A composite immune enhancer for young pike perch and its application

Abstract

A composite immunopotentiator for pike perch fry and its application, relating to the field of aquaculture, is to solve the problem that existing probiotics obtained from other species will cause intestinal damage to the tested pike perch. The composite immunopotentiator for pike perch fry of the present invention comprises Bacillus subtilis RT-BS07 and Bacillus plantarum H8, and the method is as follows: 1. Feeding Bacillus subtilis RT-BS07 feed to pike perch fry at 0-4 weeks; 2. Mixing Bacillus subtilis RT-BS07 bacterial liquid and Bacillus plantarum H8 bacterial liquid to obtain a composite immunopotentiator, spraying the composite immunopotentiator on the surface of the fish feed, and feeding the pike perch fry at 5-8 weeks after drying in the shade. The composite immunopotentiator of the present invention has a good promoting effect on the growth of pike perch fry; and has a good effect on improving the disease resistance of pike perch fry. The present invention is used for the breeding of pike perch fry.

Description

Technical Field

[0001] The invention relates to the field of aquaculture, and in particular to a composite immunoenhancer for young pike perch and application thereof. Background Art

[0002] Zander (Sander lucioperca) belongs to the order Perciformes, family Percidae, genus Zander. It is native to the rivers and lakes of the Aral Sea, Black Sea, Caspian Sea and Baltic Sea systems in central Europe. Zander is the main economic species of Percidae fish and an important species for freshwater aquaculture. It is large in size, tender in texture, delicious in taste, and has no intramuscular spines. It is known as the "king of freshwater fish". The protein content in the fish meat is as high as 20%, which has extremely high edible, nutritional and economic value.

[0003] The artificial breeding and farming technology system of zander has been constantly explored and improved. However, zander fry, juveniles and young fish are generally weak and easily frightened, which inevitably leads to rapid swimming, reduced food intake, and physical decline, resulting in growth stunting of young fish and frequent diseases, causing great trouble and obstacles to the development of the industry.

[0004] Aquatic probiotics have the effects of improving water quality, promoting feed digestion and absorption and body growth, and enhancing body immunity and disease resistance. However, most of the existing probiotics are commercialized strains for livestock and poultry. In existing aquaculture methods, the use of exogenous probiotics obtained from other species can cause safety issues such as damage to the intestinal tract of the tested fish, which affects the growth of fish. Summary of the invention

[0005] The invention aims to solve the problem that the existing probiotics obtained from other species will affect the growth of pike perch, and provides a composite immune enhancer for pike perch fry and application thereof.

[0006] The invention provides a composite immunoenhancer for young pike perch, comprising Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8, wherein the final concentration of Bacillus subtilis RT-BS07 added to fish feed is 1.0×10 7 CFU / g, the final concentration of Lactobacillus plantarum H8 added to fish feed was 1.0×10 6 CFU / g.

[0007] The plant lactobacillus H8 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the storage address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the storage date is April 7, 2024, and the storage number is CGMCC No. 30268.

[0008] The present invention also provides the use of the composite immunoenhancer in the breeding of young pike perch, and the specific method is as follows:

[0009] 1. Spray the Bacillus subtilis RT-BS07 bacterial liquid onto the surface of the fish feed, dry it in the shade to obtain the Bacillus subtilis RT-BS07 feed, and feed the Bacillus subtilis RT-BS07 feed to the pike perch fry at 0-4 weeks old; the final concentration of Bacillus subtilis RT-BS07 in the fish feed is 1.0×10 7 CFU / g;

[0010] 2. Mix Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8 to obtain a composite immunopotentiator, spray the composite immunopotentiator onto the surface of fish feed, dry it in the shade and feed it to 5-8 week old pike perch fry; the final concentration of Bacillus subtilis RT-BS07 in the fish feed is 1.0×10 7 CFU / g, the final concentration of Lactobacillus plantarum H8 was 1.0×10 6 CFU / g.

