CN114657082B - Lactococcus lactis and application thereof - Google Patents
Lactococcus lactis and application thereof Download PDFInfo
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- CN114657082B CN114657082B CN202110419707.6A CN202110419707A CN114657082B CN 114657082 B CN114657082 B CN 114657082B CN 202110419707 A CN202110419707 A CN 202110419707A CN 114657082 B CN114657082 B CN 114657082B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a lactococcus lactis with a preservation number of CGMCC No.20329, which is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) and has a preservation date of 2020, 7 and 9. The invention also provides an application of the lactococcus lactis with the preservation number of CGMCC No.20329 in preparing microbial preparations. The strain separation screen is selected from the intestinal tracts of apostichopus japonicus, can promote the healthy growth of apostichopus japonicus, improve intestinal flora, effectively resist infection of pathogenic bacteria vibrio pagoda and enhance the immune function of organisms. The lactococcus lactis is an endogenous intestinal probiotics, is not only suitable for growth and propagation in marine environment, but also has no danger to cultured animals and human beings, and has high efficiency and safety in application. The lactococcus lactis CGMCC No.20329 has the advantages of low production cost, simple process and good commercial prospect.
Description
Technical Field
The invention relates to the technical field of aquatic microorganism application, in particular to a lactococcus lactis and application thereof.
Background
Along with the rising of the aquaculture industry in China and the change of factors such as water quality environment, climate conditions and the like in the intensive culture process, various diseases mainly including bacterial diseases occur in the culture process, and the bacterial diseases are usually prevented and treated through antibiotics and chemical medicines, so that negative effects caused by the bacterial diseases are focused by a plurality of students.
Antibiotics are continuously used in the breeding industry, the water body environment is destroyed while diseases are prevented and treated, and the intestinal flora structure in a host body is disturbed, so that bacteria generate drug resistance, pathogenic bacteria with 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. In addition to chemical contamination, it has been reported that antibiotics in the environment may 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 the human body, so that the resistance of the human body to antibiotics is increased, and thus, the public health of the human body is threatened to some extent, and the public health of the human body is seriously endangered.
With the sustainable development of green foods, many scholars are devoted to the study of antibiotics and chemical drug substitutes, and probiotics (lactic acid bacteria) are becoming a research hotspot. Lactic acid bacteria are common bacteria and symbiotic bacteria of the gastrointestinal tract of humans and animals, and have been widely used as important probiotics in the industries of foods, beverages, microecologics and the like for a long time, and are recognized as safety-class microorganisms. Is a novel green additive developed in recent years, and has the effects of improving the beneficial flora in the digestive tract of cultured animals, inhibiting or killing harmful bacteria, improving the health level of animals, promoting the growth and development of animals, improving the utilization rate of feed and the like. 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 organism immunity, prevent and control pathogenic microorganisms, promote the growth capacity of cultured organisms and the like, thereby achieving the effect of preventing the diseases related to aquaculture and being valued in aquaculture. Therefore, application research of lactic acid bacteria is an important point of aquaculture.
Disclosure of Invention
The invention solves the technical problem of providing the lactococcus lactis and the application thereof, which are used as additives for effectively improving intestinal flora, preventing and controlling pathogenic microorganisms, promoting the growth capacity of cultured organisms and enhancing the immune function of a host.
In view of the above, the invention provides a lactococcus lactis with a preservation number of CGMCC No.20329.
Preferably, the strain is isolated from the intestinal tract of Apostichopus japonicus.
Correspondingly, the invention also provides application of the lactococcus lactis with the preservation number of CGMCC No.20329 in preparing microbial preparations.
