CN107964524A - A kind of Lactococcus lactis HKS2 and its separating screening method and application with lactic acid activity - Google Patents

A kind of Lactococcus lactis HKS2 and its separating screening method and application with lactic acid activity Download PDF

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CN107964524A
CN107964524A CN201810047344.6A CN201810047344A CN107964524A CN 107964524 A CN107964524 A CN 107964524A CN 201810047344 A CN201810047344 A CN 201810047344A CN 107964524 A CN107964524 A CN 107964524A
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hks2
lactococcus lactis
bacterial strain
bacterium
lactic acid
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CN107964524B (en
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谢珍玉
文金顺
�龙昊
章翔
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Hainan University
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    • 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
    • C12N1/205—Bacterial isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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    • 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
    • C12N1/02—Separating microorganisms from their culture media
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    • 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

Abstract

The present invention relates to a kind of Lactococcus lactis HKS2 with lactic acid activity and its separating screening method and application.Bacterial strain its Classification And Nomenclature of the present invention is：(Lactococus lactis) HKS2, in China typical culture collection center preservation, deposit number is CCTCC NO：M2017296, preservation date are on May 31st, 2017.The strain isolation is from Hainan Tropical breeding seawater and the mixture of litopenaeus vannamei intestines and stomach.The Lactococcus lactis HKS2 of the present invention does not have hemolytic activity, with preferable hydrophobic active, higher self-coagulation rate, compared with strong man's work gastro-intestinal Fluid tolerance, stronger cholate tolerance, preferable bacteriostasis, the Lactococcus lactis HKS2 of the present invention is fed into litopenaeus vannamei as feed addictive, verify that it can preferably improve immunity of organisms by culture experiment, promote prawn growth, and effect stability, be a kind of bacterial strain with applications well prospect.

Description

A kind of Lactococcus lactis HKS2 and its separating screening method with lactic acid activity and Using
Technical field
The invention belongs to strain field, is related to a kind of Lactococcus lactis, it is more particularly related to which a kind of have breast Hainan original inhabitants' Lactococcus lactis HKS2 of acid activity and its separating screening method and application.
Background technology
In order to pursue efficient, quick culture benefit, cultivation density is continued to increase, expands cultivation scale, causes aquatic products to move The thing speed of growth declines, feed coefficient increase, and the Common Diseases such as vibriosis take place frequently, and causes the chemical fishing medicine abuse such as antibiotic. Grave danger is constituted this not only adds aquaculture cost, and to the quality safety and human health of aquatic products.Therefore, find A kind of cultural method efficiently, healthy, safe has become the research hotspot of current aquaculture technology.
Using probiotics spice feed with promote aquatic livestock growth, improve immunity, suppress growth of pathogenic bacteria, The advantages that cost is low, without recontamination, it has also become be primarily upon one of focus in nuisanceless aquatic health aquaculture model.Its In, lactic acid bacteria (lactic acid bacteria, LAB) be during current aquaculture of aquatic animal spice is fed most common strain it One, and Lactococcus lactis (Lactococus lactis) belongs to lactic acid bacteria, is a kind of anaerobism or the Gram-positive of amphimicrobian Bacterium, to people and aquatic livestock body and Environmental security, can largely produce lactic acid, acetic acid and formic acid, and extracellular hydrolase and Promote aquatic livestock to digest and assimilate nutriment, also maintain the stabilization of archenteric flora of aquatic animals, and to environment With it is stronger it is adaptable, can be in the bacterium of the quick definite value of aquatic livestock enteron aisle.
Lactic acid bacteria, which has, improves aquatic livestock immunity, and lactic acid bacteria is produced by being colonized in aquatic livestock enteron aisle Lactic acid, various enzymes, the small-molecule substance Tiny ecosystem of bacteriocin, hydrogen peroxide and degraded amino acid produce pyridine polyamines and derive Thing, these materials can stimulate aquatic livestock to make immune response, aquatic livestock is in immune active state, so as to improve The immunocompetence of aquatic livestock.
To intestinal flora adjustment effect, the microorganism in the health status and enteron aisle of aquatic livestock has closes lactic acid bacteria closely It is that the microorganism in enteron aisle is the important component of ecological environment in aquatic livestock, influences many functions of animal.Lactic acid bacteria It is attached on by adhesin (S- layers albumen, lipoteichoicacid, lipopolysaccharides, peptide glycan etc.) in the epithelial cell of animal gastrointestinal tract, and Field planting forms the barrier of " biomembrane " together in the enteron aisle of host, on intestinal mucosa surface." biomembrane " barrier effectively hinders Field planting of other harmful bacterias in intestinal tract surface is kept off, so that the harmful intestinal tract bacteria of animal is reduced, flora ecology is more stable.Lactic acid Bacterium sticks or is colonized in GI tract, the occupy-place in enteron aisle, and surviving in the gastrointestinal tract to other harmful bacterias to form bacterium and do Disturb.
