CN116144525B - Seawater fish probiotics lactococcus lactis strain and application thereof - Google Patents
Seawater fish probiotics lactococcus lactis strain and application thereof Download PDFInfo
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- CN116144525B CN116144525B CN202211125017.0A CN202211125017A CN116144525B CN 116144525 B CN116144525 B CN 116144525B CN 202211125017 A CN202211125017 A CN 202211125017A CN 116144525 B CN116144525 B CN 116144525B
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Abstract
The invention discloses a marine fish probiotic lactococcus lactis strain and application thereof. The strain is lactococcus lactis (L.lactis) M48, and the preservation number is: GDMCC No. 62621. In vitro experiments prove that: the lactococcus lactis M48 is co-cultured with sea bass cells infected by sea bass iridovirus (SPIV), and has the characteristic of resisting the proliferation of the SPIV in the sea bass cells in vitro; the lactococcus lactis M48 also has an inhibitory effect on sea fish vibrio vulnificus, vibrio harveyi, vibrio alginolyticus, streptococcus ragmitis, citrobacter and Aeromonas. Animal experiments prove that: lactococcus lactis M48 has the characteristics of immunoregulation and immunity enhancement for sea bass, and M48 strain alone has the characteristics of anti-SPIV infection for sea bass. The lactococcus lactis M48 has a certain application prospect in the aspects of resisting viruses and pathogenic bacteria of marine fish, regulating immunity and enhancing the immune function of the marine fish.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a marine fish probiotic lactococcus lactis strain and application thereof.
Background
Fish is one of the most abundant animal protein sources for human beings, 25% of animal proteins ingested by people in China are obtained from fish and shellfish, and along with the development of domestic economy and the improvement of the living standard of people, the demands of consumers on marine fish are continuously increased. With the expansion of the culture scale of sea fish, the disease problem of sea fish is increasingly prominent, especially various bacterial and viral diseases. The problems of sea water fish iridovirus disease, streptococcicosis, vibriosis and the like are to be solved.
In order to control bacterial diseases in marine fish farming, various drugs such as antibiotics are generally used, however, the use of antibiotics adversely affects the fish body and the environment. Therefore, to expand the sea bass farming industry, it is necessary to find safe and effective antibiotic alternatives. Probiotics are a class of microorganisms that regulate the intestinal flora of animals and exert a beneficial effect on the host animal. Compared with antibiotics, the probiotics do not generate drug resistance, drug residues and substances harmful to the environment. Iridovirus disease is an important viral infectious disease of sea fish, has great harm to young fish, has a mortality rate as high as 100%, and poses a great threat to sea bass culture. The prevention and treatment of general viral diseases mainly include vaccine prevention. Compared with vaccines, probiotics are not only limited to disease control, but also have the effects of promoting growth, promoting digestion and regulating immunity. After intestinal tract colonization, the probiotics can compete with intestinal pathogenic bacteria for nutrition, and the abundance of the intestinal pathogenic bacteria is reduced. Meanwhile, probiotics utilize food undigested by a host to produce substances such as short-chain fatty acids and the like, and the substances can provide rich energy for fish. The intestinal mucosa barrier is a structure of the intestinal tract for isolating harmful substances and pathogenic invasion, and is composed of a mechanical barrier, a chemical barrier, an immune barrier and a biological barrier. The probiotics can colonize intestinal mucosa to form biological barrier of intestinal tract, and prevent intestinal pathogenic bacteria from invading the intestinal mucosa. The probiotics also have good immunoregulation effect, can promote secretion of intestinal antibacterial peptide, regulate expression of intestinal cytokines and promote integrity of intestinal mucosa barrier.
