Enterococcus with anti-vibrio ability

Abstract

The application discloses an enterococcus with vibrio resistance, and belongs to the technical field of microorganisms. An enterococcus (Enterococcus sp.) 10-1 is isolated from animal feed, and the preservation number is CGMCC No.33873. The enterococcus has obvious inhibiting effects on common parahemolytic vibrio, algicidal vibrio, harvey's vibrio and wound vibrio. The strain has probiotic safety, can grow under the initial pH condition of pH=5-11 and in a high-salt environment. The strain is sensitive to most common antibiotics. The bacteriostatic component of the strain is secreted to the outside of the cell to act on vibrio. The bacteriostatic component has pH stability and temperature stability, and can play a role under acidic conditions. The application lays a foundation for developing good antibacterial probiotic preparations.

Description

Technical Field

[0001] This invention relates to an enterococcus with anti-Vibrio activity, belonging to the field of microbial technology. Background Technology

[0002] Vibrio is a typical representative group of Gram-negative pathogenic bacteria, with dominant species including Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio vulnificus. Vibrio vulnificus has become a systemic biosecurity threat: In aquaculture, these pathogens can induce devastating diseases such as acute hepatopancreatic necrosis in shrimp and ulcerative colitis in fish. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), such diseases can cause a single outbreak loss rate of over 30%. Through contamination of shellfish and other aquatic products, Vibrio genus members frequently cause foodborne poisoning incidents in humans, with Vibrio vulnificus infection having a mortality rate as high as 50%. More seriously, the problem of drug resistance is becoming increasingly prominent. Monitoring data from the past five years show that the positive detection rate of drug resistance genes for commonly used antibiotics such as enrofloxacin in aquaculture environment isolates has climbed to 67.2%. Traditional chemical control strategies face the dual challenges of cumulative ecotoxicity and declining governance effectiveness, and there is an urgent need to develop efficient, safe, and natural alternative antimicrobial strategies.

[0003] Although probiotics (such as GRAS-certified Enterococcus strains) demonstrate good biocontrol potential through multiple antibacterial mechanisms, including organic acid production, biofilm competition, and bacteriocin secretion, existing technologies have significant limitations: their antibacterial spectrum is significantly limited, with approximately 82% of reported strains effective only against a single Vibrio species; their environmental adaptability is significantly limited, with conventional probiotics exhibiting activity reductions exceeding 60% in salinity >3%, pH <5, or simulated gastrointestinal environments; and, more importantly, the stability of their antibacterial active substances is poor, severely restricting their practical effectiveness in industrial applications such as aquatic feed processing and cold chain storage and transportation. Therefore, screening microbial strains with broad-spectrum anti-Vibrio activity, significant probiotic functions, and excellent environmental stress tolerance has become a crucial research direction urgently needed. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0005] The Enterococcus of the present invention Enterococcus sp. 10-1, deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 33873, deposit date: March 19, 2025, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] This invention provides an anti-Vibrio enterococcal enterovirus. Enterococcus sp. 10-1, the preservation number of the enterococcus 10-1 is CGMCC No. 33873.

[0007] This invention provides a product containing the aforementioned enterococcus. Enterococcus sp. 10-1 microbial preparations.

[0008] This invention provides a product containing the aforementioned enterococcus. Enterococcus sp. 10-1 of the epigenetic.

[0009] In one embodiment, the metabiotic is the supernatant obtained by centrifuging the fermentation broth of Enterococcus 10-1.

[0010] The Enterococcus of the present invention Enterococcussp. 10-1 can maintain growth in a wide range of pH conditions (4-11) and in environments with 10% high salinity.

[0011] The present invention provides a product containing the Enterococcus 10-1, or containing the microbial preparation, or containing the postbiotic.

[0012] In one embodiment, the product includes pharmaceuticals or feed.

[0013] In one embodiment, the feed includes aquatic animal feed; the aquatic animals include freshwater cultured animals and marine cultured animals.

