Freeze-dried spermaceti powder as well as preparation method and application thereof

Abstract

The invention relates to spermaceti freeze-dried powder as well as a preparation method and application thereof, and belongs to the technical field of microbial culture. The invention provides a culture medium of spermaceti, which takes water as a solvent and comprises cane sugar, peptone, yeast powder, yeast extract, sodium thioglycollate, sodium chloride, cysteine hydrochloride, resazurin, sodium bicarbonate, hemin and vitamin K. The freeze-dried powder of the spermaceti can promote growth and development of tilapia mossambica, activate specific immune response and a non-specific immune network of the tilapia mossambica and regulate and control functions of intestinal attached flora of the tilapia mossambica, so that the immune response capability and the disease resistance of the tilapia mossambica are enhanced, and the freeze-dried powder of the spermaceti has the advantages of being high in safety and remarkable in growth promoting

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, and particularly relates to a freeze-dried powder of Cetobacterium somerae, a preparation method thereof and an application thereof. Background Art

[0002] Streptococcus agalactiae ( Streptococcus agalactiae ) causes streptococcosis in fish, which has brought serious harm to the aquaculture industry. The incidence rate is increasing day by day, and the incidence rate in some aquaculture areas is as high as 100%. Using antibiotic drugs is one of the effective means to prevent and control streptococcosis in fish at present. However, the input of excessive antibiotic drugs has brought increasingly prominent negative effects to the aquaculture industry, resulting in problems such as increased drug resistance of pathogenic bacteria and drug residues in aquatic products.

[0003] Fish fatty liver is a common nutritional disease in artificially cultured fish, which can damage the liver function of fish, slow down swimming, reduce appetite, and significantly decline the meat quality and disease resistance of fish. The main causes include overfeeding and unbalanced nutritional intake. Excessive energy intake causes excessive accumulation of liver lipids in cultured fish. Coupled with the very low ability of fish to utilize carbohydrates, fish fatty liver is induced. The symptoms of fish fatty liver are relatively hidden in the early stage of the disease, and it will seriously affect the growth and development and disease resistance of cultured fish during the development process, thus causing huge economic losses to aquaculture and severely restricting the development of the aquaculture industry.

[0004] Probiotics are widely used in the aquaculture industry and have functions such as regulating the aquaculture environment, enhancing the host immunity and improving metabolism. They have become a green prevention and control means to replace antibiotics. At present, the probiotic preparations applied in the aquaculture industry mainly include microbial preparations derived from livestock and poultry such as Lactobacillus, Bacillus, Saccharomyces cerevisiae, EM bacteria, etc. They are not only not suitable for stable proliferation in aquatic animals and aquaculture water bodies, but also cause new problems in aquaculture such as immune collapse. The prior art has not disclosed a fish-derived microbial preparation that can exert the functions of promoting growth and disease resistance and improving the aquaculture efficiency. Summary of the Invention

[0005] The purpose of the present invention is to provide a freeze-dried powder of Cetobacterium somerae, a preparation method thereof and an application thereof, so as to solve the problem in the prior art that there is a lack of a fish-derived microecological preparation with high effectiveness and safety and having the functions of promoting growth and disease resistance.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides a culture medium for Cetobacterium, wherein the culture medium uses water as a solvent and comprises components in the following concentrations: sucrose 10 - 30 g / L, peptone 10 - 20 g / L, yeast powder 15 - 25 g / L, yeast extract 5 - 15 g / L, sodium thioglycollate 0.1 - 1 g / L, sodium chloride 1 - 4 g / L, cysteine hydrochloride 0.2 - 0.8 g / L, resazurin 0.0005 - 0.002 g / L, sodium bicarbonate 0.1 - 1 g / L, hemin 0.001 - 0.01 g / L, vitamin K 0.0001 - 0.001 g / L.

[0007] The present invention also provides the use of the above-mentioned culture medium in the preparation of freeze-dried powder of Cetobacterium.

[0008] The present invention also provides a method for preparing freeze-dried powder of Cetobacterium, comprising the following steps: (1) Mix Cetobacterium with the above-mentioned culture medium and culture for 24 - 60 h to obtain a Cetobacterium culture solution; (2) Centrifuge the Cetobacterium culture solution, take the precipitate to obtain bacterial cells, and mix the bacterial cells with a cryoprotectant to obtain a bacterial cell paste; (3) Dry the bacterial cell paste for 40 - 60 h to obtain the freeze-dried powder of Cetobacterium.

