A composite bacterial agent for rainbow trout juvenile breeding and its application

Abstract

A composite bacterial agent for rainbow trout fry breeding and its application, relates to the field of aquaculture, and aims to solve the problem that the existing probiotics obtained from other species will cause intestinal damage to the tested rainbow trout. The composite bacterial agent includes Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8, and the method of use is as follows: spray the bacterial liquid of Bacillus subtilis RT-BS07 and the bacterial liquid of Lactobacillus plantarum H8 onto the surface of the fish feed, and feed after drying in the shade. The composite bacterial agent of the present invention is a fish-derived mixed probiotic preparation, which can protect the intestinal tissue structure of rainbow trout fry and is more conducive to the absorption of nutrients by rainbow trout fry. The added Lactobacillus plantarum H8 and its metabolites have an inhibitory effect on pathogenic bacteria. The combination of the two strains can not only further enhance the disease resistance of rainbow trout fry, but also effectively increase the growth rate of rainbow trout fry. The present invention is used for the breeding of rainbow trout fry.

Description

Technical Field

[0001] The invention relates to the field of aquaculture, and in particular to a composite bacterial agent for rainbow trout juvenile breeding and an application thereof. Background Art

[0002] Rainbow trout is one of the important farmed fish in the world. It is a precious cold-water fish and one of my country's famous, special and new fish species. It is listed as a high-end commercial fish in the domestic and foreign markets and is a high-yield, high-efficiency and high-quality farmed species with broad development prospects. With the continuous development and improvement of my country's rainbow trout breeding and cultivation of new varieties and land-sea relay breeding models and technologies, the green and healthy aquaculture of rainbow trout is developing rapidly.

[0003] Due to the stocking density, water environment, temperature stress and other reasons, rainbow trout fry are susceptible to bacterial diseases and outbreaks, resulting in the death of a large number of fry, which has brought great obstacles to the development of the industry. Bacterial diseases that rainbow trout fry are susceptible to include rainbow trout fry syndrome caused by Flavobacterium psychrophilum (Fp), surface ulcers and bacterial sepsis caused by Aeromonas salmonicida (As) and Aeromonas hydrophilia (Ah). However, the flow pond or factory farming model cannot be effectively treated with antibiotics, and the growth of fry and environmental ecological safety issues caused by unscientific use of antibiotics and drug residues should not be underestimated. Therefore, the development of high-quality and efficient probiotic preparations that promote growth and enhance immunity as an effective preventive method for rainbow trout fry is the direction that green and healthy farming has been committed to developing.

[0004] In existing aquaculture methods, probiotics obtained from other species are used to prevent and resist diseases in rainbow trout fry. However, studies have found that the use of probiotics obtained from other species can cause safety issues such as damage to the intestinal tract of the test fish, which affects the growth of the fish. Summary of the invention

[0005] The present invention aims to solve the problem that the existing probiotics obtained from other species may cause intestinal damage to the tested rainbow trout, and provides a composite bacterial agent for juvenile rainbow trout breeding and its application.

[0006] The present invention provides a composite bacterial agent for juvenile rainbow trout breeding, comprising Bacillus subtilis RT-BS07 and Lactiplantibacillus plantarum H8, wherein the final concentration of Bacillus subtilis RT-BS07 added to fish feed is 1.0×10 6 CFU / g, the final concentration of Lactobacillus plantarum H8 added to fish feed was 1.0×10 5CFU / g.

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

[0008] The present invention also provides the use of the composite bacterial agent in the breeding of rainbow trout juveniles, and the specific method is as follows:

[0009] Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8 were sprayed on the surface of the fish feed and then fed after being dried in the shade. The final concentration of Bacillus subtilis RT-BS07 in the fish feed was 1.0×10 6 CFU / g, the final concentration of Lactobacillus plantarum H8 was 1.0×10 5 CFU / g.

[0010] Furthermore, the preparation method of the Bacillus subtilis RT-BS07 bacterial solution is:

[0011] 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.

[0012] Furthermore, the preparation method of the plant lactobacillus H8 bacterial liquid is as follows:

[0013] 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.

