Application of lactobacillus casei metagen in improvement of immune function of crucian carp

Abstract

The invention is suitable for the technical field of probiotic postbiotics, and provides an application of lactobacillus casei postbiotics in improving the immune function of crucian carps. In order to solve the problem of pollution of antibiotics to the culture water environment, the antibiotics are replaced by the postbiotics obtained by inactivating the lactobacillus casei, so that the problems of drug resistance, drug residues, edible safety and the like are solved, the growth performance of aquatic animals is improved, and the yield of the aquatic animals is increased on the basis of realizing green culture. By means of the lactobacillus casei, the immune function of the crucian is enhanced, and the immune response of the crucian is improved.

Description

Technical Field

[0001] This invention belongs to the field of probiotic postbiotic technology, and particularly relates to the application of Lactobacillus casei postbiotic in improving the immune function of crucian carp. Background Technology

[0002] In the development of modern aquaculture, the scale of farming has continued to expand, and the degree of intensification has been constantly increasing. To effectively control diseases in farmed aquatic animals and ensure production output and economic benefits, antibiotics have been widely used in aquaculture. However, the extensive use of antibiotics in aquaculture has seriously harmed the microbial community, water chemistry, and ecological balance of the aquaculture environment, causing numerous severe and undeniable hazards. Therefore, achieving green, healthy, and sustainable development of aquaculture is urgently needed.

[0003] Probiotics, as live microorganisms that can replace antibiotics, are live microorganisms implanted in the gut and reproductive tract of living organisms. The emergence of probiotics has replaced antibiotics, solving problems such as drug resistance, drug residues, and food safety, and improving the growth performance of aquatic animals. Although the use of live probiotics has many benefits, long-term high-dose use may lead to safety issues such as drug resistance gene transfer and bacterial allergic reactions. Metabiotics are a collective term for the metabolites of probiotics after processing, including bacterial cells and metabolites. Inactivated probiotic cells mainly contain teichoic acid, extracellular polysaccharides, peptidoglycans and surface proteins, lipoteichoic acid, etc. Metabolites mainly include active peptides, amino acids, short-chain fatty acids (such as butyric acid, acetic acid, propionic acid, etc.), bacteriocins, extracellular polysaccharides, secretory proteins, extracellular vesicles, bile acids, organic acids, vitamins, and some biosurfactants. The active products of microbial enzymes mainly include oligosaccharides and small peptides. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Lactobacillus casei postbiotic in improving the immune function of crucian carp, thereby addressing the problems mentioned in the background art.

[0005] The present invention is implemented as follows: the application of Lactobacillus casei postbiotic in improving the immune function of crucian carp.

[0006] Preferably, the method for preparing the Lactobacillus casei postbiotic includes the following steps: Inoculate Lactobacillus casei into the culture medium and then adjust the bacterial concentration. The bacterial culture was heat-inactivated to obtain Lactobacillus casei post-genetic agent.

[0007] Preferably, the concentration of the adjusted bacterial solution is 1×10⁻⁶. 6 -1×10 8 CFU / mL.

[0008] Preferably, the heat inactivation treatment is performed by water bath treatment at a temperature of 70°C for 10 minutes.

[0009] Preferably, the method includes the following steps: mixing the Lactobacillus casei post-biotic with fish food evenly and feeding it to crucian carp.

[0010] Preferably, the volume-to-mass ratio of the Lactobacillus casei postbiotic to the fish food is 0.8-1.2:1 (mL:g).

[0011] Another objective of this invention is to provide a crucian carp biological agent, wherein the agent comprises *Lactobacillus casei* postbiotic, and the concentration of the *Lactobacillus casei* postbiotic in the bacterial solution is 1×10⁻⁶. 6 -1×10 8 CFU / mL.

