edwards tarda* in the in vitro environment. In an in vivo infection model of *Nocardia amberjack*, a major pathogenic bacterium affecting largemouth bass, *Bacillus belye* BSG-2 significantly reduced mortality and alleviated infection symptoms in largemouth bass.

[0018] In the above applications, the pathogens of the viral diseases include, but are not limited to, Largemouth bass virus (LMBV). In vitro antiviral experiments showed that incubation of Bacillus belye var. vegetans BSG-2 with the virus significantly reduced cell invasion, thereby decreasing the viral load in cells. In vivo viral infection experiments showed that Bacillus belye var. vegetans BSG-2 significantly reduced the mortality rate of LMBV infection and decreased the proportion of moderately and severely infected fish, while also significantly reducing the viral load in the spleen and intestines.

[0019] The fourth aspect of this invention provides the application of immune enhancers in improving feed utilization and / or promoting fish growth and / or enhancing fish disease resistance in largemouth bass. Through in vivo regression experiments on largemouth bass, it was found that Bacillus belyeis BSG-2 can improve nutrient utilization, specifically by increasing feed utilization and promoting fish growth. It can also increase the activity of innate immune enzymes in the serum of largemouth bass and provide a certain degree of immune protection when largemouth bass are infected with bacterial and viral diseases.

[0020] This invention relates to preservation instructions for biological materials:

[0021] Bacterial strain name: Bacillus belyssus BSG-2;

[0022] Latin name: Bacillus velezensis BSG-2;

[0023] Preservation institution: China Center for Type Culture Collection;

[0024] Accession number: CCTCC NO: M 20222096;

[0025] Deposit date: December 29, 2022;

[0026] Location of the collection: Wuhan University, Wuhan City, Hubei Province.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The microbial immune enhancer provided by the present invention has a good ability to resist adverse environments, so as an additive it can effectively reach the intestine and colonize therein;

[0029] (2) The microbial immune enhancer provided by the present invention is a symbiotic Bacillus isolated from the intestine of California bass, which has less adverse damage to homologous species and is conducive to its intestinal colonization.

[0030] (3) Compared with other Bacillus species obtained from the intestines of California bass, the Bacillus vesiculus BSG-2 provided by this invention has good ability to antagonize pathogens and produce digestive enzymes in vitro. At the same time, it helps resist frog iridovirus infection in cell experiments. In vivo experiments show that BSG-2 can effectively improve the body's nutritional utilization and enhance the body's nutritional metabolism. After feeding, it can increase the activity of innate immune enzymes in the serum of California bass, provide immune protection for the body under bacterial and viral infections, and reduce the mortality rate of pathogen infection. Attached Figure Description

[0031] Figure 1The results of physiological and biochemical identification and ANI analysis of Bacillus bereaves BSG-2 in Example 1 are shown. A represents the physiological and biochemical identification of BSG-2, and B represents the average nucleotide sequence (ANI) similarity analysis at the genomic level of Bacillus.

[0032] Figure 2 This is a statistical analysis of the in vitro antibacterial activity of Bacillus vesiculosus BSG-2 and other Bacillus species against Aeromonas hydrophila, Nocardia amberjack, and Edwardsiella tarda in Example 2.

[0033] Figure 3 This is an analysis of the stress resistance of Bacillus belyss BSG-2 and other isolated Bacillus species in Example 3. A represents the survival rate of different Bacillus species in acidic environments of pH 2 and 4, and B represents the survival rate of different Bacillus species after treatment at 100°C for 10 min.

[0034] Figure 4 This is a comparative analysis of the ability of Bacillus vesiculosus BSG-2 in Example 4 to produce amylase, cellulase, protease and lipase compared with other Bacillus species.

[0035] Figure 5 This is an analysis of the in vitro antiviral ability of Bacillus belyssus BSG-2 in Example 5, where A is a cell image (a, c control group; b, d Bacillus belyssus treatment group), and B is an analysis of LMBV load in cells.