[0011] Furthermore, the preparation method of the Bacillus subtilis RT-BS07 bacterial solution is:

[0012] After thawing the Bacillus subtilis RT-BS07 strain stored at -80°C on ice, streak the Bacillus subtilis RT-BS07 on an LB solid culture plate under sterile conditions, and then invert and culture it at 25°C for 24 hours; pick a single colony in an LB liquid culture medium, and culture it at 120rpm for 18 hours at 25°C to obtain an RT-BS07 seed solution; take the RT-BS07 seed solution and add it to an LB liquid culture medium (inoculation amount is 1‰), and culture it at 120rpm for 24-36 hours at 25°C; collect the bacteria by centrifugation at 3000rpm for 10 minutes, wash and resuspend the bacteria with sterile phosphate buffer PBS to obtain a Bacillus subtilis RT-BS07 bacterial solution, and temporarily store it at 4°C for use.

[0013] Furthermore, the preparation method of the plant lactobacillus H8 bacterial liquid is as follows:

[0014] The plant lactobacillus H8 strain stored at -80°C is thawed on ice, and the plant lactobacillus H8 is streaked on an MRS solid culture plate under sterile conditions, and then inverted and cultured at 25°C for 24 hours; a single colony is picked in an MRS liquid culture medium, and cultured at 120 rpm for 18 hours at 25°C to obtain an H8 seed liquid; the H8 seed liquid is added to an MRS liquid culture medium (the inoculation amount is 1‰), and cultured at 120 rpm for 24-36 hours at 25°C; the bacterial bodies are collected by centrifugation at 3000 rpm for 10 minutes, washed and resuspended with a sterile phosphate buffer solution PBS to obtain a plant lactobacillus H8 bacterial liquid, and temporarily stored at 4°C for use.

[0015] The present invention provides application of the above-mentioned pike perch fry composite immunoenhancer in promoting the growth of pike perch fry.

[0016] The present invention provides application of the above-mentioned pike perch fry composite immunoenhancer in inhibiting pathogenic bacteria of pike perch fry.

[0017] Furthermore, the pathogenic bacteria are Aeromonas hydrophila, Aeromonas veronii and Aeromonas salmonicida.

[0018] Beneficial effects of the present invention:

[0019] The composite immunopotentiator of the present invention comprises Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8, and the two strains are screened from the healthy intestinal tract of rainbow trout and the healthy intestinal tract of carp, respectively. Adding the composite immunopotentiator of the present invention to feed has a good promoting effect on the growth of young pike perch; it has a good effect on improving the disease resistance of young pike perch; and the growth promoting effect is better for small-sized young fish in the first 4 weeks of feeding. It is speculated that the intestinal flora and digestive capacity of small-sized fish are not yet complete, and they are more susceptible to the regulation of probiotics. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0021] Specific implementation method 1: In this implementation method, the composite immune enhancer for young pike perch includes Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8, wherein the final concentration of Bacillus subtilis RT-BS07 added to the fish feed is 1.0×10 7 CFU / g, the final concentration of Lactobacillus plantarum H8 added to fish feed was 1.0×10 6 CFU / g.

[0022] The plant lactobacillus H8 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the storage address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the storage date is April 7, 2024, and the storage number is CGMCC No. 30268.

[0023] Specific implementation method 2: The application of the composite immune enhancer for young pike perch in the breeding of young pike perch in this implementation method is as follows:

[0024] 1. Spray the Bacillus subtilis RT-BS07 bacterial liquid onto the surface of the fish feed, dry it in the shade to obtain the Bacillus subtilis RT-BS07 feed, and feed the Bacillus subtilis RT-BS07 feed to the pike perch fry at 0-4 weeks old; the final concentration of Bacillus subtilis RT-BS07 in the fish feed is 1.0×10 7 CFU / g;

[0025] 2. Mix Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8 to obtain a composite immunopotentiator, spray the composite immunopotentiator onto the surface of fish feed, dry it in the shade and feed it to 5-8 week old pike perch fry; the final concentration of Bacillus subtilis RT-BS07 in the fish feed is 1.0×10 7 CFU / g, the final concentration of Lactobacillus plantarum H8 was 1.0×10 6 CFU / g.