[0014] The present invention also provides application of the composite bacterial agent in protecting the intestinal tissue structure of rainbow trout juveniles.

[0015] The present invention also provides application of the composite bacterial agent in improving the growth rate of rainbow trout fry.

[0016] The present invention also provides application of the composite bacterial agent in inhibiting pathogenic bacteria of rainbow trout fry.

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

[0018] Beneficial effects of the present invention:

[0019] The composite bacterial agent of the present invention comprises Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8, and the two bacterial strains are screened from the intestines of healthy rainbow trout and carp, respectively. Therefore, the composite bacterial agent of the present invention is a fish-source mixed probiotic preparation, which can protect the intestinal tissue structure of rainbow trout fry and make it benignly improved, and is more conducive to the absorption of nutrients by rainbow trout fry.

[0020] The Lactobacillus plantarum H8 and its metabolites added to the composite bacterial agent of the present invention have an inhibitory effect on pathogenic bacteria, are beneficial to preventing the invasion and growth of pathogenic bacteria, and can regulate the immunity of rainbow trout bodies.

[0021] The present invention combines the two strains, which can not only further enhance the disease resistance of rainbow trout fry, but also effectively increase the growth rate of rainbow trout fry. This shows that the addition of Bacillus subtilis RT-BS07 in the present invention can synergistically promote the growth of Lactobacillus plantarum H8 and play a prebiotic role while effectively promoting fish growth and reducing the feed coefficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the observation results of intestinal sections of experimental fish in the control group and experimental group. DETAILED DESCRIPTION

[0023] 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.

[0024] Specific implementation method 1: In this implementation method, the composite bacterial agent for rainbow trout fry farming includes 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 6 CFU / g, the final concentration of Lactobacillus plantarum H8 added to fish feed was 1.0×10 5 CFU / g.

[0025] The Lactobacillus plantarum H8 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is April 7, 2024, and the deposit number is CGMCC No.30268.

[0026] Specific implementation method 2: The application of the composite bacterial agent for juvenile rainbow trout breeding in this implementation method in juvenile rainbow trout breeding is as follows:

[0027] Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8 were sprayed on the surface of the fish feed and then fed after being dried in the shade. The final concentration of Bacillus subtilis RT-BS07 in the fish feed was 1.0×10 6 CFU / g, the final concentration of Lactobacillus plantarum H8 was 1.0×10 5 CFU / g.

[0028] 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:

[0029] 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.

[0030] 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:

[0031] 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.

[0032] Specific implementation method 5: This implementation method is the use of composite bacteria for rainbow trout fry farming in protecting the intestinal tissue structure of rainbow trout fry.

[0033] Specific implementation method six: This implementation method is to use the composite bacteria for rainbow trout fry farming to improve the growth rate of rainbow trout fry.

[0034] Specific implementation method seven: This implementation method is to use the composite bacteria for rainbow trout fry farming in inhibiting pathogenic bacteria of rainbow trout fry.

[0035] Specific embodiment eight: This embodiment differs from specific embodiment seven in that the pathogenic bacteria are Aeromonas hydrophila, Aeromonas salmonicida and Flavobacterium psychrophilum. The rest is the same as specific embodiment seven.

[0036] 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.

[0037] Embodiment 1:

[0038] The preparation method of the plant lactobacillus H8 feed of the present embodiment comprises the following steps:

[0039] 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;

[0040] 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 5 The fish feed is a commercially available Danish Aile salmon and trout feed with a particle size of 2 mm.

[0041] Embodiment 2:

[0042] The method for preparing the composite microbial agent feed for juvenile rainbow trout breeding in this embodiment comprises the following steps:

[0043] 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;

[0044] 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;

[0045] 3. Mix the bacterial solution of Bacillus subtilis RT-BS07 and the bacterial solution of Bacillus plantarum H8 to obtain a composite bacterial agent, spray the composite bacterial agent on the surface of the fish feed, and dry it in the shade to obtain a composite bacterial agent feed; wherein the final concentration of Bacillus subtilis RT-BS07 in the fish feed is 1.0×10 6 CFU / g, the final concentration of Lactobacillus plantarum H8 was 1.0×10 5 The fish feed is a commercially available Danish Aile salmon and trout feed with a particle size of 2 mm.

