Enterococcus with vibrio resistance

Abstract

The invention discloses enterococcus with vibrio resistance, and belongs to the technical field of microorganisms. The enterococcus sp. 10-1 is separated from animal feed, the preservation number of the enterococcus sp. 10-1 is CGMCC (China General Microbiological Culture Collection Center) No.33873, and the enterococcus sp. 10-1 has an obvious inhibition effect on common vibrio parahaemolyticus, vibrio alginolyticus, vibrio harveyi and vibrio vulnificus. The strain has probiotic safety, and can grow under the initial pH condition of pH = 5-11 and in a high-salt environment. And the bacterium is sensitive to most common antibiotics. The antibacterial component of the bacterial strain acts on vibrio by being secreted to the outside of a cell, and the antibacterial component has pH stability and temperature stability and plays a role under an acidic condition. The invention lays a foundation for developing a good antibacterial probiotic preparation.

Description

Technical Field

[0001] The invention relates to enterococci with anti-vibrio ability, belonging to the technical field of microorganisms. Background Art

[0002] Vibrio is a typical representative group of Gram-negative pathogens. Its dominant species, such as V. parahaemolyticus, V. alginolyticus, V. harveyi, and V. vulnificus, have posed a systemic biosecurity threat. In aquaculture, these pathogens can induce devastating diseases such as acute hepatopancreatic necrosis of shrimp and fish ulcer disease. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), It shows that such diseases can cause a single epidemic loss rate of more than 30%; by contaminating shellfish and other aquatic products, members of the genus Vibrio cause frequent foodborne poisoning incidents in humans, among which the mortality rate of Vibrio vulnificus infection is as high as 50%; what is more serious is that the problem of drug resistance is becoming increasingly prominent. Monitoring data in the past five years show that the positive detection rate of drug-resistant genes of isolates from aquaculture environments to commonly used antibiotics such as enrofloxacin has climbed to 67.2%. Traditional chemical prevention and control strategies face the dual challenges of cumulative ecotoxicity effects and diminished governance effectiveness. There is an urgent need to develop efficient, safe and natural alternative antibacterial strategies.

[0003] Although probiotics (such as GRAS-certified Enterococcus strains) have shown good biocontrol potential through multi-pathway antibacterial mechanisms such as organic acid production, biofilm competition and bacteriocin secretion, the existing technology system has significant limitations: the antimicrobial spectrum is significantly limited, with approximately 82% of reported strains being effective only against a single Vibrio species; there are significant limitations in environmental adaptability, with conventional probiotics experiencing a decrease in activity of more than 60% in salinity >3%, pH <5, or in simulated gastrointestinal fluid environments; more importantly, the stability of antimicrobial active substances is poor, which seriously restricts their actual effectiveness in industrial application scenarios 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 key research direction that needs to be carried out urgently. Summary of the Invention

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

[0005] The Enterococcus sp. 10-1 of the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No. 33873, a deposit date of March 19, 2025, and an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The invention provides an enterococcus sp. 10-1 with resistance to Vibrio. The deposit number of the enterococcus 10-1 is CGMCC No. 33873.

[0007] The invention provides a microbial preparation containing the Enterococcus sp. 10-1.

[0008] The invention provides a postbiotic containing the Enterococcus sp.10-1.

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

[0010] The Enterococcus sp. 10-1 of the present invention can maintain growth under a wide range of pH conditions (4-11) and a 10% high salt environment.

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

[0012] In one embodiment, the product includes food, medicine, health product or feed.

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

[0014] The present invention provides a Vibrio inhibitor, which contains the 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] The present invention provides the use of the enterococcus 10-1 or the microbial preparation or the postbiotic in preparing a product for inhibiting 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 the Enterococcus 10-1 strain has the ability to resist acid, alkali and high temperature.

