Lactococcus lactis strain lbm15 isolated from the intestine of a cichlid fish and uses thereof

Abstract

The application discloses a lactococcus lactis LBM15 isolated from the intestine of a micropterus salmoides and application thereof.The strain is classified and named as lactococcus lactis (Lactococcus lactis) Lactococcus lactis ), which has been preserved in the China Center for Type Culture Collection on August 31, 2023, and the preservation number is CCTCC NO: M 20231551. The lactococcus lactis LBM15 can effectively inhibit the growth of aquatic pathogenic bacteria, and can effectively inhibit the adhesion of the aquatic pathogenic bacteria to the cuticular mucus of the micropterus salmoides, wherein the substitution effect is the best.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactococcus lactis LBM15 and its applications. Background Technology

[0002] As aquaculture shifts from extensive to intensive farming, the problem of antibiotic resistance in aquatic strains has become increasingly serious due to antibiotic overuse. The emergence of these resistant strains not only hinders the development of the aquaculture industry but also endangers the safety of aquatic products and public safety. Therefore, seeking safe and effective treatment methods is urgently needed.

[0003] Probiotics, when applied to the internal and external environments of aquatic animals, can achieve the goals of disease prevention and treatment and improving the quality of aquatic products, and are considered to have the potential to replace antibiotics.

[0004] Probiotics are a class of live microorganisms that, when administered in sufficient quantities, produce beneficial effects on the health of the host. They are a new type of green additive developed in recent years, possessing the functions of improving the beneficial flora of the digestive tract of farmed animals, inhibiting or killing harmful bacteria, improving animal health, promoting animal growth and development, and increasing feed utilization. Lactic acid bacteria are common and symbiotic bacteria in the gastrointestinal tracts of humans and animals. As important probiotics, lactic acid bacteria are widely used in the food, beverage, and microecological preparation industries and are recognized as safe-grade microorganisms.

[0005] However, current research indicates that because lactic acid bacteria do not produce spores, their heat resistance and shelf life are poor, they have weak resistance to adverse environments, and their ability to resist stomach acid and bile is weak, meaning they have limited ability to enter the intestines. This restricts the beneficial functions of lactic acid bacteria. Fish-derived lactic acid bacteria have good colonization ability; therefore, screening lactic acid bacteria from the intestines of largemouth bass and further studying their biological characteristics is of positive significance for research on probiotics in fish. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Lactococcus lactis LBM15 screened from the intestine of California bass and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A strain of *Lactococcus lactis* LBM15 isolated from the intestine of California bass was named *Lactococcus lactis*. Lactococcus lactis It was deposited at the China Center for Type Culture Collection on August 31, 2023, with accession number CCTCC NO: M 20231551, and the deposit address is No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] The above-mentioned Lactococcus lactis strain LBM15 has the following characteristics: 1) It has acid-producing ability and can produce a 26.987 mm color change zone on MRS agar medium supplemented with bromocresol purple; 2) It exhibits good growth performance, with slow growth in the first 8 hours of culture, entering the logarithmic growth phase from 8 to 18 hours, and then entering the stationary phase after 18 hours. 3) It has a certain tolerance to acids, alkalis, NaCl, bile salts and intestinal fluid. It grows well in culture media with a pH of 6-8 and can grow in MRS medium with a NaCl content of 0%-6.5%. It has a strong tolerance to artificial intestinal fluid. 4) It has the ability to produce protease, and can produce a hydrolysis zone of 26.363 mm on MRS agar medium with added skim milk powder; 5) Shows sensitivity or moderate sensitivity to multiple antibiotics.

[0009] A microbial agent containing the aforementioned Lactococcus lactis LBM15.

[0010] The above-mentioned lactococcus lactis LBM15 or its bacterial agent is used in the preparation of a product that inhibits aquatic pathogens, wherein the aquatic pathogens are Vibrio alginolyticus, Vibrio harveyi, Pseudomonas proteus, Edwardsiella tarda, Vibrio anguillarum, and Aeromonas hydrophila, and the product is a product that inhibits the growth and / or adhesion of aquatic pathogens.

[0011] The above-mentioned application of Lactococcus lactis LBM15 or its inoculum in the preparation of feed additives for aquatic animals, wherein the aquatic animal is California bass.

