CN113980853B - Lactic acid-producing lactococcus garvieae WBT0008 and application thereof - Google Patents
Lactic acid-producing lactococcus garvieae WBT0008 and application thereof Download PDFInfo
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Abstract
The invention relates to lactic acid-producing lactococcus garvieae WBT0008 and application thereof, which can effectively solve the problems of high yield of lactic acid and preparation of preservative and inhibition of harmful bacteria, and one lactic acid-producing lactococcus garvieae WBT0008 is classified and named as lactococcus garvieae @Lactococcus garvieae) The application of the lactococcus garvieae WBT0008 in lactic acid production, the application in preparing the bacteria inhibiting agent and the fresh beefsteak preservative is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.22576 and the preservation date of 2021, 5 and 20. The lactococcus garvieae WBT0008 is effectively used for preparing high-yield lactic acid, inhibiting harmful bacteria and fresh-keeping fresh steak, and has the advantages of simple preparation and application methods and environmental protection.
Description
Technical Field
The invention relates to a microorganism, in particular to lactic acid-producing lactococcus garvieae WBT0008 and application thereof.
Background
Lactic acid bacteria are widely used as probiotics which are in close contact with human life in various fields such as food, feed, medicine and the like, and are recognized as food-grade safe strains. In the food industry, the application history of the lactobacillus is long, and the lactobacillus has application in various foods such as meat, eggs, milk and the like, so that the flavor of the foods can be improved, and the nutritional value of the foods can be improved. In the feed industry, lactic acid bacteria can improve the feed utilization rate, improve the cultivation efficiency and reduce the use of chemical drugs and antibiotics. In the medical field, lactobacillus can regulate the balance of intestinal flora of the organism, prevent and treat cardiovascular diseases, and improve the oxidation resistance and immunity of the organism. Lactic acid bacteria not only have various probiotic functions, but also can produce various active metabolites, wherein lactic acid as a main metabolite produced by fermentation of lactic acid bacteria can exhibit the ability to inhibit growth of various harmful bacteria. The research finds that pathogenic bacteria in food are often important factors causing food spoilage and affecting food safety, and the chemical and physical preservation means used at present have great influence on the taste of preserved food, so that the biological preservation means becomes a novel technology in food preservation application. Lactococcus garvieae is an important probiotic, has more application in food fermentation in recent years, but has less application in high yield of lactic acid, inhibition of growth of harmful bacteria and prolongation of food fresh-keeping time, so that the lactic acid and fermentation products thereof are produced by using the lactococcus garvieae to inhibit growth of harmful bacteria in food, and the prolongation of food fresh-keeping time can overcome the prior technical defects, show obvious advantages and have wide application prospect, but has no public report so far.
Disclosure of Invention
Aiming at the situation, the invention aims to overcome the defects of the prior art and provide the lactic acid-producing lactococcus garvieae WBT0008 and the application thereof, which can effectively solve the problems of high lactic acid production, preparation of preservative and inhibition of harmful bacteria.
The technical scheme is that the lactic acid-producing lactococcus garvieae WBT0008 is classified and named as lactococcus garvieae (Lactococcus garvieae), and is preserved in China general microbiological culture Collection center (CGMCC) No.22576, and the preservation date is 2021, 5 months and 20, and the preservation address is: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing;
the application of the lactococcus garvieae WBT0008 in lactic acid production;
the application of the lactococcus garvieae WBT0008 in preparing the preparation of the bacteria agent for inhibiting the harmful bacteria, wherein the harmful bacteria are staphylococcus aureus, salmonella or coliform;
the application of the lactococcus garvieae WBT0008 in preparing fresh steak preservative.
The invention is a novel screened strain of lactococcus garvieae WBT0008, can be effectively used for preparing high-yield lactic acid, inhibiting harmful bacteria and fresh-keeping fresh steak, has simple preparation and application methods, is green and environment-friendly, provides a novel strain of lactococcus garvieae, exploits a novel application of the lactococcus garvieae, and has wide application prospect and huge economic and social benefits.
