CN104593300A - Strain for intestinal improvement of aquatic livestock and application thereof - Google Patents

Abstract

The present invention isolates and screens a functional bacterial strain that can promote the improvement of the intestines of aquaculture animals from healthy large yellow croaker intestines cultured in cages in Ningde City, Fujian Province. By measuring the physiological and biochemical characteristics of the bacterial strain and identifying 16s rDNA, Finally, it was confirmed that it was a strain of Lactobacillus casei; at the same time, it was disclosed that the use of it to prepare microbial preparations that can promote the intestinal tract of aquatic animals; Monascus has a good inhibitory effect, and can quickly reduce the pH value, can reduce the bait coefficient of aquaculture animals, and can improve the survival rate of aquatic animals.

Description

A bacterial strain for intestinal improvement of aquatic animals and its application

technical field

The invention belongs to the technical field of microbes, and in particular relates to a bacterial strain for improving intestinal tract of aquatic animals and application thereof.

Background technique

The occurrence of diseases in aquaculture animals is related to the breeding environment, pathogenic bacteria and the organisms of aquatic animals themselves. Therefore, in addition to improving the breeding environment during the breeding process, it is also very important to pay attention to the intestinal health of aquatic animals. Studies have shown that lactic acid bacteria can effectively improve the intestinal health of aquatic animals, thereby promoting the growth of aquatic animals and reducing the feed coefficient. For example, the Chinese patent No. 201210465656.1 discloses a preparation method of medical stone carrier lactic acid bacteria agent for sea cucumber cultivation, which mixes lactic acid bacteria with nutritional feed, and is suitable for sea cucumber cultivation. However, the efficacy of probiotics used in aquaculture reported so far is relatively single, and screening and isolation of probiotics with multiple functions can create huge economic benefits, which is of great significance.

Contents of the invention

The technical problem to be solved by the present invention is to provide a bacterial strain for improving the intestinal tract of aquatic animals. The bacterial strain is Lactobacillus casei strain DS31 ( Lactobacillus casei ), which was preserved in the General Committee of China Microorganism Culture Collection Management Committee on November 28, 2014. Microbiology Center, the address is No. 2, No. 1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No: 10073. At the same time, the microbial preparation prepared by using the bacterial strain is disclosed.

The present invention solves the above technical problems through the following technical solutions:

The applicant isolated and screened a functional strain that can promote the intestinal improvement of aquaculture animals from healthy large yellow croaker intestines collected from cage culture in Ningde City, Fujian Province, and carried out physiological and biochemical characteristics of the strain and 16s rDNA identification , finally confirming that it is a strain of Lactobacillus casei .

At the same time, the bacterial strain is used to prepare a microbial preparation for intestinal improvement of aquatic animals, and the preparation steps of the microbial preparation are as follows:

(1) Strain expansion: take the Lactobacillus casei strain DS31 ( Lactobacillus casei ) and inoculate it into MRS medium, and activate it at 30-45°C and shaker at 150-200rpm for 5-10h; activate Once ready, the seed liquid is obtained, and then inoculate the seed liquid into the MRS medium according to the inoculation amount of 1-10%, and cultivate it for 12-24 hours at 30-45 ° C and 150-200 rpm on a shaker;

(2) Strain adsorption: add diatomite or medical stone powder or zeolite powder 50g/L-2000g/L with a crushing fineness of 120-250 mesh to the bacterial liquid after expansion in step (1), and put it at 100-150rpm Adsorption 1-5h;

(3) Concentration and drying: centrifuge the adsorbed bacterial solution at 8000-10000rpm for 5-10min in step (2), discard the supernatant to obtain the wet bacterial solution, add 0.1-1.0ml/L protective agent, and stir at 30-40°C After drying, subpackage and pack to obtain the product of the microbial preparation.

Further, the protective agent is any one of glycerin, sodium alginate and dextrin.

Further, the components of the MRS medium are: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K ₂ HPO ₄ 2.0 g, MgSO ₄ ·7H ₂ O 0.58 g, MnSO ₄ ·H ₂ O 0.25 g, H ₂ O 1000 mL, pH 6.2-6.6.

