CN114657082A

CN114657082A - Lactococcus lactis and application thereof

Info

Publication number: CN114657082A
Application number: CN202110419707.6A
Authority: CN
Inventors: 杨求华; 林琪; 周宸; 吴建绍; 肖益群; 杨福元; 王丽莉
Original assignee: Fujian Fisheries Research Institute
Current assignee: Fujian Fisheries Research Institute
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2022-06-24
Anticipated expiration: 2041-04-19
Also published as: CN114657082B

Abstract

The invention discloses lactococcus lactis, the preservation number of which is CGMCC No.20329, the lactococcus lactis is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation date is 2020, 7 and 9 days. The invention also provides application of the lactococcus lactis with the preservation number of CGMCC No.20329 in preparation of a microbial preparation. The strain separating screen is selected from Apostichopus japonicus intestinal tract, and can promote the healthy growth of Apostichopus japonicus, improve intestinal flora, effectively resist infection of pathogenic bacteria tower-type vibrio, and enhance organism immunity. The lactococcus lactis is an endogenous intestinal probiotic, is suitable for growth and propagation in marine environment, has no danger to cultured animals and human beings, and has high efficiency and safety in the aspect of application. The lactococcus lactis CGMCC No.20329 has the advantages of low production cost, simple process and good commercial prospect.

Description

Lactococcus lactis and application thereof

Technical Field

The invention relates to the technical field of aquatic microorganism application, in particular to lactococcus lactis and application thereof.

Background

With the rise of the aquaculture industry in China and the change of factors such as water quality environment, climate conditions and the like in the intensive aquaculture process, various diseases mainly comprising bacterial diseases appear in the aquaculture process, and the bacterial diseases are usually prevented and treated by antibiotics and chemical drugs, so that negative effects brought by the bacterial diseases are paid attention to by a plurality of scholars.

Antibiotics are continuously used in the breeding industry, the water body environment is also destroyed while diseases are prevented and treated, the intestinal flora structure in a host body is disturbed, bacteria generate drug resistance, pathogenic bacteria of the drug resistance can be transmitted to human beings through a food chain, and the drug resistance of normal flora in the human body is changed. There are studies reporting that in addition to chemical contamination, environmental antibiotics may also induce the production of resistant microorganisms and resistance genes in the environment and exacerbate the risk of antibiotic resistance transmission. Some resistant microorganisms may directly or indirectly participate in food chains and other ways to enter human bodies, so that the resistance of the human bodies to antibiotics is increased, and certain degree of threat to the public health of human beings is brought, and the human health is seriously harmed.

With the sustainable development of green and environment-friendly foods, a plurality of scholars are dedicated to the research on antibiotics and chemical drug substitutes, and probiotics (lactic acid bacteria) become a research hotspot. Lactic acid bacteria are common bacteria and symbiotic bacteria in human and animal gastrointestinal tracts, and have been widely used in industries such as food, beverages, microecologics and the like as important probiotics for a long time, and are generally recognized as safety-level microorganisms. Is a novel green additive developed in recent years, and has the effects of improving beneficial flora in the digestive tract of cultured animals, inhibiting or killing harmful bacteria, improving the health level of the animals, promoting the growth and development of the animals, improving the utilization rate of feed and the like. The lactic acid bacteria also have: has no side effect, no pollution and no residue, can purify the water environment, improve the feed taste, improve the immunity of the organism, prevent and control pathogenic microorganisms, promote the growth capacity of cultured organisms and the like, thereby achieving the effect of preventing related diseases of aquaculture and being paid much attention to aquaculture. Therefore, the research on the application of the lactic acid bacteria becomes the key point of the healthy aquaculture.

Disclosure of Invention

The invention aims to provide lactococcus lactis and application thereof, wherein the lactococcus lactis is used as an additive to effectively improve intestinal flora, prevent and control pathogenic microorganisms, promote the growth capacity of cultured organisms and enhance the host immune function.

In view of the above, the invention provides lactococcus lactis with the preservation number of CGMCC No. 20329.

Preferably, the strain is isolated from the intestinal tract of apostichopus japonicus selenka.

Correspondingly, the invention also provides application of the lactococcus lactis with the preservation number of CGMCC No.20329 in preparation of a microbial preparation.