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 steps of: inoculating lactococcus lactis CGMCC No.20329 on MRS solid culture medium, inoculating single colony into MRS liquid culture medium, and culturing at 30deg.C and shaking table rotation speed of 150-200rpm for 30-50 hr to obtain seed solution; transferring the seed liquid into fresh MRS liquid culture medium according to 5%, and culturing at 30deg.C and shaking table rotation speed of 150-200rpm for 40-60 hr to obtain fermentation liquid; and (3) centrifugally separating 5000g of the fermentation liquor for 30min, washing for 2 times by using PBS (phosphate buffered saline) lysate, discarding supernatant to leave precipitates, and re-suspending 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 hours.
Preferably, in the step of obtaining the seed liquid, the rotation speed of the shaking table is 180rpm.
Preferably, in the step of obtaining the fermentation broth, the cultivation time is 48 hours.
Preferably, in the step of obtaining the fermentation broth, the rotation speed of the shaking table is 180rpm.
The invention provides a lactococcus lactis with a preservation number of CGMCC No.20329, which is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) and has a preservation date of 2020, 7 and 9. The invention also provides an application of the lactococcus lactis with the preservation number of CGMCC No.20329 in preparing microbial preparations. The strain separation sieve is selected from intestinal tracts of apostichopus japonicus (Apostichopus japonicus), can promote healthy growth of apostichopus japonicus, improve intestinal flora, effectively resist infection of pathogenic bacteria vibrio pagoda and enhance organism immunity. The lactococcus lactis is an endogenous intestinal probiotics, is not only suitable for growth and propagation in marine environment, but also has no danger to cultured animals and human beings, and has high efficiency and safety in application. The lactococcus lactis CGMCC No.20329 has the advantages of low production cost, simple process and good commercial prospect.
Description of biological preservation
Lactococcus lactis, classified and named: lactococcus lactis. The strain is preserved in China general microbiological culture Collection center (CGMCC) at the address of 7.9 in 2020: the institute of microorganisms of national academy of sciences of China, no.1, no. 3, north Chen West Lu, the Korean region of 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 that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the growth of lactococcus lactis CGMCC No.20329 according to the embodiment of the invention;
FIG. 2 is a graph showing the growth of lactococcus lactis CGMCC No.20329 under different temperature conditions provided by the embodiment of the invention;
FIG. 3 is a growth chart of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under different pH conditions;
fig. 4 is a growth chart of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under the condition of different glucose concentrations;
FIG. 5 is a growth chart of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under the condition of different nitrogen source concentrations;
FIG. 6 is a growth chart of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under different liquid loading amounts;
FIG. 7 is a growth chart of lactococcus lactis CGMCC No.20329 provided by the embodiment of the invention under the conditions of pH control and non-pH control;
fig. 8 is a graph of cumulative survival rate of lactococcus lactis CGMCC No.20329 applied to apostichopus japonicus culture, provided by the embodiment of the invention.
Detailed Description
For a further understanding of the present invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are merely intended to illustrate further features and advantages of the invention, and are not limiting of the claims of the invention.
The embodiment of the invention discloses a lactococcus lactis, which has a preservation number of CGMCC No.20329. The lactococcus lactis CGMCC No.20329 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2020, 7 and 9 days, and a preservation unit address is North Chen Xili No.1 and 3 in the Korean region of Beijing city.
Correspondingly, the invention also provides application of the lactococcus lactis with the preservation number of CGMCC No.20329 in preparing microbial preparations. The microbial preparation is used for inhibiting pathogenic microorganisms, promoting the growth capacity of cultured organisms and enhancing the immune function of hosts. Preferably, the microbial preparation is liquid or powder probiotics, namely the lactococcus lactis is prepared into bacterial liquid or bacterial powder.
