Leuconostoc mesenteroides hyprobio-y3, its use and its microbial preparation

Abstract

The application discloses Leuconostoc mesenteroides HYProbio-Y3, application thereof and a microbial preparation thereof, and belongs to the technical field of microorganisms.The Leuconostoc mesenteroides is Leuconostoc mesenteroides HYProbio-Y3 strain, and the preservation number is CGMCC No.30413.Under PFOA exposure, the immunity of Ruditapes philippinarum is destroyed.After intervention of the L.mesenteroides HYProbio-Y3, the immunity damage of the Ruditapes philippinarum caused by PFOA can be significantly improved, the immunity of the Ruditapes philippinarum can be effectively improved, the Ruditapes philippinarum can be helped to cope with the harm of persistent organic pollutants, and the development of the aquaculture industry can be promoted.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Leuconostoc mesenteroides HYProbio-Y3, its application, and its microbial preparations. Background Technology

[0002] Perfluorooctanoic acid (PFOA) is a perfluorinated compound belonging to the perfluoroalkyl and polyfluoroalkyl substances (PFASs) family. PFOA has a highly stable chemical structure and is extremely difficult to degrade in the environment, classifying it as a persistent organic pollutant. It can persist in water, soil, and air for extended periods and bioaccumulates through the food chain. Studies have shown that PFOA has various toxic effects, including endocrine disruption, impaired reproductive development, immune system damage, and liver damage. In 2017, the World Health Organization classified PFOA as a Group 2B carcinogen. The continuous accumulation of PFOA in nearshore waters has led to its increasing levels, resulting in its accumulation in fish and shellfish, severely damaging their immune systems and significantly impacting aquaculture.

[0003] Current research has shown that probiotics can promote fish growth and improve immunity. However, there are no reports on whether probiotics have a restorative effect on immune damage caused by persistent pollutants. Providing probiotics that can restore immune damage caused by persistent pollutants is of great significance for aquaculture and the prevention and control of the damage of persistent pollutants to fish and shellfish. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of this invention is to provide a strain of Leuconostoc mesenteroides HYProbio-Y3, its application and its microbial preparation.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A strain of Leuconostocmesenteroides, specifically strain HYProbio-Y3, with accession number CGMCC No. 30413.

[0007] The above-mentioned application of Leuconostoc mesenteroides is selected from at least one of the following:

[0008] a. Used in the preparation of formulations with antioxidant properties;

[0009] b. Used in the preparation of formulations that enhance the immunity of aquatic animals;

[0010] c. For the preparation of immune restoration agents for aquatic animals whose immunity has been damaged by persistent organic pollutants.

[0011] Based on the above scheme, the antioxidant effect is the ability to scavenge free radicals.

[0012] Based on the above scheme, the ability to scavenge free radicals refers to the ability to scavenge DPPH free radicals and hydroxyl free radicals.

[0013] Based on the above scheme, the aquatic animals mentioned are at least one of fish, crustaceans, and mollusks.

[0014] Based on the above scheme, the aquatic animal mentioned is the Manila clam.

[0015] Based on the above scheme, the persistent organic pollutant is perfluorooctanoic acid (PFOA).

[0016] Based on the above scheme, the immune recovery refers to the increase in the total number of blood cells in aquatic animals, the slowing of apoptosis, the improvement of lymphocyte phagocytic activity, and the reduction of non-specific esterase content compared to before recovery.

[0017] A microbial preparation containing the Leuconostoc mesenteroides HYProbio-Y3 strain with accession number CGMCC No. 30413.

[0018] Based on the above scheme, the content of Leuconostoc mesenteroides HYProbio-Y3 in the preparation is ≥10. 6 CFU / mL.