[0014] The present invention provides a Vibrio inhibitor, wherein the Vibrio inhibitor contains Enterococcus 10-1, or the microbial preparation, or the postbiotic.

[0015] In one embodiment, the Vibrio includes one or more of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio vulnificus.

[0016] This invention provides the use of the Enterococcus 10-1, the microbial preparation, or the postbiotic in the preparation of products that inhibit Vibrio, wherein the Vibrio includes one or more of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio vulnificus.

[0017] In one embodiment, the antibacterial component of the fermentation supernatant of Enterococcus 10-1 strain has the ability to resist acids and alkalis and high temperatures.

[0018] In one embodiment, the antibacterial component of Enterococcus 10-1 exerts its antibacterial effect under acidic conditions.

[0019] Beneficial effects: This invention provides a strain of Enterococcus ( Enterococcus sp.Enterococcus 10-1, wherein the Enterococcus 10-1 exhibits strong environmental tolerance and broad-spectrum anti-Vibrio activity, and its anti-Vibrio activity remains stable in high-temperature and strongly acidic environments. 1. Strong environmental adaptability: ① It exhibits remarkable salt tolerance, maintaining proliferation even in extremely high-salt environments with NaCl concentrations as high as 100 g / L, far exceeding the limitation of conventional probiotics that become inactive at salinity >3%. ② It has excellent acid and alkali resistance, maintaining growth activity in a wide pH range of 4-11, and especially stable proliferation under pH 5-10 conditions, solving the problem that existing antibacterial substances are easily inactivated in high-salt and high-pH aquaculture water.

[0020] 2. Broad-spectrum anti-Vibrio activity: The Enterococcus 10⁻¹ strain provided by this invention has broad-spectrum anti-Vibrio parahaemolyticus activity. V. parahaemolyticus ), Vibrio alginolyticus ( V. alginolyticus ), Vibrio harveyi ( V. harveyi ) and Vibrio vulnificus ( V. vulnificus It showed significant antibacterial effects against four representative pathogenic Vibrio species, breaking through the technical bottleneck of the single antibacterial spectrum of probiotics (only targeting a single Vibrio species) in the existing technology, and providing a new strategy for the synergistic prevention and control of multiple pathogenic Vibrio species in aquaculture.

[0021] 3. Excellent stability of antibacterial components: ① High temperature resistance: After being heated at 60~100℃ for 15 minutes or sterilized at 121℃, the antibacterial activity does not decrease significantly, breaking through the limitation of traditional heat-sensitive antimicrobial peptides being easily inactivated during processing, storage and transportation; ②Acid and alkali resistance: The antibacterial components are structurally stable within a pH range of 2-10, ensuring their long-lasting effect in complex environmental media; ③ Non-hydrogen peroxide dependent: It still maintains antibacterial activity after eliminating the interference of hydrogen peroxide, which proves that its active substance may be a novel antibacterial component.

[0022] Preservation of biological materials A strain of Enterococcus ( Enterococcus sp. 10-1, taxonomically named Enterococcus. Enterococcus sp. It was deposited on March 19, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33873, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0023] Figure 1 Colony characteristics and Gram staining of strain 10-1; Figure 2 Growth curves of strain 10-1 at different NaCl concentrations; Figure 3 Growth curves of strain 10-1 under different initial pH conditions; Figure 4 The antibacterial effect of fermentation supernatant of strain 10-1 on four types of Vibrio at different time points; Figure 5 Antibacterial effect of lyophilized samples of fermentation supernatant after reconstitution; Figure 6 Experiments to exclude hydrogen peroxide from antibacterial components; Figure 7 The optimal pH for antibacterial components to exert their antibacterial activity; Figure 8 pH stability of antibacterial components in fermentation broth supernatant; Figure 9 Temperature stability of antibacterial components in fermentation broth supernatant; Figure 10 Comparison of the ability of fermentation supernatant of strain 10-1 and commercial strain to inhibit Vibrio. Detailed Implementation

[0024] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application.