[0009] Preferably, the volume ratio of Cetobacterium to the culture medium in step (1) is 1 - 5:100.

[0010] Preferably, the temperature of the culture in step (1) is 28 - 40 °C, and the pH of the culture is 6.0 - 8.5.

[0011] Preferably, the rotation speed of the centrifugation in step (2) is 10000 - 15000 rpm, and the centrifugation time is 5 - 20 min.

[0012] Preferably, the cryoprotectant in step (2) comprises one or more of an aqueous solution of lactose, an aqueous solution of soluble starch, an aqueous solution of glycerol, an aqueous solution of sucrose, an aqueous solution of glucose, and an aqueous solution of trehalose; The concentration of the cryoprotectant is 10 - 120 g / L; The mass-volume ratio of the bacterial cells to the cryoprotectant is 1 g:1 - 5 mL.

[0013] Preferably, the drying in step (3) is freeze-drying, and the drying temperature is -60 - -30 °C.

[0014] The present invention also provides the freeze-dried powder of Cetobacterium prepared by the preparation method described in any one of the above.

[0015] The present invention also provides the use of the above-mentioned freeze-dried powder of Cetobacterium in the preparation of a preparation for promoting the growth of fish and / or improving the disease resistance of fish.

[0016] The present invention has the following technical effects and advantages: The freeze-dried powder of Cetobacterium prepared by the present invention can promote the growth and development of tilapia, enhance the specific immune response of tilapia, significantly improve the resistance of tilapia to Streptococcus agalactiae infection, and at the same time activate the non-specific immune network of tilapia to promote the production of immune active substances such as immunoglobulins and lysozymes, thereby enhancing the immune response ability and disease resistance of tilapia; it can also change the characteristic biomarkers of the intestinal flora of tilapia, regulate the function of the intestinal adherent flora of tilapia, improve the intestinal microecological environment of tilapia by promoting the growth of beneficial flora such as Bifidobacterium and Lactobacillus, enhance the intestinal barrier function, thereby reducing the invasion ability of pathogenic bacteria, and has the characteristics of high safety, significant growth promotion and disease resistance effects, and significantly improving the aquaculture benefit, thus providing a new idea for the healthy and sustainable development of aquaculture. Description of the Drawings