Preferably, the microbial preparation is used for inhibiting pathogenic microorganisms, promoting the growth capacity of cultured organisms and enhancing the immune function of a host.

Preferably, the microbial preparation is a liquid or powder probiotic.

Preferably, the method comprises the following steps: inoculating lactococcus lactis CGMCC No.20329 on an MRS solid culture medium, inoculating a single colony in an MRS liquid culture medium, and culturing at 30 ℃ and a shaking table rotation speed of 150-; transferring the seed liquid to a fresh MRS liquid culture medium according to 5 percent, and culturing for 40-60h under the conditions of 30 ℃ and the rotating speed of a shaking table of 150-; and (3) carrying out centrifugal separation on 5000g of the fermentation liquor for 30min, washing for 2 times by using PBS lysate, discarding supernatant, retaining precipitate, and carrying out heavy suspension by using the PBS lysate to obtain the probiotic of the lactococcus lactis CGMCC No. 20329.

Preferably, in the step of obtaining the seed solution, the cultivation time is 36 h.

Preferably, in the step of obtaining the seed solution, the rotation speed of the shaker is 180 rpm.

Preferably, in the step of obtaining the fermentation broth, the culture time is 48 h.

Preferably, in the step of obtaining the fermentation broth, the rotation speed of the shaker is 180 rpm.

The invention provides lactococcus lactis, the preservation number of which is CGMCC No.20329, the lactococcus lactis is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation date is 2020, 7 and 9 days. The invention also provides application of the lactococcus lactis with the preservation number of CGMCC No.20329 in preparation of a microbial preparation. The strain separation sieve is selected from Apostichopus japonicus (Apostichopus japonicus) intestinal tract, and can promote the healthy growth of Apostichopus japonicus, improve the intestinal flora, effectively resist the infection of pathogenic bacteria vibrio Tower, and enhance the immune function of organism. The lactococcus lactis is an endogenous intestinal probiotic, is suitable for growth and propagation in marine environment, has no danger to cultured animals and human beings, and has high efficiency and safety in the aspect of application. The lactococcus lactis CGMCC No.20329 has the advantages of low production cost, simple process and good commercial prospect.

Biological preservation Instructions

Lactococcus lactis, classified name: lactococcus lactis. Is preserved in China general microbiological culture Collection center (CGMCC) in 7, 9 and 2020, with the address as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing. The preservation number is CGMCC No. 20329.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a growth curve diagram of lactococcus lactis CGMCC No.20329 according to an embodiment of the present invention;

FIG. 2 is a graph showing the growth of lactococcus lactis CGMCC No.20329 at different temperatures according to an embodiment of the present invention;

FIG. 3 is a growth diagram of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under different pH conditions;

FIG. 4 is a growth diagram of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under different glucose concentrations;

FIG. 5 is a growth diagram of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under different nitrogen source concentration conditions;

FIG. 6 is a growth diagram of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under different liquid loading amounts;

FIG. 7 is a growth diagram of lactococcus lactis CGMCC No.20329 under pH control and pH non-control conditions according to an embodiment of the invention;

FIG. 8 is a graph of the cumulative survival rate of lactococcus lactis CGMCC No.20329 applied to Apostichopus japonicus culture according to an embodiment of the present invention.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.

The embodiment of the invention discloses lactococcus lactis with the preservation number of CGMCC No. 20329. The lactococcus lactis CGMCC No.20329 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 2020, 7 and 9 days, and the preservation unit address is No. 3 of No.1 Xilu of Beijing Kogyo-Yang district.

Correspondingly, the invention also provides application of the lactococcus lactis with the preservation number of CGMCC No.20329 in preparation of a microbial preparation. The microbial preparation is used for inhibiting pathogenic microorganisms, promoting the growth capacity of cultured organisms and enhancing the host immune function. Preferably, the microbial preparation is liquid or powder probiotics, namely the lactococcus lactis is prepared into bacterial liquid or bacterial powder.