[0019] Advantages of the technical solution of this invention

[0020] This invention discloses a strain of Leuconostoc mesenteroides HYProbio-Y3. Under PFOA exposure, the immunity of Manila clams is compromised. Intervention with L. mesenteroides HYProbio-Y3 significantly increased the number of hemocytes, significantly reduced the number of apoptotic cells, significantly improved phagocytic capacity, and significantly decreased the content of nonspecific esterases in Manila clams, thus alleviating the oxidative stress response induced by PFOA. Therefore, L. mesenteroides HYProbio-Y3 can significantly improve the immune damage of Manila clams induced by PFOA, effectively enhance their immunity, help them cope with the harm of persistent organic pollutants, and promote the development of aquaculture. Attached Figure Description

[0021] Figure 1Colony morphology of L. mesenteroides HYProbio-Y3 on MRS plates;

[0022] Figure 2 Gram staining results of L. mesenteroides HYProbio-Y3 under microscopic examination;

[0023] Figure 3 The effect of L. mesenteroides HYProbio-Y3 on hemocyte count in PFOA-contaminated Manila clams;

[0024] Figure 4 Effects of L. mesenteroides HYProbio-Y3 on apoptosis in 0.2 ng / mL PFOA-contaminated Manila clam cells;

[0025] Figure 5 Effects of L. mesenteroides HYProbio-Y3 on apoptosis in Manila clam cells contaminated with 2 ng / mL PFOA;

[0026] Figure 6 Effects of L. mesenteroides HYProbio-Y3 on phagocytic activity of lymphocytes in PFOA-contaminated Manila clams;

[0027] Figure 7 Effects of L. mesenteroides HYProbio-Y3 on nonspecific esterase activity in PFOA-contaminated Manila clams. Detailed Implementation

[0028] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Example 1

[0031] Isolation, purification and identification of strains

[0032] Fresh sea bream (wild-caught sea bream from the Yellow Sea near Qingdao) samples were collected. Approximately 3g of intestinal contents were aseptically extracted and homogenized in 50mL of physiological saline. 0.1mL of the homogenate was added to 5mL of MRS medium and incubated at 37℃ for 24-48 hours. After thoroughly shaking the culture, 200μL of the bacterial solution was carefully spread evenly onto MRS solid medium using a spreader and incubated upside down at 37℃ for 24-48 hours. Colonies of different morphologies on the solid medium were carefully isolated and cultured for screening. Single colonies were selected for species identification.

[0033] The isolated and purified target bacteria were transferred to MRS solid medium and incubated at 37°C for 24-48 hours. After 24 hours of incubation on MRS medium, *Leuconostoc mesenteroides* formed round colonies with a diameter of less than 1.0 mm, a milky white color, a smooth surface, and regular edges. Figure 1 Gram staining is positive, and the cells are arranged in a beaded pattern. Figure 2 Genomic DNA was extracted from isolated single colonies, and the strain was identified by 16S rRNA sequence alignment, as shown in SEQ ID NO: 1. The sequencing results were compared with species similarity in the NCBI database. The sequence alignment results showed that the bacterium had a 99.93% similarity to Leuconostocmesenteroides, therefore the bacterium was identified as Leuconostocmesenteroides.

[0034] The isolated Leuconostoc mesenteroides HYProbio-Y3 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 24, 2024, with accession number CGMCC No. 30413; the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0035] SEQ ID NO: 1 (5'→3')

[0036]

[0037] Example 2

[0038] In vitro antioxidant activity of Leuconostoc mesenteroides HYProbio-Y3

[0039] L. mesenteroides HYProbio-Y3 was inoculated onto MRS medium at a volume fraction of 2% (V / V) for activation and cultured at 37°C for 24 h. The second-generation fermentation broth was then centrifuged at 4000 rpm for 10 min at 4°C, the supernatant was discarded, and the cells were resuspended in 5 mL of physiological saline. The bacterial concentration was then diluted to 1 × 10⁻⁶ with physiological saline. 9 CFU / mL, inoculated into MRS medium at a volume fraction of 2% (V / V) and cultured to the 3rd generation. Cultured at 37℃ for 24 h. After 24 h of culture, the 3rd generation culture was taken, centrifuged at 4℃ and 6000 rpm for 10 min, and the supernatant was collected and sterilized by passing through a 0.22 μm organic filter membrane for later use.