The organic acid that lactic acid bacteria produces, predominantly lactic acid, acetic acid, propionic acid etc..The pH value of intestines and stomach can be effectively reduced, so that Inhibit the survival of the not strong harmful bacteria of other acid resistances in the gastrointestinal tract.The hydrogen peroxide that lactic acid bacteria produces in the gastrointestinal tract With certain bactericidal effect.
Lactic acid bacteria can synthesize vitamin (folic acid, vitamin B12 etc.), various digestive ferments (protease etc.), gamma-amino It is certain that acid, exocellular polysaccharide (Capsular polysaccharides etc.), conjugated linoleic acid, these lactic acid bacteria secretion or synthetic product can provide animal Nutriment, contributes to animal to digest and assimilate nutriment, promotes the healthy growth of animal！Correlative study research shows breast Sour bacterium obtains the research such as remarkable result, Kongnum in aquatic products application aspect and shows, litopenaeus vannamei is fed using lactobacillus spice After 6 weeks, the immunity enhancing of prawn, feed conversion rate, rate of body weight gain, survival rate and the resistance to vibrios are improved. The researchs such as Dawood show, add lactobacillus in feed and Lactococcus lactis feeds Red snapper 56 days, immune indexes and growth Index is obviously improved, and feed coefficient reduces.The researchs such as Gou little Lan find that lactic acid bacteria is to Aeromonas hydrophila (Aeromonas Hydrophila), the pathogenic bacteria such as Aeromonas sobria (Aeromonas sobria) have obvious inhibitory action.
The breeding water body purification that the B. licheniformis strain of different growing environment corresponds under home environment has preferable Effect, and clean-up effect is poor, and clean-up effect is unstable etc. is then often shown to strange land environment.At present, domestic related ground breast The patent of sour bacterium probiotics is concentrated mainly on fermenting microbe and food of fertilizer production etc., related breeding feed addition Lactococcus lactis patent it is less, more have no the related patents on Lactococcus lactis Hainan Island geographical population.
The content of the invention
The present invention is directed to the problem of pointed in background technology and the shortcomings of the prior art, the first object of the present invention It is to provide a kind of Lactococcus lactis HKS2 with lactic acid activity, another object of the present invention is to provide above-mentioned to have lactic acid The separating screening method of the Lactococcus lactis HKS2 of activity, a further object of the invention are to provide Lactococcus lactis described above The application of HKS2, particularly feeds litopenaeus vannamei for breeding feed additive.
In order to realize above-mentioned first purpose of the present invention, the technical solution adopted by the present invention is：One kind has lactic acid activity Lactococcus lactis HKS2, its Classification And Nomenclature is：(Lactococus lactis) HKS2, has been preserved in Chinese Typical Representative culture Collection (CCTCC), address：Wuhan, China Wuhan University, its deposit number are：CCTCC NO:M2017296, preservation day Phase：On May 31st, 2017.
Lactococcus lactis HKS2 of the present invention, is to support shrimp waters from Wenchang, hainan respectively and Hainan Tropical live body is all The lactic acid bacteria strains that separation screening obtains in the seawater and live body shrimp intestinal samples of the interior collection of shore prawn body of receiving.The Lactococcus lactis Bacterium HKS2 bacterium achroacyte dissolubilities, have preferable hydrophobic active, higher self-coagulation rate is safe, and this bacterium also has There are stronger acid producing ability, artificial gastro-intestinal Fluid tolerance, stronger cholate tolerance and stronger antagonism pathogenic bacteria ability： Stronger inhibitory action is respectively provided with to Vibrio harveyi, vibrio alginolyticus, Streptococcusagalactiae, Wdwardsiella tarda.By institute of the present invention The Lactococcus lactis HKS2 stated feeds litopenaeus vannamei as feed addictive, verifies that it can also be preferably by culture experiment Immunity of organisms is improved, promotes prawn growth, and effect stability, is a kind of bacterial strain with applications well prospect.
Present invention Lactococcus lactis (Lactococus lactis) HKS2 described above also has following property：
1st, microbial characteristic
Lactococcus lactis HKS2 is gram-positive bacteria, and bacterial strain bacterium colony on MRS culture mediums is milky, flat, surface It is coarse, moistening, neat in edge, 37 DEG C culture 24h after colony diameter be 2~4mm, colonial morphology is as shown in Figure 1.
2nd, 16S rRNA are analyzed
Using the corresponding primer of 16S rRNA ITS sequences, upstream and downstream primer sequence is respectively：5ˊ- AGAGTTTGATCCTGGCTCA-3 ˊ and 5 ˊ-GGTTACCTTGTTACGACTT-3 ˊ carry out PCR amplification to Lactococcus lactis HKS2, The fragment of 1409bp is obtained after sequencing respectively, is MF037867 in the accession number of GenBank, the 16s rRNA gene orders of the bacterium Such as SEQ ID NO：Shown in 1.