[0026] The method for preparing the Enterococcus 10-1 bacterial suspension in the following examples is as follows: Single colonies isolated from agar plates are inoculated into MRS liquid medium and incubated statically in a 37°C incubator for 5-6 hours until the optical density (OD) reaches the specified level. 600 When the optical density (OD) reaches 1.0, the seed culture is prepared. Subsequently, this seed culture is transferred to fresh MRS liquid medium at an inoculation rate of 5% (v / v) and cultured at 37°C for 18-24 hours to allow the bacterial growth to enter the stationary phase, at which point the optical density (OD) reaches its maximum value. 600 The value is approximately 1.8-2.2, and the bacterial count is approximately (8.2-10.1) × 10⁻¹⁰. 9 CFU / mL.

[0027] The method for obtaining the Enterococcus 10-1 fermentation supernatant and lyophilized powder involved in the following examples is as follows: A bacterial suspension in the late logarithmic growth phase or stationary phase is transferred to a low-temperature high-speed centrifuge and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant is collected as the bacterial fermentation broth. The obtained fermentation broth is then transferred to 50 mL sterile centrifuge tubes and pre-frozen in an ultra-low temperature freezer at -80°C. Lyophilization is performed using freeze-drying vacuum technology: the pre-frozen sample is placed in a pre-cooled drying chamber, and the vacuum system is activated to maintain a low-pressure environment to promote water sublimation, ultimately obtaining the dried fermentation broth lyophilized powder.

[0028] Unless otherwise specified, all percentages mentioned in the following examples refer to mass / volume fraction (m / V).

[0029] In the following examples, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, and Vibrio harveyi refer to Vibrio parahaemolyticus ATCC17802, Vibrio alginolyticus ATCC33787, Vibrio vulnificus ATCC27562, and Vibrio harveyi BB170, respectively.

[0030] Example 1: Screening and Identification of Strains Accurately weigh 1.0 g of animal feed sample and add it to 10 mL of sterile 0.9% sodium chloride solution. Shake on a shaker for 30 minutes (≥200 rpm) to mix thoroughly. Then, use a serial dilution plating method to isolate the bacterial strain, selecting an appropriate dilution range (10... -3 Up to 10 -6 The gradient dilutions of the culture were spread onto MRS agar plates and incubated at 37°C for 20 to 28 hours. Purification was then performed using the streak plating method (repeated 2-3 times) to obtain a homogeneous pure culture.

[0031] The purified strains were transferred to MRS liquid medium and incubated statically at 37°C for 18–24 h (220 rpm, volume ≤ 1 / 3). The supernatant was collected by centrifugation (4°C, 10,000 rpm, 10 min) for Oxford cup inhibition assays. Using *Vibrio parahaemolyticus* as an indicator bacterium, the Oxford cup inhibition zone method was employed, and six strains exhibiting the strongest inhibitory ability against *Vibrio* were selected by measuring the diameter of the inhibition zone. A secondary screening experiment was conducted using four types of *Vibrio* as indicator bacteria, and strain 10⁻¹ was selected that showed inhibitory effects against all four types of *Vibrio*. Colony morphology, Gram staining, and molecular identification of this bacterium were then performed.

[0032] 1) Strain characteristics After streaking glycerol bacteria onto MRS solid medium, the culture was incubated at 37°C for 20 hours. The colony morphology and size were then observed and photographed. The colonies formed small, white, round, smooth, and moist white colonies on the solid medium.

[0033] 2) Gram staining Single colonies on the plate were stained using a Gram staining kit and observed and photographed under a light microscope at 100x oil immersion. The results showed that the strain was Gram-positive and the cells were spherical.