[0012] This invention addresses the problem of antibiotic pollution in aquaculture environments by using postbiotics obtained from inactivated Lactobacillus casei to replace antibiotics. This solves problems related to drug resistance, drug residues, and food safety, improves the growth performance of aquatic animals, and enhances the immune function and immune response of crucian carp by utilizing Lactobacillus casei in green aquaculture. Attached Figure Description

[0013] Figure 1 The growth characteristics and pH curve provided in Example 1 of the present invention; Figure 2 The changes in SOD and CAT levels in the serum of each experimental group provided in Example 3 of the present invention; Figure 3 The changes in LZM and AST levels in the serum of each experimental group provided in Example 3 of the present invention; Figure 4 The results of serum IgM content in each experimental group provided in Example 3 of the present invention; Figure 5 The changes in C3 and C4 content in the serum of each experimental group provided in Example 3 of the present invention; Figure 6 The PP and PI values ​​for each experimental group provided in Example 3 of this invention; Figure 7 The results of relative expression levels of α-tumor necrosis factor gene in different tissues (liver and spleen) of each experimental group provided in Example 3 of the present invention; Figure 8 The results of relative expression levels of α-tumor necrosis factor gene in different tissues (kidney, intestine) of each experimental group provided in Example 3 of the present invention; Figure 9 The results of relative expression levels of IL-10 in different tissues (liver and kidney) of each experimental group provided in Example 3 of the present invention; Figure 10 The results of relative expression levels of IL-10 in different tissues (spleen and intestine) of each experimental group provided in Example 3 of the present invention; Figure 11 The results of relative expression levels of IL-1β in different tissues (liver and kidney) of each experimental group provided in Example 3 of the present invention; Figure 12 The results of relative IL-1β expression levels in different tissues (spleen, intestine) of each experimental group provided in Example 3 of the present invention; Figure 13 The results of relative expression levels of IFN-γ in different tissues (liver and kidney) of each experimental group provided in Example 3 of the present invention; Figure 14 The results of relative expression levels of IFN-γ in different tissues (spleen and intestine) of each experimental group provided in Example 3 of the present invention; Figure 15 The results of the immune protection rate detection for each experimental group provided in Example 3 of the present invention; Figure 16 The changes in bacterial load in different tissues (liver and kidney) of each experimental group provided in Example 3 of the present invention; Figure 17 The changes in bacterial load in different tissues (spleen, intestine) of each experimental group provided in Example 3 of the present invention; In the attached figures, * indicates significance, where * means p < 0.05; ** means p < 0.01; and *** means p < 0.001. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0015] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0016] Example 1: Growth performance and pH determination of Lactobacillus casei: Lactobacillus casei ATCC393 ( Lactobacillus caseiATCC393 (purchased from Biofeng (https: / / www.biofeng.com / ) in July 2019 and stored in the Preventive Veterinary Laboratory of Jilin Agricultural University) was inoculated into MRS liquid medium at a 1% inoculum and anaerobically cultured at 37℃ for 30 hours. During the culture period, every 2 hours, the bacterial culture and MRS liquid medium (blank set to zero) were aspirated, and the OD value at 600 nm was measured using a spectrophotometer. Simultaneously, the pH value was measured using a pH meter, and the results were recorded. Figure 1 As shown, the pH generally shows a downward trend. The strain grows the fastest and the pH decreases the fastest during the period from 8h to 16h. During the period from 16h to 30h, the strain grows slowly and the pH decreases gradually slows down.

[0017] Example 2, Optimal thermal inactivation conditions: Lactobacillus casei was streaked on MRS solid medium and placed in a constant temperature incubator for anaerobic culture at 37°C. After overnight incubation, a loopful of single colony with good shape and condition was picked and inoculated into MRS liquid medium for later use. Heat inactivation was performed at 55-80°C (each 5°C gradient, for a total of 6 groups) for 10 min, 20 min, and 30 min. The heat-inactivated bacterial suspension was spread on MRS solid medium using the dilution plating method. The spread plates were then placed in an anaerobic incubator at 37°C for anaerobic culture, and the growth was observed every 12 hours. The colony growth at water bath temperatures of 55℃-80℃ is shown in Table 1 (in the table, "+" indicates "yes" and "-" indicates "no"): Table 1

[0018] As shown in Table 1, the optimal thermal inactivation conditions are water bath heating at 70℃ for 10 minutes.