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

[0019] Beneficial effects:

[0020] The present invention provides an Enterococcus sp. 10-1 strain, which has strong environmental tolerance and broad-spectrum anti-Vibrio activity, and has stable anti-Vibrio activity in high temperature and strong acid environments:

[0021] 1. Strong environmental adaptability:

[0022] ① It has remarkable salt tolerance and can continue to proliferate in extremely high-salt environments with NaCl concentrations as high as 100g / L, far exceeding the limitation of conventional probiotics that are inactivated when salinity exceeds 3%;

[0023] ② It has excellent acid and alkali resistance and maintains growth activity in a wide range of pH 4-11. In particular, it proliferates stably under pH 5-10, solving the problem that existing antibacterial substances are easily inactivated in high-salt and high-pH water bodies in aquaculture.

[0024] 2. Broad-spectrum anti-Vibrio activity: The Enterococcus 10-1 provided by the present invention has a significant antibacterial effect on four representative pathogenic Vibrio, including Vibrio parahaemolyticus (V. parahaemolyticus), Vibrio alginolyticus (V. alginolyticus), Vibrio harveyi (V. harveyi) and Vibrio vulnificus (V. vulnificus), breaking through the technical bottleneck of the existing technology that probiotics have a single antibacterial spectrum (only targeting a single Vibrio species), and provides a new strategy for the coordinated prevention and control of multi-pathogenic Vibrio in aquaculture.

[0025] 3. Outstanding stability of antibacterial ingredients:

[0026] 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 that are easily inactivated during processing, storage and transportation;

[0027] ② Acid and alkali resistance: The antibacterial component structure is stable within the pH range of 2-10, ensuring its long-term effect in complex environmental media;

[0028] ③ Non-hydrogen peroxide dependence: The antibacterial activity is still maintained after eliminating the interference of hydrogen peroxide, proving that its active substance may be a new antibacterial component.

[0029] Biomaterial Deposit

[0030] A strain of Enterococcus sp. 10-1, taxonomically named Enterococcus sp., was deposited in the General Microbiology Center of the China Culture Collection Administration on March 19, 2025, with the deposit number CGMCC No. 33873, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Colony characteristics and Gram staining of strain 10-1;

[0032] Figure 2 Growth curves of strain 10-1 at different NaCl concentrations;

[0033] Figure 3 Growth curves of strain 10-1 under different initial pH conditions;

[0034] Figure 4 The antibacterial effect of the fermentation supernatant of strain 10-1 at different times on four Vibrio species;

[0035] Figure 5 Antibacterial effect of reconstituted lyophilized samples of fermentation supernatant;

[0036] Figure 6 Antibacterial component exclusion hydrogen peroxide experiment;

[0037] Figure 7 The optimal pH for the antibacterial components to exert their antibacterial activity;

[0038] Figure 8 pH stability of antibacterial components in the fermentation broth supernatant;

[0039] Figure 9 Temperature stability of antibacterial components in fermentation broth supernatant;

[0040] Figure 10 Comparison of the inhibitory ability of fermentation supernatants of strain 10-1 and commercial strains against Vibrio. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application.

[0043] The preparation method of the Enterococcus 10-1 bacterial suspension involved in the following examples is: a single colony isolated from an agar plate is inoculated into MRS liquid culture medium, and cultured in a constant temperature incubator at 37°C for 5-6 hours until the optical density (OD 600 ) value reaches 1.0, and the seed solution is prepared. Subsequently, the seed solution is transferred to fresh MRS liquid medium at a 5% (v / v) inoculation volume and cultured in a 37°C constant temperature incubator for 18-24 hours to allow the bacterial growth to enter the stable phase. At this time, the optical density (OD 600) value is about 1.8-2.2, and the number of bacteria is about (8.2-10.1)×10 9 CFU / mL.

[0044] The method for obtaining the fermentation supernatant and lyophilized powder of Enterococcus 10-1 involved in the following embodiments is as follows: the bacterial suspension in the late logarithmic growth phase or the stable phase is transferred to a low-temperature high-speed centrifuge, centrifuged at 10,000 rpm for 10 minutes at 4°C, and the supernatant is collected as the bacterial fermentation liquid. The resulting fermentation liquid is then transferred to a 50mL sterile centrifuge tube and placed in a -80°C ultra-low temperature refrigerator for pre-freezing. The freeze-drying process is completed using freeze vacuum drying technology: the pre-frozen sample is placed in a pre-cooled drying chamber, the vacuum system is started to maintain a low-pressure environment to promote water sublimation, and finally a dry fermentation liquid freeze-dried powder is obtained.