The beneficial effect of the present invention is that it provides a bacterial strain for improving the intestinal tract of aquatic animals, that is, Lactobacillus casei ( Lactobacillus casei ) DS31, and at the same time discloses the use of it to prepare microbial preparations that can promote the intestinal tract of aquatic animals. The microbial preparations have a good inhibitory effect on aquatic pathogenic bacteria Vibrio alginolyticus and Aeromonas hydrophila, and can quickly reduce the pH value, reduce the bait coefficient of aquaculture animals, and improve the survival rate of aquatic animals; therefore It has a good application prospect.

Detailed ways

Lactobacillus casei ( Lactobacillus casei ) DS31, a bacterial strain used for improving the intestinal tract of aquatic animals, is isolated from healthy large yellow croaker intestines cultured in cages in Ningde City, Fujian Province and screened to improve the intestinal tract of aquaculture animals. A functional strain with a promoting effect, and discloses the use of the strain FA08 to prepare microbial preparations for improving the intestinal tract of aquatic animals.

1. Isolation and identification of strain DS31:

1. Primary screening:

Dissect the fresh large yellow croaker on an ice tray, scrape the mucous membrane of the intestinal wall of the fish and place it in 0.85% NaCl, stir it evenly, then dilute it in a gradient and spread it on the BCP medium, and cultivate it in a biochemical incubator at 37°C for 24-48h until When the colonies grow out, pick the colonies that turn yellow around them and save them on the MRS slope for later use.

In the present invention, the components of the BCP medium: yeast extract 25g, peptone 5.0g, glucose 5.0g, bromocresol purple 0.004g, agar 15g, water 1000ml, pH7.0; the components of the MRS medium are: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K ₂ HPO4 2.0 g, MgSO ₄ ·7H ₂ O 0.58 g, MnSO ₄ ·H ₂ O 0.25 g, H ₂ O 1000 mL, agar 15 g, pH 6.2-6.6;

2. Re-screening:

Pick the colonies saved in the primary screening and inoculate them into MRS test tubes for activation for 6 hours. The activated bacteria solution is inoculated into MRS medium at 5% inoculum, and cultured at 37°C, 180 rpm, on a shaking table for 24 hours. Samples were taken for 2 hours to measure the pH, and then the strain with the strongest acid-producing ability was screened out, which was labeled as strain DS31.

3. Identification:

The screened strain DS31 was inoculated into MRS solid medium, cultured at 37°C, the colony morphology was observed, and some physiological and biochemical characteristics were measured. The results are as follows: the colony is milky white, with smooth surface, neat edges, opaque, Gram-positive, No spores, positive for glucose fermentation, positive for lactose fermentation. At the same time, the 16S rDNA of the strain was identified, and its 16S rDNA sequence was obtained as shown in SEQ ID NO:1.

The measured 16s rDNA sequence was input into NCBI for homology search, and it was found that the highest similarity was Lactobacillus casei ( Lactobacillus casei ); combined with the results of physiological and biochemical identification and 16s rDNA sequence database comparison results, it was determined that the strain DS31 was A strain of Lactobacillus casei .

2. Preparation of microbial preparations:

Utilize bacterial strain DS31 to prepare the microbial preparation for intestinal improvement of aquatic animals, and its operation steps are as follows:

(1) Strain expansion: take the Lactobacillus casei strain DS31 ( Lactobacillus casei ) and inoculate it into MRS medium, and activate it at 30-45°C and shaker at 150-200rpm for 5-10h; activate Once ready, the seed liquid is obtained, and then inoculate the seed liquid into the MRS medium according to the inoculation amount of 1-10%, and cultivate it for 12-24 hours at 30-45 ° C and 150-200 rpm on a shaker;

(2) Strain adsorption: add diatomite or medical stone powder or zeolite powder 50g/L-2000g/L with a crushing fineness of 120-250 mesh to the bacterial liquid after expansion in step (1), and put it at 100-150rpm Adsorption 1-5h;

(3) Concentration and drying: centrifuge the adsorbed bacterial solution at 8000-10000rpm for 5-10min in step (2), discard the supernatant to obtain the wet bacterial solution, add 0.1-1.0ml/L protective agent, and stir at 30-40°C After drying, subpackage and pack to obtain the product of the microbial preparation.

In the present invention, the protective agent is selected from any one of glycerin, sodium alginate, and dextrin; the components of the MRS medium are: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, triammonium citrate 2.0 g, Glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K ₂ HPO ₄ 2.0 g, MgSO ₄ ·7H ₂ O 0.58 g, MnSO ₄ ·H ₂ O 0.25 g, H ₂ O 1000 mL, pH 6.2 -6.6.