[0019] Example 3: Analysis of the effects of Lactobacillus casei postbiotic on crucian carp: Preparation of Lactobacillus casei postbiotics and feed formulation: The concentration of Lactobacillus casei was determined by the dilution plating method, and the concentration was adjusted to 1×10⁻⁶. 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL (serially diluted with physiological saline according to known concentration), the prepared bacterial solution was placed in a water bath and heated at 70℃ for 10 minutes to obtain Lactobacillus casei post-biotic; then 100mL of Lactobacillus casei post-biotic was mixed with 100g of fish food, placed in an electric hot air drying oven at 40℃ and dried, and then sealed for later use. Animal experimental model establishment: The experimental crucian carp (purchased from Changchun Daziran Flower, Bird and Fish Market) were randomly divided into 4 groups (50 fish in each group). The first group received PBS + basal fish food (PBS group), the second group received PBS with a concentration of 1×10 6 Group A: Postbiotic + basal fish food at CFU / mL; Group B: Concentration 1×10⁻⁶ CFU / mL 7 Group B: Postbiotic + basal fish food (CFU / mL); Group 4: Concentration 1×10⁻⁶ CFU / mL 8 The feed (Group C) contains CFU / mL of post-biotic + basic fish food and is fed twice a day. Each feeding is 3% of the total weight of the crucian carp in each group. The oxygen supply in the water is maintained daily. After 28 days of continuous feeding, 20 fish from each group were randomly selected and challenged with Aeromonas hydrophila TPS (preserved in the Animal Science Laboratory of Jilin Agricultural University) via intraperitoneal injection. The protective effect of each group was observed. Each experimental group was injected intraperitoneally with a dose of 200 mL per fish. The observation period was 7 days. Dead fish were promptly removed and recorded. Based on the mortality of each group, the relative immune protection rate (RPS) was calculated: Relative immune protection rate (RPS) = (1 - mortality rate of immunized group / mortality rate of control group) × 100%; Test results: 1. Blood immunological parameter determination: The enzyme activities of SOD, CAT, LZM, and AST in crucian carp serum samples were measured. Figure 2 , Figure 3 As shown, there was little difference between the experimental groups at day 0. From day 7 to day 21 of oral feeding, SOD, CAT, LZM, and AST all showed an increasing trend. At day 21, the activities of CAT, LZM, and AST all reached their peak. The CAT level in group B was significantly higher than that in group A, the LZM level in group B was significantly higher than that in groups A and C (p < 0.001), and the AST level in group B was significantly higher than that in group C (p < 0.001). Subsequently, the levels decreased from day 28 to day 35. The SOD enzyme activity reached its peak at day 28, and the activity in group B was significantly higher than that in group A. Subsequently, the activity decreased at day 35. When SOD, CAT, LZM, and AST reached their peak, the levels in each experimental group were significantly higher than those in the PBS group (p < 0.001). Moreover, the PBS group remained unchanged throughout the entire experiment. 2. Immunoglobulin M (IgM) activity assay: Dilute the sample to be tested as required, and set up positive controls, negative controls, and standards. Add the diluted antigen that specifically binds to IgM to the wells of an ELISA plate and incubate overnight at 4°C or 1-2 hours at 37°C to allow the antigen to adsorb onto the surface of the wells. Then wash the plate to remove unbound antigen. Add blocking buffer to the 96-well plate for blocking and incubate at 37°C for 30-60 minutes. Wash the plate to remove excess blocking buffer and reduce non-specific binding. Add the diluted sample, standards, and controls to the wells and incubate at 37°C. After 1-2 hours, the IgM in the sample will specifically bind to the antigen in the well. Wash the plate to remove unbound contaminating proteins, add enzyme-labeled antibody against IgM, and incubate at 37°C for 1 hour to allow the enzyme-labeled antibody to bind to the bound IgM. Wash the plate again to remove unbound enzyme-labeled antibody, add substrate solution, and the enzyme will catalyze the substrate to produce a blue color reaction. Incubate at room temperature in the dark for 15-30 minutes, then add stop solution to terminate the reaction. The color will turn yellow. Measure the absorbance (OD value) of each well at 450nm-630nm using an ELISA reader, and plot a standard curve based on the OD values ​​of the standards. The results are as follows Figure 4 As shown, there was little difference between the experimental groups at day 0. At day 7, the IgM content of each experimental group increased. At day 14, the IgM content of group B reached the highest value and was significantly higher than that of groups A and C. From day 21 to day 35, the overall IgM content of each experimental group decreased. When the IgM reached its peak, the content of each experimental group was significantly higher than that of the PBS group (p < 0.001), and the PBS group remained unchanged throughout the entire experiment. 