[0012] The above-mentioned Lactococcus lactis LBM15 or its agent is used in the preparation of aquatic animal aquaculture water additives, wherein the aquatic animal is California bass.

[0013] The above-mentioned Lactococcus lactis LBM15 or the bacterial agent described herein is used in the study of the lactic acid production capacity, antibiotic resistance, pH resistance, bile salt resistance, NaCl resistance, gastric juice resistance, intestinal juice resistance, adhesion ability, and resistance to adhesion of aquatic pathogens by Lactococcus lactis.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are as follows: The Lactococcus lactis LBM15 of this invention can inhibit a variety of common aquatic pathogens and shows sensitivity to or moderate sensitivity to a variety of antibiotics commonly used in the aquaculture industry. Lactococcus lactis LBM15 can adhere to fish mucus, inhibiting the growth of aquatic pathogens and their adhesion to fish surface mucus. Attached Figure Description

[0015] Figure 1 Colony morphology of Lactococcus lactis LBM15 on MRS solid plates.

[0016] Figure 2 Results of acid-producing capacity assay for Lactococcus lactis LBM15.

[0017] Figure 3 Growth curve of Lactococcus lactis LBM15.

[0018] Figure 4 Results of protease hydrolysis activity assay of Lactococcus lactis LBM15.

[0019] Figure 5 Growth of Lactococcus lactis LBM15 at different pH levels.

[0020] Figure 6 Growth of Lactococcus lactis LBM15 under different NaCl contents.

[0021] Figure 7 Growth of Lactococcus lactis LBM15 in simulated gastric and intestinal fluids.

[0022] Figure 8 Growth of Lactococcus lactis LBM15 under different bile salt contents.

[0023] Figure 9 Lactococcus lactis LBM15 inhibits the adhesion of Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Aeromonas hydrophila, Edwardsiella tarda, and Pseudomonas proteus. Detailed Implementation

[0024] To make the content of this invention easier to understand, the technical solution of this invention will be further described below in conjunction with specific embodiments. However, it should be understood that the following examples are not intended to limit the scope of protection of this invention. Any changes and modifications made on the basis of this invention are within the scope of protection of this invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1: This embodiment provides the isolation and identification of Lactococcus lactis LBM15.

[0028] 1.1 Source of Lactococcus lactis strain LBM15 Healthy California bass intestines were used as samples, and homogenized with sterilized PBS. After heating at 60°C for 1 hour, the samples were spread onto MRS agar plates and incubated upside down at 37°C for 48-72 hours. Single colonies exhibiting typical lactic acid bacteria characteristics were selected and streaked onto MRS agar plates three or more times for purification until uniform single colonies appeared on the plates, thus obtaining strain LBM15. The strain morphology is as follows: Figure 1 As shown.

[0029] 1.2 Identification of Lactococcus lactis LBM15 16S rDNA sequence homology analysis: Genomic DNA was extracted from strain LBM15 using the Novizan Biospin Bacterial Genomic DNA Extraction Kit; detailed operating procedures are provided in the kit's instructions. PCR amplification was performed using Novizan 2×Phanta®Max Master Mix (Dye Plus).

[0030] Table 1 PCR amplification system

[0031] Table 2 PCR Cycling Conditions

[0032] Table 3 Primer sequence information

[0033] The PCR products were sent to Sangon Biotech Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.1. The obtained sequence results were compared with the GenBank database. BLAST alignment analysis showed that the 16S rDNA gene sequence of strain LBM15 was similar to that of *Lactococcus lactis* (…). Lactococcus lactis The sequence similarity was highest at 99.74%, therefore strain LBM15 was considered to be *Lactococcus lactis*. Lactococcus lactis ).

[0034] Example 2: This embodiment provides a determination of the acid-producing capacity of Lactococcus lactis LBM15.

[0035] Add bromocresol purple to MRS agar medium at a ratio of 1:100 m / v, sterilize at 121°C for 20 min, pour into plates, allow to solidify, and punch wells. Add 100 μL of cultured OD to each well. 600nm A bacterial suspension of *Lactococcus lactis* LBM15 with a concentration of 0.6 ± 0.01 was incubated at 37°C for 24 hours. The diameter of the discoloration point (from purple to yellow) was measured to be 26.987 mm. Figure 2 As shown.

[0036] Example 3: This embodiment provides the determination of the growth curve of Lactococcus lactis LBM15.