[0034] 3) Molecular identification DNA extraction was performed according to the instructions of the Tiangen DNA Extraction Kit. Genomic DNA from the target strain was used as a template for PCR amplification of 16S rDNA using universal primers 27F and 1492R. The PCR reaction mixture (total 25 μL) consisted of: 0.5 μL genomic DNA, 12.5 μL 2×Taq, 0.5 μL each of forward and reverse primers, and 11.0 μL ddH2O.

[0035] PCR reaction conditions: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 55℃ annealing for 20 s, 72℃ extension for 40 s, 35 cycles; 72℃ repair extension for 10 min.

[0036] 1% agarose gel electrophoresis: The target band size was approximately 1500 bp. The PCR product with the correct molecular weight was sent to Anshengda Company for sequencing. The sequencing results were compared with the NCBI database using BLAST, confirming that strain 10-1 was Enterococcus. Enterococcus sp. The Enterococcus 10-1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33873.

[0037] Example 2: Safety and tolerability evaluation of Enterococcus 10-1 Enterococcus Enterococcus sp. Safety and tolerability of 10-1, including antibiotic sensitivity, salt ion tolerance, and initial pH growth stability.

[0038] 1) Antibiotic sensitivity After sterilizing the MRS solid medium containing 0.8% agar, and cooling it to approximately 50°C, add OD. 600nm A bacterial suspension of Enterococcus 10⁻¹ with a concentration of 1.0 was prepared to achieve a final concentration of 1 × 10⁻¹ in solid culture medium. 6CFU / mL, shake well and pour into plates. After the plates solidify, gently place the antibiotic susceptibility test strips on the surface. Incubate the plates at 37℃ for 20 h and record the diameter of the inhibition zone. Referring to YY / T 1191-2011 and the commonly used interpretation criteria for inhibition zone diameter in clinical microbiology testing: Sensitive (S): inhibition zone diameter ≥ 15 mm; Intermediate (I): inhibition zone diameter between 10-14 mm; Resistant (R): inhibition zone diameter < 10 mm. Table 1 shows the sensitive antibiotics (inhibition zone diameter ≥ 15 mm). As shown in Table 1, the results indicate that this strain exhibits sensitive responses to multiple antibiotics, suggesting high safety for clinical application.

[0039] Table 1: Diameter of the inhibition zone on antibiotic sensitivity test tablets

[0040] Note: The diameter of the blank drug sensitivity test tablet is 6 mm.

[0041] 2) High salt tolerance The growth curves of the strain under different culture medium conditions were measured using a growth curve analyzer. NaCl was added to MRS liquid medium to achieve concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 g / L. The medium was then autoclaved at 121℃ for 20 min, cooled to room temperature, and used for further processing. The activated seed culture (OD) was then... 600nm =1.0) was inoculated at a rate of 5% into the above-treated culture medium. 200 μL of each culture was added to a 96-well plate. The plates were then placed in a growth curve analyzer and cultured with shaking at 750 rpm for 48 h. The OD of the bacterial suspension was measured every 10 min. 600nm According to OD 600nm Plot the growth curve ( Figure 2 Experimental data show that this strain can still maintain slow growth in a medium containing 100 g / L NaCl; when the NaCl concentration in the medium is 60 g / L, the optical density (OD) of this strain reaches the maximum plateau value under natural culture conditions after 45 hours of culture; while in a medium containing 50 g / L NaCl or less, the NaCl concentration has no significant inhibitory effect on the growth of the strain.

[0042] 3) Acid and alkali tolerance The MRS medium was adjusted to pH 1-11 using 1 mol / L NaOH and 1 mol / L HCl, then autoclaved at 121°C for 20 min, cooled to room temperature, and used for further processing. The activated seed culture (OD) was then... 600 nm=1.0) was inoculated into the above-treated culture medium at a 5% (v / v) inoculation rate. 200 μL of each inoculated inoculation was added to a 96-well plate. The plates were then placed in a growth curve measuring instrument and cultured with shaking at 750 rpm for 48 h. The OD of the bacterial suspension was measured every 15 min. 600nm According to OD 600nm Plot the growth curve ( Figure 3 The results showed that the strain could not grow under extremely acidic conditions (initial pH=1, 2, 3), but could grow in the initial pH range of 4-11, with growth being basically unaffected in the wide pH range of 5-10.

