Probiotic characteristics of fish intestinal probiotics and application of fish intestinal probiotics as feed additive to growth promotion and disease resistance

Abstract

The invention relates to a probiotic and a feed additive for fish culture and application of the probiotic and the feed additive, and the probiotic for fish culture is a Lactococcus lactis strain HNL12 (MK889240) separated from intestinal tracts of wild humpback perches by a research group. The probiotic feed additive disclosed by the invention is used for promoting the growth of the humpback perches, improving the non-specific immunity of the humpback perches and improving the antibacterial ability of the humpback perches. The method is of great significance to healthy breeding of the humpback perches and prevention and control of bacterial diseases.

Description

Technical Field

[0001] The invention relates to the field of probiotics, and in particular to a fish intestinal probiotic agent and application thereof in promoting growth and preventing and controlling bacterial diseases. Background Art

[0002] Humpback perch ( Cromileptes altivelis ) is an important marine economic fish. It has become an important part of the global food market due to its rich nutritional value. It also occupies a place in the ornamental fish market due to its diverse shapes and colors. As consumer demand for humpback bass continues to rise, its aquaculture scale continues to expand, and disease problems are becoming increasingly prominent, especially various bacterial diseases.

[0003] At present, the most commonly used method for fish disease prevention and control is still to mix feed with oral anti-pathogen drugs, but the disordered use of drugs has seriously affected the safety of the breeding environment and product quality. Probiotics are a type of microorganism that can regulate the intestinal flora of animals and have a beneficial effect on host animals. Compared with antibiotics, probiotics do not develop drug resistance, nor do they produce drug residues, let alone substances that are harmful to the environment. Compared with ordinary feed, probiotics are not only limited to controlling diseases, but also have the effects of promoting growth, promoting digestion and immunomodulation. After probiotics colonize in the intestine, they can compete with intestinal pathogens for nutrients and reduce the abundance of intestinal pathogens. At the same time, probiotics use undigested food from the host to produce substances such as short-chain fatty acids, which can provide fish with abundant energy. The intestinal mucosal barrier is a structure that isolates the intestine from harmful substances and pathogen invasion, and is composed of mechanical barriers, chemical barriers, immune barriers and biological barriers. Probiotics can colonize on the intestinal mucosa to form a biological barrier in the intestine, preventing intestinal pathogens from invading the intestinal mucosa. Probiotics also have good immunomodulatory effects, can promote the secretion of intestinal antimicrobial peptides, regulate the expression of intestinal cytokines, and promote the integrity of the intestinal mucosal barrier. Summary of the invention

[0004] The purpose of the present invention is to provide a probiotic strain for fish farming, a feed additive for fish farming, and applications of the probiotic strains for preparing fish feed and products for promoting the growth and disease resistance of humpback seabass.

[0005] The probiotic Lactococcus lactis of the present invention ( Lactococcus lactis ) The HNL12 strain was isolated from the intestine of wild humpback seabass and grew well on MRS medium. A strain of Lactococcus lactis was obtained by streaking and was named Lactococcus lactis ( L. lactis )HNL12, the NCBI accession number of its 16S rDNA sequence is: MK889240.

[0006] Probiotic Lactococcus lactis ( L. lactis )The HNL12 strain has the following characteristics: Lactococcus lactis HNL12 forms milky white, smooth and moist colonies with neat edges on MRS culture medium and is a Gram-positive coccus; the HNL12 is acid-resistant, with a survival rate of over 90% under pH 3 and a survival rate of 60% under pH 2; the HNL12 is bile-resistant, and when cultured in artificial intestinal fluid containing 0.3% and 0.5% ox bile, it shows good tolerance with only a slight decrease in proliferation ability; the HNL12 has self-agglutination properties, with a self-agglutination rate of 75% 4 hours after culture, which can form a biological barrier in the intestine to effectively resist the invasion of pathogens.

[0007] The 16SrDNA sequence of the Lactococcus lactis HNL12 strain of the present invention is more than 99% similar to the 16SrDNA sequence of the Lactococcus lactis currently published. According to the 16SrDNA sequence analysis, the Lactococcus lactis HNL12 strain is identified as Lactococcus lactis ( L. lactis ).

[0008] The present invention has developed a new strategy to increase the production and income of fish farming, namely, Lactococcus lactis ( L. lactis ) HNL12 was fed to fish as a feed additive, and it was found that the feed was effective in promoting the growth and disease resistance of fish, which was reflected in the following aspects: (1) It has good acid resistance, choline resistance and self-agglutination properties; (2) It increases the weight gain rate and specific growth rate of fish; (3) It increases the immune activity of fish; and (4) It improves the disease resistance of fish.