Drawings
FIG. 1 is a graph showing the variation of the 24-hour lactic acid content of the lactococcus garvieae of the present invention.
Detailed Description
The following describes in detail the embodiments of the present invention with reference to specific cases and examples.
Example 1
In the specific implementation of the invention, a lactic acid producing strain WBT0008, classified and named as lactococcus garvieae (Lactococcus garvieae), is prepared by taking soil samples around Henan Jiyuan pig farm, weighing 1g of the soil samples, preparing 10mL of suspension by using sterile water, and then sequentially diluting into 10 -3 、10 -4 、10 -5 Sample diluent is respectively absorbed with 0.1mL and coated on a calcium carbonate screening plate, the sample diluent is cultured for 48 hours in a constant temperature incubator at 37 ℃, a transparent circle is observed, a strain with obvious transparent circle is selected, a strain with the number of WBT0008 is obtained through screening, and the calcium carbonate screening plate: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80mL of Tween, 0.5g of magnesium sulfate,0.25g of manganese sulfate, 10g of calcium carbonate and 18g of agar, adding distilled water to 1000mL, sterilizing at 121 ℃ for 25 minutes, cooling for standby, and preparing a flat plate; screening to obtain strain with WBT0008, classified and named as lactococcus garvieae (Lactococcus garvieae), and preserving in China general microbiological culture Collection center (CGMCC) with preservation number of 22576, and preserving date of 2021, 5 months and 20, and preserving address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
Example 2
The application of the strain WBT0008 in lactic acid production comprises the following steps:
(1) Activating strains and culturing seeds: inoculating the frozen preserved strain WBT0008 into a seed culture medium according to the inoculum size of 1% after activation, and culturing at 37 ℃ for 24 hours at 180 r/min; the seed culture medium is as follows: 50g of glucose, 10g of peptone, 10g of yeast extract, 5g of sodium acetate, 0.2g of diammonium citrate, 0.2g of dipotassium hydrogen phosphate, 80mL of Tween, 0.2g of magnesium sulfate, 0.02g of manganese sulfate, 0.01g of ferrous sulfate and pH6.3-6.6, adding distilled water to 1000mL, sterilizing at 121 ℃ for 25 minutes, and cooling for later use;
(2) Lactic acid preparation: inoculating the seed solution into MRS culture medium according to volume of 2% of MRS culture medium, shake culturing at 37deg.C and 180r/min for 48 hr to obtain fermentation broth, namely lactic acid;
the MRS liquid culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80mL of Tween, 0.5g of magnesium sulfate and 0.25g of manganese sulfate, adding distilled water to 1000mL, sterilizing at 121 ℃ for 25 minutes, and cooling to obtain the beef extract;
(3) And (3) measuring the content of lactic acid:
sampling the prepared fermentation liquor lactic acid at 4h, 8h, 12h, 16h, 20h and 24h, repeating each group of the fermentation liquor lactic acid at three times, centrifuging at the temperature of 4 ℃ and under the condition of 12000r/min for 8min, taking the supernatant to measure the content of lactic acid, oxidizing the lactic acid under the action of lactic acid dehydrogenase to generate pyruvic acid, reducing MTT to generate a purple substance, wherein the substance has a characteristic absorption peak at 570nm, calculating the content of lactic acid to be 2-11 g/L/mL by utilizing the proportional relation between the lactic acid and the concentration of lactic acid, and determining the content of lactic acid by the following formula:
wherein: a represents absorbance of fermentation supernatant, A 0 The absorbance of the blank is represented by K, the slope of the standard curve, the intercept of the standard curve, and the conversion coefficient by 1176.67.
As shown in FIG. 1, the maximum lactic acid production of the strain WBT0008 within 24 hours reached 10.60g/L, indicating that the strain was able to achieve high yields of lactic acid in a short period of time, which is an excellent strain for efficient fermentative production of lactic acid.