As a preferred scheme, the probiotic bacteria of lactococcus lactis CGMCC No.20329 are prepared according to the following method, comprising: inoculating lactococcus lactis CGMCC No.20329 on MRS solid culture medium, inoculating single colony into MRS liquid culture medium, and culturing at 30deg.C and shaking table rotation speed of 150-200rpm for 30-50 hr to obtain seed solution; transferring the seed liquid into fresh MRS liquid culture medium according to 5%, and culturing at 30deg.C and shaking table rotation speed of 150-200rpm for 40-60 hr to obtain fermentation liquid; and (3) centrifugally separating 5000g of the fermentation liquor for 30min, washing for 2 times by using PBS (phosphate buffered saline) lysate, discarding supernatant to leave precipitates, and re-suspending 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 36 hours, and the rotation speed of the shaking table is preferably 180rpm. In the step of obtaining the fermentation broth, the cultivation time is preferably 48 hours, and the rotation speed of the shaking table is preferably 180rpm.
The invention preferably adopts the following method to detect the concentration of the thalli: the concentration of the cells was expressed as absorbance (OD 600) by the enzyme-labeled method. And taking the culture medium as a background control, and determining the OD600 value of the strain after growing for a certain time, wherein the OD600 value of the background is subtracted to be the value of the detected thalli. And (3) measuring the number of the thalli by using a coating plate counting method, and determining the concentration of the required thalli according to the OD600 value and the number of the thalli in the thalli to prepare the liquid probiotics.
The lactococcus lactis is derived from endogenous lactococcus lactis CGMCC No.20329 in the intestinal tract 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 lactococcus lactis CGMCC No.20329 strain information of the invention is as follows:
1. a source. The strain is separated from the intestinal canal of apostichopus japonicus, can promote the healthy growth of the apostichopus japonicus, improve intestinal flora, effectively resist the infection of vibrio tower and strengthen the immune function of an organism.
2. Morphological features. Gram positive bacteria, facultative anaerobic bacteria, no capsular and spore, are cultured on MRS solid culture medium at 30 deg.C for 48 hr to form colony with diameter of 1-2 mm, round, raised, milky white, smooth surface and regular edge.
3. Physiological and biochemical characteristics. D-amygdalin reaction positive, phosphatidylinositol phospholipase C reaction negative, D-xylose reaction positive, arginine double hydrolase 1 reaction positive, beta-galactosidase reaction negative, alpha-glucosidase reaction positive, cyclodextrin reaction positive, L-aspartic acid arylamine enzyme reaction negative, beta-galactopyranoside enzyme reaction negative, alpha-mannosidase reaction negative, phosphatase reaction negative, leucine arylamine enzyme reaction positive, L-proline arylamine enzyme reaction negative, beta-glucuronidase reaction negative, alpha-galactosidase reaction negative, L-pyrrolidone arylamine enzyme reaction negative, beta-glucuronidase reaction negative, alanine arylamine reaction positive, tyrosine arylamine reaction negative, D-sorbitol reaction positive, UREASE reaction negative, polymyxin B drug resistance 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 positive, neomycin drug resistance 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.virorio.) reaction negative, salicin reaction positive, sucrose reaction positive, d-trehalose is positive in reaction, arginine double hydrolase 2 is positive in reaction, and the olprixin drug resistance is positive.
The 16SrRNA gene sequence of the lactococcus lactis is shown as SEQ ID No. 1.
The lactococcus lactis CGMCC No.20329 has the following characteristics:
1. the probiotic feed prepared from the lactococcus lactis CGMCC No.20329 can promote the healthy growth of apostichopus japonicus, improve intestinal flora, effectively resist infection of pathogenic bacteria vibrio tower and strengthen 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 sea apostichopus japonicus and is an endogenous intestinal probiotics, so that the lactococcus lactis is not only suitable for growth and propagation in a marine environment, but also has no danger to cultured animals and human beings, and has high efficiency and safety in application.
In order to further understand the present invention, the following embodiments are provided to describe the technical solution of the present invention in detail, and the scope of protection 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 by the embodiment of the invention is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), the preservation date is 7 and 9 days in 2020, the preservation number is CGMCC No.20329, and the bacterial strain separation sieve is selected from intestinal tracts of apostichopus japonicus (Apostichopus japonicus).