3. Complement Activity Assay: Collect fresh serum samples and dilute them according to the kit requirements. Prepare standards and negative / positive controls. Dilute the C3 / C4 specific monoclonal antibody and add it to the wells of an ELISA 96 plate. Incubate overnight at 4°C or 1-2 hours at 37°C to allow the antibody to adsorb onto the surface of the wells. Wash the plate three times to remove unbound antibodies. Add blocking buffer to cover the wells and incubate at 37°C for 30-60 minutes to block blank sites and reduce non-specific binding. Wash the plate to remove excess blocking buffer. Add the diluted sample, standards, and controls to the corresponding wells and incubate at 37°C. Incubate for 1-2 hours to allow C3 / C4 in the sample to specifically bind to the antibody coating in the wells; wash the plate 3-5 times to remove unbound contaminating proteins, add biotin- or enzyme-labeled anti-human C3 / C4 secondary antibody, incubate at 37°C for 1 hour, wash the plate to remove unbound secondary antibody, then add substrate solution, incubate at 37°C in the dark for 15-30 minutes, the enzyme catalyzes the substrate to develop color (blue), add stop solution to terminate the reaction, the color turns yellow, measure the absorbance (OD value) at 450nm wavelength (reference wavelength 630nm) using an ELISA reader, and plot a standard curve based on the OD values ​​of the standards; The results are as follows Figure 5As shown, there was little difference between the experimental groups at day 0. During days 7-14, all experimental groups showed an upward trend. At day 14, the contents of C3 and C4 reached their peak, and the contents of C3 in group B were significantly higher than those in groups A and C (p < 0.001), and the contents of C4 in group B were significantly higher than those in group C (p < 0.001). During days 21-35, although the contents of C3 and C4 rebounded slightly, they were still far below the peak values ​​at day 14, showing an overall downward trend. When C3 and C4 reached their peak values, all experimental groups were significantly higher than those in the PBS group (p < 0.001), and the contents of the PBS group remained unchanged throughout the entire experimental period. 4. Leukocyte phagocytic activity: Anticoagulated peripheral blood was collected, leukocytes were separated and mixed with fluorescently stained targets in a certain proportion, and incubated at 37°C to allow leukocytes to actively phagocytose the targets. The reaction was then terminated with cold buffer, and the free targets were washed away. The proportion and number of phagocytic cells were directly counted under a microscope. The results are as follows Figure 6 As shown, the results indicated that both the percentage of white blood cells phagocytized (PP) and the phagocytic index (PI) changed. At day 0, the values ​​were not significantly different among the experimental groups. They began to gradually increase after 7 days of initial oral feeding, reaching a peak at day 28. Among the experimental groups, group B had the highest PP at 34.85%, which was significantly higher than groups A and C (p < 0.001). Among the experimental groups, group B had the highest PI value of 4.8. The phagocytic index values ​​of groups A and C were relatively close, at 3.99 and 3.92 respectively, which were significantly higher than group C (p < 0.001). After day 35, the PP and PI values ​​decreased. When PP and PI reached their peak, all experimental groups were significantly higher than the PBS group (p < 0.001), and the PBS group remained unchanged throughout the entire experiment. 5. Expression of inflammatory factors in organs: 5.1 Analysis of relative expression levels of α-tumor necrosis factor gene in different tissues: Different tissues were collected, total RNA was extracted and purified to ensure that the RNA was intact and undegraded, and the RNA was reverse transcribed into cDNA (stable and easy to amplify) using reverse transcriptase. Real-time quantitative PCR was performed on the TNF-α gene and the internal reference gene using specific primers, and the Ct value was recorded. The expression differences of TNF-α and the internal reference gene in different tissues were compared by the 2^(-ΔΔCt) method to obtain the relative expression levels. The results are as follows Figure 7 , Figure 8As shown, the expression levels of TNF-α in the tissues of each experimental group were basically the same at 0 days. After 7 days, the gene expression levels of each experimental group gradually increased. At 14 days, the expression level of TNF-α gene in group B in the intestine reached its peak, which was significantly higher than that in