In order to realize another object of the present invention, the present invention provides the separation screening of Lactococcus lactis HKS2 described above Method, the method mainly include the following steps that：
(1) sample collection
Supported respectively from Wenchang, hainan and seawater sample and work are gathered in shrimp waters and Hainan Tropical live body litopenaeus vannamei body Body shrimp intestinal samples, the seawater sample of collection take a small amount of stand-by；Live body shrimp intestinal samples are put into sterile saline, are shredded Shrimp enteron aisle, concussion are mixed into shrimp enteron aisle mixed liquor；Take a small amount of mixed liquor stand-by, whole operation process carries out on super-clean bench；
(2) sample bacterial strain expands culture
Step (1) seawater sample and shrimp enteron aisle mixed liquor are seeded to equipped with MRS fluid nutrient mediums respectively, in 30 DEG C, 180r/min, shake culture 24h in shaking table；
(3) bacterial strain initial gross separation
Separation and purifying to bacterial strain are combined using plate streak with rubbing method, take the bacterium after culture in step (2) Liquid is applied to containing 0.5%~3% lightweight CaCO3MRS solid mediums in, 48h is cultivated in 30 DEG C of insulating boxs, to bacterium The tablet fallen is observed, and picking has the bacterial strain of obvious molten calcium circle, and line extremely contains 0.5%~3% lightweight CaCO3MRS solids Culture medium, is repeated 3 and is cultivated with last time line, until the bacterium colony grown on tablet, morphological feature is completely the same, is finally separated The purpose bacterial strain of purifying；
(4) screening of bacterial strain production acid and preservation
Picking step (3) isolate and purify after purpose bacterial strain, then line is to containing 0.5%~3% lightweight CaCO3MRS On solid medium, 48h is cultivated in 30 DEG C of insulating boxs, with the molten calcium circle size of each bacterial strain of vernier caliper measurement, screens molten calcium Larger bacterial strain is enclosed, is stored in 50% glycerite in -20 DEG C of refrigerators.
Further, the Lactococcus lactis HKS2 inoculum concentrations are 1%~2%, and initial pH value is 7.0~7.2.
A kind of purposes of Lactococcus lactis HKS2 in vibrio alginolyticus growth is suppressed.
A kind of purposes of Lactococcus lactis HKS2 in Streptococcusagalactiae growth is suppressed.
A kind of purposes of Lactococcus lactis HKS2 in probiotics is prepared.
A kind of probiotics, contains above-mentioned Lactococcus lactis HKS2.
A kind of bacterium powder, is prepared using above-mentioned Lactococcus lactis HKS2.
Purposes of the above-mentioned bacterium powder as aquaculture feed additive.
A kind of feed for litopenaeus vannamei, containing above-mentioned Lactococcus lactis HKS2, the viable count of the Lactococcus lactis HKS2 For 1 × 107~1 × 109cfu/g。
It is further preferred that the prawn feed additive that the present invention is described above, described containing above-mentioned enterococcus faecalis HKF7 The viable count of Lactococcus lactis HKS2 is 1 × 108cfu/g。
Compared with prior art, the present invention has following beneficial effect：
A kind of Lactococcus lactis HKS2 achroacyte dissolubilities provided by the invention, have preferable hydrophobic active, higher Self-coagulation rate, it is safe, and this bacterium also have stronger acid producing ability, artificial gastro-intestinal Fluid tolerance, stronger cholate Tolerance and stronger antagonism pathogenic bacteria ability：To Vibrio harveyi, vibrio alginolyticus, Streptococcusagalactiae, Wdwardsiella tarda Stronger inhibitory action is respectively provided with, it is most notable to the inhibition of Streptococcusagalactiae and vibrio alginolyticus, and the lactic acid of the present invention Galactococcus HKS2 can also preferably suppress bacterial reproduction in animal body, improve immunity of organisms, promote growth of animal, it is applied Prospect is extensive, can be applied to probiotics, can also be made into bacterium powder, add as aquaculture feed additive Be added in feed, grown for suppressing in animal body pathogenic bacteria, reduce animal especially the death rate of prawn while can promote Prawn grows, and is a kind of very extensive Tiny ecosystem strain of application prospect.
Brief description of the drawings
Fig. 1 is colonial morphology figure of the excrement Lactococcus lactis HKS2 bacterial strains of the present invention on MRS solid mediums；
Fig. 2 is the aspect graphs of excrement Lactococcus lactis HKS2 of the present invention under the microscope；
Fig. 3 is that Lactococcus lactis HKS2 bacterial strains hemolysis tests design sketch in the embodiment of the present invention 4；
Fig. 4 is Lactococcus lactis HKS2 hydrophobic rate test result figures in the embodiment of the present invention 5；
Fig. 5 be in the embodiment of the present invention 6 Lactococcus lactis HKS2 from aggegation rate test result figure；
Fig. 6 is the tolerance test result figure of Lactococcus lactis HKS2 simulated gastric fluids in the embodiment of the present invention 7；
Fig. 7 is the tolerance test result figure of Lactococcus lactis HKS2 simulated intestinal fluids in the embodiment of the present invention 8；
Fig. 8 feeds litopenaeus vannamei for Lactococcus lactis HKS2 bacterial strains in the embodiment of the present invention 11 and feeds the cycle in different Infectious age compares figure.