[0036] Figure 6 This study analyzes the effects of Bacillus vesiculosus BSG-2 feeding on nutrient utilization and innate immune enzyme activity in largemouth bass in Example 6. A represents the feed conversion ratio analysis of the normal group and the Bacillus vesiculosus feeding group at 28 and 56 days; B represents the weight gain rate analysis of the normal group and the Bacillus vesiculosus feeding group at 28 and 56 days; C represents the specific growth rate analysis of the normal group and the Bacillus vesiculosus feeding group at 28 and 56 days; and D represents the statistical analysis of the serum lysozyme (LSZ), superoxide dismutase (SOD), and alkaline phosphatase (ACP) activities of the normal group and the Bacillus vesiculosus feeding group at 28 and 56 days.

[0037] Figure 7This is an analysis of the immune protection provided by Bacillus vesicles BSG-2 to the body after pathogen infection in Example 7. A shows the mortality rate after LMBV injection infection in the normal group and the Bacillus vesicles feeding group 28 days after administration; B shows the symptom phenotype of California bass in the normal group and the Bacillus vesicles feeding group 7 days after LMBV infection; C shows the viral load in the spleen and intestines of the normal group and the Bacillus vesicles feeding group 7 days after LMBV infection; D shows the mortality rate after Nocardia amberjack injection infection in the normal group and the Bacillus vesicles feeding group 56 days after administration; E shows the liver pathological changes (arrows point to Nocardia amberjack nodules) in the normal group and the Bacillus vesicles feeding group 10 days after Nocardia amberjack injection infection (56 days after administration). Detailed Implementation

[0038] To make the technical problems, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0039] Unless otherwise specified, the methods described in the following examples are conventional; the reagents and materials described are commercially available unless otherwise specified.

[0040] Example 1: Isolation and Identification of Bacillus belye BSG-2

[0041] The Bacillus belyceae BSG-2 strain provided by this invention was isolated from the intestinal sample of California bass from a fish farm in Xiantao City, Hubei Province (this farm had not fed commercially available Bacillus belyceae). The intestinal sample processing method was as follows: intestinal contents were removed, the intestine was rinsed thoroughly in PBS, and microorganisms were scraped from the intestinal wall with 1 mL of sterile PBS. The sample was then heated in an 80°C water bath for 10 min. Subsequently, the heat-treated intestinal microorganisms were plated onto BHI plates and cultured at 37°C for 24 h. Single colonies of different morphologies were randomly selected, and these colonies were continuously selected for expansion culture and preservation.

[0042] The finally isolated strain underwent whole-genome sequencing, followed by physiological and biochemical identification analysis and genome-level ANI analysis. Analysis results... Figure 1 As shown, the results clearly indicate that the strain is *Bacillus belye*. Figure 1 The sample (A, B) was named BSG-2 and deposited at the China Center for Type Culture Collection.

[0043] Example 2: Identification of the in vitro antibacterial activity of Bacillus belyssus BSG-2

[0044] This example compares the in vitro antibacterial activity of Bacillus belyss BSG-2 with other Bacillus species also isolated from the intestines of California bass, as detailed below:

[0045] (1) Obtaining other Bacillus species.

[0046] Intestinal samples were collected from California bass from 11 different farms in 5 cities (districts) of Hubei and Hunan provinces. The intestinal sample processing method was the same as in Example 1. The isolated strains were sequenced using 16S sequencing, and the number of Bacillus bacteria collected from different locations was counted, as shown in Table 1.

[0047] Table 1. Statistical table of other Bacillus species.

[0048]

[0049] (2) In vitro antibacterial experiment.

[0050] Common aquatic pathogens, namely Aeromonas hydrophila and Edwardsiella tarda, as well as Nocardia amberjack, a major pathogenic bacterium affecting largemouth bass, were used as representative pathogens. The in vitro antibacterial activity of this strain was compared with that of other Bacillus species using in vitro inhibition plates. The specific experimental steps were as follows:

[0051] ① Inoculate the Bacillus spp. culture solution to be tested into 10 mL of liquid nutrient medium and incubate at 37℃ for 200 rpm / min for 6 h. Adjust the bacterial concentration to 1×10⁻⁶. 8 cfu / mL.