[0045] Unless otherwise specified, the percentages mentioned in the following examples refer to mass / volume fractions (m / V).

[0046] The Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, and Vibrio harveyi involved in the following embodiments refer to Vibrio parahaemolyticus ATCC17802, Vibrio alginolyticus ATCC33787, Vibrio vulnificus ATCC27562, and Vibrio harveyi BB170, respectively.

[0047] Example 1: Screening and identification of strains

[0048] 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 (speed ≥ 200 rpm) to mix evenly. Then, apply the gradient dilution coating method to isolate the strains. Select the appropriate dilution range (10 -3 to 10 -6 ) were spread onto MRS agar plates and incubated in a 37°C incubator for 20 to 28 hours. Purification was then performed using the plate streak method (repeated 2-3 times) to obtain a pure culture with uniform morphology.

[0049] The purified strain was transferred to MRS liquid culture medium and cultured in a 37°C incubator for 18-24 hours (220 rpm, liquid volume ≤ 1 / 3). Centrifugation (4°C, 10,000 rpm, 10 min) was performed to collect the supernatant for the Oxford cup antibacterial test. Vibrio parahaemolyticus was used as the indicator bacteria, and the Oxford cup inhibition zone method was used to screen out 6 strains with the strongest antibacterial ability to Vibrio by measuring the diameter of the inhibition zone. In the rescreening experiment, four Vibrio species were set as indicator bacteria, and the strain 10-1 with an inhibitory effect on all four Vibrio species was selected through the antibacterial experiment. The bacteria were subjected to colony morphology, Gram staining and molecular identification.

[0050] 1) Strain characteristics

[0051] Streak the glycerol stock onto MRS solid medium and incubate in a 37°C incubator for 20 hours. Observe the colony morphology and size and take photos. White, round, smooth, moist microcolonies should form on the solid medium.

[0052] 2) Gram staining

[0053] Single colonies on the plates 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 with spherical cell morphology.

[0054] 3) Molecular identification

[0055] DNA extraction was performed according to the Tiangen DNA Extraction Kit instructions. 16S rDNA was amplified by PCR using genomic DNA of the target strain as a template, using universal primers 27F and 1492R. The PCR reaction system (25 μL total) consisted of 0.5 μL genomic DNA, 12.5 μL 2× Taq, 0.5 μL each of the upstream and downstream primers, and 11.0 μL ddH2O.

[0056] PCR reaction conditions: pre-denaturation at 95°C for 4 min; denaturation at 94°C for 30 s, annealing at 55°C for 20 s, extension at 72°C for 40 s, 35 cycles; repair and extension at 72°C for 10 min.

[0057] 1% agarose gel electrophoresis revealed a band of approximately 1500 bp. The PCR product with the correct molecular weight was sequenced by Anshengda. BLAST comparison of the sequencing results against the NCBI database confirmed that strain 10-1 was Enterococcus sp. Enterococcus 10-1 was deposited with the General Microbiology Center of the China General Culture Collection Administration under the accession number CGMCC No. 33873.

[0058] Example 2: Safety and Tolerability Evaluation of Enterococcus 10-1

[0059] Safety and tolerability of Enterococcus sp.10-1, including antibiotic sensitivity, salt ion tolerance, and initial pH growth stability.

[0060] 1) Antibiotic sensitivity

[0061] After the MRS solid medium containing 0.8% agar was sterilized and cooled to about 50°C, OD 600nm =1.0, so that the final concentration in the solid culture medium is 1×10 6CFU / mL, shake well, and pour onto the plate. After the plate solidifies, gently place the antibiotic-containing susceptibility plate on the surface. Incubate the plate in a 37°C incubator for 20 hours and record the diameter of the inhibition zone. (Referring to YY / T 1191-2011 and the Interpretation Standards for Inhibition Zone Diameters of Commonly Used Drug Susceptibility Tests in Clinical Microbiology): 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 antibiotics to which it is susceptible (inhibition zone diameter ≥ 15 mm). As shown in Table 1, the results indicate that this strain is sensitive to multiple antibiotics, suggesting a high safety profile for clinical use.