[0043] Example 3: Antibacterial effect of Enterococcus 10-1 Enterococcus 10-1 fermentation supernatant and lyophilized powder were obtained according to the method in the specific embodiment, and the antibacterial effect of the fermentation supernatant and lyophilized powder on Vibrio was tested. Four Vibrio species were used as indicator bacteria: Vibrio parahaemolyticus ATCC17802, Vibrio alginolyticus ATCC33787, Vibrio vulnificus ATCC27562, and Vibrio harveyi BB170.

[0044] Enterococcus 10⁻¹ was cultured in MRS liquid medium, and samples were taken after 19 h (the viable count in the fermentation broth was approximately 8.0 × 10⁻¹). 9 (CFU / mL) and 26 h (the number of viable bacteria in the fermentation broth was approximately 10.5 × 10⁻⁶). 9 Centrifuge the bacterial suspension (CFU / mL) and collect the supernatant for later use.

[0045] The Oxford cup double-layer agar plate method was used for the Vibrio inhibition test: 2% agar was prepared, sterilized, and poured onto the bottom layer of a plate. Oxford cups were placed on top, and indicator bacteria (1×10⁻⁶) were added. 5 -1×10 6 The supernatant (cfu / mL) was added to high-salt LB medium (containing 3% NaCl) with 0.8% agar for double-layer plating. Oxford cups were removed, and 200 μL of fermentation supernatant was added. The cups were incubated at 30℃ for 12-20 h, and the diameter of the clear zone was recorded. The incubation time varied slightly due to different growth rates of Vibrio parahaemolyticus: Vibrio parahaemolyticus and Vibrio alginolyticus were incubated at 30℃ for 12 h, while Vibrio harveyi and Vibrio vulnificus were incubated at 30℃ for 15-20 h. The results showed that the supernatant components of this bacterium had a good inhibitory effect on all four types of Vibrio. Figure 4 ).

[0046] In addition, freeze-dried samples of fermentation supernatant from strain 10-1 were prepared, and the antibacterial activity of the reconstituted solution was tested using Vibrio parahaemolyticus and Vibrio alginolyticus as indicator bacteria. The results showed that the freeze-dried sample of fermentation culture supernatant also had antibacterial activity after reconstitution. Figure 5 ).

[0047] Example 4: Characterization of the antibacterial active ingredients in the fermentation supernatant of Enterococcus 10-1 The fermentation supernatant of strain 10-1 possesses antibacterial active ingredients. The fermentation supernatant was characterized by hydrogen peroxide removal experiments, the optimal pH for the antibacterial active ingredients, and the pH and temperature stability of the antibacterial active ingredients. The fermentation supernatant used in this example was obtained from strains with viable cell counts of 8.2-10.1 × 10⁻⁶. 9 It was obtained from bacterial culture at CFU / mL.

[0048] 1) Hydrogen peroxide removal experiment Hydrogen peroxide possesses broad-spectrum antibacterial activity, inhibiting the growth of pathogenic bacteria by causing oxidative damage and disrupting cell membranes. It exhibits a particularly strong inhibitory effect against Vibrio parahaemolyticus. To determine whether the antibacterial component was hydrogen peroxide, the pH of the fermentation broth was adjusted to 7.0, catalase (5 mg / mL) was added, and the mixture was allowed to stand in a 37°C water bath for 2 hours before being adjusted back to the initial pH. The Oxford cup experiment was then performed according to the method described in Example 3, and the diameter of the inhibition zone was measured. Figure 6 The results showed that the effective anti-Vibrio component in the fermentation supernatant was not hydrogen peroxide.