Embodiment
Form is described in further detail the above of the present invention again by the following examples, but should not manage this The scope solved as the above-mentioned theme of the present invention is only limitted to following embodiment, and all technologies for being realized based on the above of the present invention are equal Belong to the scope of the present invention.
Culture medium prescription is used in following embodiments：
MRS culture mediums of the present invention are references《The manufacture and application of microbiological culture media》Made from method.
MRS fluid nutrient mediums：
Take peptone 10g, powdered beef 10g, dusty yeast 5g, glucose 20g, dipotassium hydrogen phosphate 5g, sodium chloride 5g, citric acid Three ammonium 5g, magnesium sulfate 0.5g, manganese sulfate 0.2g, Tween 80 1.0g add 121 DEG C of autoclavings after distilled water 1000ml dissolvings, It is spare to put 4 DEG C of refrigerators.
MRS solid mediums：
Take peptone 10g, powdered beef 10g, dusty yeast 5g, glucose 20g, dipotassium hydrogen phosphate 5g, sodium chloride 5g, citric acid Three ammonium 5g, magnesium sulfate 0.5g, manganese sulfate 0.2g, Tween 80 1.0g add distilled water 1000ml to add agar powder 20g after dissolving, and 121 DEG C autoclaving, it is spare to put 4 DEG C of refrigerators.
The MRS solid mediums that separating screening method of the present invention uses are gone back on the basis of above-mentioned MRS solid cultures based formulas It with the addition of the precipitated calcium carbonate (CaCO of 1% (V/W)3).MRS tablets are the special screening flat boards of lactic acid bacteria, and other tablet ratios are more Be conducive to the growth of lactic acid bacteria.Sterile glass beads are conducive to enteron aisle and grind more abundant, and the bacteria concentration higher in such liquid, has Beneficial to screening more favourable microorganisms.Again draw plate to be conducive to select the more preferable bacterium colony of activity, optimize strain.
The separation screening of 1 Lactococcus lactis HKS2 of embodiment.
The separating screening method of present invention Lactococcus lactis HKS2 described above, mainly includes the following steps that：
(1) sample collection
Supported respectively from Wenchang, hainan and seawater and live body shrimp are gathered in shrimp waters and Hainan Tropical live body litopenaeus vannamei body Intestinal contents, the seawater sample of collection take a small amount of stand-by；Take live body shrimp enteron aisle to be put into sterile saline, shred shrimp intestines Road, concussion are mixed into shrimp enteron aisle mixed liquor；Take a small amount of mixed liquor stand-by, whole operation process carries out on super-clean bench；
(2) sample bacterial strain expands culture
Step (1) seawater sample and shrimp enteron aisle mixed liquor are seeded to equipped with MRS fluid nutrient mediums respectively, in 30 DEG C, 180r/min, shake culture 24h in shaking table；
(3) bacterial strain initial gross separation
Separation and purifying to bacterial strain are combined using plate streak with rubbing method, to cultured bacterial strain, take 0.1mL Bacterium solution is applied to MRS solid mediums and (contains 1% lightweight CaCO3, V/W, similarly hereinafter) in, 48h is cultivated in 30 DEG C of insulating boxs, to length The tablet for having bacterium colony is observed, and picking has the bacterial strain of obvious molten calcium circle, rules and (contains 1% lightweight to MRS solid mediums CaCO3) V/W, similarly hereinafter, repeat 3 and cultivated with last time line, until the bacterium colony grown on tablet, morphological feature is completely the same, separation Purify purpose bacterial strain.
The production acid screening of 2.2 bacterial strains and preservation
The bacterial strain of picking after purification, then rules to MRS solid mediums (added with 1% light caco3), in 30 DEG C of perseverances 48h is cultivated in incubator.With the molten calcium circle size of each bacterial strain of vernier caliper measurement, the larger bacterial strain of molten calcium circle is screened, it is sweet with 50% Oil solution is stored in -20 DEG C of refrigerators.
Colony characteristics：
Lactococcus lactis HKS2 is gram-positive bacteria, bacterial strain bacterium colony on MRS solid mediums is milky, it is flat, Rough surface, moistening, neat in edge, after 37 DEG C are cultivated 24h, colony diameter is 2~4mm, and colonial morphology is as shown in Figure 1.
The Molecular Identification of 2 Lactococcus lactis HKS2 of embodiment.
To identify bacterial strain, first using scanning electric mirror observing cell form and the part biochemical reactions of test strain, Scanning electron microscope (SEM) photograph is as shown in Fig. 2, can tentatively judge the bacterial strain for Lactococcus lactis.
PCR identifications are carried out to the bacterial strain screened using the universal primer of bacterium：Said by the operation of DNA of bacteria extracts kit Bright book extracts template DNA.Using the sense primer 5 '-AGAGTTTGATCCTGGCTCA-3 ' of 16S rRNA conserved sequences, SEQ is seen 5 '-GGTTACCTTGTTACGACTT-3 of ID No.2 and anti-sense primer, is shown in SEQ ID No.3, to the 16s of Lactococcus lactis HKS2 RRNA genetic fragments carry out PCR amplification, clone and are sequenced, obtain the fragment of 1409bp after sequencing respectively, in the login of GenBank Number it is MF037867, obtain the Pseudomonas by sequencing sees SEQ ID No.1 in Lactococcus lactis, the sequencing result of amplified fragments.