The sea bass has delicious meat quality, has the effects of tonifying liver and kidney, tonifying spleen and stomach, resolving phlegm and relieving cough, has good tonifying effect on liver and kidney deficiency, and is popular with more and more consumers. Meanwhile, the sea bass has the advantages of strong adaptability to water environment, good growth performance and wide feed sources, and is an excellent artificial sea fish culture variety in China. Since 2014, the culture scale of the sea bass in China is increased year by year, and the yield is continuously increased, so that the sea bass has become the second largest sea farming fish in China. Although the intensive culture of the sea bass can maintain a higher yield, the sea bass cultured in high density is more easily attacked by various pathogens and is also more sensitive to the change of water environment. Probiotics have great application potential in aquaculture and become an important means for promoting the growth of aquatic animals and controlling diseases. In recent years, research on the anti-pathogenic bacteria and antiviral effects of probiotics has become a hotspot, and great research results are obtained.
Screening probiotics in specific areas has a better effect on specific species, as aquatic animals will constantly interact with a variety of microorganisms in their habitat during growth.
Disclosure of Invention
The invention aims to provide a probiotic lactococcus lactis (Lactococcus lactis) M48 strain with antiviral property and immunoregulation and immunity function enhancing properties for sea fish.
The probiotic lactococcus lactis (Lactococcus lactis) M48 strain is separated from intestinal mucosa of young semi-wild sea bass, grows well on an MRS culture medium, and is named as lactococcus lactis (Lactococcus lactis) M48 which is obtained by streaking and pure separation and is preserved in the microorganism strain preservation center of Guangdong province at the month of 2022 and the preservation number is: GDMCC No. 62621, the preservation address is: building 5, no. 59, guangdong province, guangzhou City, first, in the first, guangdong province, post code: 510070.
the lactococcus lactis M48 strain has the following characteristics:
the microorganism is characterized in that: aerobic or facultative anaerobes grow well on solid and liquid MRS media, and the colonies on the MRS media are large colonies which are milky, neat in edge, smooth and moist in surface, raised and opaque; gram staining is typically positive, and the thalli are medium-sized morphologically irregular cocci; the M48 strain grows well at 20-28 ℃, can grow at 37 ℃ and does not grow at more than 40 ℃; the M48 strain has acid resistance, can grow on an MRS culture medium with the pH value of 4.0, and can well grow between the pH value of 5.0 and 6.0; the M48 strain has the characteristic of cholate resistance, can be rapidly proliferated on an MRS culture medium with the cholate concentration of 0.0-0.2%, and can be rapidly proliferated on an MRS culture medium with the cholate concentration of 0.3-0.5%.
The 16SrDNA sequence of the lactococcus lactis M48 strain provided by the invention has more than 99% similarity with the 16SrDNA sequence of the currently published lactococcus lactis, and the lactococcus lactis M48 strain is identified as lactococcus lactis (L.lactis) according to 16SrDNA sequence analysis, wherein the 16SrDNA sequence is shown as SEQ ID NO.1 in the nucleotide sequence.
In vitro cell experiments prove that: the screened lactococcus lactis M48 strain is co-cultured with virus-infected SPF cells (sea bass cells), has the characteristic of resisting the proliferation of sea bass iridovirus (SPIV) in SPF cells in vitro, and also has the effect of inhibiting vibrio vulnificus, vibrio harveyi, vibrio alginolyticus, streptococcus iniae, citric acid bacillus and aeromonas. Animal experiments prove that: the lactococcus lactis M48 strain screened by the invention has the characteristics of immunoregulation and immunity enhancement for the sea bass, and the lactococcus lactis M48 strain screened by the invention is independently fed to the sea bass, and has the characteristic of anti-SPIV infection.
It is therefore a second object of the present invention to provide the use of said lactococcus lactis (l.lactis) M48 for the preparation of a marine fish feed additive.
A third object of the invention is to provide the use of said lactococcus lactis (L.lactis) M48 in the preparation of an iridovirus vaccine formulation.
The fourth object of the invention is to provide the application of the lactococcus lactis (L.lactis) M48 in preparing a seawater fish vaccine immunomodulator or immunopotentiator.