[0037] Growth curve: The overnight culture of Lactococcus lactis LBM15 in MRS medium was adjusted to OD. 600nm =0.6±0.01, and diluted 10000 times. 200 μL of the diluted bacterial culture was added to each well of a 96-well plate, while 200 μL of LMR medium was added to each well of the control group. The plates were incubated at 37°C for 24 hours, and the OD of the bacterial culture was monitored hourly. 600nm Value. For example... Figure 3 As shown, Lactococcus lactis LBM15 grows slowly in the first 8 hours of culture, which is the lag phase; from 8 to 18 hours it is the logarithmic growth phase; and after 18 hours it enters the stationary phase.

[0038] Example 4: This embodiment provides an assay for the protease hydrolysis activity of Lactococcus lactis LBM15.

[0039] Add skim milk powder to sterilized MRS agar medium at a ratio of 1:50 m / v, mix well, pour into plates, and after the plates have solidified, punch wells. Add 100 μL of cultured OD to each well. 600nm A bacterial suspension of *Lactococcus lactis* LBM15 with a concentration of 0.6 ± 0.01 was incubated at 37°C for 24 hours. The diameter of the hydrolysis zone was measured to be 26.363 mm. Figure 4 As shown.

[0040] Example 5: This embodiment provides a determination of the tolerance of Lactococcus lactis LBM15 to pH, NaCl, artificial gastric juice, artificial intestinal juice, and bile salts.

[0041] Growth of Lactococcus lactis LBM15 at different pH values: The pH of MRS medium was adjusted to 2, 3, 4, 5, 6, 8, 10, and 12, respectively. OD 600nm Lactococcus lactis LBM15 bacterial suspension with a concentration of 0.6 ± 0.01 was added to each culture medium at a ratio of 1:50 v / v, and cultured at 37°C and 220 rpm for 4 hours. The absorbance at 600 nm was then measured. Figure 5 As shown: Within 4 hours, in a culture medium with a pH of 6-8, the OD of LBM15 culture medium increased with increasing pH. 600 The OD value gradually increased, reaching its maximum at pH 8, indicating that LBM15 grew best at this pH. Growth slowed at pH values ​​of 2, 4, 10, and 12.

[0042] Growth of Lactococcus lactis LBM15 under different NaCl concentrations: The NaCl concentration in MRS medium was adjusted to 0.5%, 1.5%, 2.5%, 3.5%, 4.5%, 5.5%, and 6.5%, respectively, while a control group was established with a NaCl concentration of 0.0% in MRS medium. OD 600nm Lactococcus lactis LBM15 bacterial suspension with a concentration of 0.6 ± 0.01 was added to each culture medium at a ratio of 1:50 v / v, and the absorbance was measured at 600 nm after culturing for 24 h. The results are as follows: Figure 6 As shown: LBM15 can grow in MRS medium with NaCl concentrations ranging from 0% to 6.5%. With increasing NaCl concentration, the OD of the LBM15 culture medium... 600 The value gradually decreases.

[0043] Growth of *Lactococcus lactis* LBM15 in simulated gastric and intestinal fluids: Simulated gastric fluid: MRS medium was supplemented with pepsin at a ratio of 100:1 v / m, pH adjusted to 2, and filtered through a 0.22 μm membrane before use. Simulated intestinal fluid: MRS medium was supplemented with KH₂PO₄ at a ratio of 100:0.68 v / m, sterilized, pH adjusted to 6.80 with NaOH, and trypsin was added at a ratio of 100:1 v / m. The medium was then filtered through a 0.22 μm membrane before use. A control group was set up using MRS medium. OD₂... 600nm Lactococcus lactis LBM15 bacterial suspension with a concentration of 0.6 ± 0.01 was added to each culture medium at a ratio of 1:50 v / v and cultured for 4 h. The absorbance at 600 nm was monitored every 1 h. The results are as follows: Figure 7 As shown, within 4 hours, *Lactococcus lactis* LBM15 exhibited strong tolerance in the artificial intestinal fluid, which had no effect on its growth. However, in the artificial gastric fluid, the OD of the LBM15 culture medium... 600 The value hardly increases.