[0062] Table 1: Diameter of inhibition zone of antibiotic sensitive tablets

[0063]

[0064] Note: The diameter of the blank drug sensitive tablet is 6 mm.

[0065] 2) High salt tolerance

[0066] The growth curve of the strain under different culture medium conditions was measured using a growth curve analyzer. NaCl was added to the MRS liquid medium to make its mass concentration 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 g / L, respectively. The medium was sterilized by high pressure steam at 121°C for 20 min, cooled to room temperature and then used. The activated seed solution (OD 600nm =1.0) was inoculated into the treated culture medium at a 5% inoculum volume, and 200 μL was taken and added to a 96-well plate. The plate was placed in a growth curve analyzer for incubation at 750 rpm for 48 h. The OD value of the bacterial suspension was measured every 10 min. 600nm , according to OD 600nm Draw a growth curve ( Figure 2 Experimental data showed that the strain could maintain slow growth in a culture medium containing 100 g / L NaCl. When the NaCl concentration in the culture medium was 60 g / L, the optical density (OD) of the strain reached the maximum platform value under natural culture conditions after 45 hours of culture. In a culture medium containing 50 g / L NaCl concentration or less, NaCl concentration had no significant inhibitory effect on the growth of the strain.

[0067] 3) Acid and alkali tolerance

[0068] The pH of the MRS medium was adjusted to 1-11 with 1 mol / L NaOH and 1 mol / L HCl, and then sterilized by high pressure steam at 121°C for 20 min. The medium was cooled to room temperature for later use. The activated seed solution (OD 600nm=1.0) was inoculated into the treated culture medium at a volume of 5% (v / v), and 200 μL was added to each 96-well plate. The plate was placed in a growth curve analyzer for incubation at 750 rpm for 48 h, and the OD value of the bacterial suspension was measured every 15 min. 600nm , according to OD 600nm Draw a 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 within the initial pH range of 4-11, and was basically unaffected in the broad pH range of 5-10.

[0069] Example 3: Antibacterial effect of Enterococcus 10-1

[0070] Enterococcus 10-1 fermentation supernatant and lyophilized powder were obtained according to the methods described in the specific embodiments, and the antibacterial effects of the fermentation supernatant and lyophilized powder on Vibrio spp. The four Vibrio spp. used as indicator bacteria were Vibrio parahaemolyticus ATCC17802, Vibrio alginolyticus ATCC33787, Vibrio vulnificus ATCC27562, and Vibrio harveyi BB170.

[0071] Enterococcus 10-1 was cultured in MRS liquid medium, and the samples were taken for 19 h (the number of viable bacteria in the fermentation broth was about 8.0×10 9 CFU / mL) and 26h (the number of viable bacteria in the fermentation broth was about 10.5×10 9 CFU / mL) of bacterial suspension was centrifuged and the supernatant was taken for use.

[0072] The Vibrio inhibition test was performed using the Oxford cup double-layer agar plate method: 2% agar was prepared and sterilized and poured onto the bottom of the plate, and the Oxford cup was placed on top. The indicator bacteria (1×10 5 -1×10 6 cfu / mL) was added to a high-salt LB medium (NaCl containing 3%) containing 0.8% agar for double-layer plating. Remove the Oxford cup, add 200μL of the fermentation supernatant, and incubate at 30℃ for 12-20h, and record the diameter of the transparent zone. Among them, due to the different growth rates of Vibrio, the incubation time is slightly different: Vibrio parahaemolyticus and Vibrio alginolyticus are incubated at 30℃ for 12h, Vibrio harveyi and Vibrio vulnificus are incubated at 30℃ for 15-20h. The results showed that the supernatant components of the bacteria have a good inhibitory effect on all four Vibrio species ( Figure 4 ).

[0073] In addition, freeze-dried samples of the fermentation supernatant of the 10-1 strain were prepared, and the reconstituted solution was tested for its antibacterial activity against Vibrio parahaemolyticus and Vibrio alginolyticus. The results showed that the reconstituted freeze-dried sample of the fermentation culture supernatant also had antibacterial activity ( Figure 5 ).