[0052] ② Take 500 μL of a concentration of approximately 10 8 Add the pathogenic bacterial suspension at cfu / mL to 50mL of heated and melted solid BHI medium, mix well, pour into plates, and after the plates have dried, use a pipette to transfer 1μL of the Bacillus suspension (10... 8 The bacterial colony (cfu / mL) was inoculated into a plate of pathogenic bacteria, and then incubated upside down in a 37°C incubator. After 24 hours, the colony diameter (d) and the inhibition zone diameter (D) were counted, and the ratio of the two (D / d) was calculated.

[0053] The in vitro antibacterial activity of BSG-2 and other Bacillus strains against Aeromonas hydrophila, Edwardsiella tarda, and Nocardia amberjack is shown in the following results. Figure 2 As shown in the figure, different Bacillus strains have varying degrees of inhibitory ability against Aeromonas hydrophila, Edwardsiella tarda, and Nocardia amberjack, while strain BSG-2 has excellent inhibitory effects against all three common aquatic pathogens.

[0054] Example 3: Stress resistance assessment of Bacillus belyssus BSG-2

[0055] Given that microorganisms used as feed additives must overcome the challenges of the acidic environment of the gastrointestinal tract and the high-temperature processing of feed, and that long-term colonization of the body and its beneficial effects on health are prerequisites for probiotics to exert their effects, it is necessary to evaluate the acid and high-temperature resistance of Bacillus belyssum BSG-2. The specific process in this case is as follows:

[0056] (1) The bacterial strain was inoculated into 100 mL of liquid nutrient medium and cultured at 37℃ for 96 h at 200 rpm / min. After centrifugation at 8000 rpm / min for 5 min, the supernatant was discarded, and the bacterial cells were resuspended in PBS. The cells were treated with 200 μg / mL lysozyme for 10 min, washed three times with PBS, and the concentration of the spores in the final resuspended spores was adjusted to 1 × 10⁻⁶. 8 After obtaining the cfu / mL concentration, proceed to the next experimental step.

[0057] (2) Acid resistance test. To simulate the pH environment in the stomach, different pH treatment groups were set up with pH=2 and 4. PBS was used as the control group. The spore suspension was treated with pH=2, 4 and PBS for 2 h, respectively. Then, it was serially diluted and plated to calculate the spore survival rate.

[0058] (3) High temperature resistance test. The spore suspension was treated at 100℃ for 10 min, while the control group was not treated with high temperature. Then, the suspension was serially diluted and plated. The number of spores was counted to calculate the survival rate.

[0059] The survival rates of BSG-2 and five other Bacillus strains with strong overall antibacterial activity at pH 2 and 4 are shown in the table below. Figure 3 As shown in Figure A, the six Bacillus strains have different tolerances to low pH, while BSG-2 has excellent tolerance to low pH.

[0060] The survival rates of BSG-2 and the aforementioned Bacillus spp. after treatment at 100℃ are shown in the following results. Figure 3 As shown in Figure B, the six Bacillus strains have different resistance to high temperatures, while BSG-2 has good tolerance to high temperatures of 100℃.

[0061] Example 4: Enzyme production capacity analysis of Bacillus belyssus BSG-2

[0062] This example demonstrates the enzyme-producing ability of Bacillus belyssus BSG-2 using a plate enzyme production experiment. The specific procedure is as follows:

[0063] (1) Prepare the corresponding protein plates, starch plates, cellulose plates and fat plates in advance.

[0064] The formula for BHI medium is as follows: 4.0g of bovine brain extract powder, 4.0g of bovine heart extract powder, 5.0g of peptone, 16.0g of casein peptone, 5.0g of sodium chloride, 2.0g of glucose, 2.5g of disodium hydrogen phosphate, and 13.5g of agar are dissolved in 1L of ultrapure water and sterilized.

[0065] Preparation of protein plates: Dissolve skim milk in ultrapure water to prepare 2% milk liquid, and at the same time prepare BHI solid culture medium at twice the concentration and natural pH. Mix well and pour into plates.