[0044] Growth of Lactococcus lactis LBM15 under different bile salt concentrations: The bile salt concentration in MRS medium was adjusted to 0.15%, 0.30%, and 0.45%, respectively, while a control group was set up with 0% bile salt content in MRS medium. OD 600nm Lactococcus lactis LBM15 bacterial suspension with a concentration of 0.6 ± 0.01 was added to each culture medium at a ratio of 1:50 v / v, and the absorbance was measured at 600 nm after culturing for 24 h. The results are as follows: Figure 8 As shown, compared with the control group, the growth of strains in all bile salt concentration groups was inhibited. At low bile salt concentration (0.1%), the OD of LBM15 culture medium was significantly lower. 600 The values ​​continued to increase; at 0.30% and 0.45% bile salt concentrations, the OD values ​​of LBM15 culture medium... 600 The value hardly increases.

[0045] Example 6: This embodiment provides the determination of the antibacterial activity of Lactococcus lactis LBM15 against aquatic pathogens.

[0046] The inhibitory activity of Lactococcus lactis LBM15 against aquatic pathogens was determined using the perforation method. Overnight cultured OD... 600nm 100 μL of each of the following bacterial suspensions (0.3 g / mL): Vibrio alginolyticus, Vibrio harveyi, Pseudomonas proteus, Edwardsiella tarda, Vibrio anguillarum, and Aeromonas hydrophila. Spread evenly onto LB agar plates, then punch three wells in each plate and add 100 μL of OD245 solution to each well. 600nm The diameter of the inhibition zone was measured after incubating a 0.6% Lactococcus lactis LBM15 bacterial suspension at 36℃ for 24 hours.

[0047] The results are shown in the table below. LBM15 inhibited the growth of Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Aeromonas hydrophila, and Edwardsiella tarda, with the strongest inhibitory effect on Vibrio alginolyticus.

[0048] Table 4. Antagonistic effect of LBM15 against aquatic pathogens

[0049] Example 7: This embodiment provides a determination of the adhesion inhibition ability of Lactococcus lactis LBM15.

[0050] Construction of standard curves for California bass mucus and protein: Scrape the mucus from the skin of California bass onto a glass slide, place it in a 4°C refrigerator overnight, then centrifuge at 12000×g for 30 min at 4°C. Collect the supernatant and filter it through a 0.1μm filter membrane for sterilization. Measure the protein standard curve using the Bradford Protein Quantitative Kit and adjust the mucus protein concentration to 1 mg / mL. Aliquot the mucus and store it at -20°C for later use.

[0051] Inhibition of adhesion assay: Mucus protein (1 mg protein / mL) from California bass was taken out from -20℃, thawed at 4℃, and 100 μL was added to a 96-well black polystyrene culture plate. After fixing at 4℃ for 24 h, 200 μL of Hepes-Hanks solution was added to each well and the plate was gently washed twice to remove unadhered mucus protein.

[0052] ① Competitive inhibition of adhesion assay: Add 100 μL of stained pathogenic bacteria and unstained LBM15 to each well. A positive control was set up by adding 100 μL of stained pathogenic bacteria to each well. After incubation at 37℃ for 1 h, gently wash twice with Hepes-Hanks solution. Finally, add 200 μL of 1% SDS-0.1 mol / L NaOH lysis buffer to each well and incubate at 60℃ for 1 h to elute the adhered bacteria. A negative control was set up by adding 100 μL of unstained pathogenic bacteria and LBM15 to each well. Fluorescence values ​​were measured using a fluorescence microplate reader with an excitation wavelength of 340 nm and an emission wavelength of 460 nm.

[0053] ② Repulsion Inhibition Adhesion Assay: Add 100 μL of unstained LBM15 to each well, incubate at 37℃ for 1 h, gently wash twice with Hepes-Hanks solution, then add 100 μL of stained pathogenic bacteria to each well; simultaneously, add 100 μL of stained pathogenic bacteria to each of the other wells as a positive control; incubate at 37℃ for 1 h, gently wash twice with Hepes-Hanks solution, then add 200 μL of 1% SDS-0.1 mol / L NaOH lysis buffer to each well, and incubate at 60℃ for 1 h to elute the adhered bacteria. The negative control consists of 100 μL of unstained pathogenic bacteria and LBM15 per well. Measure the fluorescence value using a fluorescence microplate reader; the excitation wavelength is 340 nm, and the emission wavelength is 460 nm.