In order to better illustrate the microbial preparation in the present invention, the applicant cites the following examples.

Example 1

Strain expansion: take Lactobacillus casei strain DS31 ( Lactobacillus casei ) and inoculate it into MRS medium, and activate and cultivate it at 30°C and shaker at 150rpm for 10 hours; after activation, the seed liquid is obtained, and then the inoculation amount is 10%. The seed solution was inoculated into the MRS medium, and cultured at 30°C and a shaker at 200rpm for 12h;

Strain adsorption: Add 2000g/L of diatomaceous earth with a crushing fineness of 120-250 mesh to the expanded bacterial liquid, and adsorb at 150rpm for 1h;

Concentration and drying: the adsorbed bacterial solution was centrifuged at 10,000 rpm for 8 minutes, the supernatant was discarded to obtain the wet bacterial solution, and 0.1ml/L glycerin was added, stirred and dried at 40°C, then subpackaged and packaged to obtain the product of the microbial preparation.

Example 2

Strain expansion: take the Lactobacillus casei strain DS31 ( Lactobacillus casei ) and inoculate it into MRS medium, and activate and cultivate it at 45°C and shaker at 150rpm for 5 hours; after activation, the seed liquid is obtained, and then press 1 % Inoculum Amount Inoculate the seed liquid into the MRS medium, and cultivate it for 24 hours at 34°C and shaker at 150rpm;

Strain adsorption: Add 1000g/L of medical stone powder with a crushing fineness of 120-250 mesh to the bacterial liquid after expansion, and absorb at 100rpm for 5h;

Concentration and drying: centrifuge the adsorbed bacterial solution at 9000rpm for 5 minutes, discard the supernatant to obtain the wet bacterial solution, add 1.0ml/L sodium alginate, stir and dry at 30°C, then sub-package to obtain the product of the microbial preparation .

Example 3

Strain expansion: take the Lactobacillus casei strain DS31 ( Lactobacillus casei ) and inoculate it into MRS medium, and activate and cultivate it at 37°C and shaker at 180rpm for 7 hours; after activation, the seed liquid is obtained, and then press 5 % inoculum amount Inoculate the seed liquid into the MRS medium, and cultivate it for 18 hours at 35°C and 180rpm on a shaker;

Strain adsorption: Add 50g/L of zeolite powder with a crushing fineness of 120-250 mesh to the expanded bacterial liquid, and adsorb at 140rpm for 3h;

Concentration and drying: centrifuge the adsorbed bacterial solution at 8000rpm for 10 minutes, discard the supernatant to obtain the wet bacterial solution, add 0.5ml/L dextrin, stir and dry at 35°C, then sub-package to obtain the product of the microbial preparation.

3. Effect test of strain DS31 and microbial preparations:

Test 1:

Take the strain DS31 and inoculate it in the MRS medium, activate it at 37°C and 170rpm for 5h; inoculate the activated bacterial solution into the MRS medium at 5% (the components are: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g , triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K ₂ HPO ₄ 2.0 g, MgSO ₄ ·7H ₂ O 0.58 g, MnSO ₄ ·H ₂ O 0.25 g, H ₂ O 1000 mL, pH 6.2-6.6), 37°C, 170rpm, cultured for 20h; after cultivation, centrifuged at 10000rpm for 6min, discarded the precipitate, and obtained the fermentation supernatant; made antibacterial plates, punched holes, and added 50uL fermentation supernatant to the wells After the supernatant was cultured overnight at 37°C; and the antibacterial plate: add 1% activated pathogenic bacteria (Vibrio alginolyticus, Aeromonas hydrophila) to molten SLB at 45°C; observe and record the inhibition zone on the antibacterial plate path.

test results:

Indicator bacteria Inhibition zone (mm) Vibrio alginolyticus 13.92±0.88 Aeromonas hydrophila 13.27±0.53

Test 2:

Take the strain DS31 and inoculate it in the MRS medium, activate it at 37°C and 170rpm for 5h; inoculate the activated bacterial liquid into the MRS medium with 1% inoculation amount, and ferment and cultivate it at 37°C and 170rpm for 20h; detect and record the fermentation culture Changes in the pH of the fermentation broth during the process.