Preferably, the vaccine immunomodulator or immunopotentiator can achieve the aim of adjusting or enhancing the immune function of the sea water fish by improving the levels of the sea water fish IL, IFN, MHC and IgM.
The fifth object of the invention is to provide the application of the lactococcus lactis (L.lactis) M48 in preparing live carrier vaccines for sea water fishes.
Preferably, the lactococcus lactis (L.lactis) M48 is used as a live carrier host strain for secretory expression of the foreign protein.
A sixth object of the present invention is to provide a marine fish feed additive, an iridovirus vaccine formulation, a marine fish vaccine immunomodulator/immunopotentiator, or a marine fish live vector vaccine comprising the lactic acid lactococcus (l.lactis) M48 described above.
A seventh object of the present invention is to provide a method for improving immunity of marine fish by applying the above-mentioned lactococcus lactis (l.lactis) M48 to marine fish.
Preferably, the lactococcus lactis (l.lactis) M48 is used alone, or as a feed additive in combination with feed, or as an immunomodulator/immunopotentiator in combination with vaccine.
The invention has the following beneficial effects:
the isolated strain of the sea bass probiotics lactococcus lactis (L.lactis) M48 has antibacterial and antiviral properties and immunoregulation and immunity enhancement properties on sea bass, and has a certain application prospect in the aspect of sea bass antivirus and immunoregulation and immunity enhancement on sea bass.
Lactococcus lactis (Lactococcus lactis) M48 of the invention was deposited at the microorganism strain collection in Guangdong province at 7.15 of 2022 under the accession number: GDMCC No. 62621, the preservation address is: building 5, no. 59, guangdong province, guangzhou City, first, in the first, guangdong province, post code: 510070.
drawings
FIG. 1 is a morphological structure diagram of the lactococcus lactis M48 strain of the invention, wherein, A is a colony characteristic diagram of the lactococcus lactis M48 strain, and B is a morphological characteristic diagram of the lactococcus lactis M48 strain under a G staining microscope.
FIG. 2 is a graph showing the proliferation of the M48 strain of lactococcus lactis of the present invention in MRS liquid media at different pH values.
FIG. 3 is a graph showing proliferation of the lactococcus lactis M48 strain of the present invention in MRS liquid media of different cholate concentrations.
FIG. 4 is a graph showing proliferation of the M48 strain of Lactobacillus according to the present invention in MRS liquid media with different NaCl concentrations.
FIG. 5 is a diagram showing an oxford cup experiment of the interaction of the lactococcus lactis M48 strain of the invention with common pathogenic bacteria of marine fish.
Fig. 6 is a graph showing experimental results of in vitro antiviral properties of co-culture of lactococcus lactis M48 strain and SPF cells (sea bass cells) according to the present invention, wherein: p <0.05.
FIG. 7 is a graph showing the results of a test of the immunoregulation and immunoenhancement of marine fish by the lactococcus lactis M48 strain of the present invention, wherein: p <0.05,: p <0.01.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
EXAMPLE 1 isolation, purification and characterization of the lactococcus lactis (L.lactis) M48 Strain
(1) Preparation of a culture medium:
MRS solid Medium formulation (g/L): 10.0 portions of casein enzyme digest, 10.0 portions of beef extract powder, 4.0 portions of yeast extract powder, 2.0 portions of tri-ammonium citrate, 5.0 portions of sodium acetate, and magnesium sulfate (MgSO) 4 ·7H 2 O) 0.2, manganese sulfate (MnSO) 4 ·4H 2 O) 0.05, dipotassium hydrogen phosphate 2.0, glucose 20.0, tween 80 1.08 and agar 15, wherein the solvent is water. Preparing: after dissolving the ingredients in water, sterilizing at 121 ℃ for 15 minutes, adjusting the pH to 5.7, and pouring the mixture into a flat plate for later use.