groups A and C (p < 0.001). At 21 days, the expression level of TNF-α gene in group B in the kidney reached its peak, which was significantly higher than that in groups A and C (p < 0.001). The gene expression levels in the liver and spleen continued to increase. At 28 days, the expression level of TNF-α gene in group B in the liver and spleen reached its peak, and the expression level of group B in the spleen was significantly higher than that in groups A and C (p < 0.001). Subsequently, like the kidney and intestine, after reaching the peak, it began to gradually decrease. When each tissue reached its peak, the expression levels of each experimental group were significantly higher than those in the PBS group (p < 0.001), and the PBS group remained unchanged throughout the experiment. 5.2 Analysis of relative expression levels of interleukin-10 gene in different tissues: Different tissue samples were collected, and total RNA was extracted after rapid freezing. The purity and integrity of the RNA were detected. Using RNA as a template, cDNA was synthesized by reverse transcriptase. Specific primers for the IL-10 gene and the internal reference gene were designed, and a qRT-PCR system was constructed. Amplification was performed, and the fluorescence signal was monitored in real time. The Ct value was recorded, and the expression level of the IL-10 gene relative to the internal reference gene was calculated using the 2^(-ΔΔCt) method. The relative expression differences of this gene in different tissues were compared. The results are as follows Figure 9 , Figure 10 As shown, the expression levels of IL-10 in the tissues of each experimental group were basically the same at day 0. After day 7, the gene expression levels of each experimental group gradually increased. At day 14, the expression level of group B IL-10 gene in the spleen and intestine reached its peak, and the spleen group B was significantly higher than group A and group C (p < 0.001), while the intestine group B was significantly higher than group C (p < 0.001). The expression levels in other tissues continued to increase. At day 28, the expression level of group B IL-10 gene in the kidney and liver reached its peak, and the liver group B was significantly higher than group A and group C (p < 0.001). Subsequently, it began to gradually decrease. When each tissue reached its peak, the expression levels of each experimental group were significantly higher than those of the PBS group (p < 0.001), and the PBS group remained unchanged throughout the experiment. 5.3 Analysis of relative expression levels of interleukin-1β gene in different tissues: Different tissue samples were collected, and total RNA was extracted after rapid processing. The purity and integrity of the RNA were tested. The extracted RNA was converted into cDNA through reverse transcription. Specific primers for IL-1β gene and internal reference gene were designed, and a reaction system was constructed. The fluorescence signal during the amplification process was monitored in real time using a PCR instrument, and the Ct value was recorded. The expression level of IL-1β gene relative to the internal reference gene was calculated using the 2^(-ΔΔCt) method, and the relative expression differences in different tissues were compared. The results are as follows Figure 11 , Figure 12As shown, the expression levels of IL-1β in the tissues of each experimental group were basically the same at day 0. From day 7 to day 21, the gene expression levels of each experimental group gradually increased. At day 21, the expression level of group B IL-1β in the spleen, liver, and kidney reached its peak. Group B in the liver and spleen was significantly higher than that in group A (p < 0.001), and group B in the kidney was significantly higher than that in groups A and C (p < 0.001). The expression level in the intestine continued to increase. At day 28, the expression level of group B IL-1β in the intestine reached its peak and was significantly higher than that in groups A and C (p < 0.001). Then it began to gradually decrease. When each tissue reached its peak, the expression level of each experimental group was significantly higher than that in the PBS group (p < 0.001), and the PBS group remained unchanged throughout the experiment. 