Example 3
The application method of the lactococcus garvieae WBT0008 in preparing the bacteria agent for inhibiting harmful bacteria comprises the following steps:
(1) Lactic acid bacteria preparation
The WBT0008 of the lactococcus garvieae is inoculated in an MRS liquid culture medium for shaking culture at 37 ℃ for 48 hours, and the concentration of the cultured thalli is 1 multiplied by 10 8 Centrifuging the CFU/mL and then the bacterial liquid at the temperature of 4 ℃ and 12000r/min for 20min to obtain a supernatant, thereby obtaining a bacteriostatic agent for inhibiting harmful bacteria;
the harmful bacteria are staphylococcus aureus, salmonella or coliform bacteria.
(2) Antibacterial Activity test
The antibacterial activity test adopts a filter paper sheet method, harmful bacteria are inoculated in LB liquid culture medium, shaking culture is carried out for 24 hours at 37 ℃, 200 mu L of bacteria liquid is absorbed and evenly coated on a nutrient agar plate, then a sterilized filter paper sheet with the diameter of 5mm is placed on the coated plate, 10 mu L of a centrifugal supernatant of a lactococcus garvieae agent is dripped on the filter paper sheet, and the culture is carried out for 24 hours at 37 ℃, and the diameter of an antibacterial ring is observed and measured.
Example 4
The application of the lactococcus garvieae WBT0008 in inhibiting harmful bacteria comprises the following steps:
(1) Lactic acid bacteria preparation
The WBT0008 of the lactococcus garvieae is inoculated in an MRS liquid culture medium for shaking culture at 37 ℃ for 48 hours, and the concentration of the cultured thalli is 1 multiplied by 10 8 CFU/mL, then the bacterial liquid is processed at 4 DEG CCentrifuging at 12000r/min for 20min, and collecting supernatant for antibacterial test.
(2) Antibacterial Activity test
The antibacterial activity test adopts a filter paper sheet method, salmonella ATCC14028 is inoculated in LB liquid medium for shaking culture at 37 ℃ for 24 hours, 200 mu L of bacteria liquid is absorbed and uniformly coated on a nutrient agar plate, then two sterilized filter paper sheets with the diameter of 5mm are placed on the coated plate, 10 mu L of strain WBT0008 centrifugal supernatant is dripped on the filter paper sheet by an experimental group, 10 mu L of sterile water is dripped by a control group for culturing at 37 ℃ for 24 hours, the diameter of a bacteria inhibition zone is observed and measured, and the outer diameter of the salmonella inhibition zone of the experimental group is found to reach 12.6 mm, so that the growth of salmonella can be truly inhibited by the strain WBT0008 supernatant, and the effect is obvious.
Example 5
The application of the strain lactococcus garvieae WBT0008 in inhibiting harmful bacteria comprises the following steps:
(1) Lactic acid bacteria preparation
The WBT0008 of the lactococcus garvieae is inoculated in an MRS liquid culture medium for shaking culture at 37 ℃ for 48 hours, and the concentration of the cultured thalli is 1 multiplied by 10 8 CFU/mL, and then the bacterial liquid is centrifuged for 20min at the temperature of 4 ℃ and 12000r/min to obtain supernatant for bacteriostasis test.
(2) Antibacterial Activity test
The antibacterial activity test adopts a filter paper sheet method, staphylococcus aureus ATCC25923 is inoculated in LB liquid culture medium for shaking culture at 37 ℃ for 24 hours, 200 mu L of bacteria liquid is absorbed and uniformly coated on a nutrient agar plate, then two sterilized filter paper sheets with the diameter of 5mm are placed on the coated plate, 10 mu L of strain WBT0008 centrifugal supernatant is dripped on the filter paper sheet by an experimental group, 10 mu L of sterile water is dripped by a control group, the culture is carried out for 24 hours at 37 ℃, the diameter of a bacteria inhibition zone is observed and measured, and the outer diameter of the bacteria inhibition zone of the staphylococcus aureus of the experimental group reaches 10.7 mm, so that the growth of staphylococcus aureus can be truly inhibited by the supernatant of the strain WBT0008, and the effect is obvious.