MRS liquid culture Medium formulation (g/L): 10.0 portions of casein enzyme digest, 10.0 portions of beef extract powder, 4.0 portions of yeast extract powder, 2.0 portions of tri-ammonium citrate, 5.0 portions of sodium acetate, and magnesium sulfate (MgSO) 4 ·7H 2 O) 0.2, manganese sulfate (MnSO) 4 ·4H 2 O) 0.05, dipotassium hydrogen phosphate 2.0, glucose 20.0 and Tween 80 1.08, and the solvent is water. Preparing: dissolving the above components in water, sterilizing at 121deg.C for 15 min, and adjusting pH to 5.7.
(2) Isolation and purification of strains:
the experimental juvenile fish is collected from a pollution-free natural water area, the stress of sea bass is reduced as much as possible, living fish samples are firstly anesthetized, and then the fish bodies are wiped and disinfected by 75% alcohol in an ultra-clean workbench. Then dissecting the fish body, separating and shearing the complete intestinal canal of the fish, completely squeezing the content of the intestinal canal into a sterile centrifuge tube, and lightly scraping mucus on the inner wall of the intestinal canal into the centrifuge tube. Adding sterile PBS with the volume of three times of the intestinal contents into a centrifuge tube for dilution, vibrating, grinding and uniformly mixing to obtain an intestinal microorganism sample. Then, diluting the intestinal microorganism sample by 10 times and 100 times with PBS, respectively coating the diluted liquids with different proportions on a MRS solid culture medium flat plate, culturing the coated flat plate at the constant temperature of 28 ℃ for 1-3 days, and observing the colony growth state every 12 hours. Observing the growth state and colony morphology of the coated plate, selecting proper dilution (about 10-200 colonies), selecting single colony numbers of different morphologies, andscribing and culturing at constant temperature of 28 ℃ for 12-24 h. Observing whether the morphological characteristics of the streak culture colonies are single, and continuously picking single colonies on a plate with a non-single colony morphological structure and streak culturing until pure culture single bacteria with consistent colony morphological characteristics are obtained. The isolated and purified pure culture single colony is selected and inoculated into a liquid culture medium, and is subjected to shaking culture at a constant temperature of 28 ℃ at 200rpm until bacterial liquid OD is obtained 600 The value reaches 0.6-1.0, and the purified isolate strain is obtained. The culture characteristics are as follows: aerobic or facultative anaerobes grow well on solid and liquid MRS media, and colonies on MRS media are milky white, clean in edge, smooth and moist in surface, raised, opaque and smaller colonies without obvious odors; gram staining was typically positive and the cells were medium-sized morphologically irregular cocci, and finally pure cultures of the MRS were kept. The colony characteristics of the lactococcus lactis M48 strain are shown in FIG. 1A, and the gram staining microscopic observation is shown in FIG. 1B.
(3) Physiological and biochemical characteristics of the strain
The lactococcus lactis M48 strain can grow well at the temperature of between 20 and 28 ℃ and does not grow at the temperature of more than 40 ℃, and the M48 strain has the acid resistance, can grow on an MRS culture medium with the pH value of 4.0 and can grow well between the pH value of 5.0 and 6.5; the M48 strain has cholate resistance and can be proliferated on MRS culture medium with cholate concentration of 0.1-0.5%. The physiological and biochemical characteristics of the lactococcus lactis M48 strain are as follows: phosphatidyl phospholipase C (-); d-xylose (+); arginine bishydrolase 1 (+); beta-D-galactosidase (-); alpha-glucosidase (-); alanine-phenylalanine-proline arylamidase (+); cyclodextrin (+); l-aspartate arylamidase (+); beta-galactopyranosidase (-); alpha-mannosidase (-); phosphatase (-); leucine arylamidase (+); l-proline arylamidase (-); beta-glucuronidase (-); alpha-galactosidase (-); pyrrolidinyl arylamidase (-); beta-glucosidase (-); alanine arylamidase (+); tyrosine arylamidase (+); d-sorbitol (-); urease (-); polymyxin B tolerance (-); d-galactose (+); d-ribose (+); lactate producing alkali (-); lactose (+); N-acetyl-D-glucosamine (+); d-maltose (+); bacitracin tolerance (+); novobiocin tolerance (+); 6.5% NaCl growth (+); d-mannitol (-); d-mannose (+); methyl-B-D-glucopyranoside (+); amylopectin (-); d-raffinose (-); o/129 tolerance (+); salicin (+); sucrose (+); d-trehalose (+); arginine bishydrolase 2 (+); olprixin tolerance (+). (note: positive for the experimental result, negative for the experimental result).