[0009] Therefore, a second object of the present invention is to provide the Lactococcus lactis ( L. lactis ) Application of HNL12 in the preparation of marine fish feed additives.

[0010] The third object of the present invention is to provide a method for promoting the growth and disease resistance of fish, which comprises the following steps: L. lactis ) HNL12 is used in marine fish.

[0011] Preferably, the Lactococcus lactis ( L. lactis ) HNL12 is used in combination with feed as a feed additive.

[0012] The present invention has the following beneficial effects: A strain of humpback sea bass probiotic Lactococcus lactis isolated by the present invention L. lactis) The HNL12 strain has antibacterial and immune-enhancing properties against humpback seabass, and has certain application prospects in the disease resistance and immune enhancement of humpback seabass. Specifically, it has the following characteristics: (1) It has good acid resistance, choline resistance and self-agglutination properties; (2) It improves the weight gain rate and specific growth rate of fish; (3) It improves the immune activity of fish; (4) It improves the disease resistance of fish. Therefore, the use of this probiotic in the preparation of fish feed additives or fish feed has a good prospect. At the same time, the above strain is a probiotic, the product is highly safe, and it is not easy to produce resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the self-agglutination rate of Lactococcus lactis HNL12 strain at different incubation times; Figure 2 This is the effect of feeding different concentrations of HNL12 strain on superoxide dismutase in humpback seabass; Figure 3 This is a graph showing the effect of feeding different concentrations of HNL12 strain on the acid phosphatase of humpback seabass; Figure 4 This graph shows the effect of feeding different concentrations of HNL12 strain on the lysozyme of humpback seabass.

[0014] Figure 5 This graph shows the effect of different concentrations of Lactococcus lactis HNL12 strain on the immune protection of humpback seabass. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further explained below by examples, but the technical solution of the present invention is not limited in any form by the examples.

[0016] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0017] Example 1, Lactococcus lactis ( L. lactis )Study on acid resistance of HNL12 strain: Experimental method: Add 0.5 g sodium chloride and 30 mg pepsin to 60 mL of sterile PBS, adjust the pH to 2, 3, or 4 with 1 M HCl or 1 M NaOH to prepare simulated artificial gastric juice with different pH values, and finally adjust the volume to 100 mL and filter with a 0.22 μm microporous filter membrane for sterilization. The HNL12 bacterial solution cultured overnight at 30°C was centrifuged at 4000 rpm / min for 10 min, washed twice with sterile PBS (pH 7.4), and then resuspended with sterile PBS to adjust the bacterial solution concentration to 108 CFU / mL. The bacterial solution was then inoculated into the simulated artificial gastric fluid with different pH values ​​prepared above (5%, v / v), and cultured at 30°C for different time periods (0h, 2h, 4h). 100 μL was taken out in each culture time period, and after 10-fold gradient dilution, it was evenly spread on MRS-CaCO3 solid culture medium. There were 3 gradients at each time point and 3 parallels for each dilution. The culture was cultured in a constant temperature incubator at 30°C overnight. The next day, the plates with single colonies showing calcium dissolution circles and colony counts between 30 and 300 were selected for colony counting, and the number of viable bacteria per unit volume was calculated.

[0018] Experimental results: The results of the study on the tolerance of HNL12 to simulated artificial gastric fluid with different pH values ​​showed (Table 1) that when the pH was 2, the number of viable bacteria of HNL12 after incubation in simulated artificial gastric fluid (SGF) for 0h, 2h and 4h were (1.053±0.017)×108, (5.693±0.082)×107 and (3.850±0.081)×107, respectively, and the concentration of viable bacteria gradually decreased with time; when the pH was 4, the number of cells of HNL12 after incubation in simulated artificial gastric fluid for 2h and 4h were (1.054±0.023)×108 and (1.034±0.009)×108, respectively, and the number of viable bacteria was not significantly different from that of the control group (0h, (1.085±0.062)×108) (p>0.05). The above results show that lactic acid bacteria HNL12 has strong survival ability in simulated artificial gastric fluid of different pH values ​​and has the ability to colonize in the acidic environment of the intestine.