Test results: After culturing for 12 hours, the pH of the fermentation broth dropped to 4.1; after 24 hours, the pH of the fermentation broth dropped to below 3.5.

Test 3:

Get the microbial preparation of the present invention (obtained as an example with embodiment 3) and use it in the cultured soft-shelled turtle pond: design a control group and an experimental group; In the experimental group, add the microorganism of the present invention by the bait mixing amount of 4g/kg in soft-shelled turtle feed The preparation was mixed with bait once a day and fed; the control group was a blank control group, which fed the same bait but did not add the microbial preparation of the present invention; after 5 months of the test, the bait coefficient and survival rate of soft-shelled turtles in the soft-shelled turtle pond were calculated.

The test results obtained by calculation are: the control group without using the probiotic preparation of the present invention has a bait coefficient of 1.56 and a survival rate of 80.1%; while the experimental group has a bait coefficient of 1.40 and a survival rate of 87.5%.

Test 4:

Get the microbial preparation of the present invention (obtained as an example with embodiment 3) and use it in the cultured soft-shelled turtle pond: design a control group and an experimental group; In the experimental group, add the microorganism of the present invention by the bait mixing amount of 8g/kg in soft-shelled turtle feed The preparation was mixed with bait once a day and fed; the control group was a blank control group, which fed the same bait but did not add the microbial preparation of the present invention; after 5 months of the test, the bait coefficient and survival rate of soft-shelled turtles in the soft-shelled turtle pond were calculated.

The calculated test results are as follows: the control group without using the probiotic preparation of the present invention has a bait coefficient of 1.56 and a survival rate of 80.1%; while the experimental group has a bait coefficient of 1.37 and a survival rate of 89.3%.

Comparing Test 3 with Test 4, it can be seen that increasing the dosage of this product can reduce the bait coefficient and slightly increase the survival rate.

In summary, the present invention provides a bacterial strain for improving the intestinal tract of aquatic animals, namely Lactobacillus casei ( Lactobacillus casei ) DS31. It has good inhibitory effect, can quickly reduce the pH value, can reduce the bait coefficient of aquaculture animals, and can improve the survival rate of aquatic animals; therefore, it has a good application prospect.

SEQUENCE LISTING

the

<110> The Third Institute of Oceanography, State Oceanic Administration

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Claims (4)

1. A bacterial strain for intestinal improvement of aquatic animals, characterized in that: the bacterial strain is Lactobacillus casei strain DS31 ( Lactobacillus casei ), which was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on November 28, 2014 , and the deposit number is CGMCC No:10073. 2. A microbial preparation for intestinal improvement of aquatic animals, characterized in that: the preparation steps of the microbial preparation are as follows: (1) Strain expansion: take the Lactobacillus casei strain DS31 ( Lactobacillus casei ) described in claim 1 and inoculate it into MRS medium, and activate and cultivate it at 30-45°C and shaker at 150-200rpm 5- 10h; After activation, the seed solution is obtained, and then the seed solution is inoculated into the MRS medium according to the inoculum amount of 1-10%, and cultivated at 30-45°C and shaker at 150-200rpm for 12-24h; (2) Strain adsorption: add diatomite or medical stone powder or zeolite powder 50g/L-2000g/L with a crushing fineness of 120-250 mesh to the bacterial liquid after expansion in step (1), and put it at 100-150rpm Adsorption 1-5h; (3) Concentration and drying: centrifuge the adsorbed bacterial solution at 8000-10000rpm for 5-10min in step (2), discard the supernatant to obtain the wet bacterial solution, add 0.1-1.0ml/L protective agent, and stir at 30-40°C After drying, the product of the microbial preparation is obtained by subpackaging and packaging. 3. A probiotic preparation for improving intestinal tract of aquaculture animals according to claim 2, characterized in that: the protective agent is any one of glycerin, sodium alginate, and dextrin. 4. A probiotic preparation for improving the intestinal tract of aquaculture animals according to claim 2, characterized in that: the components of the MRS medium are: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g , triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K ₂ HPO ₄ 2.0 g, MgSO ₄ ·7H ₂ O 0.58 g, MnSO ₄ ·H ₂ O 0.25 g, H ₂ O 1000 mL, pH 6.2-6.6.