(4) 16SrDNA sequence analysis of strains
The purified PCR product was sequenced by amplifying 16SrDNA from the genome of M48 strain using the general primer 27F/1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 142R: 5 '-TACGGCTACCTTGTTACGACTT-3') of 16 SrDNA: the homology of the 16SrDNA sequence (shown in SEQ ID NO. 1) of the strain M48 with the 16SrDNA of the currently published lactococcus lactis reaches more than 99%. Combining morphological characteristics with 16SrDNA sequence identification, the strain M48 is lactococcus lactis.
The strain selected above was designated as lactococcus lactis (Lactococcus lactis) M48, and the lactococcus lactis (Lactococcus lactis) M48 was deposited at the microorganism strain deposit center of Guangdong province at 2022, 7 and 15, accession numbers: GDMCC No. 62621, the preservation address is: building 5, no. 59, guangdong province, guangzhou City, first, in the first, guangdong province, post code: 510070.
the 16SrDNA sequence of lactococcus lactis (L.lactis) M48 (shown in SEQ ID NO. 1) is specifically: GGCGGTGTGCTATGATGCAAGTTGAGCGCTGAAGGTTGGTACTTGTACCGACTGGATGAGCAGCGAACGGGTGAGTAACGCGTGGGGAATCTGCCTTTGAGCGGGGGACAACATTTGGAAACGAATGCTAATACCGCATAAAAACTTTAAACACAAGTTTTAAGTTTGAAAGATGCAATTGCATCACTCAAAGATGATCCCGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGATGATACATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGGTAGAGAAGAACGTTGGTGAGAGTGGAAAGCTCATCAAGTGACGGTAACTACCCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTATTAAGTCTGGTGTAAAAGGCAGTGGCTCAACCATTGTATGCATTGGAAACTGGTAGACTTGAGTGCAGGAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGCCTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGATGTAGGGAGCTATAAGTTCTCTGTATCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACTCGTGCTATTCCTAGAGATAGGAAGTTCCTTCGGGACACGGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAACGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACAGTGATGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTGGGAGTACCCGAAGTAGGTTGCCTAACCGCAAGGAGGGCGCTCCTAAGGTAGACCGAGTCC.
EXAMPLE 2 acid resistance Property study of lactococcus lactis (L.lactis) M48 Strain
The lactic acid lactococcus M48 strain has unique acid resistance:
(1) The experimental method comprises the following steps: the lactococcus lactis M48 strain was cultured at 28℃and the growth of the strain in MRS liquid media at different pH (pH 3.0, pH4.0, pH5.5, pH6.0 and pH 6.5) was observed.
(2) Experimental results: the lactococcus lactis M48 strain has good adaptability in culture mediums with different pH values, and the acidic MRS culture medium with the pH value of 3.0-4.0 can be proliferated; proliferation was faster in MRS media at ph5.5 and ph 6.5; proliferation was rapid in MRS medium at pH 6.0. The proliferation curve shown in FIG. 2 shows that the lactococcus lactis M48 has tolerance capability in the low pH environment of the intestinal tract, and the lactococcus lactis M48 strain has colonization capability in the acidic environment of the intestinal tract.
EXAMPLE 3 study of cholate resistance of lactococcus lactis (L.lactis) M48 Strain
The lactic acid lactococcus M48 strain has unique cholate resistance:
(1) The experimental method comprises the following steps: the lactococcus lactis M48 strain was cultured at 28℃and observed for growth in MRS liquid medium containing different cholate concentrations (cholate concentrations of 0.10%, 0.20%, 0.30%, 0.40% and 0.50% by mass).