[0019] Table 1 Test results of Lactococcus lactis HNL12 in artificial simulated gastric fluid Example 2, Lactococcus lactis ( L. lactis )Study on the bile resistance of HNL12 strain: Experimental method: Add 10 mg trypsin and 0.3 g ox bile salt to 60 mL of sterile PBS, respectively, and then adjust the pH to 6.8 and 8.0 with 1 M HCl or 1 M NaOH to prepare artificial intestinal fluid with different pH values. Finally, make the volume to 100 mL and filter with a 0.22 μm microporous filter membrane for sterilization. The HNL12 bacterial solution cultured overnight at 30°C was centrifuged at 4000 rpm / min for 10 min, washed twice with sterile PBS (pH 7.4), and then resuspended with sterile PBS to adjust the bacterial solution concentration to 108 CFU / mL. The bacterial solution was then inoculated into simulated artificial intestinal fluid with different pH values ​​(5%, v / v) and cultured at 30°C for different time periods (0h, 2h, 4h). Similarly, 100 μL was taken out in each culture time period, diluted 10 times in a gradient dilution, and then spread on MRS-CaCO3 solid culture medium. There were 3 dilutions at each time point, and 3 parallels for each dilution. The culture was cultured in a constant temperature incubator at 30°C overnight. The next day, the plates with single colonies showing calcium dissolution circles and colony counts between 30 and 300 were selected for colony counting, and the number of viable bacteria per unit volume was calculated.

[0020] Experimental results: The results of the study on the tolerance of HNL12 to simulated artificial intestinal fluids of different pH values ​​showed (Table 2) that in simulated artificial intestinal fluids (SIG) of pH 6.8 and 8.0, the number of HNL12 cells decreased after 2 hours of incubation, but at pH 6.8, the number of cells increased by 144.29% after 4 hours of incubation. The above results show that lactic acid bacteria HNL12 have strong survival ability in simulated artificial intestinal fluids of different pH values ​​and have the ability to colonize in high bile salt in the intestine.

[0021] Table 2 Detection results of Lactococcus lactis HNL12 artificial simulated intestinal fluid Example 3, Lactococcus lactis ( L. lactis )Study on the self-agglutination characteristics of HNL12 strain: (1) Experimental method: The overnight cultured HNL12 was centrifuged at 4000 rpm / min for 10 min, then washed twice with sterile PBS (pH 7.4), and the cells were resuspended with sterile PBS to OD600≈1. Then, 4 mL of the PBS resuspension was turbine-oscillated for 10 s, incubated at room temperature, and then 100 μL of the upper liquid was aspirated and dropped into the microwells of the ELISA plate at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 10 h, and the absorbance at 600 nm was measured. Three parallels were performed each time. The calculation formula is: Autoagglutination rate = 1-At / A0×100%, where At is the absorbance at 1 h, 2 h, 3 h, 4 h, 5 h, and 10 h, and A0 is the absorbance at 0 h.

[0022] (2) Experimental results: The experimental results are as follows Figure 1As shown in the figure, the autoagglutination rate is related to the adhesion ability of bacteria on epithelial cells. The results of autoagglutination test under different incubation times showed that the autoagglutination of Lactococcus lactis HNL12 increased with the extension of incubation time. After 1 hour of incubation, its autoagglutination rate was 35.57%, and after 10 hours of incubation, its autoagglutination rate increased to 79.2%, showing a high degree of autoagglutination.

[0023] Example 4, Lactococcus lactis ( L. lactis )Study on the growth-promoting properties of HNL12 strain: (1) Experimental methods: The average body weight of humpback seabass for growth index determination was 3.97±0.54 g. Before the experiment, the fish were temporarily kept in a circular cement pool with a radius of 45 cm and a water depth of 50 cm with all-weather circulating water. Commercial feed was fed at a fixed time and place every day. After adaptation, the experimental fish were randomly divided into 4 groups, 30 fish in each group, and the groups were numbered A, B, C, and D. Group A was the blank control group without HNL12 added, which was fed with commercial basic feed without HNL12; Group B was fed with HNL12 feed with a live bacteria count of 1×106 CFU / g; Group C was fed with HNL12 feed with a live bacteria count of 1×108 CFU / g; Group D was fed with HNL12 feed with a live bacteria count of 1×1010 CFU / g; each group had 2 parallels. The feed amount was 3% of the fish body weight, and the fish were fed twice a day, at 7:30 am and 6:00 pm. After feeding, the fish feeding situation was observed and the water quality changes were monitored. The experiment was carried out for 4 consecutive weeks. After feeding each group of experimental humpback bass with feed containing different concentrations of HNL12, samples were taken at the beginning of the experiment and every other week after the experiment. Ten humpback bass were randomly selected from each group to measure their weight and length. The weight gain rate and specific growth rate were calculated according to the following formula: Weight gain rate (%) (PWG) = [(final weight – initial weight) / (initial weight)] × 100% Specific growth rate (% / day) (SGR) = 100 × [ln (final weight) - ln (initial weight)] / number of days x 100% (2) Experimental results: After feeding humpback seabass with different concentrations of HNL12 for 4 weeks, the growth of each group of experimental humpback seabass is shown in Table 3. The results show that the body weight and length of group C fed with HNL12 containing a final concentration of 108 CFU / g of lactococcus lactis were the best; the weight gain rate (PWG) of group C fed with a final concentration of 108 CFU / g of lactococcus lactis and group D fed with a final concentration of 1010 CFU / g of lactococcus lactis were significantly higher than that of the control group and group B fed with a final concentration of 106 CFU / g of lactococcus lactis (p<0.05). In terms of specific growth rate (SGR), groups C and D were also significantly higher than the control group and group B (p<0.05).