[0017] Figure 1 It is the screening result of the culture conditions of Cetobacterium NK01 strain, where A is the screening result of the carbon source type, B is the screening result of the nitrogen source type, C is the screening result of the inoculation amount of Cetobacterium seed liquid, D is the screening result of the culture temperature, and E is the screening result of the culture pH; Figure 2 It is the determination result of the growth physiology and disease resistance physiology indexes of each group of tilapia, where A is the determination result of the body weight of each group of tilapia, B is the determination result of the body length of each group of tilapia, C is the determination result of the blood glucose concentration of each group of tilapia, D is the determination result of the insulin content of each group of tilapia, E is the result of the carcass ratio of each group of tilapia, F is the result of the hepatosomatic index of each group of tilapia, G is the determination result of the serum triglyceride content of each group of tilapia, and H is the determination result of the serum total cholesterol content of each group of tilapia; in the figure, CK is the control group and NK is the Cetobacterium group; Figure 3 It is the determination result of the acetic acid content in the intestinal contents of each group of tilapia. In the figure, CK is the control group and NK is the Cetobacterium group; Figure 4 It is the cumulative survival rate of each group of tilapia. In the figure, CK is the control group and NK is the Cetobacterium group; Figure 5 It is the determination result of the immune indexes of each group of tilapia after intragastric administration of the freeze-dried powder of Cetobacterium. Among them, A is the determination result of the Streptococcus agalactiae IgM antibody level of each group of tilapia, B is the determination result of the immunoglobulin M level of each group of tilapia, C is the determination result of the complement C3 level of each group of tilapia, D is the determination result of the alkaline phosphatase level of each group of tilapia, and E is the determination result of the lysozyme level of each group of tilapia; in the figure, CK0 is the control group before challenge, NK0 is the Cetobacterium group before challenge, CK12 is the control group 12 h after challenge, and NK12 is the Cetobacterium group 12 h after challenge; Figure 6 Determination results of immune indexes of tilapia in each group after intragastric administration of sodium acetate. Among them, A is the determination result of Streptococcus agalactiae IgM antibody level of tilapia in each group, B is the determination result of immunoglobulin M level of tilapia in each group, C is the determination result of complement C3 level of tilapia in each group, D is the determination result of alkaline phosphatase level of tilapia in each group, and E is the determination result of lysozyme level of tilapia in each group; in the figure, CK0 is the control group before challenge, AC0 is the sodium acetate group before challenge, CK12 is the control group 12 h after challenge, and AC12 is the sodium acetate group 12 h after challenge; Figure 7 Cumulative survival rate of tilapia in each group after challenge. In the figure, CK is the control group, NK is the Cetobacterium group, and AC is the sodium acetate group; Figure 8 KEGG pathway enrichment analysis results of differentially expressed genes between the Cetobacterium group and the control group of tilapia; Figure 9 KEGG pathway enrichment analysis results of differentially expressed genes between the sodium acetate group and the control group of tilapia; Figures 10 to 11 Relative abundances of intestinal flora of tilapia in each group at the genus level before challenge. In the figure, CI is the intestinal mucosa of tilapia in the control group before challenge, NI is the intestinal mucosa of tilapia in the Cetobacterium group before challenge, AI is the intestinal mucosa of tilapia in the sodium acetate group before challenge, CC is the intestinal content of tilapia in the control group before challenge, NC is the intestinal content of tilapia in the Cetobacterium group before challenge, and AC is the intestinal content of tilapia in the sodium acetate group before challenge; Figure 12 Comparison results of adherent flora in intestinal mucosa of tilapia in each group before challenge. In the figure, CI is the intestinal mucosa of tilapia in the control group before challenge, NI is the intestinal mucosa of tilapia in the Cetobacterium group before challenge, AI is the intestinal mucosa of tilapia in the sodium acetate group before challenge, CC is the intestinal content of tilapia in the control group before challenge, NC is the intestinal content of tilapia in the Cetobacterium group before challenge, and AC is the intestinal content of tilapia in the sodium acetate group before challenge; Figure 13 Comparison results of adherent flora in intestinal contents of tilapia in each group before challenge. In the figure, CI is the intestinal mucosa of tilapia in the control group before challenge, NI is the intestinal mucosa of tilapia in the Cetobacterium group before challenge, AI is the intestinal mucosa of tilapia in the sodium acetate group before challenge, CC is the intestinal content of tilapia in the control group before challenge, NC is the intestinal content of tilapia in the Cetobacterium group before challenge, and AC is the intestinal content of tilapia in the sodium acetate group before challenge; Figure 14KEGG pathway comparison results of the intestinal mucosal adherent flora among groups of tilapia before challenge. Among them, A shows the KEGG pathway comparison results of the intestinal mucosal adherent flora between the Cetobacterium group and the control group of tilapia before challenge, and B shows the KEGG pathway comparison results of the intestinal mucosal adherent flora between the sodium acetate group and the control group of tilapia before challenge; Figure 15 Relative abundances of intestinal flora of tilapia in each group at the genus level after challenge. In the figure, CS represents the intestinal contents of tilapia in the control group after challenge, NS represents the intestinal contents of tilapia in the Cetobacterium group after challenge, and AS represents the intestinal contents of tilapia in the sodium acetate group after challenge; Figure 16 Comparison results of the adherent flora in the intestinal contents of tilapia in each group after challenge. In the figure, CS represents the intestinal contents of tilapia in the control group after challenge, NS represents the intestinal contents of tilapia in the Cetobacterium group after challenge, and AS represents the intestinal contents of tilapia in the sodium acetate group after challenge. Detailed implementation methods

[0018] The present invention provides a culture medium for Cetobacterium. The culture medium uses water as a solvent and includes components with the following concentrations: sucrose 10 - 30 g / L, preferably 20 g / L; peptone 10 - 20 g / L, preferably 15 g / L; yeast powder 15 - 25 g / L, preferably 20 g / L; yeast extract 5 - 15 g / L, preferably 10 g / L; sodium thioglycollate 0.1 - 1 g / L, preferably 0.5 g / L; sodium chloride 1 - 4 g / L, preferably 2.5 g / L; cysteine hydrochloride 0.2 - 0.8 g / L, preferably 0.5 g / L; resazurin 0.0005 - 0.002 g / L, preferably 0.001 g / L; sodium bicarbonate 0.1 - 1 g / L, preferably 0.4 g / L; hemin 0.001 - 0.01 g / L, preferably 0.005 g / L; vitamin K 0.0001 - 0.001 g / L, preferably 0.0005 g / L.