(2) Experimental results: can be rapidly proliferated in MRS culture medium with cholate concentration of 0.0-0.2% by mass fraction; can be rapidly proliferated in MRS culture medium with cholate concentration of 0.3-0.5% by mass. See the proliferation curve of FIG. 3, which shows that the lactococcus lactis M48 strain has the ability to colonize intestinal hypercholesterate.
EXAMPLE 4 osmotic pressure resistance Property study of lactococcus lactis (L.lactis) M48 Strain
The lactococcus lactis M48 strain has unique osmotic pressure resistance:
(1) The experimental method comprises the following steps: the lactococcus lactis M48 strain is cultured at 28 ℃, and the growth condition of the strain in MRS liquid culture medium containing different NaCl concentrations (the NaCl concentration is 0.0 per mill, 15.0 per mill, 25.0 per mill and 35.0 per mill) is observed.
(2) Experimental results: can be rapidly proliferated in MRS culture medium with NaCl concentration of 0.0-25.0 per mill; can be proliferated rapidly in MRS culture medium with NaCl concentration of 35.0 per mill. See the proliferation curve of FIG. 4, which shows that the lactococcus lactis M48 strain has the ability to withstand the high osmotic pressure of seawater.
EXAMPLE 5 in vitro anti-pathogenic property study of lactococcus lactis (L.lactis) M48 Strain
The lactococcus lactis M48 strain has unique in-vitro anti-pathogenic bacteria characteristics:
(1) The experimental method comprises the following steps: the vibrio vulnificus, vibrio harveyi, vibrio alginolyticus, streptococcus iniae, citric acid bacillus and aeromonas bacteria liquid which are cultivated for 12 hours are diluted to OD600 = 0.3, 100 mu L of the six diluted bacteria liquids are respectively absorbed and evenly coated on LB solid culture medium, and a sterile oxford cup is placed in the middle of the culture medium. After that, the lactococcus lactis M48 bacterial liquid cultured for 12 hours was diluted to od600=0.3, and 120 μl of the bacterial liquid was aspirated and added to the oxford cup. The medium was placed in a 28℃incubator and the results were observed for 24 hours.
(2) Experimental results: the lactococcus lactis M48 strain can form an obvious inhibition zone on vibrio vulnificus (figure 5A), vibrio harveyi (figure 5B), vibrio alginolyticus (figure 5C), streptococcus iniae (figure 5D), citric acid bacillus (figure 5E) and aeromonas (figure 5F). The lactococcus lactis M48 strain has the effects of inhibiting vibrio vulnificus, vibrio harveyi, vibrio alginolyticus, streptococcus iniae, citric acid bacillus and aeromonas.
Example 6 in vitro antiviral Property Studies of lactococcus lactis (L.lactis) M48 Strain
The lactococcus lactis has unique in vitro antiviral properties:
(1) The experimental method comprises the following steps: SPF cells (sea bass cells, constructed by the present laboratory) were grown in L15 medium supplemented with 10% fetal bovine serum (41300039, gibco TM ) Medium passage, cell density was adjusted to 1X 10 7 Per mL, 100. Mu.L of cell suspension per well in 96-well plates, and cultured at 28 ℃. The cell growth state was continuously observed, and the experiment was performed after the cells were full (1.8X10 6 And/well) starts the next day after. Culturing lactococcus lactis M48 strain to be tested 3 days before the start of the experiment, taking out 1.5mL of bacterial liquid, measuring OD600 value, adjusting the bacterial liquid to be tested to reach OD value of 1, taking out 300 mu L of bacterial liquid in a sterilizing centrifuge tube according to the infection complex number (MOI) of 1:40, centrifuging for 10 minutes at 6000g, discarding the supernatant of the residual culture liquid, adding the bacterial cells into 500 mu L of cell culture medium, and uniformly mixing, wherein the cell culture medium contains fetal bovine serum. The final cell-cell culture medium mixture contained fetal bovine serum at a final concentration of 15%. The culture solution in the 96-well plate with the grown cells was aspirated, taking care to be gentle and to ensure consistent cell numbers, 100. Mu.L of the mixture of the bacterial culture supernatant of the strain to be tested and the cell culture medium (100. Mu.L of the mixture of PBS and the cell culture medium) was added to each well (control group) while adding the SPIV virus solution (titer 10) at 1:1000 to the co-culture medium 8 TCID 50). The culture supernatants of the test group and the control group were aspirated respectively, and TCID50 value was measured and compared in a 28℃incubator for 24 hours.