Preferably, the probiotic of lactococcus lactis CGMCC No.20329 is prepared by the following method: inoculating lactococcus lactis CGMCC No.20329 on an MRS solid culture medium, inoculating a single colony in an MRS liquid culture medium, and culturing at 30 ℃ and a shaking table rotation speed of 150-; transferring the seed liquid to a fresh MRS liquid culture medium according to 5 percent, and culturing for 40-60h under the conditions of 30 ℃ and the rotating speed of a shaking table of 150-; and (3) carrying out centrifugal separation on 5000g of the fermentation liquor for 30min, washing for 2 times by using PBS lysate, discarding supernatant, retaining precipitate, and carrying out heavy suspension by using the PBS lysate to obtain the probiotic of the lactococcus lactis CGMCC No. 20329.

Preferably, in the step of obtaining the seed solution, the cultivation time is preferably 36h, and the rotation speed of the shaker is preferably 180 rpm. In the step of obtaining the fermentation broth, the cultivation time is preferably 48h, and the rotation speed of the shaker is preferably 180 rpm.

The invention preferably adopts the following method to detect the thallus concentration: the concentration of the cells was expressed by absorbance (OD600) using a microplate reader method. And (3) determining the OD600 value of the strain after a certain period of growth by taking the culture medium as a background control, and subtracting the background OD600 value to obtain the value of the measured thallus. And (3) measuring the number of the thalli by using a coating plate counting method, and determining the required bacteria liquid concentration according to the OD600 value and the number of the thalli in the bacteria liquid to prepare the liquid probiotics.

The lactococcus lactis is derived from endogenous lactococcus lactis CGMCC No.20329 in intestinal tracts of Apostichopus japonicus (Apostichopus japonicus), and the strain not only has the effect of inhibiting pathogenic bacteria, but also can enhance the nonspecific immunity level of cultured organisms and the capability of resisting pathogenic bacteria infection.

The information of the lactococcus lactis CGMCC No.20329 strain of the invention is as follows:

1. and (4) source. The strain is separated from the intestinal tract of the apostichopus japonicus, can promote the healthy growth of the apostichopus japonicus, improve the intestinal flora, effectively resist the infection of tower vibrio and enhance the immune function of organisms.

2. Morphological characteristics. Gram-positive bacteria, facultative anaerobe, no capsula or spore, and the colony cultured in MRS solid culture medium at 30 deg.c for 48 hr has the form diameter of 1-2 mm, circular shape, raised shape, milk white color, smooth surface and regular edge.

3. Physiological and biochemical characteristics. D-amygdalin reaction is positive, phosphatidylinositol phospholipase C reaction is negative, D-xylose reaction is positive, arginine double hydrolase 1 reaction is positive, beta-galactosidase reaction is negative, alpha-glucosidase reaction is positive, cyclodextrin reaction is positive, L-aspartic acid arylamine reaction is negative, beta-galactopyranosidase reaction is negative, alpha-mannosidase reaction is negative, phosphatase reaction is negative, leucine arylamine reaction is positive, L-proline arylamine reaction is negative, beta-glucuronidase reaction is negative, alpha-galactosidase reaction is negative, L-pyrrolidone arylamine reaction is negative, beta-glucuronidase reaction is negative, alanine arylamine reaction is positive, tyrosine arylamine reaction negative, D-sorbitol reaction positive, UREASE reaction negative, polymyxin B drug resistance reaction positive, D-galactose reaction positive, D-ribose reaction positive, L-lactate alkalization reaction negative, lactose reaction negative, N-acetyl-D-glucosamine reaction positive, D-maltose reaction positive, bacitracin drug resistance reaction positive, neomycin drug resistance reaction positive, growth reaction positive in 6.5% NaCl, D-mannitol reaction negative, D-mannose reaction positive, methyl-B-D-glucopyranoside reaction positive, amylopectin reaction negative, D-raffinose reaction negative, O/129 drug resistance (comp.vibrio) reaction negative, saligenin reaction positive, sucrose reaction positive, D-trehalose reaction positive, arginine double hydrolase 2 reaction is positive, and alprotoxin drug resistance reaction is positive.

The gene sequence of the lactococcus lactis 16SrRNA is shown as SEQ ID No. 1.

The lactococcus lactis CGMCC No.20329 has the following characteristics:

1. the probiotic feed prepared from lactococcus lactis CGMCC No.20329 can promote the healthy growth of apostichopus japonicus, improve intestinal flora, effectively resist the infection of pathogenic bacteria tower vibrios and enhance the immune function of organisms.

2. The lactococcus lactis CGMCC No.20329 has the advantages of low production cost, simple process and good commercial prospect.