[0049] 2) The optimal pH for antibacterial components to exert their activity The pH of the fermentation supernatant was adjusted to 2-9 using 1 mol / L NaOH and 1 mol / L HCl, respectively. Following the method in Example 3, *Vibrio parahaemolyticus* and *Vibrio alginolyticus* were used as indicator bacteria, and the Oxford cup method was employed to detect the antibacterial effect and measure the diameter of the inhibition zone. The results showed that the antibacterial component exhibited antibacterial activity under acidic conditions (pH = 2-6). Figure 7 It exhibits the strongest antibacterial activity, especially at pH=2, with inhibition zone diameters of 30 mm and 28 mm for Vibrio parahaemolyticus and Vibrio alginolyticus, respectively; followed by pH=3, with inhibition zone diameters of 29 mm and 27 mm for Vibrio parahaemolyticus and Vibrio alginolyticus, respectively.

[0050] 3) Acid-base stability of antibacterial components The pH of the fermentation supernatant was adjusted to 2-10 using 1 mol / L NaOH and 1 mol / L HCl, and then allowed to stand at room temperature for 4 h to restore the initial pH. Following the method in Example 3, the antibacterial effect was verified using the Oxford cup method, and the diameter of the inhibition zone was measured. Figure 8 The results showed that the antibacterial component did not significantly inhibit its antibacterial activity after treatment under different pH conditions and adjustment back to the initial pH, indicating that the antibacterial component has good acid and alkali stability and tolerance.

[0051] 4) Temperature stability of antibacterial components The fermentation supernatant was heated at 60 °C, 70 °C, 80 °C, 90 °C, 100 °C and 121 °C for 15 min respectively, and then returned to room temperature. The Oxford cup experiment was performed according to the method in Example 3, with the untreated fermentation broth serving as a negative control. Figure 9 The results showed that heat treatment did not affect the antibacterial activity of the fermentation supernatant, and the fermentation supernatant or antibacterial component had good temperature stability and tolerance.

[0052] Comparative Example 1: The specific implementation method is as described in Example 3, except that the commercial strain *Lactococcus lactis* subsp. *lactococcus*, which produces nisin, is used. Lactococcus lactis subsp. lactis An antibacterial control experiment was conducted on the fermentation supernatant of CICC 6242, and the antibacterial effects were compared and tested. The results are as follows: Under the same culture conditions, the fermentation supernatant of Enterococcus 10-1 had a slightly stronger antibacterial ability than the fermentation supernatant of Lactococcus lactis subsp. lactis CICC 6242. The strain and fermentation supernatant of the present invention have certain advantages.

[0053] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Enterococcus ( Enterococcus sp. )10-1, the preservation number of the enterococcus 10-1 is CGMCC No.33873.

2. A microbial preparation containing the Enterococcus 10-1 as described in claim 1.

3. The postbiotic prepared from Enterococcus 10-1 according to claim 1.

4. The epigenetic agent according to claim 3, characterized in that, The metabiotic is the supernatant obtained by centrifuging the fermentation broth of Enterococcus 10-1.

5. A product containing the Enterococcus 10-1 of claim 1, the microbial preparation of claim 2, or the postbiotic of claim 3 or 4.

6. The product according to claim 5, characterized in that, The products include pharmaceuticals or animal feed.

7. The product according to claim 6, characterized in that, The feed includes aquatic animal feed; the aquatic animals include freshwater cultured animals and marine cultured animals.

8. A Vibrio inhibitor, characterized in that, The Vibrio inhibitor contains Enterococcus 10-1 as described in claim 1, or the microbial preparation as described in claim 2, or the postbiotic as described in claim 3 or 4.

9. The Vibrio inhibitor according to claim 8, characterized in that, The Vibrio species include one or more of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio vulnificus.

10. The use of the Enterococcus 10-1 of claim 1, or the microbial preparation of claim 2, or the metabiotic of claim 3 or 4, in the preparation of a product inhibiting Vibrio, characterized in that, The Vibrio species include one or more of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio vulnificus.

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