[0046] Three different sizes of rainbow trout juveniles of 7.5 g, 36 g and 70 g were selected as experimental subjects. A control group, an experimental group I and an experimental group II were set for each size of rainbow trout juveniles, with 3 parallels in each group and 30 fish in each parallel. The experimental group I was fed with the plant lactobacillus H8 feed prepared in Example 1, and the experimental group II was fed with the composite bacterial agent feed prepared in Example 2. The specific grouping and feeding method are shown in Table 1:

[0047] Table 1

[0048]

[0049] Under the same culture conditions, the juvenile rainbow trout were fed with feed at 2% of their body weight, twice a day, morning and evening, for 56 consecutive days, and the growth data of each group of experimental fish were collected. After the feeding, the intestines of the experimental fish in the control group, experimental group I and experimental group II were sliced ​​for observation; 30 experimental fish in each group were randomly selected for intraperitoneal injection infection experiments with Aeromonas hydrophila (Ah), Aeromonas salmonicida (As) and Flavobacterium psychrophilum (Fp), and continuous observation was performed, and the status and mortality of the experimental fish were recorded to evaluate the differences in disease resistance.

[0050] Experimental results:

[0051] During the experiment, no mortality occurred in all control groups and experimental groups. No abnormalities were found in the swimming behavior, body color, appearance, feeding and excretion of the tested rainbow trout. The initial body weight of the experimental fish was measured before feeding, and other growth parameters were measured and calculated after feeding. The specific results are shown in Table 2:

[0052] Table 2

[0053]

[0054] As can be seen from Table 2, after feeding the feed with the compound bacterial agent, the final body weight, weight gain rate and fatness of the experimental fish were significantly higher than those of the control group and experimental group I, and the feed conversion rate was significantly reduced. Such results show that the growth-promoting effect of Lactobacillus plantarum added to the fish feed in experimental group I is not obvious, but the Bacillus subtilis added to experimental group II has a good promoting effect on the growth of experimental fish.

[0055] The results of the intestinal slice observation of the experimental fish in the control group A and experimental group II C are as follows Figure 1 As shown. Figure 1 It can be seen that, like the control group, the intestinal tissue of the fish in the experimental group was intact and the structure was clear. There was no damage to the folds, mucosal epithelium and lamina propria, and there was no purulent secretion filling, inflammation or blood cell infiltration. The length and width of the intestinal villi of the fish in the experimental group increased, and the intestinal wall muscle layer was slightly thickened. This structural feature is more conducive to the intestinal absorption of nutrients.

[0056] The results of the intraperitoneal injection infection experiment are shown in Table 3:

[0057] Table 3

[0058]

[0059] As shown in Table 3, the resistance of rainbow trout fry to Ah, Fp and As infection in the experimental group fed with bacterial agent feed was significantly enhanced, and the survival rate increased by 6.67%-30%, and the effect of experimental group II with compound bacterial agent was better. At the same time, the results showed that among the three experimental groups of different sizes, the disease resistance of small size fry (7.5g) was the most significantly improved, reaching 13.33%-30%.

[0060] From the above experimental results, it can be concluded that: ① after feeding the feed added with the fish-derived composite bacterial agent of the present invention, the intestinal tissue structure of rainbow trout fry is protected and benignly improved, which is more conducive to the absorption of nutrients; ② the feed added with Lactobacillus plantarum H8 alone has no significant beneficial effect on the growth of rainbow trout fry, but can effectively reduce the mortality of rainbow trout fry caused by infection with Aeromonas hydrophila, Aeromonas salmonicida and Flavobacterium psychrophilum. This is because the plant lactobacillus plantarum H8 and its metabolites added in the present invention have an inhibitory effect on pathogenic bacteria, which is beneficial to preventing the invasion and growth of pathogenic bacteria, and can regulate the immune system of the body. ③ The fish feed to which the composite bacterial agent of Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8 is added at the same time can not only further enhance the disease resistance, but also effectively improve the growth rate of the experimental rainbow trout fry, which indicates that the Bacillus subtilis RT-BS07 added in the composite bacterial agent of the present invention can synergistically promote the growth of Lactobacillus plantarum H8 and play a probiotic role while effectively promoting the growth of fish and reducing the feed coefficient; ④ The composite bacterial agent of the present invention has a better effect on small-sized fish. It is speculated that this is because the intestinal flora and digestive ability of small-sized fish are not yet complete and are more susceptible to the regulation of probiotics.