[0074] Example 4: Characterization of the antibacterial active ingredients in the fermentation supernatant of Enterococcus 10-1

[0075] The fermentation supernatant of strain 10-1 has antibacterial active ingredients. The fermentation supernatant was characterized by hydrogen peroxide exclusion test, optimal pH of the antibacterial active ingredients, pH stability and temperature stability of the antibacterial active ingredients. The fermentation supernatant used in this example was obtained from a strain with a viable cell count of 8.2-10.1×10 9 CFU / mL of bacterial solution was obtained.

[0076] 1) Hydrogen peroxide elimination experiment

[0077] Hydrogen peroxide has a broad spectrum of antibacterial activity and can inhibit the growth of pathogenic bacteria by causing oxidative damage and destroying cell membranes. It has a particularly strong inhibitory effect on Vibrio parahaemolyticus. In order to determine whether the antibacterial component is hydrogen peroxide, the pH of the fermentation broth was adjusted to 7.0, and catalase (5 mg / mL) was added. After standing in a 37°C water bath for 2 hours, the pH was adjusted to the initial pH. The Oxford cup test was performed according to the method of Example 3 and the diameter of the inhibition zone was measured ( Figure 6 The results showed that the active ingredient against Vibrio in the fermentation supernatant was not hydrogen peroxide.

[0078] 2) The optimal pH for the antibacterial components to be active

[0079] The fermentation supernatant was adjusted to pH 2-9 with 1 mol / L NaOH and 1 mol / L HCl, respectively. The antibacterial effect was detected by the Oxford cup method using Vibrio parahaemolyticus and Vibrio alginolyticus as indicator bacteria, and the diameter of the inhibition zone was measured. The results showed that the antibacterial component had antibacterial activity under acidic conditions (pH = 2-6) ( Figure 7 ), especially at pH = 2, it has the strongest antibacterial activity, with the diameters of the inhibition zones for Vibrio parahaemolyticus and Vibrio alginolyticus being 30 mm and 28 mm, respectively; followed by pH = 3, with the diameters of the inhibition zones for Vibrio parahaemolyticus and Vibrio alginolyticus being 29 mm and 27 mm, respectively.

[0080] 3) Acid-base stability of antibacterial ingredients

[0081] The fermentation supernatant was adjusted to pH 2-10 with 1 mol / L NaOH and 1 mol / L HCl, and the initial pH was restored after standing at room temperature for 4 h. Referring to the method of Example 3, the antibacterial effect was verified by the Oxford cup method and the diameter of the inhibition zone was measured ( Figure 8 The results showed that after the antibacterial components were treated under different pH conditions, there was no obvious inhibitory effect on their antibacterial activity after the initial pH was adjusted back to the original pH, indicating that the antibacterial components had good acid-base stability and tolerance.

[0082] 4) Temperature stability of antibacterial ingredients

[0083] 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 test was carried out according to the method of Example 3, and the fermentation broth without high temperature treatment was used as the 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 the antibacterial component had good temperature stability and tolerance.

[0084] Comparative Example 1:

[0085] Specific embodiments are as in Example 3, except that the fermentation supernatant of the commercial strain Lactococcus lactis subsp. lactis CICC 6242, which produces nisin, was used to perform an antibacterial control experiment, 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 has 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.

[0086] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Enterococcus sp. 10-1, the deposit number of which is CGMCC No. 33873.

2. A microbial preparation containing the Enterococcus 10-1 according to claim 1.

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

4. The postbiotic according to claim 3, characterized in that The postbiotics are the supernatant obtained by centrifuging the fermentation broth of the Enterococcus 10-1.

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

6. The product according to claim 5, characterized in that: The products include food, medicine, health products or feed.

7. The product according to claim 6, characterized in that The feed includes feed for aquatic animals; the aquatic animals include freshwater farmed animals and seawater farmed animals.

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

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

10. Use of the enterococcus 10-1 according to claim 1, the microbial preparation according to claim 2, or the postbiotic according to claim 3 or 4 in preparing a product for inhibiting Vibrio, characterized in that: The Vibrio includes one or more of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio vulnificus.

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