[0019] The present invention also provides the application of the above-mentioned culture medium in the preparation of freeze-dried powder of Cetobacterium.

[0020] The present invention also provides a preparation method of freeze-dried powder of Cetobacterium, including the following steps: (1) Mix Cetobacterium with the above-mentioned culture medium and culture for 24 - 60 h, preferably 48 h, to obtain a Cetobacterium culture solution; (2) Centrifuge the Cetobacterium culture solution, take the precipitate to obtain bacterial cells, and mix the bacterial cells with a cryoprotectant to obtain a bacterial sludge; (3) Dry the bacterial sludge for 40 - 60 h, preferably 55 h, to obtain freeze-dried powder of Cetobacterium.

[0021] In the present invention, the Cetobacterium is preferably Cetobacterium sp. NK01 strain, which is purchased from Guangdong Provincial Microbial Culture Collection Center (GDMCC), and the preservation number is GDMCC No. 61502.

[0022] In the present invention, the volume ratio of the Cetobacterium described in step (1) to the culture medium is 1-5:100, preferably 2:100.

[0023] In the present invention, the cultivation described in step (1) is preferably anaerobic cultivation; the temperature of the cultivation is 28-40 °C, preferably 40 °C; the pH of the cultivation is 6.0-8.5, preferably 7.0.

[0024] In the present invention, the rotation speed of the centrifugation described in step (2) is 10000-15000 rpm, preferably 12000 rpm; the time of the centrifugation is 5-20 min, preferably 10 min.

[0025] In the present invention, the cryoprotectant described in step (2) includes one or more of lactose aqueous solution, soluble starch aqueous solution, glycerol aqueous solution, sucrose aqueous solution, glucose aqueous solution and trehalose aqueous solution, preferably glucose aqueous solution; The concentration of the cryoprotectant is 10-120 g / L, preferably 100 g / L; The mass-volume ratio of the bacterial cells to the cryoprotectant is 1 g:1-5 mL, preferably 1 g:4 mL.

[0026] In the present invention, the drying described in step (3) is preferably freeze-drying, and the temperature of the drying is -60 to -30 °C, preferably -45 °C.

[0027] The present invention also provides a freeze-dried powder of Cetobacterium prepared by the preparation method described in any one of the above.

[0028] The present invention also provides the application of the freeze-dried powder of Cetobacterium in the preparation of a preparation for promoting the growth of fish and / or improving the disease resistance of fish.

[0029] In the present invention, the fish is preferably tilapia, and the disease resistance is preferably resistance to Streptococcus disease.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

[0031] Among the test materials of the present invention, the Cetobacterium sp. NK01 strain is purchased from Guangdong Provincial Microbial Culture Collection Center (GDMCC), and the preservation number is GDMCC No. 61502; the virulent strain WC1535 of Streptococcus agalactiae is from Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences; tilapia is purchased from Guangzhou, Guangdong Province; In the reagent of the present invention, the FAB liquid medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the kits for measuring blood glucose content, insulin content, triglyceride (TG) content, and total cholesterol (TC) content were purchased from Nanjing Jiancheng Bioengineering Institute; the Streptococcus agalactiae IgM antibody (GBS-IgM) ELISA kit, immunoglobulin M (IgM) ELISA kit, alkaline phosphatase (AKP) ELISA kit, lysozyme (LYS) ELISA kit, and complement C3 ELISA kit were purchased from Jiangsu Enzyme Immunoassay Industry Co., Ltd.; the tilapia feed was purchased from Guangzhou Panyu Lingchuan Feed Co., Ltd.