[0066] Preparation of fat plates: Tributyrate agar medium was used, the formula of which includes 5g beef extract, 15g agar powder, 5g sodium chloride, 10g peptone, 20mL tributyrate, and 1000mL distilled water.

[0067] Preparation of starch plates: The starch concentration is 0.3%. After weighing the starch, add it to BHI solid medium and sterilize. Pour the medium into plates and allow it to reach the desired pH value.

[0068] Cellulose plates: K2HCO3 2.5g, Na2HPO4 2.5g, sodium carboxymethyl cellulose 10g, peptone 2g, yeast extract 0.5g, agar 15g, sterilized and poured into plates.

[0069] (2) Measurement.

[0070] Bacillus berberis bacterial suspension and five other Bacillus bacterial suspensions were inoculated onto starch plates and cultured at 37°C for 24 hours. An appropriate amount of iodine solution was then dropped onto the plate, and a transparent hydrolysis zone was formed around the colony.

[0071] Bacillus belye bacterial suspension and five other Bacillus bacterial suspensions were inoculated onto cellulose plates and incubated at 37°C for 24 h. The plates were stained with Congo red (1 mg / mL) at room temperature for 1 h, destained with 1 M NaCl (58.4) for 1 h, and then the background was turned blue with 1 M HCl, revealing a clear transparent zone.

[0072] Bacillus bellis bacterial suspension and five other Bacillus bacterial suspensions were inoculated into protein plates and fat plates respectively, and the diameter of the transparent zone was directly counted.

[0073] The specific method is to use a pipette to transfer 1 μL of Bacillus bacterial suspension after the plate has dried (10 μL). 8 The bacteria were inoculated into enzyme plates with a concentration of cfu / mL, and then incubated upside down in a 37°C incubator. After 24 hours, the colony diameter (d) and the diameter of the clear zone (D) were counted, and the ratio of the two (D / d) was calculated to statistically analyze the enzyme production capacity of Bacillus.

[0074] The results of BSG-2 and five other Bacillus strains in the digestive enzyme identification plate assay are shown below. Figure 4As shown in the figure, several different Bacillus strains have different abilities to produce amylase, cellulase, protease and lipase, while BSG-2 has excellent ability to produce lipase.

[0075] Example 5: Identification of in vitro antiviral activity of Bacillus belyssus BSG-2

[0076] This embodiment uses frog iridovirus as an example and employs two virus inoculation methods to conduct in vitro antiviral experiments on strain BSG-2, specifically including the following steps:

[0077] (1) Inoculate a single colony into 10 mL of liquid nutrient medium and grow it to 10^ 8 Bacterial culture was collected at cfu / mL. In vitro antiviral experiments were conducted in EPC cells. Cells were seeded into 24-well plates one day in advance, and the next step of the experiment was performed once the cells were fully colonized.

[0078] (2) The first method of virus inoculation is co-treatment. The bacterial culture and frog iris virus culture were mixed at a ratio of 1:1 and added to the cell culture plate. After 2 hours, the virus mixture was removed and the plate was treated with gentamicin (1:1000 dilution) for 30 minutes. After washing with PBS 3 times, the plate was inoculated into normal M199 medium and cultured in an incubator at 28°C for 12 hours.

[0079] (3) The second method of virus inoculation is the pre-treatment group. The bacterial culture and frog iris virus were mixed at a ratio of 1:1 and placed in a 15 mL centrifuge tube for co-incubation at 28°C for 2 h. After incubation, the mixture was filtered through a 0.22 μm filter membrane. The filtered virus solution was added to a cell culture plate. After 2 h, the virus solution was removed, washed 3 times with PBS, and then incubated in normal M199 medium at 28°C for 12 h.

[0080] (4) After the above culture is completed, observe the cell state under a microscope, collect the cells and extract the cell DNA, and use specific primers to detect the LMBV load in the cells (see Table 2 for probe primers).