Example 6
The application of the strain lactococcus garvieae WBT0008 in inhibiting harmful bacteria comprises the following steps:
(1) Lactic acid bacteria preparation
The WBT0008 of the lactococcus garvieae is inoculated in an MRS liquid culture medium for shaking culture at 37 ℃ for 48 hours, and the concentration of the cultured thalli is 1 multiplied by 10 8 CFU/mL, and then the bacterial liquid is centrifuged for 20min at the temperature of 4 ℃ and 12000r/min to obtain supernatant for bacteriostasis test.
(2) Antibacterial Activity test
The antibacterial activity test adopts a filter paper sheet method, escherichia coli ATCC25922 is inoculated in an LB liquid culture medium for shake cultivation for 24 hours at 37 ℃, 200 mu L of bacteria liquid is absorbed and uniformly coated on a nutrient agar plate, then two sterilized filter paper sheets with the diameter of 5mm are placed on the coated plate, 10 mu L of strain WBT0008 centrifugal supernatant is dripped on the filter paper sheet by an experimental group, 10 mu L of sterile water is dripped on a control group for cultivation for 24 hours at 37 ℃, the diameter of a bacteria inhibition zone is observed and measured, and the outer diameter of the bacteria inhibition zone of the experimental group gold escherichia coli reaches 11.2 mm, so that the growth of escherichia coli can be truly inhibited by the supernatant of the strain WBT0008, and the effect is obvious.
Example 7
The application of the strain WBT0008 in fresh steak preservation comprises the following steps:
(1) Lactic acid bacteria preparation
The WBT0008 of the lactococcus garvieae is inoculated in an MRS liquid culture medium for 48 hours at 37 ℃ according to an inoculum size of 2 percent, and the concentration of the cultured bacteria is 3 multiplied by 10 8 CFU/mL, and then the bacterial liquid is centrifuged for 20min at the temperature of 4 ℃ and 12000r/min to obtain supernatant for bacteriostasis test.
(2) Steak fresh-keeping test
Taking steak with consistent freshness, uniformly spraying fermentation supernatant on the surface of fresh steak by using a clean sterile watering can, uniformly spraying sterile water on the surface of steak by using comparison, and then completely covering by using a sterile preservative film. Staphylococcus aureus, salmonella and coliform counts are carried out at 6h, 12h, 18h, 24h and 30h according to national standards GB4789.10-2016 staphylococcus aureus test, GB 4789.4-2016 salmonella test and GB 4789.3-2010 coliform count. The results are shown in tables 1, 2 and 3, compared with the control group, the quantity of staphylococcus aureus, salmonella and escherichia coli of the experiment group sprayed with the supernatant is obviously lower than that of the control group, the fresh-keeping effect within 30 hours is improved by 2-6 times, and the strain WBT0008 has better effect in the beef steak fresh-keeping process and higher application value.
TABLE 1 beef steak fresh test for Staphylococcus aureus number variation (lg cfu/mL)
Note that: the control group and the experimental group in the table are the average value of three repetitions ± standard deviation
Table 2 fresh keeping test of steak Salmonella quantity Change (lg cfu/mL)
Note that: the control group and the experimental group in the table are the average value of three repetitions ± standard deviation
TABLE 3 fresh-keeping test of E.coli quantity variation (lg cfu/mL)
Note that: the control group and the experimental group in the table are the average value of three repetitions ± standard deviation
The strain of the invention is a newly screened strain WBT0008 for producing lactic acid, which is classified and named as lactococcus garvieae (Lactococcus garvieae), and has very good beneficial technical effects through field application, and the related data are as follows:
1. screening of Strain WBT0008
Taking a soil sample around a pig farm in Henan Jiyuan City, weighing 1g of the soil sample, preparing 10mL of suspension by using sterile water, and sequentially diluting to 10 -3 、10 -4 、10 -5 Sample dilution, respectively sucking 0.1mL, coating on a calcium carbonate screening plate, culturing in a constant temperature incubator at 37deg.C for 48 hr, observing transparent rings, selecting strain with obvious transparent rings, and screeningThe strain designated WBT0008 was selected and the calcium carbonate screen plates: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80mL of Tween, 0.5g of magnesium sulfate, 0.25g of manganese sulfate, 10g of calcium carbonate and 18g of agar, adding distilled water to 1000mL, sterilizing at 121 ℃ for 25 minutes, cooling for later use, and preparing a flat plate.