Example 1
Preparation of probiotics of lactococcus lactis CGMCC No. 20329:
the lactobacillus strain is stored at-80 ℃, lactobacillus CGMCC No.20329 is inoculated on an MRS solid culture medium, a single colony is inoculated in the MRS liquid culture medium, and the culture is carried out for 36 hours under the condition of 180rpm of a shaking table at 30 ℃ to obtain seed liquid;
transferring the seed solution into a fresh MRS liquid culture medium according to 5%, culturing for 48h at the rotation speed of a shaking table of 180rpm at 30 ℃ to obtain a fermentation liquor, centrifugally separating 5000g of the fermentation liquor for 30min, washing 2 times with PBS lysate, discarding supernatant to leave precipitate, and re-suspending with PBS lysate to obtain probiotic bacteria of lactococcus lactis CGMCC No.20329.
And (3) detecting the concentration of the thalli:
the concentration of the cells was expressed as absorbance (OD 600) by the enzyme-labeled method. And taking the culture medium as a background control, and determining the OD600 value of the strain after a certain time of growth, wherein the OD600 value of the background is subtracted to obtain the number of thalli.
The number of cells was measured by a coating plate count method, and the number of cells in the bacterial liquid was determined based on OD 600.
The PBS lysate is prepared according to the following method:
potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.24g, 0.2g of potassium chloride (KCl), disodium hydrogen phosphate dodecahydrate (Na 2 HPO 4 〃12H 2 O) 3.63g and 8.0g of sodium chloride (NaCl) are mixed, the volume is fixed to 1000mL by double distilled water, the pH value is regulated to 7.4, and the mixture is sterilized and stored at the temperature of 4 ℃ for standby.
MRS solid medium and MRS liquid medium are purchased from Beijing land bridge technology service Co., ltd, MRS liquid medium is MRS broth, and are prepared according to the usage on the instruction book.
Fig. 1 is a growth curve of lactococcus lactis CGMCC No.20329 provided in this example. The culture medium was MRS broth and the culture temperature was 37 ℃. Fig. 2 is a graph showing the growth of lactococcus lactis CGMCC No.20329 under different temperature conditions, wherein the culture medium is MRS broth. Fig. 3 is a growth chart of lactococcus lactis CGMCC No.20329 provided by the embodiment under different pH conditions, wherein the culture medium is MRS broth, and the culture temperature is 30 ℃. Fig. 4 is a growth chart of lactococcus lactis CGMCC No.20329 provided in the example under the condition of different glucose concentrations, wherein the culture medium is MRS broth, the culture temperature is 30 ℃, and the pH value is 6.5. Fig. 5 is a growth chart of lactococcus lactis CGMCC No.20329 provided by the embodiment under the condition of different nitrogen source concentrations, wherein the culture medium is MRS broth, and the culture temperature is 30 ℃. FIG. 6 is a growth chart of lactococcus lactis CGMCC No.20329 provided by the embodiment under different liquid loading, wherein the culture medium is MRS broth, and the culture temperature is 30 ℃. FIG. 7 is a growth chart of lactococcus lactis CGMCC No.20329 provided in the example under the conditions of pH control and non-pH control, wherein the culture medium is MRS broth, the culture temperature is 30 ℃, and the pH value of the pH group is controlled to be 6.5.
Example 2
Application of lactobacillus CGMCC No.20329 probiotic feed:
step 1) preparation of probiotic feed:
mixing the bacterial liquid with the stichopus japonicus feed uniformly, wherein the ratio of bacterial colony (mL) to feed (g) is 1:2, making into soft dough, the content of CGMCC No.20329 reaches 1 x 10 8 CFU/g。
Step 2) apostichopus japonicus culture:
after starving 240 healthy apostichopus japonicus for 2 days, the feed is used for domestication for 7 days, and the feed is randomly divided into 2 groups: experimental group (CGMCC No.20329 group) and control group (CK group), 100 per group. The apostichopus japonicus is cultivated in a water tank with the volume of 1.2 multiplied by 0.5 multiplied by 0.8m for 24 hours for aeration, and the quantitative feeding is adopted, and the feeding is carried out for 2 times a day: the daily feeding amount is about 3% of the weight of the animal, the running water is not flowed during the feeding period, the siphon tube is used for removing residual feed and excrement before the feeding, the experimental group is fed with CGMCC No.20329 bacterial feed, and the control group is fed with basic feed. The feeding and state conditions of the apostichopus japonicus are observed every day, and the test period is 8 weeks.