[0024] Table 3 Growth of humpback seabass fed with different concentrations of HNL12 Note: Data are expressed as (mean ± standard error), different letters in each group at the same sampling time point indicate significant differences (p < 0.05), n=10.

[0025] Example 5, Lactococcus lactis ( L. lactis )Study on the immunoenhancing properties of HNL12 strain: (1) Experimental methods: The average body weight of humpback seabass for disease resistance determination was 23.98±0.71 g. Before the experiment, the fish were temporarily kept in a circular cement pool with a radius of 45 cm and a water depth of 50 cm with all-weather circulating water. Commercial feed was fed at a fixed time and place every day. After adaptation, the experimental fish were randomly divided into 4 groups, 30 fish in each group, and the groups were numbered A, B, C, and D. Group A was the blank control group without HNL12 added, which was fed with commercial basic feed without HNL12; Group B was fed with HNL12 feed with a live bacteria count of 1×106 CFU / g; Group C was fed with HNL12 feed with a live bacteria count of 1×108 CFU / g; Group D was fed with HNL12 feed with a live bacteria count of 1×1010 CFU / g; each group had 2 parallels. The feed amount was 3% of the fish body weight, and the fish were fed twice a day, at 7:30 am and 6:00 pm. After feeding, the fish feeding situation was observed and the water quality changes were monitored. The experiment was carried out for 4 consecutive weeks. In the 1st, 2nd, 3rd and 4th week after the start of the experiment, 5 humpback seabass were randomly selected from each experimental group for non-specific determination, and blood was collected from the caudal vein with a disposable syringe after anesthesia. The extracted blood was slowly transferred to a 1.5mL sterile centrifuge tube, allowed to stand at room temperature for 40 minutes, and then slightly tilted and placed in a refrigerator (4°C) overnight. After centrifugation at 800g / min and 4°C for 15 minutes, the supernatant was aspirated and stored at -80°C for later use. The following disease resistance indicators were then determined using a commercial kit: ① Use the lysozyme detection kit to measure the activity of lysozyme (LZM) in serum; ② Use the acid phosphatase detection kit to measure the activity of acid phosphatase (ACP) in serum; ③Use superoxide dismutase detection kit to determine the activity of superoxide dismutase (SOD) in serum.

[0026] (2) Experimental results: ① After adding different concentrations of HNL12 to the feed and feeding humpback seabass for four weeks, the test results of superoxide dismutase (SOD) activity of each group of experimental fish are shown in Figure 2. The results showed that after one week of feeding, the SOD activity of groups C and D was significantly higher than that of the control group (p<0.05), and the SOD activity of group B was significantly lower than that of the control group (p<0.05); at the same time, the superoxide dismutase activity of group C was significantly better than that of groups B and D (p<0.05). After continuous feeding of different concentrations of HNL12 bacterial solution for 2-4 weeks, the SOD activity of each group of experimental fish was not significantly different from that of the control group (p>0.05). The above results show that the Lactococcus lactis HNL12 strain has the ability to increase the superoxide dismutase activity of fish and has the characteristics of enhancing the immunity of fish.

[0027] ② After adding different concentrations of HNL12 to the feed and feeding humpback seabass for four weeks, the test results of acid phosphatase (ACP) activity of each group of experimental fish are shown in Figure 3 . One-way ANOVA showed that after two weeks of feeding HNL12, the activity of acid phosphatase in each experimental group was significantly enhanced (p<0.05), and multiple comparison analysis showed that the acid phosphatase activity of group C was significantly better than that of group B (p<0.05). In the first, third and fourth weeks after feeding HNL12, there was no significant difference in the ACP activity of the experimental fish and the control group (p>0.05). The above results show that the Lactococcus lactis HNL12 strain has the ability to increase the activity of acid phosphatase in fish and has the characteristics of enhancing the immunity of fish.