2. Identification of strains
(1) Morphological observation
Bacterial colony of strain WBT0008 is smaller, white, smooth and glossy in surface, nearly round, orderly in edge, arranged in a chain, free of secretion and positive in gram staining.
(2) Physiological and biochemical analysis
Through physiological and biochemical experiments, the strain is found to be capable of decomposing esculin, cellobiose, maltose, mannitol, salicin, sucrose, inulin, lactose and sodium hippurate, and not capable of decomposing sorbitol and raffinose, and specific results are shown in Table 4.
TABLE 4 results of the biochemical reactions of lactococcus garvieae WBT0008
Note that: + indicates positive, -indicates negative
(3) Molecular biological analysis
The genome of WBT0008 strain was extracted by 16S rDNA sequence analysis using bacterial genome extraction kit. PCR was then performed, primer sequences: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' 14992R 5'-GGTTACCTTGTTACGACTT-3'. Reaction system 50 μl: ddH2O 21. Mu.L, 1499r1.5. Mu.L, 27F 1.5. Mu.L, taqmix enzyme 25. Mu.L, and template DNA of the fungus sample 1. Mu.L. PCR procedure: pre-denaturation at 94℃for 4min, denaturation at 96℃for 30s, annealing at 55℃for 30s, extension at 72℃for 60s,32 cycles, extension at 72℃for 10min. And then sending the PCR product to a large gene company for sequencing, wherein the sequence information is shown in a sequence table. BLAST comparison is carried out on the 16S rDNA sequence in a database of NCBI functional network, a phylogenetic tree is constructed, and homology comparison analysis is carried out, so that the strain is found to have similar homology with the lactococcus garvieae. Combining morphological results, physiological and biochemical results and 16S rDNA sequence analysis results, determining that the strain is lactococcus garvieae, and classifying and naming the strain as lactococcus garvieae (Lactococcus garvieae), and preserving the strain in China general microbiological culture Collection center (CGMCC) with a preservation number of 22576, a preservation date of 2021, 5 months and 20 numbers and a preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
16S rDNA sequence:
the screened lactococcus garvieae WBT0008 has good high-yield lactic acid after being applied in the field, and specific test conditions are shown in example 2, the highest yield of lactic acid in 24 hours reaches 10.60g/L, which indicates that the strain can realize high yield of lactic acid in a short time; the application of the antibacterial agent for inhibiting harmful bacteria in preparation of the antibacterial agent for inhibiting salmonella has the outer diameter of inhibition zone of 12.6 mm, proves that the supernatant of the bacterial strain WBT0008 can actually inhibit the growth of salmonella, has the outer diameter of inhibition zone of staphylococcus aureus of 10.7 mm, and has obvious effect, and the supernatant of the bacterial strain WBT0008 can actually inhibit the growth of staphylococcus aureus and the outer diameter of inhibition zone of escherichia coli of 11.2 mm, so that the supernatant of the bacterial strain WBT0008 can actually inhibit the growth of escherichia coli; the antibacterial fresh-keeping agent has remarkable antibacterial fresh-keeping effect when applied to fresh steak fresh keeping, the unit lg cfu/mL is within 30 hours, the number of staphylococcus aureus is 2.66+/-0.01, the number of salmonella is 1.28+/-0.01, and the number of escherichia coli is 1.39+/-0.03, and the fresh-keeping effect is improved by 2-6 times within 30 hours as shown in tables 1-3.