[0040] (1) DPPH free radical scavenging rate determination:

[0041] 0.1 mL of the fermentation supernatant of L. mesenteroides HYProbio-Y3 prepared by the above method was mixed with 3.9 mL of 25 mg / L DPPH ethanol solution. After incubation at room temperature in the dark for 30 min, the absorbance of the reactants at 517 nm was measured. Each treatment group was set up in triplicate. 0.1 mL of deionized water and 3.9 mL of DPPH reaction solution were used as blank controls.

[0042] Clearance rate (%) = (1 - AS / AB) × 100%

[0043] Wherein, "AS" represents the absorbance of the fermentation supernatant and DPPH reaction solution; "AB" represents the absorbance of the blank control.

[0044] The results showed that the fermentation supernatant of L. mesenteroides HYProbio-Y3 had a strong scavenging effect on DPPH free radicals, with a scavenging rate of 63.13% ± 1.67%, indicating that L. mesenteroides HYProbio-Y3 can metabolize to produce antioxidant metabolites.

[0045] (2) Hydroxyl radical scavenging rate determination

[0046] The hydroxyl radical scavenging activity assay kit (purchased from Beijing Solarbio Biotechnology Co., Ltd., catalog number: BC1325) was used, and the procedure was performed according to the instructions.

[0047] Add 0.15 mL of Reagent I, 0.3 mL of Reagent II, and 0.3 mL of Reagent III sequentially to the blank tube, control tube, and test tube, and mix thoroughly. Then add 0.75 mL of water to the blank tube; add 0.15 mL of Reagent IV and 0.6 mL of water to the control tube; and add 0.15 mL of the L. mesenteroides HYProbio-Y3 fermentation supernatant prepared by the above method, 0.15 mL of Reagent IV, and 0.45 mL of water to the test tube. Vortex each tube to mix thoroughly and place them in a 37°C water bath or incubator for 60 min. After the reaction is complete, centrifuge at 10000 rpm at room temperature for 10 min, and measure the absorbance at 536 nm for each supernatant. Record the results for the blank tube, control tube, and test tube as A. 空 A 对 and A 测 Blank and control tubes only need to be measured 1-2 times. Calculate the hydroxyl radical scavenging rate (%) using the following formula.

[0048] Hydroxyl radical scavenging rate % = (A 测 -A 对 )÷(A 空 -A 对 )×100%

[0049] The results showed that the fermentation supernatant of L. mesenteroides HYProbio-Y3 had a certain scavenging effect on hydroxyl radicals, with a scavenging rate of 14.23% ± 2.84%, indicating that L. mesenteroides HYProbio-Y3 can metabolize to produce antioxidant metabolites.

[0050] Example 3

[0051] Effects of L. mesenteroides HYProbio-Y3 on hemocyte count in PFOA-contaminated Manila clams

[0052] Blood cells play an important role in the immune defense of bivalves, and the number of blood cells is an important indicator of the immune status of shellfish.

[0053] Live Manila clams (R. philippinarum, shell length 28.5 ± 1.4 mm) were collected from the Yellow Sea, China. After being transported from the coast to the laboratory, the clams were acclimatized for 7 days in 30-liter tanks under 16-19℃ temperature and 12-hour light / dark cycles, fed daily with spirulina. At the end of acclimatization, healthy clams were randomly selected and placed in different groups of culture tanks for biological exposure experiments. The treatment methods for each group are as follows:

[0054] Blank group: Clams were cultured in fresh seawater and under simulated natural marine environmental conditions;

[0055] 0.2 ng / L PFOA treatment group: Add PFOA solution according to the set concentration to make the final concentration 0.2 ng / L.

[0056] 2 ng / L PFOA treatment group: Add PFOA solution according to the set concentration to make the final concentration 2 ng / L.

[0057] Treatment group with 0.2 ng / L PFOA + probiotics: PFOA and Leuconostoc mesenteroides were added simultaneously, with a final concentration of 0.2 ng / L for PFOA and 10 ng / L for Leuconostoc mesenteroides. 6 CFU / mL.

[0058] 2 ng / L PFOA + Probiotic Treatment Group: PFOA and Leuconostoc mesenteroides were added simultaneously, with a final PFOA concentration of 2 ng / L and a final Leuconostoc mesenteroides concentration of 10 ng / L. 6 CFU / mL.