[0028] ③ After adding different concentrations of HNL12 to the feed and feeding humpback seabass for four weeks, the test results of lysozyme (LZM) activity of each group of experimental fish are shown in Figure 4 . One-way ANOVA showed that after two weeks of feeding HNL12, the lysozyme activity of each experimental group was significantly higher than that of the control group (p<0.05). After four weeks of feeding HNL12, the lysozyme activity of group C was significantly higher than that of the control group (p<0.05). Multiple comparison analysis showed that after two weeks of feeding HNL12, the lysozyme activity of groups C and D was significantly better than that of group B (p<0.05); after four consecutive weeks of feeding, group C was significantly better than groups B and D (p<0.05). There was no significant difference in the LZM activity of each group in the first and third weeks of feeding (p>0.05). The above results show that the Lactococcus lactis HNL12 strain has the ability to increase the lysozyme activity of fish and has the characteristics of enhancing the immunity of fish.

[0029] Example 6, Lactococcus lactis ( L. lactis )Study on the disease resistance of HNL12 strain: Experimental method: After activation, a single colony of Vibrio harveyi QT520 stored at -80℃ was taken and cultured at 30℃, 180rpm shaking for 8h. The OD600 value was determined by spectrophotometer. The bacterial solution with OD600≈0.8 was centrifuged at 3000 rpm / min at room temperature. The precipitate was resuspended twice with sterile PBS. Finally, the cell suspension was made with sterile PBS and the OD600 value was accurately determined. According to the results of the pre-experimental challenge, a bacterial suspension with a concentration of 1.0×106CFU / mL was prepared with sterile PBS. The challenge was intraperitoneally injected with 100μL / tail, 25 tails per group. The activity and death of each group of challenged fish were recorded every day after the challenge, and the records were observed for two weeks after the challenge. The relative protection rate (RPS) of each experimental group was calculated using the following formula: RPS = (1-mortality rate of experimental group / mortality rate of control group) × 100%.

[0030] (2) Experimental results: After feeding humpback sea bass with different concentrations of HNL12 for one month, the results of the challenge with the virulent strain QT520 of Harvey showed that ( Figure 5 ), the survival rates of experimental fish in groups B, C and D fed with different concentrations of Lactococcus lactis HNL12 were 56%, 70% and 68%, respectively, while the survival rate of the control group was 36%. The survival rates of groups C and D were significantly higher than those of the control group (p<0.05), and the relative protection rates of groups B, C and D were 30.98%, 53.14% and 50.20%, respectively. The results showed that Lactococcus lactis HNL12 had a good protective effect on humpback seabass, among which the protective effect of group C with a concentration of 108CF / g was the best. The above results show that the Lactococcus lactis HNL12 strain has the ability to improve the survival of fish and has the characteristics of enhancing the disease resistance of fish.

[0031] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A probiotic for fish farming, characterized in that: The probiotics for fish farming are Lactococcus lactis strain HNL12 (MK889240).

2. A feed additive for fish farming, characterized in that: The feed additive contains the probiotics according to claim 1.

3. The feed additive for fish farming according to claim 2, characterized in that: The fish feed containing the probiotic is prepared according to a method comprising the following steps: (A1) Lactococcus lactis strain HNL12 (MK889240) was inoculated into a culture medium for activation and fermentation to obtain a fermentation broth, which was centrifuged and diluted with PBS to obtain a cell suspension. (A2) Use a spray bottle to spray the diluted cell suspension obtained in (A1) on the surface of the basic feed (51.7% crude protein, 8.4% crude fat, 12.9% ash, 9.0% humidity) to fully mix it with the feed. After stirring evenly, air-dry it in an ultra-clean workbench to obtain the feed. The dried feed is stored at 4°C.

4. A fish feed, characterized in that: The fish feed comprises a basic feed and the probiotics according to claim 1 or the feed additive according to claim 2.

5. Use of the probiotics according to claim 1 or the feed additive according to claim 2 in the preparation of a product for promoting fish growth and / or improving fish disease resistance.

6. The fish feed, method or use according to any one of claims 1 to 5, characterized in that: The fish disease resistance is a fish disease caused by Vibrio harveyi; The fish is a marine fish; Furthermore, the marine fish is humpback seabass.

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