Application of bacillus coagulans metagen in improving immune function of crucian carp

Abstract

The invention is suitable for the technical field of probiotic postbiotics, and provides application of bacillus coagulans postbiotics in improving the immune function of crucian carp. According to the embodiment of the invention, the bacillus coagulans are subjected to thermal inactivation treatment to prepare the bacillus coagulans metagen, the crucian is fed with the bacillus coagulans metagen, the immune index, the leukocyte phagocytic activity and the level of tissue immune related cytokine expression quantity of the crucian are detected, and animal experiments are carried out. The method is used for evaluating the immunological enhancement effect of the bacillus coagulans metagen, and a new idea is provided for later research and development of animal preparations.

Description

Technical Field

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

[0002] Aquaculture is a crucial industry for ensuring protein supply. However, under high-density intensive farming models, bacterial and viral diseases are prevalent in fish, with the frequent emergence of pathogenic bacteria in aquatic animals, such as Aeromonas hydrophila, hindering the sustainable development of aquaculture. While antibiotics are effective in preventing Aeromonas hydrophila infections in farmed fish, their overuse has led to serious consequences, including increased environmental stress, the development of drug resistance, and disruption of the fish's gut microbiota. Therefore, developing ecologically safe and feasible antibiotic alternatives is urgently needed to avoid the overuse of antibiotics.

[0003] In an environment where antibiotic resistance is becoming increasingly serious, probiotics have attracted much attention due to their functions of regulating gut microbiota, enhancing host immunity, and inhibiting pathogens. Among them, Bacillus coagulans (Bacillus) Bacillus coagulans As a Gram-positive, spore-forming, heat-resistant probiotic, it has the characteristics of high stability, acid and bile salt resistance, and strong enzyme activity. It is regarded as a potential strain to replace antibiotics in aquaculture. It can significantly improve the disease resistance and growth performance of fish by secreting antimicrobial peptides (such as coagulins), competitively eliminating pathogens, and activating host immune signaling pathways.

[0004] In recent years, the concept of "postbiotics" has emerged in the field of probiotic research. These are the bioactive metabolites or cell components of probiotics that remain biologically active after inactivation treatment (such as peptidoglycans, short-chain fatty acids, and extracellular polysaccharides). Compared with live bacteria preparations, postbiotics avoid the risk of colonization by live bacteria and have advantages such as clearly defined components, high stability, and ease of storage and transportation. Postbiotics, with their unique advantages, have shown broad application potential in fish and crustacean aquaculture. Studies have shown that adding postbiotics to feed can significantly reduce the content of malondialdehyde in tilapia, thereby alleviating cell damage caused by propylene glycol, and enhance the activity of various antioxidant enzymes in the body, thus improving the overall antioxidant performance of the body. In another study, a 56-day feeding experiment with Bacillus subtilis LCBS1 postbiotics on bullfrogs showed a significant increase in serum complement C4 levels and alkaline phosphatase activity. In studies on striped catfish and freshwater sharks, feeding 1.42-1.66 g / kg of Bacillus subtilis postbiotics for 60 days significantly increased lysozyme and phagocytic activity, and enhanced the activities of superoxide dismutase, catalase, and glutathione peroxidase.

[0005] At present, the prior art focuses on lactic acid bacteria and bacillus, and the immune regulation mechanism of Bacillus coagulans probiotics needs to be further analyzed. SUMMARY

[0006] The purpose of the embodiment of the present application is to provide the application of Bacillus coagulans probiotics in improving the immune function of crucian carp, aiming to solve the problems raised in the above background art.

[0007] The embodiment of the present application is implemented in this way, the application of Bacillus coagulans probiotics in improving the immune function of crucian carp.

[0008] Preferably, the preparation method of the Bacillus coagulans probiotics comprises the following steps: The Bacillus coagulans is inoculated into the culture medium for culture, and then the bacterial solution is subjected to centrifugal treatment, and after the supernatant is discarded, PBS (phosphate buffered saline) is added to adjust the concentration of the bacterial solution. The bacterial solution is subjected to heat inactivation treatment to obtain the Bacillus coagulans probiotics.

[0009] Preferably, the concentration of the bacterial solution is adjusted to 1×10 6 -1×10 8 CFU / mL.

[0010] Preferably, the heat inactivation treatment adopts water bath treatment, the temperature is 99℃, and the time is 20 min.