[0059] On day 7 of treatment, hemolymph was collected from the adductor muscle of each group of Manila clams using a 1 mL syringe. 2 mL of hemolymph was collected from 10 clams randomly selected from each aquarium, pooled, and stored on ice until treatment to minimize inter-individual variability. Untreated hemolymph was used directly to determine total blood cell count (THC).

[0060] Take 200 μL of blood cell suspension, filter it through a 300-mesh sieve, and immediately test it. Plot an FSC-SSC scatter plot by detecting forward scatter (FSC) and side scatter (SSC), select the cell population, and record the number of intact cells.

[0061] The results are as follows Figure 3 As shown, perfluorooctanoic acid (PFOA) exposure for 7 days caused a significant reduction in the total blood cell count of Manila clams at both concentration gradients. Supplementation with the probiotic Leuconostoc mesenteroides HYProbio-Y3 significantly increased the total blood cell count in the low-concentration PFOA exposure group (0.2 ng / mL). The high-concentration PFOA exposure group (2 ng / mL) showed no significant change in the total blood cell count.

[0062] Example 4

[0063] Effects of L. mesenteroides HYProbio-Y3 on apoptosis in PFOA-contaminated Manila clam cells

[0064] Apoptosis is an important immune pathway for the body to clear abnormal and damaged cells, but an excessively high apoptosis rate can lead to risks such as a reduction in the number of blood cells and a decrease in the body's immunity.

[0065] The processing method and blood collection method for the Philippine clam R. philippinarum are the same as in Example 3.

[0066] Blood cell apoptosis rate was detected using an Annexin V-FITC / Propidium Iodide (Annexin V-FITC / PI) apoptosis detection kit. Blood cell suspensions were diluted and centrifuged, and the pellet was resuspended in buffer. 5 μL of Annexin V-FITC was added to every 100 μL of blood cell suspension, and the mixture was incubated at room temperature in the dark for 5 minutes. Then, 5 μL of PI solution was added, followed by 400 μL of buffer. The mixture was filtered through a 300-mesh sieve and immediately analyzed. 10,000 cells were collected for each sample. A scatter plot was plotted with Annexin V-FITC on the x-axis and PI on the y-axis. The scatter plot was divided into four quadrants using a cross-gate: FITC+ / PI+ represented late-apoptotic or necrotic cells, FITC+ / PI- represented early-apoptotic cells, and FITC- / PI- represented normal (viable) cells.

[0067] The results are as follows Figure 4 , 5 As shown, perfluorooctanoic acid (PFOA) exposure induced apoptosis in Manila clam hemolymphocytes after 7 days at both concentration gradients. After initial PFOA exposure, the proportion of viable cells in clam hemolymphocytes significantly decreased, while the proportions of early and late apoptotic cells significantly increased. In the low-concentration PFOA exposure group, the addition of the probiotic *Leuconostoc mesenteroides* slightly increased the proportion of viable cells and alleviated the degree of apoptosis, but the change was not significant. However, in the high-concentration PFOA exposure group, the number of late apoptotic cells significantly decreased after the addition of *Leuconostoc mesenteroides*, indicating that *Leuconostoc mesenteroides* can effectively inhibit PFOA-induced apoptosis.

[0068] Example 5

[0069] Effects of L. mesenteroides HYProbio-Y3 on phagocytic activity of lymphocytes in PFOA-contaminated Manila clams

[0070] The phagocytic activity of shellfish lymphocytes is an important indicator for assessing the immune defense of shellfish hematopoietic cells.

[0071] The processing method and blood collection method for the Philippine clam R. philippinarum are the same as in Example 3.

[0072] An equal volume of amine-modified polystyrene latex bead suspension (yellow-green fluorescence, purchased from Sigma, catalog number L1030) was added to the blood cell suspension of each treatment group as the phagocytic material. The mixture was incubated in the dark at 18°C ​​for 1 hour, and the reaction was terminated by adding 1 mL of 4% paraformaldehyde. After filtration through a 300-mesh sieve, the samples were analyzed. The phagocytic rate was calculated by determining the percentage of phagocytes in the total number of cells.