[0054] ③ Substitution inhibition adhesion assay: Add 100 μL of stained pathogenic bacteria to each well, incubate at 37℃ for 1 h, gently wash twice with Hepes-Hanks solution, then add 100 μL of unstained LBM15 to each well; simultaneously, add 100 μL of stained pathogenic bacteria to each of the other wells as a positive control; incubate at 37℃ for 1 h, gently wash twice with Hepes-Hanks solution, then add 200 μL of 1% SDS-0.1 mol / L NaOH lysis buffer to each well, and incubate at 60℃ for 1 h to elute the adhered bacteria. A negative control is set as follows: add 100 μL of unstained pathogenic bacteria and LBM15 to each well. Measure the fluorescence value using a fluorescence microplate reader; the excitation wavelength is 340 nm, and the emission wavelength is 460 nm.

[0055] Adhesion inhibition rate % = (positive control fluorescence intensity - experimental group fluorescence intensity) / (positive control fluorescence intensity - negative control fluorescence intensity) × 100.

[0056] The effect of competition is as follows Figure 9 As shown in Figure A, LBM15 inhibited all six pathogenic bacteria, with the highest inhibitory effect against Vibrio anguillarum. The competitive inhibition rates against the other pathogenic bacteria were all above 40%. The results of the exclusion inhibition are as follows: Figure 9As shown in B, LBM15 exhibited varying degrees of inhibitory effects against all six pathogenic bacteria, with the highest inhibitory capacity against Vibrio anguillarum, followed by Edwardsiella tarda; the substitution inhibition results are as follows. Figure 9 As shown in C, among the three modes of adhesion inhibition—competition, repulsion, and substitution—substitution has a greater impact than the other two. Among them, LBM15 has the strongest substitution inhibition ability against Aeromonas hydrophila, followed by Vibrio anguillarum.

[0057] Example 8: This embodiment provides an antibiotic susceptibility assay for Lactococcus lactis LBM15.

[0058] The susceptibility of Lactococcus lactis LBM15 to antibiotics was determined using the paper disc method. Overnight cultured OD... 600nm 100 μL of 0.3 LBM15 was evenly spread on an MRS agar plate, and standard drug discs were placed on them with tweezers. The plate was then incubated at 37°C for 24 h, and the diameter of the inhibition zone was measured.

[0059] The results are shown in the table below.

[0060] Table 5 Results of LBM15 drug susceptibility testing

[0061] "S" indicates sensitivity, "I" indicates mediation, and "R" indicates drug resistance. In summary, the *Lactococcus lactis* LBM15 strain of this invention can inhibit a variety of common aquatic pathogens and exhibits sensitivity to or moderate sensitivity to several antibiotics commonly used in the aquaculture industry. *Lactococcus lactis* LBM15 can adhere to fish mucus, inhibiting the adhesion of aquatic pathogens to fish mucus. The use of this *Lactococcus lactis* strain lays the foundation for accurate research into its biological characteristics and clarifies its application scope.

[0062] The above description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed. Many changes and variations can be made based on the above embodiments. The exemplary embodiments were selected and described to explain the specific principles and practical applications of the invention. Equivalent modifications and variations made by those skilled in the art in accordance with the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A strain of Lactococcus lactis ( Lactococcus lactis LBM15, characterized in that: The Lactococcus lactis LBM15 was deposited at the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with accession number CCTCC NO: M 20231551, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial agent, characterized in that: The bacterial agent contains Lactococcus lactis LBM15 as described in claim 1.

3. The application of the Lactococcus lactis LBM15 according to claim 1 or the bacterial agent according to claim 2 in the preparation of products inhibiting aquatic pathogens, characterized in that: The aquatic pathogen is Vibrio alginolyticus ( Vibrio alginolyticus ), Vibrio harveyi ( Vibrio harveyi Edwardsiella tarda ( ), Edwardsiella tarda ( Edwardsiella tarda ), Vibrio anguillarum ( Vibrio anguillarum Aeromonas hydrophila ( ) Aeromonas hydrophila ).

4. The application of the Lactococcus lactis LBM15 according to claim 1 or the bacterial agent according to claim 2 in the preparation of aquatic animal feed additives, characterized in that: The aquatic animal mentioned is the California bass ( Micropterus salmoides ).

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