5.4 Analysis of relative expression levels of interferon-γ gene in different tissues: Different tissue samples were collected, and total RNA was extracted from the tissues using Trizol or a kit. The purity and integrity of the RNA were verified by UV spectrophotometer and agarose gel electrophoresis. Using total RNA as a template, reverse transcriptase, primers, etc. were added, and the RNA was converted into stable complementary DNA (cDNA) through reverse transcription reaction. This cDNA served as the template for subsequent PCR. Specific primers for the IFN-γ gene and internal reference gene were designed, and a reaction system was constructed for amplification. Simultaneously, fluorescence signals were monitored, and Ct values ​​(the number of cycles when fluorescence reaches the threshold, which is negatively correlated with the initial template amount) were recorded. The relative expression level was calculated using the 2^(-ΔΔCt) method, where relative expression level = 2^(-ΔΔCt). The higher the value, the more active the IFN-γ gene expression in the tissue. The results are as follows Figure 13 , Figure 14 As shown, the expression levels of IFN-γ in the tissues of each experimental group were basically the same at day 0. After day 7, the gene expression levels of each experimental group gradually increased, and the expression level of group B IFN-γ gene in the intestine reached its peak at day 14. The expression levels in other tissues continued to increase. At day 21, the expression levels of group B IFN-γ gene in the kidney and liver reached their peak, and the expression level of group B in the kidney was significantly higher than that of group A (p < 0.001). At day 28, the expression level of group B IFN-γ gene in the spleen reached its peak, and the expression level of group B was significantly higher than that of group C (p < 0.001). Subsequently, it began to gradually decrease. When each tissue reached its peak, the expression levels of each experimental group were significantly higher than those of the PBS group (p < 0.001), and the expression level of the PBS group remained unchanged throughout the experiment. 6. Immunoprotection rate detection: After oral feeding of crucian carp in each experimental group for 28 days, a challenge protection test was conducted using Aeromonas hydrophila. The results are as follows: Figure 15As shown, after the virus challenge, observation of the dead fish revealed congestion on the body surface, fins, and around the eyes, redness and swelling of the anus, and abdominal distension. Dissection revealed turbid ascites and hemolysis. Fish died in all experimental groups after the virus challenge, with death symptoms similar to the control group. On day 7 after the virus challenge, the survival rate of group A was 35%, group B was 45%, and group C was 40%. The results indicate that heat-inactivated Lactobacillus casei can activate the immune system of crucian carp and enhance its resistance to Aeromonas hydrophila. Group B was the most suitable concentration for activating the immune system of crucian carp. 7. Changes in bacterial load in various tissues after challenge: After oral feeding, challenge experiments were conducted by intraperitoneal injection of Aeromonas hydrophila to simulate the situation that crucian carp may encounter pathogen infection in actual aquaculture. Liver, spleen, kidney and intestinal tissues of dying crucian carp in groups A, B, C and PBS were collected at 24h, 48h and 72h after infection, respectively. The bacterial load in each tissue was detected by plate count method. The results are as follows Figure 16 , Figure 17 As shown, within 48 hours post-infection, the bacterial load in the spleen and kidney tissues of crucian carp in groups A, B, and C decreased significantly. Within 72 hours, the bacterial load in the liver and spleen tissues of crucian carp in groups A, B, and C decreased significantly, and was lower than that in the PBS group. The experimental results indicate that oral feeding of crucian carp with heat-inactivated Lactobacillus casei provides a certain degree of protection against Aeromonas hydrophila infection.

[0020] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of Lactobacillus casei postbiotic in improving the immune function of crucian carp.

2. The application according to claim 1, characterized in that, The method for preparing the Lactobacillus casei postbiotic includes the following steps: Inoculate Lactobacillus casei into the culture medium and then adjust the bacterial concentration. The bacterial culture was heat-inactivated to obtain Lactobacillus casei post-genetic agent.

3. The application according to claim 2, characterized in that, The bacterial suspension concentration was adjusted to 1×10⁻⁶. 6 -1×10 8 CFU / mL.

4. The application according to claim 2, characterized in that, The heat inactivation treatment is performed by water bath treatment at a temperature of 70°C for 10 minutes.

5. The application according to claim 1, characterized in that, Includes the following steps: The Lactobacillus casei post-biotic was mixed evenly with the fish food and then fed to the crucian carp.

6. The application according to claim 5, characterized in that, The volume-to-mass ratio of *Lactobacillus casei* postbiotic to fish food is 0.8-1.2:1 (mL:g).

7. A biological agent for crucian carp, characterized in that, The formulation includes *Lactobacillus casei* postbiotic, and the bacterial concentration of the *Lactobacillus casei* postbiotic is 1 × 10⁻⁶. 6 -1×10 8 CFU / mL.

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