[0032] Example 1: Screening of the culture conditions for Cetobacterium somerae

[0033] (1) Basic culture conditions: The basic medium was set as the FAB liquid medium, which used water as the solvent and included 15 g / L of peptone, 10 g / L of yeast extract, 0.5 g / L of sodium thioglycollate, 2.5 g / L of sodium chloride, 0.5 g / L of cysteine hydrochloride, 0.001 g / L of resazurin, 0.4 g / L of sodium bicarbonate, 0.005 g / L of hemin, and 0.0005 g / L of vitamin K; the basic culture conditions were set as anaerobic culture at 37 °C and pH = 6.5 for 48 h to obtain the Cetobacterium somerae culture solution. (2) Strain activation: The Cetobacterium somerae NK01 strain was inoculated into the basic medium, and repeated activation culture was carried out 3 generations under the basic culture conditions to obtain the Cetobacterium somerae seed solution. (3) Screening of the medium components: Carbon source screening: Glucose, maltose, sucrose, and trehalose were respectively added to 100 mL of the basic medium until the final concentration of each carbon source was independently 20 g / L, and the corresponding screening media A, B, C, and D were obtained. 3 mL of the Cetobacterium somerae seed solution was respectively inoculated into 100 mL of the screening media A - D, cultured under the basic culture conditions, and the viable cell count of each Cetobacterium somerae culture solution was measured. Nitrogen source screening: Yeast powder, yeast peptone, beef extract powder, and soy peptone were respectively added to 100 mL of the basic medium until the final concentration of each nitrogen source was independently 20 g / L, and the corresponding screening media E, F, G, and H were obtained. 3 mL of the Cetobacterium somerae seed solution was respectively inoculated into 100 mL of the screening media E - H, cultured under the basic culture conditions, and the viable cell count of each Cetobacterium somerae culture solution was measured. (4) Screening of the culture conditions: Screening of the inoculation amount of the Cetobacterium somerae seed solution: 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the Cetobacterium somerae seed solution were respectively inoculated into 100 mL of the basic medium, cultured under the basic culture conditions, and the viable cell count of each Cetobacterium somerae culture solution was measured. Cultivation temperature screening: Take 3 mL of the Cetobacterium seed liquid and inoculate it into 100 mL of the basal medium, and cultivate it at 28 °C, 31 °C, 34 °C, 37 °C, and 40 °C respectively. Other cultivation conditions are the basal cultivation conditions, and the viable cell count of each Cetobacterium culture solution is measured; Cultivation pH screening: Take 3 mL of the Cetobacterium seed liquid and inoculate it into 100 mL of the basal medium, and cultivate it at pH 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 respectively. Other cultivation conditions are the basal cultivation conditions, and the viable cell count of each Cetobacterium culture solution is measured. The screening results are as Figure 1 shown.

[0034] The results show that the Cetobacterium NK01 strain has the highest utilization rate of sucrose and yeast powder. Therefore, sucrose and yeast powder are selected as the optimal carbon source and nitrogen source respectively; the optimal inoculation amount of the Cetobacterium seed liquid is 2 mL, the optimal cultivation temperature is 40 °C, and the optimal cultivation pH is 7.0.

[0035] Example 2: Screening of the preparation conditions of Cetobacterium freeze-dried powder

[0036] (1) Basic freeze-drying conditions: Take 3 mL of the Cetobacterium seed liquid prepared in Example 1 and inoculate it into 100 mL of the basal medium, cultivate it under the basal cultivation conditions described in Example 1 to obtain the Cetobacterium culture solution, centrifuge at 12,000 rpm for 10 min, take the precipitate to obtain the cells, add the cryoprotectant, and then obtain the Cetobacterium freeze-dried powder after freeze-drying; Set the basic freeze-drying conditions as: freeze-dry at -45 °C for 40 h; (2) Screening of freeze-drying conditions: Cryoprotectant screening: Add 10 g / L lactose aqueous solution, 100 g / L soluble starch aqueous solution, 10 g / L glycerol aqueous solution, 40 g / L sucrose aqueous solution, 100 g / L glucose aqueous solution, and 10 g / L trehalose aqueous solution to 30 mL of the Cetobacterium culture solution respectively, and conduct freeze-drying under the basic freeze-drying conditions, and measure the viable cell survival rate of each Cetobacterium freeze-dried powder. The results are shown in Table 1; Cryoprotectant addition ratio screening: Add 100 g / L glucose aqueous solution to the cells according to the mass-volume ratio of cells to cryoprotectant of 1 g:1 mL, 1 g:2 mL, 1 g:3 mL, 1 g:4 mL, and 1 g:5 mL respectively, and conduct freeze-drying under the basic freeze-drying conditions, and measure the viable cell survival rate of each Cetobacterium freeze-dried powder. The results are shown in Table 2; Freeze-drying time screening: Add 100 g / L glucose aqueous solution to the cells according to the mass-volume ratio of cells to cryoprotectant of 1 g:4 mL, and then conduct freeze-drying at -45 °C for 40 h, 45 h, 50 h, 55 h, and 60 h respectively, and measure the viable cell survival rate of each Cetobacterium freeze-dried powder. The results are shown in Table 3.