[0026] Specific embodiment 3: This embodiment is different from the specific embodiment 2 in that: the preparation method of the Bacillus subtilis RT-BS07 bacterial liquid is:

[0027] After thawing the Bacillus subtilis RT-BS07 strain stored at -80°C on ice, streak the Bacillus subtilis RT-BS07 on an LB solid culture plate under sterile conditions, and then invert and culture it at 25°C for 24 hours; pick a single colony in an LB liquid culture medium, shake and culture it at 120rpm for 18 hours at 25°C to obtain RT-BS07 seed liquid; take the RT-BS07 seed liquid and add it to the LB liquid culture medium (inoculation amount is 1‰), shake and culture it at 120rpm for 24-36 hours at 25°C; collect the bacteria by centrifugation at 3000rpm for 10 minutes, wash and resuspend the bacteria with sterile phosphate buffer PBS, obtain the Bacillus subtilis RT-BS07 bacterial liquid, and temporarily store it at 4°C for use. The rest is the same as the second specific implementation method.

[0028] Specific embodiment 4: This embodiment is different from specific embodiment 2 or 3 in that: the preparation method of the plant lactobacillus H8 bacterial liquid is:

[0029] After thawing on ice, plant lactobacillus H8 strains stored at -80°C are streaked on MRS solid culture plates under aseptic conditions, and then inverted and cultured at 25°C for 24h; a single colony is picked in MRS liquid culture medium, and cultured at 120rpm for 18h at 25°C to obtain H8 seed liquid; the H8 seed liquid is added to MRS liquid culture medium (inoculation amount is 1‰), and cultured at 120rpm for 24-36h at 25°C; the bacterial bodies are collected by centrifugation at 3000rpm for 10min, washed and resuspended with sterile phosphate buffer PBS, and plant lactobacillus H8 bacterial liquid is obtained, and stored at 4°C for standby use. Others are the same as the second or third specific embodiments.

[0030] Specific implementation method 5: The application of the composite immunoenhancer for young pike perch in this implementation method in promoting the growth of young pike perch.

[0031] Specific implementation method six: the application of the composite immunoenhancer for young pike perch in this implementation method in inhibiting pathogenic bacteria of young pike perch.

[0032] Specific embodiment 7: This embodiment is different from specific embodiment 6 in that the pathogenic bacteria are Aeromonas hydrophila, Aeromonas veronii and Aeromonas salmonicida. The rest is the same as specific embodiment 6.

[0033] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0034] The present invention compares and analyzes the intestinal microbial diversity of pike perch fry and adult fish fed with different baits (live bait and artificial feed), and finds that the dominant bacterial phylum of microorganisms in the intestine of pike perch fry that have not opened their mouths is Firmicutes, while the dominant bacterial phylum of adult fish fed with live bait or artificial feed is Proteobacteria and Fusobacteria. The abundance of Fusobacteria in the intestine of adult fish fed with artificial feed is higher (35.68%), while the abundance of Acidobacteria in adult fish fed with live bait is higher (4.84%).

[0035] Therefore, it is planned to add fish-derived Firmicutes probiotic Bacillus subtilis after the young zander start eating to help them digest and absorb nutrients and buffer the impact of changes in intestinal microbial richness. In addition, Lactobacillus plantarum of the Acidobacteria phylum, which has antibacterial and immunity-enhancing effects, is added to regulate its disease resistance.

[0036] Embodiment 1:

[0037] The preparation method of Bacillus subtilis RT-BS07 feed in this embodiment comprises the following steps:

[0038] 1. Thaw the Bacillus subtilis RT-BS07 strain stored at -80°C on ice, streak the Bacillus subtilis RT-BS07 on an LB solid culture plate under sterile conditions, and then invert and culture it at 25°C for 24 hours; pick a single colony in an LB liquid culture medium, shake and culture it at 120rpm for 18 hours at 25°C to obtain RT-BS07 seed liquid; add the RT-BS07 seed liquid to an LB liquid culture medium (inoculation amount is 1‰), shake and culture it at 120rpm for 24-36 hours at 25°C; collect the bacteria by centrifugation at 3000rpm for 10 minutes, wash and resuspend the bacteria with sterile phosphate buffer PBS to obtain Bacillus subtilis RT-BS07 bacterial liquid, and store it at 4°C for later use;