[0011] Preferably, the following steps are included: the Bacillus coagulans probiotics is uniformly mixed with daily ration feed, and the crucian carp is fed.

[0012] Preferably, the volume to mass ratio mL: g of the Bacillus coagulans probiotics to the daily ration feed is 0.8-1.2:1.

[0013] Another purpose of the embodiment of the present application is to provide a crucian carp biological preparation, which comprises the Bacillus coagulans probiotics, and the concentration of the bacterial solution of the Bacillus coagulans probiotics is 1×10 6 -1×10 8 CFU / mL.

[0014] The embodiment of the present application prepares the Bacillus coagulans probiotics by heat inactivation treatment of Bacillus coagulans, feeds the crucian carp with the Bacillus coagulans probiotics, detects the immune indexes, white blood cell phagocytic activity, and expression level of tissue immune related cytokines, and performs animal test to evaluate the immune enhancement effect of the Bacillus coagulans probiotics, thereby providing a new idea for the research and development of animal preparation in the later stage. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The growth characteristics and pH curve provided for the embodiment 1 of the present application; Figure 2 The results of serum IgM (immunoglobulin M) content in each experimental group provided in Example 3 of the present invention; Figure 3 The changes in the levels of AST (aspartate aminotransferase), LZM (lysozyme), SOD (superoxide dismutase), and GPX (glutathione peroxidase) in the serum of each experimental group provided in Example 3 of the present invention. Figure 4 The changes in complement C3 and C4 levels in the serum of each experimental group provided in Example 3 of the present invention; Figure 5 The results of relative expression levels of IL-10 (interleukin-10) in different tissues of each experimental group provided in Example 3 of the present invention; Figure 6 The results of relative expression levels of IFN-γ (interferon-γ) in different tissues of each experimental group provided in Example 3 of the present invention; Figure 7 The results of relative expression levels of IL-1β (interleukin-1β) in different tissues of each experimental group provided in Example 3 of the present invention; Figure 8 The results of relative expression levels of TNF-α (tumor necrosis factor-α) in different tissues of each experimental group provided in Example 3 of the present invention; Figure 9 The results of the one-week survival rate of each experimental group provided in Example 3 of the present invention. Detailed Implementation

[0016] 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.

[0017] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0018] Example 1, strain culture: Will Bacillus coagulans yb07 (Bacillus coagulans) yb07 (Purchased from the China Center for Type Culture Collection) After being removed from the refrigerator, the culture was inoculated onto MRS liquid medium for activation and incubated overnight at 37°C at 160 rpm. The culture was then aliquoted into centrifuge tubes and centrifuged at 4500 rpm for 15 min. After discarding the supernatant, PBS was added to adjust the culture volume to 1×10⁻⁶. 8 CFU / mL, store at 4℃; Growth characteristics and pH curve determination: the strain was inoculated into MRS liquid medium at a ratio of 1%, and cultured in a 37°C incubator. The bacterial solution was taken out every 2h to determine the pH and growth curve. The OD value was determined and the curve was plotted as shown in 600nm Figure 1 Figure 1 The culture time of the strain was determined to be 14h.

[0019] Example 2, optimal heat inactivation conditions: After the strain was cultured for the optimal time, it was placed in a 85°C, 90°C, 95°C, 99°C water bath for 10, 20, 30min, respectively. The heat-inactivated bacterial solution was plated on MRS solid medium by dilution and plating method. The growth of colonies on the plate was observed every 12h, as shown in Table 1: Table 1

[0020] It was determined that there was no strain growth on the 36h plate after 99°C treatment for 20min and 30min. The optimal heat inactivation condition was 99°C for 20min.