[0037] Table 1 Screening Results of Protective Agents

[0038] Table 2 Screening Results of the Addition Ratio of Protective Agents

[0039] Table 3 Screening Results of Freeze-Drying Time

[0040] The results showed that when using 100 g / L glucose aqueous solution as the protective agent, the viable cell survival rate of the freeze-dried powder of Cetobacterium was the highest; when the mass-volume ratio (g:mL) of the bacteria to the freeze-drying protective agent was between 1:1 and 1:4, the viable cell survival rate of the freeze-dried powder of Cetobacterium gradually increased, and the viable cell survival rate was the highest when the mass-volume ratio (g:mL) was 1:4. Continuing to increase the protective agent afterwards would cause the viable cell survival rate to decrease; the viable cell survival rate of the freeze-dried powder of Cetobacterium reached the maximum value when the freeze-drying time was 55 h.

[0041] Example 3: Preparation of Freeze-Dried Powder of Cetobacterium (1) Take 20 g of sucrose, 15 g of peptone, 20 g of yeast powder, 10 g of yeast extract, 0.5 g of sodium thioglycollate, 2.5 g of sodium chloride, 0.5 g of cysteine hydrochloride, 0.001 g of resazurin, 0.4 g of sodium bicarbonate, 0.005 g of hemin, and 0.0005 g of vitamin K, dissolve them in 1 L of water, adjust the pH to 7.0, and obtain the culture medium after sterilization; (2) Take 2 mL of the Cetobacterium seed solution prepared in Example 1 and inoculate it into 100 mL of the culture medium prepared in step (1), and anaerobically culture it at 40 °C for 48 h to obtain the Cetobacterium culture solution; (3) Centrifuge the Cetobacterium culture solution at 12,000 rpm for 10 min, take the precipitate to obtain the bacteria, and add 100 g / L glucose aqueous solution to the bacteria according to the mass-volume ratio of 1 g:4 mL to obtain the bacterial sludge; (4) Freeze-dry the bacterial sludge at -45 °C for 55 h to obtain the freeze-dried powder of Cetobacterium, and its initial viable cell concentration is 1.0×10 8 CFU / g.

[0042] Experimental Example 1: Effects of Freeze-Dried Powder of Cetobacterium on the Growth Promotion and Disease Resistance of Tilapia 180 tilapia with a body weight of 20 g were divided into two groups, namely the Cetobacterium somerae group (NK) and the control group (CK), with 90 tilapia in each group. The tilapia in the NK group were fed tilapia feed added with freeze-dried powder of Cetobacterium somerae, and the addition amount of the freeze-dried powder of Cetobacterium somerae was 0.02 g / g tilapia feed. The tilapia in the CK group were fed tilapia feed without added freeze-dried powder of Cetobacterium somerae. They were fed twice a day, with a single feeding amount of 27 g, and fed for 42 days in total. After that, the tilapia in each group were starved for 24 h, and the whole fish body weight and body length of the tilapia in each group were measured. After sacrificing the tilapia in each group, the liver was taken out and weighed, and the hepatosomatic index was calculated. After removing the head, tail and fin rays of the tilapia in each group, the carcass mass was weighed and the carcass ratio was calculated. The blood of the tilapia in each group was drawn and centrifuged at 1000×g for 5 min to obtain serum, and the blood glucose, insulin, triglyceride (TG) and total cholesterol (TC) contents in the serum of the tilapia in each group were measured using a blood glucose content assay kit, an insulin content assay kit, a triglyceride (TG) content assay kit and a total cholesterol (TC) content assay kit, respectively. The acetic acid content in the intestinal contents of the tilapia in each group was measured by GC-MS method. 45 tilapia were selected from each group and intraperitoneally injected with a virulent strain WC1535 of Streptococcus agalactiae at a concentration of 1.0×10 7 CFU / mL, the injection volume was 100 μL, and the cumulative survival rate of the tilapia in each group within 144 h after challenge was calculated. The results are as Figures 2 to 4 shown; The formula for calculating the hepatosomatic index is: ; The formula for calculating the carcass ratio is: .

[0043] The results showed that the freeze-dried powder of Cetobacterium somerae of the present invention could increase the carcass ratio of tilapia, increase the serum insulin level and the acetic acid content in the intestinal contents, decrease the hepatosomatic index, blood glucose and TG levels, and significantly improve the cumulative survival rate of tilapia. It shows that the freeze-dried powder of Cetobacterium somerae of the present invention can promote the growth and development of tilapia.