[0081] Table 2. Primers for frog iridovirus-specific probes

[0082]

[0083] Test results are shown Figure 5 This indicates that, regardless of whether it is in the co-treatment group or the pre-treatment group, co-incubation of Bacillus belye BSG-2 with frog iridovirus can significantly reduce the cell death damage caused by the virus, reduce viral invasion, and thus reduce the viral load in the cells.

[0084] Example 6: Live feeding of Bacillus belyssus BSG-2

[0085] In this embodiment, Bacillus belye BSG-2 was fed as a supplementary feed to detect the effect of Bacillus belye on the nutrient utilization of largemouth bass. The experimental procedure is as follows:

[0086] (1) Inoculate the bacterial strain into 10 mL of liquid nutrient medium and wait for it to grow to 10 mL. 8 The bacterial cells were transferred to 500 mL of culture medium at CFU / mL and cultured at 37°C for 96 h at 200 rpm / min. After the culture, the spore production rate was measured, the bacterial suspension was collected and centrifuged at 8000 rpm / min for 5 min, the supernatant was discarded, the cells were washed three times with PBS and resuspended, and the number of cells was counted under a microscope.

[0087] (2) Dilute the Bacillus berberis BSG-2 bacterial solution obtained in step (1) and spray it onto the feed to ensure a final concentration of 10. 7 A feeding experiment was conducted on largemouth bass using cfu / g. The bass were weighed before feeding to record their initial weight. They were weighed again after 28 and 56 days of feeding to analyze their feed conversion ratio, weight gain rate, and specific growth rate.

[0088] Analysis results as follows Figure 6 As shown in the results, after 28 and 56 days of dietary feeding, compared with the normal group (fed only basal feed, with other operations the same as the Bacillus vesiculosus BSG-2 feeding group), the feed conversion ratio (FCR) of the BSG-2 feeding group was significantly lower in both the 28-day and 56-day periods. Specifically, the FCR of the normal group was approximately 1.06 at 28 days, while the FCR of the BSG-2 group was 0.95. In the 56-day feeding period, the FCR of the normal group was approximately 1.14, while the FCR of the BSG-2 group was 1.07. This indicates that the absorption and conversion efficiency between food and body weight differs at different growth stages in largemouth bass. Simultaneously, compared with the normal group, the weight gain rate and specific growth rate of the BSG-2 feeding group were significantly higher. These results indicate that feeding with this strain of Bacillus vesiculosus BSG-2 can effectively improve nutrient utilization and thus promote the growth of largemouth bass.

[0089] Meanwhile, the detection of lysozyme (LSZ), superoxide dismutase (SOD), and alkaline phosphatase (ACP) in serum revealed that, compared with the normal group, the 28-day and 56-day BSG-2 feeding groups showed increased activity of the innate immune enzymes LSZ and SOD in serum, while the activity of ACP enzyme only increased significantly at 28 days and showed no significant difference at 56 days. This indicates that feeding with this strain of Bacillus belysin BSG-2 can promote the body's immune response to a certain extent.

[0090] Example 7: Immunoprotective effect of Bacillus belycetamol BSG-2 during pathogen infection

[0091] Based on Example 6, frog iridovirus and yellowtail nocardiosis were administered to the Bacillus belysus BSG-2 feeding group and the normal group 28 and 56 days after feeding, respectively. Mortality rate, pathological symptoms, and tissue load were recorded.

[0092] The method of viral infection is as follows: pre-enriched LMBV virus in the laboratory, and detection of the virus's TCID. 50 10 -6.4 Both the Bacillus baileyi BSG-2 feeding group and the normal group were injected with 100 μL of LMBV virus suspension, while their respective control groups were injected with an equal volume of M199 culture medium. The physiological status of the largemouth bass and the mortality rate were observed and recorded after injection.

[0093] The method of bacterial infection is as follows: adjust the concentration of Nocardia amberjack bacterial solution to 10. 5 cfu / mL, after centrifugation, the supernatant was discarded, and the suspension was resuspended in PBS. The Bacillus berberis BSG-2 feeding group and the normal group were injected with 100 μL of Nocardia suspension, and the respective control groups were injected with an equal amount of PBS.