[0073] The results are as follows Figure 6 As shown, perfluorooctanoic acid (PFOA) exposure for 7 days induced changes in the phagocytic activity of hemolymphocytes in Manila clams at both concentration gradients. PFOA exposure resulted in a certain degree of decrease in the phagocytic capacity of clam hemolymphocytes. The addition of the probiotic *Leuconostoc mesenteroides* improved the phagocytic capacity of the cells to some extent. This indicates that *Leuconostoc mesenteroides* can promote the phagocytic activity of lymphocytes. Figure 6 ).

[0074] Example 6

[0075] Effects of L. mesenteroides HYProbio-Y3 on nonspecific esterase activity in PFOA-contaminated Manila clams

[0076] Nonspecific esterases are synthesized and secreted by blood cells into the immune system, where they participate in the body's immune defense. The activity of nonspecific esterases can, to some extent, reflect the immune defense capabilities of shellfish against stimuli.

[0077] The processing method and blood collection method for the Philippine clam R. philippinarum are the same as in Example 3.

[0078] Nonspecific esterases were specifically labeled using fluorescein diacetate working solution (FDA, Sigma). 2 μL of FDA working solution was added to 400 μL of blood cell suspension and incubated at room temperature in the dark for 15 minutes. A histogram was plotted with FL1 (FDA fluorescence intensity) on the x-axis and cell number (Events) on the y-axis. The percentage of cells producing fluorescence was used to assess the activity of nonspecific esterases.

[0079] The results are as follows Figure 7 As shown, perfluorooctanoic acid (PFOA) exposure for 7 days altered the levels of nonspecific esterases in the hemolymphocytes of Manila clams under both concentration gradients. Manila clams exposed to low PFOA concentrations (0.2 ng / mL) and high PFOA concentrations (2 ng / mL) exhibited strong oxidative stress responses after PFOA stress, with a significant increase in nonspecific esterase activity. The addition of the probiotic *Leuconostoc mesenteroides* significantly reduced the levels of nonspecific esterases. Figure 7 ).

[0080] In summary, PFOA exposure impairs the immunity of Manila clams. Intervention with *L. mesenteroides* HYProbio-Y3 significantly increased hematopoietic cell count, decreased apoptosis rate, enhanced phagocytic capacity, and reduced nonspecific esterase levels in Manila clams, thus alleviating PFOA-induced oxidative stress. These findings demonstrate that *L. mesenteroides* HYProbio-Y3 can significantly improve PFOA-induced immune damage in Manila clams, effectively enhance their immunity, help them cope with persistent organic pollutants, and promote the development of aquaculture.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Leuconostoc mesenteroides strain, characterized in that, The Leuconostoc mesenteroides is Leuconostoc mesenteroides HYProbio-Y3 strain with a preservation number of CGMCC No.30413.

2. Use of Leuconostoc mesenteroides according to claim 1, characterized in that, at least one selected from a-c: a. for preparing a preparation with antioxidant efficacy; b. for preparing a preparation with improved immunity of aquatic animals; c. for preparing a preparation for recovering the immunity of aquatic animals after the immunity is destroyed by persistent organic pollutants; The aquatic animals are Ruditapes philippinarum. The persistent organic pollutants are perfluorooctanoic acid.

3. Use according to claim 2, characterized in that, The antioxidant efficacy is the ability of scavenging free radicals.

4. Use according to claim 3, characterized in that, The ability of scavenging free radicals is the ability of scavenging DPPH free radicals and hydroxyl free radicals.

5. Use according to claim 2, characterized in that, The recovery of immunity refers to that the total number of blood cells of aquatic animals is increased, cell apoptosis is slowed down, lymphocyte phagocytic activity is improved, and non-specific esterase content is reduced after the recovery.

6. A microbial preparation, characterized in that, The Leuconostoc mesenteroides is Leuconostoc mesenteroides HYProbio-Y3 strain with a preservation number of CGMCC No.30413.

7. The microbial preparation according to claim 6, characterized in that, The content of Leuconostoc mesenteroides HYProbio-Y3 in the preparation is ≥10 6 CFU / mL.

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