Claims

1. A composite bacterial agent for rainbow trout juvenile breeding, characterized in that: The composite bacterial agent includes Bacillus subtilis ( Bacillus subtilis )RT-BS07 and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) H8, in which Bacillus subtilis RT-BS07 was added to the fish feed at a final concentration of 1.0 × 10 6 CFU / g, the final concentration of Lactobacillus plantarum H8 added to fish feed was 1.0×10 5 CFU / g; The Lactobacillus plantarum ( Lactiplantibacillus plantarum )H8 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 7, 2024, and the deposit number is CGMCC No.30268.

2. The use of the composite bacterial agent for juvenile rainbow trout breeding as claimed in claim 1 in juvenile rainbow trout breeding, characterized in that: This method excludes use in the prevention and treatment of diseases, specifically: Bacillus subtilis RT-BS07 and Lactobacillus plantarum H8 were sprayed on the surface of the fish feed and then fed after being dried in the shade. The final concentration of Bacillus subtilis RT-BS07 in the fish feed was 1.0×10 6 CFU / g, the final concentration of Lactobacillus plantarum H8 was 1.0×10 5 CFU / g.

3. The use according to claim 2, characterized in that: The preparation method of the Bacillus subtilis RT-BS07 bacterial liquid is as follows: 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 h; pick a single colony in an LB liquid culture medium, and culture it at 120 rpm for 18 h at 25°C to obtain the RT-BS07 seed solution; take the RT-BS07 seed solution and add it to the LB liquid culture medium with an inoculation amount of 1‰, and culture it at 120 rpm for 24-36 h at 25°C; collect the bacteria by centrifugation at 3000 rpm for 10 min, wash and resuspend the bacteria with sterile phosphate buffered saline (PBS) to obtain the Bacillus subtilis RT-BS07 bacterial solution, and store it at 4°C for later use.

4. The use according to claim 2, characterized in that: The preparation method of the plant lactobacillus H8 bacterial liquid is as follows: The Lactobacillus plantarum H8 strain stored at -80°C was thawed on ice, and the Lactobacillus plantarum H8 was streaked on an MRS solid culture plate under sterile conditions, and then inverted and cultured at 25°C for 24 h; a single colony was picked in an MRS liquid culture medium, and cultured at 120 rpm for 18 h at 25°C to obtain an H8 seed solution; the H8 seed solution was added to an MRS liquid culture medium with an inoculation amount of 1‰, and cultured at 120 rpm for 24-36 h at 25°C; the bacteria were collected by centrifugation at 3000 rpm for 10 min, washed and resuspended with sterile phosphate buffered saline (PBS) to obtain a Lactobacillus plantarum H8 bacterial solution, which was temporarily stored at 4°C for later use.

5. Use of the composite bacterial agent as claimed in claim 1 in protecting the intestinal tissue structure of rainbow trout fry, excluding the use of this method in disease prevention and treatment.

6. Use of the composite bacterial agent as claimed in claim 1 in increasing the growth rate of rainbow trout fry, excluding the use of this method in disease prevention and treatment.

7. Use of the composite bacterial agent as claimed in claim 1 in inhibiting pathogenic bacteria of rainbow trout fry; excluding the use of this method in disease prevention and treatment; the pathogenic bacteria are Aeromonas hydrophila, Aeromonas salmonicida and Flavobacterium psychrophilum.

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