The acid producing ability test of 3 Lactococcus lactis HKS2 of embodiment.
Acid producing ability is tested：The aimed strain of oese picking primary dcreening operation is first used, bacterial strain sterile working is inoculated in MRS liquid In culture medium, then the 180rpm shaken cultivations 24h in 30 DEG C of incubators, spare；Prepare MRS solid mediums and (add 1% lightweight CaCO3), each culture dish culture medium of falling 20mL, using after dilution 108The 100 μ L of bacterium solution of cfu/mL are coated on consolidating in culture dish On body culture medium, the quiescent culture 48h in 30 DEG C of incubators, with the molten calcium loop diameter of vernier caliper measurement, each sample sets 3 weights It is multiple, it is averaged, finally the definite molten calcium loop diameters of Lactococcus lactis HKS2 are up to 5.00~7.00mm, as shown in Figure 1, molten by Fig. 1 Calcium loop diameter size understands that Lactococcus lactis HKS2 acid producing abilities are stronger.
4 Lactococcus lactis HKS2 hemolysis of embodiment is tested.
Hemolysis is tested：Using haemolysis culture medium (horse blood), lined using bacterial strain after purification on blood plate, 30 DEG C Incubator quiescent culture 48h, observes culture dish haemolysis situation.Hemolytic test result is unchanged for blood plate color, no haemolysis Activity, is γ haemolysis.
Fig. 3 tests design sketch for hemolysis.
5 Lactococcus lactis HKS2 hydrophobic performances of embodiment are tested.
Hydrophobic performance is tested：By primary dcreening operation aimed strain in 30 DEG C, 180r/min shaking tables shaken cultivation 24h.Take bacterium solution into Row centrifugation, temperature are 4 DEG C, rotating speed 4000rpm centrifugations 10min.It is resuspended and is centrifuged with PBS (pH=7.4) solution, is repeated twice. It is resuspended again with PBS solution, the OD600 values of bacterium solution is approximately equal to 1.00；Then isometric bacterium solution is mixed with dimethylbenzene, turbine shakes 2min is swung, stands 1h, water intaking mutually measures OD600 values, calculates the hydrophobic rate of purpose bacterial strain.The calculation formula of hydrophobic rate is：Dredge Water rate (%)=[(A0-AT)]/A0] × 100.
Fig. 4 is the present embodiment Lactococcus lactis HKS2 hydrophobic performance test result figures, as shown in Figure 4, excrement Lactococcus lactis HKS2 has preferable hydrophobic performance.
6 Lactococcus lactis HKS2 of embodiment is from aggegation performance test.
From aggegation performance test：4mL bacteria suspensions are taken in 5mL test tubes, vortex 10s, room temperature is stood, stand respectively 1h, 2h, 1mL clear liquids measure OD600 values are drawn after 3h, 4h, 5h, 10h, are as a result calculated as A1, A2, A3, A4, A5, A6.From aggegation (%)=1- (At/A0) light absorption value of OD600 when × 100, At 1h, 2h, 3h, 4h, 5h, 10h, A0 are the OD600 values before mixing.
Fig. 5 is the present embodiment Lactococcus lactis HKS2 from aggegation the performance test results figure, as shown in Figure 5, Lactococcus lactis HKS2 has preferably from agglutinating performance.
Artificial gastric juice resistance's property test of 7 Lactococcus lactis HKS2 of embodiment.
Test method is as follows：
1) purpose bacterial strain is cultivated：With 50mL/250mL (V/V) MRS fluid nutrient medium culture purpose bacterial strains, 30 DEG C, 180r/ 24h is cultivated in min shaking tables；
2) simulated gastric fluid is configured：Sodium chloride (0.5%) and pepsin (0.3mg/mL) are added to the PBS solution of sterilizing In, packing is mixed, pH to 3.5 is adjusted with the HCl solution of 1mol/L, is then filtered out with 0.22 μm of sterilised membrane filter miscellaneous in gastric juice Bacterium；
3) purpose bacteria suspension is prepared：First by cultured purpose bacterium solution, 4000rpm is centrifuged 10 minutes, then molten with PBS Liquid is washed and centrifuged, and is repeated 2 times, and it is concentration 10 to be finally resuspended again with PBS solution9The bacterium solution of cfu/mL；
4) inoculated and cultured and count plate are by 5% inoculum concentration inoculation purpose bacterium solution 20mL/50mL (V/ into simulated gastric fluid V).30 DEG C, cultivate in 180r/min shaking tables, when cultivating to 0h, 2h, 4h, carry out count plate, calculate purpose bacterial strain in different people Survival rate under work gastric juice and different incubation times.