[0094] The experimental results are shown in Figure 7 The specific analysis is as follows:

[0095] In the established LMBV injection infection model of largemouth bass, the mortality rate of the Bacillus baileyi BSG-2-fed group was significantly lower (40%) than that of the normal group (fed a basal diet), while the mortality rate of the normal group was 60%. Figure 7 A) Observation of symptoms in largemouth bass 7 days after viral infection revealed that, compared with the normal group, the group fed with Bacillus vesiculosus BSG-2 had milder clinical viral infection symptoms. Simultaneously, sampling of infected spleen and intestines revealed significantly reduced LMBV viral load in the intestinal and spleen tissues. These results indicate that feeding largemouth bass with this strain of Bacillus vesiculosus BSG-2 has a certain protective effect against viral infection.

[0096] In the constructed Nocardia infection model, compared with the normal group (fed a basal diet), the mortality rate of yellowtail fed with Bacillus belyceae BSG-2 was significantly lower, i.e., the mortality rate of the normal group was 36%, while the mortality rate of the Bacillus belyceae BSG-2 fed group was 64%. Figure 7D). The primary target organ for Nocardia amberjack infection is the liver. Therefore, pathological analysis of liver sections from infected largemouth bass revealed that the normal group exhibited greater Nocardia amberjack invasion, with multiple granulation tissue formations within the liver tissue (red arrows indicate granulation tissue proliferation caused by pathogen invasion). The Bacillus subtilis (BSG-2) feeding group showed milder bacterial infection, with granulation tissue mainly concentrated on the surface of the liver tissue. Figure 7 E). The above results indicate that, in cases of Nocardia infection, feeding California bass with Bacillus vesiculosus BSG-2, derived from California bass itself, provided by this invention, can provide California bass with a certain degree of immune protection, thereby resisting the threat of bacterial pathogens.

[0097] In summary, the embodiments of this invention comprehensively evaluated the performance of Bacillus vesiculosus provided by this invention through in vitro antibacterial and antiviral function analysis, live feeding experiments on largemouth bass to analyze the nutritional utilization of largemouth bass after feeding with Bacillus vesiculosus BSG-2, and further injection infection experiments with bacteria and viruses. The experimental results show that Bacillus vesiculosus BSG-2 provided by this invention can improve the body's nutritional utilization and simultaneously generate immune protection in largemouth bass infected with bacterial and viral pathogens. At the same time, the probiotic Bacillus vesiculosus from the same species participates in regulating the body's nutritional immunity while avoiding the safety issues of non-homologous species, thus providing more effective immune protection. Therefore, Bacillus vesiculosus BSG-2 provided by this invention has good application prospects in fish farming.

[0098] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. An immune enhancer suitable for aquatic animals, characterized in that, The active ingredient of the immune enhancer is Bacillus belyssus BSG-2, and the preservation number of Bacillus belyssus BSG-2 is CCTCC NO: M 20222096.

2. The use of the immune enhancer as a feed additive in the preparation of fish feed according to claim 1.

3. The application according to claim 2, characterized in that, The method for preparing the fish feed is as follows: Bacillus belye BSG-2 is inoculated into liquid culture medium and cultured for 96 hours. After centrifugation and discarding of supernatant, the bacteria are washed with PBS, and the resulting bacterial resuspended is sprayed onto the feed.

4. The application according to claim 2 or 3, characterized in that, The fish in question is a California bass.

5. The use of the immune enhancer of claim 1 in the preparation of a drug for the prevention and / or treatment of bacterial and / or viral diseases in aquaculture, wherein the pathogen of the bacterial disease is Aeromonas hydrophila, Nocardia amberjack, or Edwardsiella tarda, and the pathogen of the viral disease is California bass iridovirus.

6. The application according to claim 5, characterized in that, The fish used in the aquaculture is California bass.

7. The use of the immune enhancer according to claim 1 in improving fish feed utilization and / or promoting fish growth.

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