(2) Experimental results: lactococcus lactis M48 strain interaction experimental group with average viral titer of 10 4.84 TCID50. Control group was 10 6.48 TCID50, statistical analysis showed that the differences were very significant (P<0.05 (see fig. 6). The lactococcus lactis M48 strain and SPF cells (sea bass cells) can prevent the proliferation of viruses after being co-cultured, and has the characteristic of in vitro antivirus (SPIV).
EXAMPLE 7 immunomodulating and immune function enhancing Properties of lactococcus lactis (L.lactis) M48 Strain
Animal experiments prove the characteristics of the lactococcus lactis M48 strain on immunoregulation and immunity enhancement of the sea bass.
(1) Experimental animal
Young fishes with the average body length of 5cm are selected, and are fed into a constant-temperature oxygen supply circulation aquarium at 28 ℃ for one month before the experiment, and 50 fish are fed into each independent water tank.
(2) Experimental method
Before the test starts, the probiotic candidate strain lactococcus lactis M48 is subjected to rejuvenation and subculture, 1:100 transfer culture is carried out 2 days before the test, and when the strain reaches the logarithmic phase, the bacterial liquid is taken out to measure the OD600 value. The small-sized pellet feed machine is used for preparing the sea bass pellet feed and the bacterial liquid coated feed, and the prepared feed is slightly dried in a blast constant temperature incubator at 20 ℃. Each group of fish is fed with 1X 10 bacteria 8 cfu/g pellet feed (12.5 g/50 tails daily fed with bacteria-containing feed, on-line) was set up with parallel control group 1 (12.5 g/50 tails daily fed with normal feed), each group being 50 tails. Feed intake, mental state, etc. were recorded daily, after continuous feeding for one week, the head kidney and part of middle kidney and spleen of the fish were aseptically collected and placed in sterilized disposable EP tubes, respectively, after addition of sterilized steel balls, tissues were ground and pulverized with an oscillator to extract RNA, the extracted RNA was immediately reverse transcribed into cDNA, qPCR was performed using primers designed according to the IL-1 β, IL-8, MHC, igM, and IFN of sea bass (table 1), the relative expression amounts of the respective mRNA of sea bass were calculated, and the experimental group and the control group were compared.
TABLE 1 antiviral Property study results of lactococcus lactis M48 Strain on sea bass
(3) Experimental results
The results show that: the feeding group has no difference in feed intake and mental state from the control group, the feed intake is normal (all the feed intake is complete within 2 minutes), and the mental state is good; compared with the control group, the expression quantity of mRNA of the spleen IFN and IL-8 of the experimental group is extremely higher than that of the control group, and the difference is extremely remarkable (P < 0.01); the expression levels of mRNA of kidney IgM, IL-1 beta, IL-8 and MHC in the experimental group were significantly higher than those in the control group, and the difference was significant (P < 0.05) compared with the control group (see FIG. 7). The result shows that the lactococcus lactis M48 strain has excellent immunoregulation and immunity enhancement properties for sea fish.
EXAMPLE 8 antiviral Property study of lactococcus lactis (L.lactis) M48 Strain on sea bass
Animal experiments prove that the lactococcus lactis M48 strain has antiviral property on sea bass.