Survival rate (%)=(Nt/N0) × 100, wherein：N0 represents viable count during purpose strain culturing 0h, and Nt is represented not With the viable count of incubation time.
Fig. 6 is the tolerance test result figure of the present embodiment Lactococcus lactis HKS2 simulated gastric fluids.It will be appreciated from fig. 6 that after 2h Survival rate be 38.16%, 4h after survival rate be 35.84%, illustrate that Lactococcus lactis HKS2 has stronger simulated gastric fluid Tolerance.
The simulated intestinal fluid tolerance test of 8 Lactococcus lactis HKS2 of embodiment.
Test method is as follows：
Simulated intestinal fluid is configured, trypsase (0.1mg/L) is added in the PBS solution of sterilizing, then with 1mol/L's NaOH solution adjusts pH to 6.8, finally with 0.22 μm of sterile membrane filtration simulated intestinal fluid, removes miscellaneous bacteria；
2) with the bacteria suspension prepared, purpose bacterium solution 20mL/50mL (V/V) into simulated intestinal fluid is inoculated with by 5% inoculum concentration. 30 DEG C, cultivate in 180r/min shaking tables, when cultivating to 0h, 2h, 4h, carry out count plate.Purpose bacterial strain is calculated different artificial Survival rate under intestinal juice and different incubation times.
Survival rate (%)=(Nt/N0) × 100, wherein：N0 represents viable count during purpose strain culturing 0h, and Nt is represented not With the viable count of incubation time.
Fig. 7 is the tolerance test result figure of the present embodiment Lactococcus lactis HKS2 simulated intestinal fluids, as shown in Figure 7, after 2h Survival rate be 105.66%, 4h after survival rate be 110.38%, illustrate that Lactococcus lactis HKS2 has stronger artificial intestines The tolerance of liquid.
The artificial Bile salt resistance test of 9 Lactococcus lactis HKS2 of embodiment.
Test method is as follows：
With 50mL/250mL (V/V) MRS fluid nutrient medium culture purpose bacterial strains, 30 DEG C, vibrate training in 180r/min shaking tables Support 24h.Concentration is configured to as 109Cfu/mL bacteria suspensions.Prepare containing 0%, 0.15%, 0.3%, 0.6% (W/V) cholate MRS fluid nutrient mediums, liquid amount are 20mL (20mL/50mL, V/V).Purpose bacteria suspension is seeded to containing courage with 1% inoculum concentration In the MRS fluid nutrient mediums of salt, 30 DEG C, cultivate 4h in 180r/min shaking tables.Using colony counting method, testing goal bacterial strain is not With the viable count after culture 4h in gallbladder salinity, and calculate survival rate.
Survival rate (%)=(Nt/N0) × 100, wherein：N0 represents viable count during strain culturing 0h, and Nt represents culture th Viable count afterwards, the present embodiment Lactococcus lactis HKS2 cholate tolerance test result are as shown in table 1.
1 Lactococcus lactis HKS2 bacterial strains of table cultivate the survival rate situation table after 4h under different gallbladder salinities
As can be drawn from Table 1, Lactococcus lactis HKS2 cultivates 4h in concentration is 0.15%~0.3% cholate solution Afterwards, can still survive, explanation can survive under the conditions of high bile salt concentiration, have certain Bile salt resistance.
It is stronger to can be seen that the resistance of the Lactococcus lactis HKS2 of the present invention from the test result of embodiment 7~9, can It to be resistant to simulated gastric fluid, simulated intestinal fluid, artificial cholate, therefore can use, can ensure as aquaculture feed additive Substantial amounts of thalline passes through the enteron aisle of animal, survives in enteron aisle, plays its prebiotic function.
The antagonistic experiment of 10 Lactococcus lactis HKS2 of embodiment.
Test method is as follows：
1) tablet is prepared：Antagonistic effect is carried out using lysoplate assay, prepares 2216E seawater solid mediums, it is high Warm autoclaving 121min, is cooled to 40~45 DEG C.Respectively toward the bacterium solution that 4% indicator strain is added in culture medium, indicator strain For Vibrio harveyi, Streptococcusagalactiae (using the culture of BHI solid mediums), vibrio alginolyticus, Wdwardsiella tarda, bacterial concentration For 6.0 × 108cfu/mL.It is uniformly mixed, is down flat plate, each flat-plate inverted culture medium 20mL.It is made and is inoculated with different indicator strains Tablet；
2) punching plus antagonistic strain：Punched using aseptic card punch (bore 5mm), each tablet makes a call to 5 holes.Toward hole 50 μ L Lactococcus lactis HKS2 of middle addition, Lactococcus lactis HKS2 is separately added into the different holes of same tablet, and 1 hole adds Sterile saline compares；
3) insulating box culture：Tablet added with Antagonistic Fungi is put into 30 DEG C of insulating boxs and just puts culture 24h, each tests 3 A repetition, is averaged；
4) inhibition zone measurement：With vernier caliper measurement inhibition zone, there are significantly circle as inhibition zone, measurement with control wells Antibacterial circle diameter, its fungistatic effect is assessed according to the size of inhibition zone.