[0044] Experimental Example 2: Effect of freeze-dried powder of Cetobacterium somerae on the immune mechanism of tilapia The freeze-dried powder of Cetobacterium somerae prepared in Example 3 was mixed with water to obtain a suspension of Cetobacterium somerae with a concentration of 1.0×10 8 CFU / mL. 180 tilapia with a body weight of 41.5±0.5 g were divided into three groups, namely the Cetobacterium somerae group (NK), the sodium acetate group (AC) and the control group (CK), with 60 tilapia in each group. The tilapia in the NK group were gavaged with the suspension of Cetobacterium somerae, the tilapia in the AC group were gavaged with a 50 mg / mL sodium acetate solution, and the tilapia in the CK group were gavaged with PBS. They were gavaged once every 3 days, with a gavage volume of 200 μL each time. After continuous gavage for 3 times, serum samples of the tilapia in each group were collected. 24 h after gavage, the tilapia in each group were intraperitoneally injected with a concentration of 1.0×10 7The strong strain of Streptococcus agalactiae WC1535 at CFU / mL was used for challenge, with an injection volume of 0.1 mL. After 12 h of challenge, serum samples of tilapia in each group were collected again. The GBS-IgM, IgM, AKP, LYS, and complement C3 contents in the serum of tilapia in each group were measured using a Streptococcus agalactiae IgM antibody (GBS-IgM) ELISA kit, an immunoglobulin M (IgM) ELISA kit, an alkaline phosphatase (AKP) ELISA kit, a lysozyme (LYS) ELISA kit, and a complement C3 ELISA kit, respectively. The cumulative survival rates of tilapia in each group within 144 h of challenge were calculated, and Suzhou Panomic Biopharmaceutical Technology Co., Ltd. was commissioned to perform transcriptome sequencing analysis and KEGG pathway enrichment analysis on the serum of tilapia in each group. The results are as Figures 5 to 9 shown.

[0045] The results showed that compared with the tilapia in the CK group, the levels of immune indexes such as GBS-IgM, IgM, AKP, LYS, complement C3 in the tilapia in the NK group and GBS-IgM, IgM, AKP, complement C3 in the tilapia in the AC group were all significantly increased ( P <0.05), indicating that the freeze-dried powder of Cetobacterium somerae and its metabolite acetic acid of the present invention can simultaneously enhance the specific immune response and non-specific immune defense ability of tilapia; the cumulative survival rate of tilapia in the CK group was lower than that of tilapia in the NK group and the AC group, indicating that the freeze-dried powder of Cetobacterium somerae and its metabolite acetic acid of the present invention can significantly improve the resistance of tilapia to Streptococcus agalactiae infection; compared with the tilapia in the CK group, the differentially expressed genes (DEGs) in the tilapia in the NK group and the AC group were significantly enriched in signal pathways such as "intestinal immune network for immunoglobulin A production" and "Toll-like receptor signaling", indicating that the freeze-dried powder of Cetobacterium somerae and its metabolite acetic acid of the present invention can activate the non-specific immune network of tilapia, promote the production of immune active substances such as immunoglobulins and lysozymes, and thus enhance the immune response ability and disease resistance of tilapia.

[0046] Experimental Example 3: Effect of freeze-dried powder of Cetobacterium somerae on the intestinal flora structure of tilapia Intestinal mucosa and intestinal content samples of tilapia in each group in Experimental Example 2 were collected before and after challenge, and Suzhou Panomic Biopharmaceutical Technology Co., Ltd. was commissioned to perform high-throughput sequencing analysis of the V4 region of the 16S rRNA gene. The results are as Figures 10 to 16 shown.