(1) Experimental animal
Young fishes with the average body length of 6cm and the average weight of 2.5g are selected and fed into a constant-temperature oxygen supply circulation aquarium at 28 ℃ for one month before the experiment, and 50 fish are in each independent water tank.
(2) Experimental method
Before the test starts, the probiotic candidate strain lactococcus lactis M48 is subjected to rejuvenation and subculture, 1:100 transfer culture is carried out 2 days before the test, and when the strain reaches the logarithmic phase, the bacterial liquid is taken out to measure the OD600 value. The small-sized pellet feed machine is used for preparing the sea bass pellet feed and the bacterial liquid coated feed, and the prepared feed is slightly dried in a blast constant temperature incubator at 20 ℃. Each group of fish is fed with 1X 10 bacteria 8 cfu/g pellet feed (12.5 g/50 tails daily fed with bacteria-containing feed, on-line) was set up with parallel control group 1 (12.5 g/50 tails daily fed with normal feed), each group being 50 tails. Feed intake, mental state and the like are recorded every day, after continuous feeding for one week, detoxification (sea bass iridovirus, SPIV) is carried out, fasted for one day on the day of detoxification, test fishes are soaked in batches for anesthesia by using MS-222 (anesthetic), and immediately, intraperitoneal injection detoxification is carried out by using 8 LD50 doses of SPIV virus liquid, and the volume of the fluid is 100 mu L/tail. Removing the jewfish 24h after virus attack, continuously observing the rest of jewfish for 3 days, counting the accumulated death number of each group, and calculating the relative immune protection rate (Relative Percent Survival, RPS): rps= (1-Mi/Mc) ×100%, where: mi is immune group mortality; mc is mortality in the control group.
(3) Experimental results
The results show that: the experimental group fed lactococcus lactis M48 can result in 75% of the toxicity protection of the sea bass, while 40% of the control group dies. The results are shown in Table 2.
TABLE 2 antiviral Property study results of lactococcus lactis M48 Strain on sea bass
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (8)
1. Probiotic lactococcus lactisLactococcus lactis) M48, with deposit number: GDMCC No. 62621.
2. The lactococcus lactis of claim 1Lactococcus lactis) Application of M48 in preparing sea bass feed additive.
3. The lactococcus lactis of claim 1Lactococcus lactis) Application of M48 in preparing sea bass iridovirus vaccine preparation.
4. The lactococcus lactis of claim 1Lactococcus lactis) Application of M48 in preparing sea bass vaccine immunomodulator or immunopotentiator.
5. The use according to claim 4, wherein the vaccine immunomodulator or immunopotentiator is used for regulating or enhancing the immune function of sea bass by increasing the levels of sea bass IL, IFN, MHC and IgM.
6. The lactococcus lactis of claim 1Lactococcus lactis) Application of M48 in preparing live carrier vaccine of sea bass.
7. The use according to claim 6, wherein the lactococcus lactis is @ or @ mLactococcus lactis) M48 is used as a live vector host strain for secretory expression of foreign proteins.
8. A feed additive for sea bass, an iridovirus vaccine preparation for sea bass, an immunomodulator/immunopotentiator for sea bass vaccine or a live carrier vaccine for sea bass, which is characterized by comprising the lactococcus lactis of claim 1Lactococcus lactis）M48。
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| JP2006050915A (en) * | 2004-08-10 | 2006-02-23 | Nippon Suisan Kaisha Ltd | Feed additive comprising seaweed lactic acid fermentation substance, and feed |
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| CN114657082A (en) * | 2021-04-19 | 2022-06-24 | 福建省水产研究所 | Lactococcus lactis and application thereof |
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| CN107418905A (en) * | 2015-08-07 | 2017-12-01 | 中国农业科学院哈尔滨兽医研究所 | One plant of cold water fish probiotic lactic acid lactococcus strain and application thereof |
| CN114657082A (en) * | 2021-04-19 | 2022-06-24 | 福建省水产研究所 | Lactococcus lactis and application thereof |
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