3. The lactococcus lactis CGMCC No.20329 is derived from the intestinal tract of the marine apostichopus japonicus, is endogenous intestinal probiotics, is suitable for growth and propagation in marine environment, has no danger to cultured animals and human beings, and has high efficiency and safety in the application aspect.

For further understanding of the present invention, the following embodiments are provided to illustrate the technical solutions of the present invention in detail, and the scope of the present invention is not limited by the following embodiments.

The raw materials adopted in the embodiment of the invention are all commercially available.

The Lactococcus lactis (Lactococcus lactis) CGMCC No.20329 adopted in the embodiment of the invention is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation date is 2020, 7 and 9 days, the preservation number is CGMCC No.20329, and the strain separation sieve is selected from the intestinal tracts of Apostichopus japonicas (Apostichopus japonicus).

Example 1

Preparation of probiotics of lactococcus lactis CGMCC No. 20329:

the lactococcus lactis strain is preserved at-80 ℃, lactococcus lactis CGMCC No.20329 is inoculated on an MRS solid culture medium, a single colony is inoculated in an MRS liquid culture medium, and the single colony is cultured for 36 hours at the condition of 30 ℃ and 180rpm of shaking table rotation speed to obtain seed liquid;

transferring the seed solution to a fresh MRS liquid culture medium according to the proportion of 5%, culturing for 48h under the condition of the rotation speed of a shaking table of 180rpm at the temperature of 30 ℃ to obtain fermentation liquor, centrifugally separating 5000g of the fermentation liquor for 30min, washing for 2 times by using PBS lysate, discarding supernatant and leaving precipitate, and re-suspending by using the PBS lysate to obtain the probiotic of lactococcus lactis CGMCC No. 20329.

And (3) detecting the concentration of the thallus:

the concentration of the cells was expressed by absorbance (OD600) using a microplate reader method. And (3) determining the OD600 value of the strain after a certain period of growth by taking the culture medium as a background control, and subtracting the background OD600 value to obtain the number of the thalli.

The number of cells was measured by spread plate counting method based on OD600 and the number of cells in the bacterial suspension.

The PBS lysate is prepared according to the following method:

mixing potassium dihydrogen phosphate (KH)₂PO₄)0.24g, potassium chloride (KCl)0.2g, disodium hydrogen phosphate dodecahydrate (Na)₂HPO₄〃12H₂O)3.63g and sodium chloride (NaCl)8.0g, using double distilled water to fix the volume to 1000mL, adjusting the pH value to 7.4, sterilizing and storing at 4 ℃ for later use.

MRS solid culture medium and MRS liquid culture medium are purchased from Beijing land bridge technology service company Limited, and the MRS liquid culture medium is MRS broth and is prepared according to the use method in the specification.

FIG. 1 is a growth curve diagram of lactococcus lactis CGMCC No.20329 provided in this example. The culture medium is MRS broth, and the culture temperature is 37 ℃. FIG. 2 is a graph showing the growth of lactococcus lactis CGMCC No.20329 provided in this example under different temperature conditions, wherein the culture medium is MRS broth. FIG. 3 is a graph showing the growth of lactococcus lactis CGMCC No.20329 provided in this example under different pH conditions, wherein the medium is MRS broth and the culture temperature is 30 ℃. FIG. 4 is a graph showing the growth of lactococcus lactis CGMCC No.20329 in this example under different glucose concentrations in MRS broth at 30 deg.C and pH 6.5. FIG. 5 is a growth diagram of lactococcus lactis CGMCC No.20329 provided in this example under different nitrogen source concentrations, wherein the culture medium is MRS broth and the culture temperature is 30 ℃. FIG. 6 is a graph showing the growth of lactococcus lactis CGMCC No.20329 in different liquid loading amounts, wherein the culture medium is MRS broth and the culture temperature is 30 ℃. FIG. 7 is the growth diagram of lactococcus lactis CGMCC No.20329 provided in this example under pH-controlled and pH-uncontrolled conditions, wherein the culture medium is MRS broth, the culture temperature is 30 ℃, and the pH value of the pH-controlled group is 6.5.

