A strain of Enterococcus faecalis with high polyphenol oxidase production and its application

Abstract

The present application provides a strain of Enterococcus faecalis that produces high levels of polyphenol oxidase and its application. The Enterococcus faecalis is named Enterococcus faecalis Z2MX and was deposited in the China Center for Type Culture Collection on May 14, 2024, with a deposit number of CCTCC NO: M 2024920. The Enterococcus faecalis has the ability to produce high levels of polyphenol oxidase, and has good antibacterial and antioxidant abilities, and can improve and protect intestinal oxidative damage.

Description

Technical Field

[0001] The present application relates to the field of microorganisms, and in particular to a strain of Enterococcus faecalis that produces high amounts of polyphenol oxidase and its application. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Polyphenol oxidase (PPO) is a type of metalloproteinase that exists in various animals, plants and microorganisms and participates in their metabolic reactions. It can specifically catalyze the oxidation of polyphenol compounds to form corresponding quinone substances and is the first major type of oxidoreductase.

[0004] The strains that have been reported to produce polyphenol oxidase include bacteria, molds, yeasts and actinomycetes. Among bacteria, such as Pseudomonas ( Pseudomonas spp.) and Bacillus ( Bacillus spp.) can produce polyphenol oxidase; molds, such as Aspergillus niger and Rhizopus Rhizopus spp.) can also secrete polyphenol oxidase; yeast, such as Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) and Kava Saccharomyces cerevisiae ( Kluyveromyces spp.) can also produce a certain amount of polyphenol oxidase; among actinomycetes, such as Streptomyces spp., which are common in the soil, they can also produce polyphenol oxidase. The main functions of polyphenol oxidase include catalyzing the oxidation of phenolic compounds to produce quinone compounds, which further polymerize to form brown pigments, causing plants or fruits and vegetables to turn brown after cutting or damage. In plants, polyphenol oxidase participates in injury response and pathogen resistance, producing antimicrobial quinones to help plants resist pathogens and pests. In the food industry, polyphenol oxidase is used in fermentation processes such as tea and coffee to enhance flavor and color, and is also used to treat phenolic pollutants in wastewater, using its enzymatic oxidation reaction to degrade harmful substances. In addition, polyphenol oxidase is also used in the pharmaceutical and cosmetic industries for the biosynthesis of certain drug ingredients and the development of whitening and antioxidant products, using its catalytic effect to reduce free radical damage. Therefore, polyphenol oxidase has important application value in many fields, and strains producing polyphenol oxidase also provide an important enzyme source for related industrial production. Summary of the invention

[0005] The present invention provides a strain of Enterococcus faecalis with high polyphenol oxidase production and application thereof. The Enterococcus faecalis is separated from the intestinal contents of spiders, has the ability to produce high polyphenol oxidase and inhibit bacteria, and has good antioxidant properties, and can be used to improve obesity and alleviate intestinal damage.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect of the present invention, a strain of Enterococcus faecalis is provided, which is named Enterococcus faecalis ( Enterococcus faecalis )Z2MX, and was deposited in the China Center for Type Culture Collection on May 14, 2024. Its deposit number is CCTCC NO: M 2024920, and the deposit address is Wuhan University, Wuhan, China.

[0008] The Enterococcus faecalis ( Enterococcus faecalis ) Z2MX has a high efficiency in producing polyphenol oxidase and exhibits significant antioxidant activity. For example, in some embodiments of the present invention, the strain is cultured in MRS medium at 37°C in an anaerobic environment for 48 hours, and its polyphenol oxidase activity can be as high as 15.00 U / mL fermentation supernatant.

[0009] The Enterococcus faecalis ( Enterococcus faecalis ) Z2MX has good antibacterial ability and a wide antibacterial spectrum. It exhibits good antibacterial effects on at least Escherichia coli, Staphylococcus aureus, Salmonella, Aeromonas hydrophila, Clostridium perfringens, Vibrio parahaemolyticus and Vibrio alginolyticus, and its inhibition zone is all above 13.5 mm.

[0010] And, the Enterococcus faecalis ( Enterococcus faecalis ) Z2MX showed good antioxidant activity. According to the test results, it has the ability to resist superoxide anions, scavenge DPPH free radicals, inhibit hydroxyl free radicals, reduce malondialdehyde content, increase glutathione peroxidase activity, increase total superoxide dismutase activity, increase polyphenol oxidase (PPO) activity, increase total antioxidant capacity and increase catalase activity.

[0011] In a second aspect of the present invention, the Enterococcus faecalis ( Enterococcus faecalis ) Fermentation of Z2MX.