[0039] 2. Spray the Bacillus subtilis RT-BS07 bacterial liquid onto the surface of the fish feed and dry it in the shade to obtain the Bacillus subtilis RT-BS07 feed; wherein the concentration of Bacillus subtilis RT-BS07 in the Bacillus subtilis RT-BS07 feed is 1.0×10 7 The fish feed is a commercially available California bass feed from Zhejiang Dongyu Biotechnology Co., Ltd., with a particle size of 1 mm.

[0040] Embodiment 2:

[0041] The preparation method of the plant lactobacillus H8 feed of the present embodiment comprises the following steps:

[0042] 1. After thawing on ice, plant lactobacillus H8 strains stored at -80°C were streaked on MRS solid culture plates under aseptic conditions, and then inverted and cultured at 25°C for 24 hours; single colonies were picked in MRS liquid culture medium, and cultured at 120rpm for 18 hours at 25°C to obtain H8 seed liquid; the H8 seed liquid was added to MRS liquid culture medium (inoculation amount was 1‰), and cultured at 120rpm for 24 hours at 25°C; the bacterial cells were collected by centrifugation at 3000rpm for 10 minutes, washed and resuspended with sterile phosphate buffered saline (PBS), and the bacterial cell liquid was obtained, which was temporarily stored at 4°C for standby use;

[0043] 2. Spraying the Lactobacillus plantarum H8 bacterial liquid onto the surface of the fish feed and drying in the shade to obtain the Lactobacillus plantarum H8 feed; wherein the concentration of the Lactobacillus plantarum H8 in the Lactobacillus plantarum H8 feed is 1.0×10 6 The fish feed is a commercially available California bass feed from Zhejiang Dongyu Biotechnology Co., Ltd., with a particle size of 1 mm.

[0044] Embodiment 3:

[0045] The preparation method of the composite immune enhancer feed for young pike perch of this embodiment comprises the following steps:

[0046] 1. Thaw the Bacillus subtilis RT-BS07 strain stored at -80°C on ice, streak the Bacillus subtilis RT-BS07 on an LB solid culture plate under sterile conditions, and then invert and culture it at 25°C for 24 hours; pick a single colony in an LB liquid culture medium, shake and culture it at 120rpm for 18 hours at 25°C to obtain RT-BS07 seed liquid; add the RT-BS07 seed liquid to an LB liquid culture medium (inoculation amount is 1‰), shake and culture it at 120rpm for 24-36 hours at 25°C; collect the bacteria by centrifugation at 3000rpm for 10 minutes, wash and resuspend the bacteria with sterile phosphate buffer PBS to obtain Bacillus subtilis RT-BS07 bacterial liquid, and store it at 4°C for later use;

[0047] 2. Thaw the plant lactobacillus H8 strain stored at -80°C on ice, streak the plant lactobacillus H8 on an MRS solid culture plate under sterile conditions, and then invert and culture it at 25°C for 24 hours; pick a single colony in an MRS liquid culture medium, and culture it at 120rpm for 18 hours at 25°C to obtain an H8 seed solution; add the H8 seed solution to an MRS liquid culture medium (inoculation size is 1‰), and culture it at 120rpm for 24 hours at 25°C; collect the bacterial cells by centrifugation at 3000rpm for 10 minutes, wash and resuspend the bacterial cells with sterile phosphate buffer PBS to obtain a plant lactobacillus H8 bacterial solution, and store it at 4°C for standby use;

[0048] 3. Mix the bacterial solution of Bacillus subtilis RT-BS07 and the bacterial solution of Bacillus plantarum H8 to obtain a composite immunopotentiator, spray the composite immunopotentiator on the surface of the fish feed, and dry it in the shade to obtain a composite immunopotentiator feed; wherein the final concentration of Bacillus subtilis RT-BS07 in the fish feed is 1.0×10 7 CFU / g, the final concentration of Lactobacillus plantarum H8 was 1.0×10 6 The fish feed is a commercially available California bass feed from Zhejiang Dongyu Biotechnology Co., Ltd., with a particle size of 1 mm.