[0021] Example 3, effect of Bacillus coagulans probiotics on crucian carp: Test process: Bacillus coagulans probiotic preparation and feed preparation: the strain was diluted and plated to determine the concentration of the bacterial solution, and the concentration of the bacterial solution was adjusted to 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, respectively, as low concentration group, medium concentration group, and high concentration group. The prepared probiotic was inactivated in a water bath (99°C, 20min); then the probiotic was mixed with ordinary feed at a ratio of 1ml:1g, and the mixed feed was dried in an oven at 45°C, sealed in a sealed bag, and stored in a 4°C refrigerator for subsequent experiments. Animal experiment grouping and feeding scheme: before the experiment, the fish tank was completely disinfected, and the water was filled in advance. The crucian carp (purchased from Changchun Nature Flower and Bird Fish Market) was disinfected before being put into the tank, and was fed with basic feed for 1 week. The water temperature was 15°C~20°C, and the health condition was observed. 200 fish were selected. They were fed for 4 weeks, and were divided into four tanks (50 fish per tank). The first tank was the control group, and was fed with PBS and ordinary feed mixed at a ratio of 1ml:1g (PBS group). The last three tanks were fed with feed mixed with different concentrations of probiotics, which were low concentration group (A group), medium concentration group (B group), and high concentration group (C group), respectively. The daily feeding amount was 3% of the fish body weight. ​​Sample collection: 5 tails of Carassius auratus were randomly selected from each of the PBS, A, B, and C groups for sampling after anesthesia (every 7 days for a total of 4 times), tail vein blood was collected, the collected blood was placed in a 37℃ incubator for 0.5 h, then stored in a 4℃ refrigerator overnight, centrifuged at 5500 r / min and 4℃ for 15 min, serum was extracted and stored in a -80℃ refrigerator for subsequent detection; 5 tails of Carassius auratus were randomly selected from each of the PBS, A, B, and C groups (every 7 days for a total of 4 times), the body length and weight were measured and recorded and a table was drawn to detect the effect of heat-killed Bacillus coagulans on the growth performance of Carassius auratus; After sampling on the 28th day, 20 tails were randomly selected from each tank for challenge test, 0.2 mL (1×10 7 CFU / mL concentration) A. hydrophila TPS (Aeromonas hydrophila-TPS, preserved in the Animal Science Laboratory of Jilin Agricultural University) was injected into each fish, the protection rate was recorded, the basic diet was fed every day, 5 tails were randomly selected from each tank after one week of feeding, serum was collected, and liver, spleen, kidney, and intestinal fluid were collected under sterile conditions, then frozen with nitrogen and stored in a -80℃ refrigerator for subsequent detection; Test results: 1. The growth performance determination results are shown in Tables 2 and 3: Table 2