[0012] In a third aspect of the present invention, a composition is provided, comprising the Enterococcus faecalis ( Enterococcus faecalis ) Z2MX or its fermentation product.

[0013] In a fourth aspect of the present invention, a product is provided, comprising the Enterococcus faecalis ( Enterococcus faecalis) Z2MX or a fermentation product thereof, and at least one auxiliary material.

[0014] In some embodiments of the present invention, the product is a medicine, an antibacterial agent, an antioxidant, a feed additive or a microecological preparation.

[0015] In some embodiments of the present invention, the auxiliary materials are safe and stable, and their types may include but are not limited to the following categories as needed: lyoprotectants, stabilizers, solvents, excipients, antioxidants, fillers, carriers, etc.

[0016] The freeze-drying protective agent is used to protect the activity and stability of the strain or its fermentation product during the freeze-drying process. Specific ingredients include but are not limited to lactose, maltodextrin, pectin, sodium alginate, sodium caseinate, mannitol, milk powder, etc.

[0017] The stabilizer is used to maintain the activity of the strain or its fermentation product during storage and use. Specific ingredients include but are not limited to polyethylene glycol, glycerol, propylene glycol, hypromellose, polysorbate, etc.

[0018] The solvent is used to dissolve and dilute the strain or its fermentation product so as to prepare different dosage forms. Specific ingredients include but are not limited to ethanol, propylene glycol, water, etc.

[0019] The excipient is used to give the product a suitable physical form for easy preparation and use. Specific ingredients include but are not limited to starch, microcrystalline cellulose, talc, magnesium stearate, etc.

[0020] The antioxidant is used to prevent the oxidative degradation of the strain or its fermentation product during storage. Specific ingredients include but are not limited to ascorbic acid (vitamin C), tocopherol (vitamin E), citric acid, sodium citrate, sodium ascorbate, etc.

[0021] The filler is used to increase the volume of the product, making it easier to handle and use. Specific ingredients include but are not limited to corn starch, wheat flour, soybean meal, fish meal, etc.

[0022] The carrier is used to carry and stabilize the active ingredients of the strain or its fermentation product. Specific ingredients include but are not limited to lactose, maltodextrin, pectin, sodium alginate, sodium caseinate, mannitol, etc.

[0023] In addition, the composition or product may also contain other therapeutically effective ingredients.

[0024] It should be understood that the above list of auxiliary materials is not exhaustive but exemplary. Those skilled in the art can select other appropriate auxiliary materials according to actual needs, and these selections should be included in the protection scope of the present invention without departing from the spirit and scope of the present invention.

[0025] The product of the present invention can be prepared into different dosage forms or states, including but not limited to tablets, capsules, oral liquids, injections, powders, solutions, sprays, ointments, creams, gels, granules, powders, granular feeds, premixes, liquid preparations, etc. Those skilled in the art will appreciate that the dosage form and state of the product can be adjusted and optimized according to specific applications and usage requirements, and these adjustments and optimizations should all be included in the protection scope of the present invention.

[0026] The product of the present invention can be prepared according to any conventional method known in the art. In some embodiments of the present invention, the composition can be prepared into bacterial powder, and the bacterial powder can further contain a lyophilization protectant, and the amount of the lyophilization protectant added is 0.5-95% of the weight of the raw material, preferably 5-85%. The lyophilization protectant can adopt a conventional lyophilization protectant in the art, such as skimmed milk powder, etc. When preparing other products, bacterial powder can be directly added as a raw material. Of course, this is just an example. The Enterococcus faecalis described in the present invention can also be used as a raw material in the form of bacterial liquid, bacterial body or its fermentation product to prepare products.

[0027] For example, when preparing a drug, the method may include:

[0028] (1) Drying the bacterial liquid or bacterial fermentation product;

[0029] (2) then mixing with the auxiliary materials in proportion;

[0030] (3) Prepare dosage forms such as tablets, capsules, oral solutions or injections according to the pharmaceutical process;

[0031] (4) Conduct quality inspection on finished products to ensure that they meet pharmaceutical standards.

[0032] For example, when preparing an antibacterial agent, the method may include:

[0033] (1) Mixing the bacterial solution or bacterial fermentation product with a solvent (such as ethanol or propylene glycol);

[0034] (2) Add other excipients or excipients (such as polysorbate, sodium benzoate) and stir evenly;

[0035] (3) preparing the mixture into a solution, spray, ointment, cream or gel;

[0036] (4) Packaging and stability testing.