[0049] Pickerel fry weighing about 3 g were selected as experimental subjects, and a control group, experimental group I, experimental group II and experimental group III were set up respectively. The control group was fed with ordinary feed, experimental group I was fed with the Bacillus subtilis RT-BS07 feed of Example 1, experimental group II was fed with the plant lactobacillus H8 feed of Example 2, and experimental group III was fed with the composite immunopotentiator feed of Example 3. At 5-8 weeks, the feed fed to the experimental group was changed compared with that at 0-4 weeks, and the specific grouping and feeding methods are shown in Table 1:

[0050] Table 1

[0051]

[0052] At a water temperature of 15±0.2℃, the fish were fed with feed continuously for 56 days according to the grouping method, with feeding once in the morning and evening every day. The growth data of each group of experimental fish were collected at 28 days and 56 days after feeding.

[0053] After feeding, common and susceptible conditional pathogens of the genus Aeromonas, including Aeromonas hydrophila (Ah), Aeromonas veronii (Av) and Aeromonas salmonicida (As), were selected for intraperitoneal injection infection experiments on the experimental fish in the control group and experimental group. Continuous observation was performed, and the status and mortality of the experimental fish were recorded to evaluate the differences in their disease resistance.

[0054] Experimental results:

[0055] During the experiment, about 2% of the control group and the experimental group died. The dead fish were thin or had obvious collision injuries, which should be caused by accidental death or individual differences. The other tested zander fry swam quickly, and there was no abnormality in body color, appearance, feeding and excretion. The weight of the experimental fish was measured before and after feeding. The specific growth conditions are shown in Table 2:

[0056] Table 2

[0057]

[0058] As shown in Table 2, feeding feed supplemented with probiotics can effectively promote the growth of pike perch fry. The effect of group D, which was fed with Bacillus subtilis RT-BS07 feed from 0 to 4 weeks and with compound immune enhancer feed from 5 to 8 weeks, was obvious. The growth-promoting effect of probiotics was more significant in the first 4 weeks of addition.

[0059] The mortality of each group of experimental fish in the intraperitoneal injection infection experiment is shown in Table 3:

[0060] Table 3

[0061]

[0062] As shown in Table 3, the resistance of the pike perch fry in the experimental group fed with the compound immune enhancer feed to Ah, Av and As infection was significantly enhanced, and the survival rate increased by 6.67%-20%; and the order of adding probiotic preparations had a certain influence on the anti-disease effect.

[0063] From the above experimental results, it can be concluded that: ① After feeding with fish-derived composite immune enhancers, the growth of pike perch fry was effectively promoted; ② Adding Bacillus subtilis RT-BS07 to the feed has a good promoting effect on the growth of pike perch fry; ③ Adding Lactobacillus plantarum H8 to the feed has a good effect on improving the disease resistance of pike perch fry; ④ The growth-promoting effect of small-sized fry in the first 4 weeks of feeding is better. It is speculated that the intestinal flora and digestive ability of small-sized fish are not yet complete, and they are more susceptible to the regulation of probiotics; ⑤ Considering the probiotic effects of promoting the growth of pike perch fry and improving disease resistance, Bacillus subtilis RT-BS07 feed is first fed to promote fish growth, and then mixed probiotic feed containing composite immune enhancers is fed to further improve the growth efficiency and disease resistance of pike perch fry.

Claims

1. A composite immunopotentiator for young pike perch, characterized in that: The composite immune enhancer is composed of Bacillus subtilis RT-BS07 and Lactiplantibacillus plantarum H8, wherein the final concentration of Bacillus subtilis RT-BS07 added to the fish feed is 1.0×10 7 CFU / g, the final concentration of Lactobacillus plantarum H8 added to fish feed was 1.0×10 6 CFU / g; The plant lactobacillus H8 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the storage address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the storage date is April 7, 2024, and the storage number is CGMCC No. 30268.

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