Step 3) intestinal immunity index of apostichopus japonicus:
and (3) taking 3 intestinal mixed samples from each group of the apostichopus japonicus culture to the 6 th week, aseptically grinding, and detecting the contents of intestinal nitric oxide synthase, acid phosphatase, lysozyme, superoxide dismutase, catalase and protein. The results are shown in Table 1, the contents of nitric oxide synthase, acid phosphatase, lysozyme and protein are obviously higher than those of a control group, which shows that CGMCC No.20329 can improve the non-specific immune factor of the intestinal tract of apostichopus japonicus.
Table 1 influence of Strain CGMCC No.20329 on intestinal immunoenzymatic Activity of Apostichopus japonicus
Step 3) relative weight gain rate detection:
each group was weighed weekly for 8 weeks, and was all non-detoxified apostichopus japonicus. The relative weight gain rate (Weight gain ratio, WGR,%) =100× (final body weight-initial body weight)/initial body weight was calculated, and as can be seen from table 2, the weight gain of the experimental group apostichopus japonicus was significantly better than that of the control group, and the relative weight gain rate of each group was calculated, CGMCC No. 20329: 122.12% of CK group 109.41%. The lactobacillus CGMCC No.20329 has the effect of promoting the growth of apostichopus japonicus.
Table 2 shows the weight map of the lactococcus lactis CGMCC No.20329 applied to the cultivation of apostichopus japonicus. CGMCC No.20329 is used for feeding lactococcus lactis, and CK is used for feeding basic feed for a control group.
TABLE 2 influence of the Strain CGMCC No.20329 on the growth of Apostichopus japonicus
Step 4) high throughput sequencing analysis of intestinal flora composition:
9 apostichopus japonicus intestinal tract samples were randomly selected every week, 3 apostichopus japonicus intestinal tracts were aseptically sampled, 3 biological replicates were treated each, 7 th week and 8 th week were each group of post-challenge intestinal tracts, intestinal microbial diversity was high-throughput sequenced, at the portal level: the CGMCC No.20329 group and the CK group are mainly of the phylum of the Firmicutes (Firmides), the Proteus (Proteus); at the door level: the relative abundance of Lactococcus (Lactococcus) varies from 0.00% to 76.23% in the CGMCC No.20329 group and from 0.00% to 7.33% in the CK group within 1-8 weeks. The method shows that after the apostichopus japonicus is fed with the lactococcus lactis, the amount of the apostichopus japonicus in the intestinal tract is obviously increased, and the composition of intestinal flora is improved. The analysis of the colony structure of this example is shown in tables 3 to 6.
Table 3 structural composition of intestinal flora in CGMCC No.20329 group for experimental feeding at the level of gate
Table 4 structural composition of intestinal flora of CK group fed horizontally in comparison
Table 5 structural composition of intestinal flora of CGMCC No.20329 group for experimental feeding on level
Table 6 structural composition of intestinal flora of horizontally-controlled fed CK group
Step 5) disease resistance of apostichopus japonicus:
suspending Vibrio talus with PBS to a concentration of 1×10 8 CFU/mL is the virus-killing bacteria liquid, 30 experimental apostichopus japonicus groups in the step 2) are selected randomly, each group is divided into three biological repetition randomly, the cumulative survival rate of the apostichopus japonicus groups is counted after each injection dose is 100uL and 14d by adopting a coelom injection method, the CGMCC No.20329 group is 73.33%, and the CK group is 33.33%. Therefore, the lactococcus lactis CGMCC No.20329 can improve the resistance of the apostichopus japonicus to the infection of vibrio tower. Fig. 8 is a graph of cumulative survival rate of lactococcus lactis CGMCC No.20329 provided in the embodiment applied to apostichopus japonicus culture, wherein CGMCC No.20329 is lactococcus lactis fed, and CK is basic feed fed to a control group.