[0037] For example, when preparing an antioxidant, the method may include:

[0038] (1) Drying the bacterial liquid or bacterial fermentation product into powder;

[0039] (2) mixing the dry powder with antioxidant excipients such as ascorbic acid and tocopherol;

[0040] (3) in the form of tablets, capsules, oral liquids or powders;

[0041] (4) Test antioxidant activity to ensure product quality.

[0042] For example, when preparing a feed additive, the method may include:

[0043] (1) Drying the bacterial liquid or bacterial fermentation product into powder;

[0044] (2) Mixing the dry powder with corn starch, wheat flour, soybean meal, etc.;

[0045] (3) Made into pellets, powder or granular feed;

[0046] (4) Conduct nutritional analysis to ensure compliance with feed standards.

[0047] For example, when preparing a microecological preparation, the method may include:

[0048] (1) freeze-drying the bacterial solution or bacterial fermentation product;

[0049] (2) mixing the dry powder with a carrier such as lactose or maltodextrin;

[0050] (3) in the form of powder, granules, liquid preparation or capsule;

[0051] (4) Perform colony counting and activity testing to ensure product quality.

[0052] In a fifth aspect of the present invention, the Enterococcus faecalis ( Enterococcus faecalis ) Application of Z2MX or its fermentation product in the production of polyphenol oxidase.

[0053] In a sixth aspect of the present invention, the Enterococcus faecalis ( Enterococcus faecalis ) Use of Z2MX or its fermentation products in the preparation of antibacterial agents, antioxidants, products for controlling weight, improving obesity, or preventing and / or improving intestinal damage.

[0054] In some embodiments of the present invention, the antibacterial agent has an antibacterial effect on at least Escherichia coli, Staphylococcus aureus, Salmonella, Aeromonas hydrophila, Clostridium perfringens, Vibrio parahaemolyticus and Vibrio alginolyticus.

[0055] In some embodiments of the present invention, the antioxidant has at least the ability to resist superoxide anions, scavenge DPPH free radicals, inhibit hydroxyl free radicals, reduce malondialdehyde content, increase glutathione peroxidase activity, increase total superoxide dismutase activity, increase polyphenol oxidase activity, increase total antioxidant capacity and increase catalase activity.

[0056] In some embodiments of the present invention, the weight control is to avoid weight gain caused by a high-iron diet.

[0057] In some embodiments of the present invention, the obesity is obesity caused by a high-iron diet.

[0058] In some embodiments of the present invention, the intestinal damage is intestinal oxidative damage caused by a high-iron diet.

[0059] In some embodiments of the present invention, the product is a medicine, a feed additive or a microecological preparation.

[0060] In a seventh aspect of the present invention, a method for producing polyphenol oxidase is provided, comprising: fermenting and culturing the Enterococcus faecalis ( Enterococcus faecalis ) Z2MX to produce polyphenol oxidase, and the polyphenol oxidase is isolated. If necessary, the method further comprises a purification step.

[0061] The Enterococcus faecalis of the present invention ( Enterococcus faecalis ) Z2MX is a facultative anaerobic bacterium that can grow under aerobic or anaerobic conditions and can grow well at temperatures between 30-37°C.

[0062] For example, in one embodiment of the present invention, the method includes:

[0063] Inoculate and culture the Enterococcus faecalis ( Enterococcus faecalis )Z2MX, continue to culture for 24-48 hours until the strain reaches the logarithmic growth phase or stable phase. Samples can be taken regularly during the fermentation process to detect the production and enzyme activity of polyphenol oxidase.

[0064] After the fermentation is completed, the supernatant is collected by centrifugation (e.g., 4°C, 8000 rpm, 10 minutes) to obtain a crude enzyme extract.

[0065] If necessary, the crude enzyme extract can be purified, for example, by salting out, dialysis, ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc.

[0066] In an eighth aspect of the present invention, a method for protecting and / or improving intestinal oxidative damage is provided, which comprises providing an effective amount of the Enterococcus faecalis ( Enterococcus faecalis) Z2MX or a fermentation product thereof, or containing the Enterococcus faecalis ( Enterococcus faecalis ) A composition or product of Z2MX or a fermentation thereof.

[0067] In the present invention, the "subject" refers to an animal that has been or will be the subject of treatment, observation or experiment, such as a pig, especially a piglet. The methods described herein can be used in veterinary applications.

[0068] In the present invention, the effective amount required to achieve the above functions can be routinely determined by considering one's own knowledge, the existing technical level and those skilled in the art, such as determining or demonstrating the amount through in vivo, in vitro or ex vivo experiments, and then applying the amount to better implement the method described in the present invention. For example, in some embodiments of the present invention, when mice are treated by gavage with the fermentation broth of the strain described in the present invention, the daily dose received by each mouse is 1×10 7 CFU fermentation broth.