From the above, it is clear that the invention is a new screened strain of lactococcus garvieae WBT0008, can be effectively used for preparing high-yield lactic acid, inhibiting harmful bacteria and fresh-keeping fresh steak, has simple preparation and application methods, can effectively improve the food fresh-keeping effect, is environment-friendly, provides a new strain of lactococcus garvieae, exploits the new application of the lactococcus garvieae, and has wide application prospect and huge economic and social benefits.
Sequence listing
<110> biological research all of the Limited liability company of the academy of sciences of Henan province
<120> lactic acid-producing lactococcus garvieae WBT0008 and use thereof
<130> 2021
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1437
<212> DNA
<213> lactococcus garvieae (Lactococcus garvieae)
<400> 1
gagtttggat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60
gatgattaaa gatagcttgc tatttttatg aagagcggcg aacgggtgag taacgcgtgg 120
gaaatctgcc gagtagcggg ggacaacgtt tggaaacgaa cgctaatacc gcataacaat 180
gagaatcgca tgattcttat ttgaaagaag caattgcttc actacttgat gatcccgcgt 240
tgtattagct agttggtagt gtaaaggact accaaggcga tgatacatag ccgacctgag 300
agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360
gggaatcttc ggcaatgggg gcaaccctga ccgagcaacg ccgcgtgagt gaagaaggtt 420
ttcggatcgt aaaactctgt tgttagagaa gaacgttaag tagagtggaa aattacttaa 480
gtgacggtat ctaaccagaa agggacggct aactacgtgc cagcagccgc ggtaatacgt 540
aggtcccaag cgttgtccgg atttattggg cgtaaagcga gcgcaggtgg tttcttaagt 600
ctgatgtaaa aggcagtggc tcaaccattg tgtgcattgg aaactgggag acttgagtgc 660
aggagaggag agtggaattc catgtgtagc ggtgaaatgc gtagatatat ggaggaacac 720
cggaggcgaa agcggctctc tggcctgtaa ctgacactga ggctcgaaag cgtggggagc 780
aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctag ctgtagggag 840
ctataagttc tctgtagcgc agctaacgca ttaagcactc cgcctgggga gtacgaccgc 900
aaggttgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960
ttcgaagcaa cgcgaagaac cttaccaggt cttgacatac tcgtgctatc cttagagata 1020
aggagttcct tcgggacacg ggatacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080
agatgttggg ttaagtcccg caacgagcgc aacccttatt actagttgcc atcattaagt 1140
tgggcactct agtgagactg ccggtgataa accggaggaa ggtggggatg acgtcaaatc 1200
atcatgcccc ttatgacctg ggctacacac gtgctacaat ggatggtaca acgagtcgcc 1260
aacccgcgag ggtgcgctaa tctcttaaaa ccattctcag ttcggattgc aggctgcaac 1320
tcgcctgcat gaagtcggaa tcgctagtaa tcgcggatca gcacgccgcg gtgaatacgt 1380
tcccgggcct tgtacacacc gcccgtcaca ccacggaagt tgggagtacc caaagta 1437
Claims (4)
1. A strain of lactococcus garvieae WBT0008, classified and named as lactococcus garvieae @Lactococcus garvieae) The culture medium is preserved in China general microbiological culture Collection center (CGMCC) No.22576, and the preservation date is 2021, 5 and 20, and the preservation address is: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
2. A method of lactic acid production by a strain of lactococcus garvieae WBT0008 as defined in claim 1 comprising the steps of:
(1) Activating strains and culturing seeds: inoculating the frozen preserved strain WBT0008 into a seed culture medium according to the volume of 1% of the culture medium after activation, and culturing at 37 ℃ for 24 hours at 180r/min to obtain seed liquid;
the seed culture medium is as follows: 50g of glucose, 10g of peptone, 10g of yeast extract, 5g of sodium acetate, 0.2g of diammonium citrate, 0.2g of dipotassium hydrogen phosphate, 80mL of tween, 0.2g of magnesium sulfate, 0.02g of manganese sulfate and 0.01g of ferrous sulfate, adding distilled water to 1000mL, sterilizing at 121 ℃ for 25 minutes, and cooling to obtain the finished product;
(2) Lactic acid preparation: inoculating the seed solution into MRS culture medium according to volume of 2% of MRS culture medium, shake culturing at 37deg.C and 180r/min for 48 hr to obtain fermentation broth, namely lactic acid;