[0022] Table 3

[0023] 2. The serum sample collection results of the IgM content in the serum of each test group are shown in Table 4: Figure 2 After 7 days of feeding, the IgM concentration in the serum of the high concentration group, the medium concentration group, and the low concentration group increased, then gradually decreased in the following days, the PBS blank control group had little change; after challenge test, the IgM content in the serum of the high concentration group and the medium concentration group increased significantly, the low concentration group had a slight increase, the IgM content in the serum of the PBS blank control group decreased significantly, and the difference in the IgM content in the serum between the medium concentration group and the PBS blank control group was extremely significant ( P P<0.001), the difference between the high concentration group and the PBS blank control group was significant ( P P<0.01); The changes in the AST, LZM, SOD, and GPX contents in the serum of each test group are shown in Table 5: Figure 3AST: The AST content of the high concentration group, the medium concentration group and the low concentration group decreased after 21 days of feeding, and the PBS control group had no significant change. Until 28 days, the high concentration group had no significant difference with the PBS group (P<0.05), the medium concentration group had significant difference (P<0.01), and the low concentration group had extremely significant difference (P<0.001). After challenge, the AST content of each concentration group increased, but was lower than that of the PBS group and had significant difference (P<0.01); SOD: The SOD content of the high concentration group, the medium concentration group and the low concentration group increased significantly after 14 days of feeding, and the high concentration group and the low concentration group had significant difference with the PBS group (P<0.01), and the medium concentration group had extremely significant difference (P<0.001). After 28 days, the SOD content of the medium concentration group and the low concentration group had extremely significant difference (P<0.001), and each concentration group had extremely significant difference with the PBS group after challenge; GPX: The GPX content of the high concentration group, the medium concentration group and the low concentration group had extremely significant difference (P<0.001) after 28 days of feeding; LZM: The LZM content of the low concentration group was higher than that of the PBS group after 7 days of feeding, and the LZM content of the medium concentration group was higher than that of the high concentration group and the low concentration group after 21 days of feeding. After challenge, the LZM content of each concentration group was higher than that of the PBS group; The changes of C3 and C4 contents in the serum of each test group are shown in the following table: Figure 4 After 21 days of feeding, the C3 content in the serum of the high concentration group, the medium concentration group and the low concentration group was higher than that of the PBS control group, and the C3 content in the serum of the medium concentration group had extremely significant difference with that of the other three groups (P<0.001). After challenge, the C3 content in the serum of the PBS control group decreased, and the C3 content in the serum of the high concentration group had extremely significant difference with that of the PBS group (P<0.001); After 14 days of feeding, the C4 content in the serum of the high concentration group, the medium concentration group and the low concentration group increased significantly, and the C4 content in the serum of the high concentration group increased most obviously, which had significant difference with that of the PBS control group (P<0.01). After 21 days of feeding, the C4 content in the serum of the high concentration group, the medium concentration group and the low concentration group had extremely significant difference with that of the PBS control group (P<0.001), and the C4 content in the serum of the high concentration group had no significant difference with that of the medium concentration group and the low concentration group. After challenge, the C4 content in the serum of each test group decreased, but was still higher than that of the PBS control group and had significant difference (P<0.05); 3. Expression of IL-10 in liver, spleen, kidney and intestine: The relative expression amount of IL-10 in different tissues of each test group is shown in the following table: Figure 5The relative expression of IL-10 in the intestinal tissue of the middle concentration group was higher than that of the high concentration group, the low concentration group and the PBS blank control group after 14 days of feeding and after challenge, and the difference was extremely significant (P<0.001); in the kidney tissue, the high concentration group increased rapidly after 14 days of feeding, and the middle concentration group increased significantly after 21 days and was higher than the high concentration group, and the middle concentration group was still higher than the other groups and the PBS blank control group after challenge, and the difference was extremely significant (P<0.001); the spleen tissue reached the peak from 14 days to 21 days of feeding, and the middle concentration group was higher than the high concentration group, the low concentration group and the PBS blank control group, and the situation was the same after challenge, and the difference was extremely significant (P<0.001); the liver tissue was higher than the low concentration group, the high concentration group and the PBS blank control group after 21 days of feeding, and the high concentration group, the middle concentration group and the low concentration group were higher than the PBS blank control group after challenge, and the difference was extremely significant (P<0.001); 4. Expression of IFN-γ in liver, spleen, kidney and intestine: The relative expression of IFN-γ in different tissues of each test group is shown in Figure 6 In the intestinal tissue, the high concentration group was higher than the low concentration group after 7 days of feeding, and the high concentration group and the middle concentration group were higher than the low concentration group and the control group after 14 days, and the middle concentration group was higher than the high concentration group after 21 days, and the middle concentration group was the highest after challenge, and the difference was extremely significant (P<0.001); in the kidney tissue, the increase was slow after 7 days of feeding, and the high concentration group was higher than the middle concentration group after 14 days, and the middle concentration group was higher than the high concentration group after 21 days, and the high concentration group, the middle concentration group and the low concentration group were higher than the control group after challenge, and the difference was extremely significant (P<0.001); in the spleen tissue, the low concentration group increased rapidly in the first week, the high concentration group was the highest after 14 days, and the middle concentration group was the highest after 28 days, and the high concentration group, the middle concentration group and the low concentration group were higher than the control group after challenge, and the difference was extremely significant (P<0.001); in the liver tissue, each concentration group increased after 7 days of feeding, and reached the highest after 21 days, and the middle concentration group was the highest at this time, and the high concentration group, the middle concentration group and the low concentration group were higher than the control group after challenge, and the difference was extremely significant (P<0.001); 5. Expression of IL-1β in liver, spleen, kidney and intestine: The relative expression of IL-1β in each tissue is shown in Figure 7The results showed that in the intestinal tissue, the expression of IL-1β increased steadily in each concentration group in the first 14 days, and the increase in the medium concentration group was the largest and higher than that in the blank control group. After 21 days, the expression tended to be stable and was still higher than that in other groups. After challenge, the expression decreased significantly, and the expression in the medium concentration group was still significantly higher than that in other groups (P<0.001). In the kidney tissue, the expression in the medium and high concentration groups increased significantly after 14 days, and the expression in the medium concentration group was the highest. After 28 days, the expression reached the peak and was higher than that in other groups. After challenge, the expression decreased significantly, and the expression in the medium concentration group was still significantly higher than that in other groups (P<0.001). In the spleen tissue, the expression in each test group increased significantly after 7 days of feeding, and the expression in the medium concentration group was higher than that in the blank control group. After 21 days, the expression in the medium concentration group was higher than that in the high concentration group. After 28 days, the expression in the low and medium concentration groups was higher than that in the high concentration group. After challenge, the expression decreased, but the expression in each concentration group was still significantly higher than that in the blank control group (P<0.001). In the liver tissue, the expression in each concentration group was the highest after 14 days, and the expression in the medium concentration group was higher than that in the high concentration group. After 21 days, the expression in the low and medium concentration groups was higher than that in the high concentration group and the blank control group. After challenge, the expression in the medium concentration group was significantly higher than that in other groups (P<0.001). 6. Expression of TNF-α in the liver, spleen, kidney and intestine: The relative expression of TNF-α in different tissues in each test group is shown in Figure 8 The results showed that in the intestinal tissue, the expression of IL-1β increased steadily in each concentration group in the first 14 days, and the increase in the medium concentration group was the largest and higher than that in the blank control group. After 21 days, the expression tended to be stable and was still higher than that in other groups. After challenge, the expression decreased significantly, and the expression in the medium concentration group was still significantly higher than that in other groups (P<0.001). In the kidney tissue, the expression in the medium and high concentration groups increased significantly after 14 days, and the expression in the medium concentration group was the highest. After 28 days, the expression reached the peak and was higher than that in other groups. After challenge, the expression decreased significantly, and the expression in the medium concentration group was still significantly higher than that in other groups (P<0.001). In the spleen tissue, the expression in each test group increased significantly after 7 days of feeding, and the expression in the medium concentration group was higher than that in the blank control group. After 21 days, the expression in the medium concentration group was higher than that in the high concentration group. After 28 days, the expression in the low and medium concentration groups was higher than that in the high concentration group. After challenge, the expression decreased, but the expression in each concentration group was still significantly higher than that in the blank control group (P<0.001). In the liver tissue, the expression in each concentration group was the highest after 14 days, and the expression in the medium concentration group was higher than that in the high concentration group. After 21 days, the expression in the low and medium concentration groups was higher than that in the high concentration group and the blank control group. After challenge, the expression in the medium concentration group was significantly higher than that in other groups (P<0.001). 7. Detection of immune protection rate: After 28 days of feeding in each test group and the PBS blank control group, challenge test was performed, and 20 tails were randomly selected from each group. Each tail was injected with 0.2 ml of Aeromonas hydrophila (stored in the Animal Science Laboratory of Jilin Agricultural University) with a concentration of 1×10 7 CFU / mL into the abdominal cavity. The survival rate was recorded for one week, and the results are shown in Table 6. Figure 9The relative protection rates of the low concentration group, the medium concentration group and the high concentration group are 10%, 25% and 15% respectively, and there are still 15% of the survival rate in the PBS group after one week, and the results show that the biofertilizer of Bacillus coagulans has an immune protection effect on the crucian, and the heat-inactivated Bacillus coagulans in the medium concentration group has the best protection effect.