[0069] Compared with the prior art, the advantages of the present invention include:

[0070] The present invention provides a strain of Enterococcus faecalis with high polyphenol oxidase production and application thereof. The Enterococcus faecalis is separated from the intestinal contents of spiders, has the ability to produce high polyphenol oxidase and inhibit bacteria, and has good antioxidant properties, and can protect and improve intestinal oxidative damage and improve obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The drawings constituting part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. The implementation scheme of the present application is described in detail below in conjunction with the drawings, wherein:

[0072] Figure 1 The colony morphology of strain Z2MX after being cultured at 37 °C for 48 h is shown.

[0073] Figure 2 The morphological characteristics of strain Z2MX are shown.

[0074] Figure 3 The staining results of paraffin sections of colon tissues of mice in each group are shown (×100). DETAILED DESCRIPTION

[0075] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0076] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0077] Example 1 Strain isolation, screening and identification

[0078] 1 Materials and methods

[0079] 1.1 Separation source

[0080] Black soldier fly larvae, earthworms, spiders and mealworm intestinal contents.

[0081] 1.2 Experimental methods

[0082] 1.2.1 Strain isolation and screening

[0083] After the black soldier fly larvae, earthworms, spiders and mealworms were rinsed with clean water, they were soaked in 75% alcohol for 1 min, repeated twice, and then rinsed with sterile water for 3 times. The intestinal contents of the above insect bodies were taken out, placed in sterile saline and mixed evenly, and then spread on MRS culture medium plates with 0.5% CaCO3, and cultured anaerobically at 37 °C for 48 h, and the colonies with larger transparent circles were selected. (MRS culture medium was purchased from Qingdao Haibo Biological)

[0084] 1.2.2 Preparation of fermentation supernatant

[0085] The strains with larger transparent zones selected in 1.2.1 of the present invention were picked up with an inoculation loop and streaked onto new MRS solid slant medium to activate the strains. The activated strains were inoculated into MRS liquid medium and cultured at 37°C for 48 hours to obtain fermentation broth, which was centrifuged at 4°C and 8000 r / min for 10 minutes, and the supernatant was collected for later use.

[0086] 1.2.3 Determination of polyphenol oxidase activity

[0087] According to the instructions for use of the commercial test kit (A136-1-1, Nanjing Jiancheng Bioengineering Institute), the fermentation supernatants of the colonies with larger transparent zones screened above were respectively subjected to the polyphenol oxidase activity assay.

[0088] 1.2.4 Strain identification

[0089] 1.2.4.1 Morphological identification

[0090] The pure culture of the strain with a large transparent zone and high polyphenol oxidase production was inoculated into an MRS medium plate and cultured anaerobically at 37°C for 48 h, and the colony morphology was observed.

[0091] 1.2.4.2 Molecular biological identification

[0092] The target strain was inoculated into fresh MRS liquid medium and cultured for 48 h. The bacterial DNA was extracted using the kit of Tiangen Company and the 16 S rDNA sequence was amplified. The primers used were universal primers:

[0093] 1492r: 5'-GGTTACCTTGTTACGACTT-3';

[0094] 27f: 5' - AGAGTTTGATCCTGGCTCA - 3'.

[0095] The PCR reaction system (50 μL) was as follows: Mixture 25 μL (containing Taq DNA polymerase and dNTP, Tiangen Biochemical Technology Co., Ltd.), upstream and downstream primers 1 μL each, template DNA 2 μL, ultrapure water 21 μL. The PCR amplification program was 94 °C pre-denaturation for 5 min, 94 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 90 s, 30 cycles, and 72 °C extension for 10 min. The PCR product was sent to Beijing Boshang Biotechnology Co., Ltd. for sequence determination.

[0096] 2 Results

[0097] 2.1 Strain isolation and screening

[0098] A total of 23 lactic acid bacteria with transparent zones were isolated from the intestinal contents of black soldier fly larvae, earthworms, spiders and mealworms. They were inoculated into MRS liquid culture medium and cultured anaerobically at 37 ℃ for 48 h. The polyphenol oxidase activity in the fermentation supernatant was detected using a commercial test kit, and a strain with high polyphenol oxidase production was obtained, which was numbered Z2MX.

[0099] 2.2 Identification of Z2MX strain

[0100] 2.2.1 Morphological identification

[0101] After strain Z2MX was cultured anaerobically at 37 °C on MRS medium for 48 h, the colonies on the medium were milky white, raised, smooth, and easy to pick up (e.g. Figure 1 Observed under a microscope, the bacteria are spherical in shape and arranged singly, in pairs or in short chains (as shown in Figure 2 The colony morphology and bacterial morphology showed that it was preliminarily judged to be Lactococcus.