the MRS culture medium is as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80mL of Tween, 0.5g of magnesium sulfate and 0.25g of manganese sulfate, adding distilled water to 1000mL, sterilizing at 121 ℃ for 25 minutes, and cooling to obtain the beef extract;
(3) And (3) measuring the content of lactic acid:
sampling the prepared fermentation liquor lactic acid at 4h, 8h, 12h, 16h, 20h and 24h, repeating each group of the fermentation liquor lactic acid at three times, centrifuging at the temperature of 4 ℃ and under the condition of 12000r/min for 8min, taking the supernatant to measure the content of lactic acid, oxidizing the lactic acid under the action of lactic acid dehydrogenase to generate pyruvic acid, reducing MTT to generate a purple substance, wherein the substance has a characteristic absorption peak at 570nm, calculating the content of lactic acid to be 2-11 g/L by utilizing the proportional relation between the lactic acid and the concentration of lactic acid, and determining the formula as follows:
wherein: a represents absorbance of fermentation supernatant, A 0 The absorbance of the blank is represented by K, the slope of the standard curve, the intercept of the standard curve, and the conversion coefficient by 1176.67.
3. Use of lactococcus garvieae WBT0008 according to claim 1 for the preparation of a preparation for inhibiting harmful bacteria, characterized in that it comprises the steps of:
(1) Lactic acid bacteria preparation
The WBT0008 of the lactococcus garvieae is inoculated in an MRS liquid culture medium for shaking culture at 37 ℃ for 48 hours, and the concentration of the cultured thalli is 1 multiplied by 10 8 CFU/mL, centrifuging the bacterial liquid at 4 ℃ and 12000r/min for 20min to obtain a supernatant, thus obtaining a bacteriostatic agent for inhibiting harmful bacteria;
the harmful bacteria are staphylococcus aureus, salmonella or escherichia coli;
(2) Antibacterial Activity test
The antibacterial activity test adopts a filter paper sheet method, harmful bacteria are inoculated in LB liquid culture medium, shaking culture is carried out for 24 hours at 37 ℃, 200 mu L of bacteria liquid is absorbed and evenly coated on a nutrient agar plate, then a sterilized filter paper sheet with the diameter of 5mm is placed on the coated plate, 10 mu L of a centrifugal supernatant of a lactococcus garvieae agent is dripped on the filter paper sheet, and the culture is carried out for 24 hours at 37 ℃, and the diameter of an antibacterial ring is observed and measured.
4. The use of the lactococcus garvieae WBT0008 in preparing fresh steak preservative according to claim 1, wherein the lactococcus garvieae WBT0008 is inoculated in MRS liquid culture medium according to 2% of the volume of the MRS liquid culture medium, and cultured for 48 hours at 37 ℃ in a shaking table, and the concentration of cultured bacteria is 3 multiplied by 10 8 CFU/mL, then the bacterial liquid is centrifuged for 20min at the temperature of 4 ℃ and 12000r/min to obtain supernatant, the supernatant is uniformly sprayed on the surface of the fresh steak by a clean sterile watering can, and the fresh steak is completely covered by a sterile preservative film.
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| WO2015071502A1 (en) * | 2013-11-18 | 2015-05-21 | Amadeite | Extract of algae for use as an antibacterial agent |
| CN105385640A (en) * | 2015-12-18 | 2016-03-09 | 广州市康优元生物科技有限公司 | Lactococcus garviea and application thereof |
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| CN105385640A (en) * | 2015-12-18 | 2016-03-09 | 广州市康优元生物科技有限公司 | Lactococcus garviea and application thereof |
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