The antibacterial test results of the present embodiment Lactococcus lactis HKS2 are as shown in table 2, as can be seen from Table 2, Lactococcus lactis HKS2 has pathogenic bacteria Vibrio harveyi several frequently seen in aquaculture, Wdwardsiella tarda, Streptococcusagalactiae, vibrio alginolyticus There is obvious inhibition zone, illustrate that Lactococcus lactis HKS2 is respectively provided with preferable fungistatic effect to these types of pathogenic bacteria.
The 2 antibacterial result of Lactococcus lactis HKS2 bacterial strains of table
Application Example of the embodiment 11 using Lactococcus lactis HKS2 as feed addictive raising litopenaeus vannamei.
Experimental method：
(1) 12 200mL cultivation flumes are taken, if 4 groups, it is respectively Lactococcus lactis HKS2 low concentration groups (1 × 107cfu/ G), concentration group (1 × 10 in HKS28Cfu/g), HKS2 high concentrations group (1 × 109Cfu/g), negative control group (0.9% physiology salt Water), 3 repetitions, water salinity 20~22 are each organized, stocking size is 0.73 ± 0.20g of weight, and body grows 4.30 ± 1.2cm shrimps Seedling, each cultivation flume put 45 tails in a suitable place to breed, and spice is fed 60 days, extract 10 tail shrimps, flesh from every group of cultivation flume at random within every 14 days Meat injecting method carries out artificial challenge's vibrios experiment, specifically feeds parameter and is shown in Table 3, and the whole weight (final of survey calculation shrimp Weight), whole body length (final length), feed coefficient, rate of body weight gain (Weight gain rate), specific growth rate (specific Weight gain rate SGR), survival rate (survival rate SR), concrete outcome is shown in Table 4；
(2) feed and daily management：The use of spice concentration is 10 according to experimental design8Cfu/g feeds are fed, often It 4 times, Feeding time and feeding volume are respectively：6：30 (1%), 12:00 (0.5%), 18:00 (1.5%), 23:00 (1%). The cycle is fed as 60 days, changes within every two days water 30%, salinity is 20~22, more than dissolved oxygen content 6.5mg/L, water temperature 28~30 DEG C, pH value is 7.5~8.2.
The present embodiment Lactococcus lactis HKS2 bacterial strains feed each experimental group parameter list of litopenaeus vannamei referring to table 3, prawn life Long situation is shown in Table 4, in addition, the present embodiment prawn sees Fig. 8 in the different situations for feeding the cycle infection death rate,
As known from Table 4, the growth rate (WGR) of the obvious low negative group of the feed coefficient of the prawn of Lactococcus lactis HKS2 groups, Specific growth rate (SGR), whole weight, whole body length are above negative control group, less than positive controls.Show Lactococcus lactis HKS2 spices are fed to promoting prawn growth to have good effect.
As can be seen from Figure 8, fed experiment at 60 days, at the 15th day fed and 30 days, the prawn death rate compared with Steadily；It is more obvious to feed the infectious age decline of the 45th day, and is less than negative control group, illustrates that Lactococcus lactis HKS2 is mixed Material is fed to promoting prawn growth to have good effect.
3 Lactococcus lactis HKS2 bacterial strains of table feed each experimental group parameter list of litopenaeus vannamei
4 Lactococcus lactis HKS2 bacterial strain difference spice concentration of table feeds each experimental group growth indexes table of litopenaeus vannamei
Note：a,b,cFor the otherness of same column data, (α=0.05).