[0024] All data of the embodiments of the present application are analyzed by SPSS 27.0 and GraphPad Prism 8 software, and are expressed as mean ± standard error (SE) of 3 replicates, and the variance difference analysis among each treatment is evaluated by one-way analysis of variance (ANOVA), p Values <0.05 are considered statistically significant.

[0025] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Application of Bacillus coagulans probiotics in improving immune function of crucian.

2. Use according to claim 1, characterized in that, The preparation method of the Bacillus coagulans probiotics comprises the following steps: The Bacillus coagulans is inoculated into a culture medium for culture, and then the bacterial solution is subjected to centrifugal treatment, the supernatant is discarded, PBS is added, and the concentration of the bacterial solution is adjusted. The bacterial solution is subjected to heat inactivation treatment to obtain the Bacillus coagulans probiotics.

3. Use according to claim 2, characterized in that, The adjusted bacteria solution concentration is 1 x 10 6 -1 x 10 8 CFU / mL.

4. Use according to claim 2, characterized in that, The heat inactivation treatment is water bath treatment, the temperature is 99 DEG C, and the time is 20 min.

5. The use according to claim 1, characterized in that, The following steps are included: The Bacillus coagulans probiotics are uniformly mixed with daily ration feed, and the crucian is fed.

6. Use according to claim 5, characterized in that, The volume to mass ratio of the Bacillus coagulans probiotics to daily ration feed is 0.8-1.2:

1.

7. A carassius auratus bioformulation characterized in that, The preparation includes the bacillus coagulans probiotics, and a bacterial liquid concentration of the bacillus coagulans probiotics is 1×10 6 -1×10 8 CFU / mL.

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