Example 2

The application of the lactococcus lactis CGMCC No.20329 probiotic feed comprises the following steps:

step 1) preparation of probiotic feed:

mixing the required concentration of the bacteria solution with the Apostichopus japonicus feed, and mixing the bacteria colony (mL) with the feed(g) In a ratio of 1: 2, preparing into soft dough with CGMCC No.20329 content of 1 x 10⁸ CFU/g。

Step 2) cultivating the apostichopus japonicus:

starving 240 healthy apostichopus japonicus for 2 days, domesticating the apostichopus japonicus for 7 days by using basic feed, and randomly dividing the apostichopus japonicus into 2 groups: experimental group (CGMCC No.20329 group) and control group (CK group), 100 pieces per group. The apostichopus japonicus is cultured in a 1.2 × 0.5 × 0.8m water tank for 24h aeration, and the apostichopus japonicus is fed quantitatively for 2 times a day: feeding at 9:00 am and 5:00 pm, wherein the daily feeding amount is about 3% of the body weight, culturing in running water, removing residual feed and excrement by using a siphon before feeding, feeding CGMCC No.20329 bacteria feed to an experimental group, and feeding basic feed to a control group. The apostichopus japonicus feeding and state conditions are observed every day, and the test period is 8 weeks.

Step 3), the intestinal immunity index of the apostichopus japonicus:

and (3) testing the apostichopus japonicus culture to 6 weeks, taking 3 intestinal mixed samples per jar, performing sterile grinding, and detecting the content of nitric oxide synthase, acid phosphatase, lysozyme, superoxide dismutase, catalase and protein in the intestinal tracts. The results are shown in Table 1, the contents of nitric oxide synthetase, acid phosphatase, lysozyme and protein are obviously higher than those of the control group, which shows that the CGMCC No.20329 can improve the nonspecific immune factor of the intestinal tract of the apostichopus japonicus.

TABLE 1 influence of strain CGMCC No.20329 on intestinal immunoenzyme activity of Apostichopus japonicus

Step 3) detecting the relative weight gain rate:

each group was weighed every week for 8 weeks and was made of non-challenge Apostichopus japonicus selenka. Calculating the relative Weight gain ratio (Weight gain ratio, WGR,%) of 100 × (final Weight-initial Weight)/initial Weight, and as can be seen from table 2, the Weight gain of the apostichopus japonicus in the experimental group is obviously better than that of the control group, and the relative Weight gain ratio of each group is calculated, wherein the CGMCC No.20329 group is: 122.12% and CK group 109.41%. The result shows that lactococcus lactis CGMCC No.20329 has a promoting effect on the growth of the apostichopus japonicus.

Table 2 shows the weight chart of lactococcus lactis CGMCC No.20329 applied to apostichopus japonicus culture provided by the embodiment. CGMCC No.20329 is used as the feed for lactococcus lactis, and CK is used as the feed for the control group.

TABLE 2 influence of strain CGMCC No.20329 on growth of Apostichopus japonicus

Step 4) high-throughput sequencing analysis of the composition of the intestinal flora:

randomly selecting 9 apostichopus japonicus in each group every week, aseptically taking 3 apostichopus japonicus intestinal mixed samples, treating 3 biological replicates in each group, performing high-throughput sequencing on intestinal microbial diversity of each group of detoxified intestinal tracts in week 7 and week 8, and on a phylum level: the CGMCC No.20329 group and the CK group are mainly the phylum Firmicutes and the phylum Proteobacteria; at the door level: the relative abundance of Lactococcus (Lactococcus) in CGMCC No.20329 group varied from 0.00% to 76.23% and in CK group varied from 0.00% to 7.33% within 1-8 weeks. The result shows that the quantity of the apostichopus japonicus in the intestinal tract is obviously increased after the apostichopus japonicus is fed with the lactococcus lactis, and the composition of the intestinal flora is improved. The bacterial flora structure analysis in this example is shown in tables 3 to 6.