Note:a,b,c for the same,The difference ofthe column data, (α=0.05)
Sequence table
<110>University Of Hainan
<120>A kind of Lactococcus lactis HKS2 and its separating screening method and application with lactic acid activity
<130>Nothing
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1409
<212> DNA
<213>Lactococcus lactis (Lactococus lactisHKS2)
<400> 1
gcagttgagc gctgaaggtt ggtacttgta ccgactggat gagcagcgaa cgggtgagta 60
acgcgtgggg aatctgcctt tgagcggggg acaacatttg gaaacgaatg ctaataccgc 120
ataaaaactt taaacacaag ttttaagttt gaaagatgca attgcatcac tcaaagatga 180
tcccgcgttg tattagctag ttggtgaggt aaaggctcac caaggcgatg atacatagcc 240
gacctgagag ggtgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300
cagcagtagg gaatcttcgg caatggacga aagtctgacc gagcaacgcc gcgtgagtga 360
agaaggtttt cggatcgtaa aactctgttg gtagagaaga acgttggtga gagtggaaag 420
ctcatcaagt gacggtaact acccagaaag ggacggctaa ctacgtgcca gcagccgcgg 480
taatacgtag gtcccgagcg ttgtccggat ttattgggcg taaagcgagc gcaggtggtt 540
tattaagtct ggtgtaaaag gcagtggctc aaccattgta tgcattggaa actggtagac 600
ttgagtgcag gagaggagag tggaattcca tgtgtagcgg tgaaatgcgt agatatatgg 660
aggaacaccg gtggcgaaag cggctctctg gcctgtaact gacactgagg ctcgaaagcg 720
tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctagat 780
gtagggagct ataagttctc tgtatcgcag ctaacgcaat aagcactccg cctggggagt 840
acgaccgcaa ggttgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatactc gtgctattcc 960
tagagatagg aagttccttc gggacacggg atacaggtgg tgcatggttg tcgtcagctc 1020
gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccctattgt tagttgccat 1080
cattaagttg ggcactctaa cgagactgcc ggtgataaac cggaggaagg tggggatgac 1140
gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg atggtacaac 1200
gagtcgcgag acagtgatgt ttagctaatc tcttaaaacc attctcagtt cggattgtag 1260
gctgcaactc gcctacatga agtcggaatc gctagtaatc gcggatcagc acgccgcggt 1320
gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgggagttg ggagtacccg 1380
aagtaggttg cctaaccgca aggagggcg 1409
<210> 2
<211> 19
<212> DNA
<213>Expand Lactococcus lactis (Lactococus lactisHKS2 sense primers)
<400> 2
agagtttgat cctggctca 19
<210> 3
<211> 19
<212> DNA
<213>Expand Lactococcus lactis (Lactococus lactisHKS2 anti-sense primers)
<400> 3
ggttaccttg ttacgactt 19

Claims (10)

  1. A kind of 1. Lactococcus lactis HKS2 with lactic acid activity, it is characterised in that：It is preserved in China typical culture collection The heart, deposit number are CCTCC NO:M2017296, preservation date：On May 31st, 2017, the Lactococcus lactis HKS2 bacterium 16sRNA gene orders such as SEQ ID No：Shown in 1.
  2. 2. a kind of separating screening method of the Lactococcus lactis HKS2 according to claim 1 with lactic acid activity, it is special Sign is：The method mainly includes the following steps that：
    (1) sample collection
    Supported respectively from Wenchang, hainan and seawater sample and live body shrimp are gathered in shrimp waters and Hainan Tropical live body litopenaeus vannamei body Intestinal samples, the seawater sample of collection take a small amount of stand-by；Live body shrimp intestinal samples are put into sterile saline, shred shrimp intestines Road, concussion are mixed into shrimp enteron aisle mixed liquor；Take a small amount of mixed liquor stand-by, whole operation process carries out on super-clean bench；
    (2) sample bacterial strain expands culture
    Step (1) seawater sample and shrimp enteron aisle mixed liquor are seeded to equipped with MRS fluid nutrient mediums respectively, in 30 DEG C, 180r/min, shake culture 24h in shaking table；
    (3) bacterial strain initial gross separation
    Separation and purifying to bacterial strain are combined using plate streak with rubbing method, take the bacterium solution in step (2) after culture to apply Cloth extremely contains 0.5%~3% lightweight CaCO3MRS solid mediums in, 48h is cultivated in 30 DEG C of insulating boxs, to bacterium colony Tablet is observed, and picking has the bacterial strain of obvious molten calcium circle, and line extremely contains 0.5%~3% lightweight CaCO3MRS solid cultures Base, is repeated 3 and is cultivated with last time line, until the bacterium colony grown on tablet, morphological feature is completely the same, isolates and purifies purpose bacterium Strain；
    (4) screening of bacterial strain production acid and preservation
    Picking step (3) isolate and purify after purpose bacterial strain, then line is to containing 0.5%~3% lightweight CaCO3MRS solids training Support on base, cultivate 48h in 30 DEG C of insulating boxs, with the molten calcium circle size of each bacterial strain of vernier caliper measurement, it is larger to screen molten calcium circle Bacterial strain, be stored in 50% glycerite in -20 DEG C of refrigerators.
  3. 3. the separating screening method of the Lactococcus lactis HKS2 according to claim 1 with lactic acid activity, its feature exist In：The Lactococcus lactis HKS2 inoculum concentrations are 1%~2%, and initial pH value is 7.0~7.2.
  4. 4. purposes of the Lactococcus lactis HKS2 according to claim 1 in vibrio alginolyticus growth is suppressed.
  5. 5. purposes of the Lactococcus lactis HKS2 according to claim 1 in probiotics is prepared.
  6. A kind of 6. probiotics, it is characterised in that：The probiotics contains the Lactococcus lactis described in claim 1 HKS2。
  7. 7. purposes of the probiotics according to claim 5 as aquaculture feed additive.
  8. A kind of 8. bacterium powder, it is characterised in that：The bacterium powder is using made of the Lactococcus lactis HKS2 described in claim 1.
  9. A kind of 9. feed for litopenaeus vannamei, it is characterised in that：Contain the Lactococcus lactis described in claim 1 in the feed The viable count of HKS2, the Lactococcus lactis HKS2 are 1 × 107~1 × 109cfu/g。
  10. 10. feed for litopenaeus vannamei according to claim 9, it is characterised in that：The viable bacteria of the Lactococcus lactis HKS2 Number is 1 × 108cfu/g。
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