The above description of the embodiments is only for aiding in the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.
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> applicant's institute for aquatic products, fujian province
<120> A lactic acid bacterium and use thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1000
<212> DNA
<213> Lactococcus lactis CGMCC No.20329
<400> 1
cccctgggtt ctaactttac gagcgccctc cttgcggtta ggcaacctac ttcgggtact 60
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 (9)
1. A strain of lactococcus lactis is characterized in that the preservation number is CGMCC No.20329.
2. The application of lactococcus lactis with a preservation number of CGMCC No.20329 in preparing microbial preparations is provided.
3. The use according to claim 2, wherein the microbial preparation is used for inhibiting pathogenic microorganisms, promoting the growth capacity of farmed organisms and enhancing the immune function of the host.
4. The use according to claim 2, wherein the microbial agent is a liquid or powder probiotic.
5. The use according to claim 2, characterized by the steps of:
inoculating lactococcus lactis CGMCC No.20329 on MRS solid culture medium, inoculating single colony into MRS liquid culture medium, and culturing at 30deg.C and shaking table rotation speed of 150-200rpm for 30-50 hr to obtain seed solution;
transferring the seed liquid into MRS liquid culture medium according to 5%, and culturing at 30deg.C and shaking table rotation speed of 150-200rpm for 40-60 hr to obtain fermentation liquor;
centrifuging 5000g of the fermentation liquor for 30min, washing with PBS, discarding supernatant to leave precipitate, and re-suspending with PBS to obtain probiotic bacteria of lactococcus lactis CGMCC No.20329.
6. The method according to claim 5, wherein the seed liquid is obtained for a period of time of 36 hours.
7. The method according to claim 5, wherein the rotation speed of the shaking table is 180rpm in the step of obtaining the seed liquid.
8. The method according to claim 5, wherein the fermentation broth is obtained for a period of 48 hours.
9. The method according to claim 5, wherein the rotation speed of the shaking table is 180rpm in the step of obtaining the fermentation broth.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103815150A (en) * | 2012-11-16 | 2014-05-28 | 大连德通生物技术开发有限公司 | Lactic acid bacterium nutrient enhancer for holothurian culture |
| CN107964524A (en) * | 2018-01-18 | 2018-04-27 | 海南大学 | A kind of Lactococcus lactis HKS2 and its separating screening method and application with lactic acid activity |
| CN112251371A (en) * | 2020-09-08 | 2021-01-22 | 福建大北农水产科技有限公司 | Lactococcus lactis, microecological preparation and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103815150A (en) * | 2012-11-16 | 2014-05-28 | 大连德通生物技术开发有限公司 | Lactic acid bacterium nutrient enhancer for holothurian culture |
| CN107964524A (en) * | 2018-01-18 | 2018-04-27 | 海南大学 | A kind of Lactococcus lactis HKS2 and its separating screening method and application with lactic acid activity |
| CN112251371A (en) * | 2020-09-08 | 2021-01-22 | 福建大北农水产科技有限公司 | Lactococcus lactis, microecological preparation and application |
Non-Patent Citations (2)
| Title |
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| Effects of dietary supplementation of four strains of lactic acid bacteria on growth,immune-related response and genes expression of the juvenile sea cucumber Apostichopus japonicus Selenka;Chao Li et al.;《Fish and Shellfish Immunology》;69-75 * |
| 乳酸乳球菌 Q-9 胞外多糖对鲤先天免疫应答、抗氧化活性和抗病性能的影响;冯军厂 等;《水产学报》;1477-1487 * |
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