Table 3 experimental feeding CGMCC No.20329 group intestinal flora structure composition at level

TABLE 4 intestinal flora structure composition of control CK group fed on level

Table 5 composition of intestinal flora in CGMCC No.20329 group fed on level

TABLE 6 intestinal flora structure composition of control CK group fed on genus level

Step 5), simulating the disease resistance of the apostichopus japonicus:

taking tower type vibrio and suspending with PBS to concentration of 1 x 10⁸CFU/mL is the virus counteracting bacterial liquid, 30 experimental apostichopus japonicus in each group in the step 2) are selected randomly, each group is divided into three biological repetitions randomly, the injection dosage of each group is 100uL by adopting a body cavity injection method, the cumulative survival rate of the experimental apostichopus japonicus in each group is counted after 14 days, the total survival rate of the experimental apostichopus japonicus in each group is 73.33% in a CGMCC No.20329 group, and the total survival rate of the experimental apostichopus japonicus in a CK group is 33.33%. Therefore, the lactococcus lactis CGMCC No.20329 can improve the resistance of the apostichopus japonicus to the infection of the tower vibrio. Fig. 8 is a cumulative survival rate graph of lactococcus lactis CGMCC No.20329 applied to stichopus japonicus culture provided by the embodiment, wherein the CGMCC No.20329 is used for feeding lactococcus lactis, and the CK is used for feeding basic feed for a control group.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> research institute for aquatic products of Fujian province of applicant

<120> lactococcus lactis and application thereof

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cccaactccc gtggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc 120

gtgctgatcc gcgattacta gcgattccga cttcatgtag gcgagttgca gcctacaatc 180

cgaactgaga atggttttaa gagattagct aaacatcact gtctcgcgac tcgttgtacc 240

atccattgta gcacgtgtgt agcccaggtc ataaggggca tgatgatttg acgtcatccc 300

caccttcctc cggtttatca ccggcagtct cgttagagtg cccaacttaa tgatggcaac 360

taacaatagg ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 420

gacaaccatg caccacctgt atcccgtgtc ccgaaggaac ttcctatctc taggaatagc 480

acgagtatgt caagacctgg taaggttctt cgcgttgctt cgaattaaac cacatgctcc 540

accgcttgtg cgggcccccg tcaattcctt tgagtttcaa ccttgcggtc gtactcccca 600

ggcggagtgc ttattgcgtt agctgcgata cagagaactt atagctccct acatctagca 660

ctcatcgttt acggcgtgga ctaccagggt atctaatcct gtttgctccc cacgctttcg 720

agcctcagtg tcagttacag gccagagagc cgctttcgcc accggtgttc ctccatatat 780

ctacgcattt caccgctaca catggaattc cactctcctc tcctgcactc aagtctacca 840

gtttccaatg catacaatgg ttgagccact gccttttaca ccagacttaa taaaccacct 900

gcgctcgctt tacgcccaat aaatccggac aacgctcggg acctacgtat taccgcggct 960

gctggcacgt agttagccgt ccctttctgg gtagttaccg 1000

Claims

1. Lactococcus lactis with the preservation number of CGMCC No. 20329.

2. A lactococcus lactis according to claim 1, wherein the strain is isolated from the intestinal tract of apostichopus japonicus.

3. An application of lactococcus lactis with a preservation number of CGMCC No.20329 in preparation of a microbial preparation.

4. The use according to claim 2, wherein the microbial preparation is used for inhibiting pathogenic microorganisms, promoting the growth of farmed organisms and enhancing host immune function.

5. Use according to claim 2, characterized in that the microbial preparation is a liquid or a powder probiotic.

6. Use according to claim 2, characterized in that it comprises the following steps:

inoculating lactococcus lactis CGMCC No.20329 on an MRS solid culture medium, inoculating a single colony in an MRS liquid culture medium, and culturing at 30 ℃ and a shaking table rotation speed of 150-;

transferring the seed liquid to an MRS liquid culture medium according to 5 percent, and culturing for 40-60h under the conditions of 30 ℃ and the rotating speed of a shaking table of 150-;

and (3) carrying out centrifugal separation on 5000g of the fermentation liquor for 30min, washing with PBS (phosphate buffer solution), discarding supernatant, retaining precipitate, and carrying out heavy suspension with the PBS to obtain the probiotic of lactococcus lactis CGMCC No. 20329.

7. The use according to claim 6, wherein in the step of obtaining a seed solution, the cultivation time is 36 h.

8. The use according to claim 6, wherein in the step of obtaining the seed solution, the rotational speed of the shaker is 180 rpm.

9. The use according to claim 6, wherein in the step of obtaining the fermentation broth, the cultivation time is 48 h.

10. The use according to claim 6, wherein in the step of obtaining the fermentation broth, the rotation speed of the shaker is 180 rpm.