[0047] The results showed that before challenge, there was no significant difference in the relative abundance of Cetobacterium somerae in the intestinal mucosa-attached flora of tilapia in each group; compared with the tilapia in the CK group, the relative abundance of Cetobacterium somerae in the intestinal contents of tilapia in the NK group and the AC group was significantly increased; Before challenge, compared with CK group tilapia, NK group tilapia had higher levels of Bifidobacterium ( Bifidobacterium ) and Lactobacillus in intestinal contents ( Lactobacillus ) showed an upward trend in relative abundance. This indicates that the lyophilized powder of Bacillus cereus of the present invention can change the characteristic biomarkers of the intestinal flora of tilapia, and improve the intestinal microecological environment of tilapia by promoting the growth of beneficial flora such as bifidobacteria and lactobacilli; Before the challenge, compared with the CK group tilapia, the functional characteristics of the intestinal mucosal attached flora of the NK group and the AC group tilapia changed significantly. In the NK group and the AC group tilapia, the bacterial invasion of epithelial cells pathway showed a significant downward trend. This shows that the lyophilized powder of Bacillus cereus and its metabolite acetic acid of the present invention can enhance the intestinal barrier function by regulating the function of the intestinal attached flora of tilapia, thereby reducing the invasion ability of pathogens; After infection, the relative abundance of Bacillus subtilis in the attached flora of the intestinal contents of tilapia in the AC group increased compared with that in the CK group; at the same time, no difference was found in the relative abundance of Streptococcus agalactiae in the intestinal contents of tilapia in each group.

[0048] As can be seen from the above embodiments, the present invention provides a lyophilized powder of Bacillus ceti and its preparation method and application. The lyophilized powder of Bacillus ceti of the present invention can promote the growth and development of tilapia, enhance the specific immune response of tilapia, significantly improve the resistance of tilapia to infection with Streptococcus agalactiae, and at the same time, by activating the nonspecific immune network of tilapia, promote the production of immunoactive substances such as immunoglobulins and lysozymes, thereby enhancing the immune response ability and disease resistance of tilapia; it can also change the characteristic biomarkers of the intestinal flora of tilapia, regulate the function of the intestinal attached flora of tilapia, improve the intestinal microecological environment of tilapia by promoting the growth of beneficial flora such as bifidobacteria and lactobacilli, enhance the intestinal barrier function, thereby reducing the invasion ability of pathogens, and has the advantages of high effectiveness and safety.

[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A culture medium for Bacteroides cetorum, characterized in that, The culture medium uses water as a solvent and includes components with the following concentrations: sucrose 10 - 30 g / L, peptone 10 - 20 g / L, yeast powder 15 - 25 g / L, yeast extract 5 - 15 g / L, sodium thioglycollate 0.1 - 1 g / L, sodium chloride 1 - 4 g / L, cysteine hydrochloride 0.2 - 0.8 g / L, resazurin 0.0005 - 0.002 g / L, sodium bicarbonate 0.1 - 1 g / L, hemin 0.001 - 0.01 g / L, vitamin K 0.0001 - 0.001 g / L.

2. Use of the culture medium according to claim 1 in the preparation of freeze-dried Cetobacterium.

3. A preparation method of Bacteroides cetorum freeze-dried powder, characterized in that, It includes the following steps: (1) Mix Cetobacterium and the culture medium according to claim 1, and culture for 24 - 60 h to obtain a Cetobacterium culture solution; (2) Centrifuge the Cetobacterium culture solution, take the precipitate to obtain thalli, and mix the thalli with a cryoprotectant to obtain a bacterial sludge; (3) Dry the bacterial sludge for 40 - 60 h to obtain freeze-dried Cetobacterium.

4. The preparation method according to claim 3, characterized in that, In step (1), the volume ratio of the mixture of Cetobacterium and the culture medium is 1 - 5:

100.

5. The preparation method according to claim 4, wherein In step (1), the temperature of the culture is 28 - 40 °C, and the pH of the culture is 6.0 - 8.

5.

6. The preparation method according to claim 5, characterized in that, In step (2), the rotation speed of the centrifugation is 10000 - 15000 rpm, and the centrifugation time is 5 - 20 min.

7. The preparation method according to claim 6, characterized in that, The cryoprotectant in step (2) includes one or more of an aqueous lactose solution, an aqueous soluble starch solution, an aqueous glycerol solution, an aqueous sucrose solution, an aqueous glucose solution, and an aqueous trehalose solution; The concentration of the cryoprotectant is 10 - 120 g / L; The mass-volume ratio of the mixture of the thalli and the cryoprotectant is 1 g:1 - 5 mL.

8. The preparation method according to claim 7, wherein The drying in step (3) is freeze-drying, and the temperature of the drying is -60 - -30 °C.

9. Freeze-dried Cetobacterium prepared by the preparation method according to any one of claims 3 - 8.

10. Use of the freeze-dried Cetobacterium according to claim 9 in the preparation of a preparation for promoting the growth of fish and / or improving the disease resistance of fish.

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