[0102] 2.2.2 Molecular biological identification

[0103] The electrophoresis results of the 16S rDNA PCR product of strain Z2MX showed that a band with good specificity was obtained at a molecular weight of about 1500bp, which was consistent with the expected results, and sequencing was performed. The sequencing sequence was compared with the 16S rDNA gene sequences of some strains registered on the website http: / / www.ncbi.nlm.nih.gov, and the strain Z2MX was identified as Enterococcus faecalis ( Enterococcus faecalis ), whose sequence is shown in SEQ ID NO: 1.

[0104] Example 2 Comparison of polyphenol oxidase activity, antibacterial activity and antioxidant activity of different Enterococci faecalis

[0105] 1 Materials and methods

[0106] 1.1 Materials

[0107] 1.1.1 Test strains

[0108] The Enterococcus faecalis obtained by screening in Example 1 of the present invention ( Enterococcus faecalis ) Z2MX. Enterococcus faecalis BLCC2-0311, BLCC2-0323 and BLCC2-0591 were provided by the strain resource bank of the Science and Technology Innovation Center of Shandong Baolai Lilai Bioengineering Co., Ltd.

[0109] 1.1.2 Indicator bacteria

[0110] Escherichia coli BLCC8-0135, Staphylococcus aureus BLCC8-0138, Aeromonas hydrophila BLCC8-0121, Salmonella pullorum BLCC8-0103, Salmonella Enteritidis BLCC8-0129, Clostridium perfringens BLCC8-0136, Vibrio alginolyticus BLCC8-0146 and Vibrio parahaemolyticus BLCC8-0145 were all isolated and preserved in the strain resource bank of the Science and Technology Innovation Center of Shandong Baolai Lilai Bioengineering Co., Ltd.

[0111] 1.2 Experimental methods

[0112] 1.2.1 Determination of polyphenol oxidase activity produced by strains

[0113] Use an inoculation loop to pick up single colonies of the test strains, streak them on new MRS solid slant medium, and activate the strains. The activated strains were inoculated into MRS liquid medium and cultured at 37 °C for 48 h to obtain fermentation broth. The fermentation broth was centrifuged at 4 °C and 8000 r / min for 10 min, and the fermentation supernatant of each test strain was collected for later use. According to the instructions for use of the commercial test kit (A136-1-1, Nanjing Jiancheng Bioengineering Institute), the fermentation supernatant of the test strains was tested for polyphenol oxidase activity.

[0114] 1.2.2 Determination of antibacterial properties of strains

[0115] The antibacterial activity of the tested strains against indicator bacteria was determined by the punch method.

[0116] Escherichia coli BLCC8-0135, Staphylococcus aureus BLCC8-0138, Salmonella pullorum BLCC8-0103, Salmonella enterica serovar Enteritidis BLCC8-0129, and Aeromonas hydrophila BLCC8-0121 were inoculated into NB broth medium, cultured at 37 °C, 180 rpm for 12-18 h, and then adjusted to 1.0×10 7 CFU / mL for later use. At the same time, take 1 mL of the diluted indicator bacteria and 17 mL of NA medium into the culture dish, mix well, and let it stand until it solidifies. Then use a hole puncher to make holes, add 90 µL of fermentation supernatant (preparation method see 1.2.2 in Example 1), and place it in a 37 ℃ incubator for 12 h.

[0117] Inoculate Clostridium perfringens BLCC8-0136 in liquid thioglycollate medium, culture in a 42°C incubator for 12-18 h, directly take 1 mL of the cultured Clostridium perfringens and 17 mL of iron sulfite agar into a culture dish, mix evenly, and let it stand until it solidifies. Then use a hole puncher to punch holes, add 90 µL of fermentation supernatant (preparation method see 1.2.2 in Example 1), and place in a 37°C incubator for anaerobically cultured 12 h.

[0118] Vibrio alginolyticus BLCC8-0146 and Vibrio parahaemolyticus BLCC8-0145 were inoculated into 2216E liquid medium, cultured at 37 °C, 180 rpm for 12-18 h, and then adjusted to 1.0×10 7 CFU / mL for later use. At the same time, take 1 mL of the diluted indicator bacteria and 17 mL of 2216E agar medium into the culture dish, mix well, and let it stand until it solidifies. Then use a hole puncher to make holes, add 90 µL of fermentation supernatant (preparation method see 1.2.2 in Example 1), and place it in a 37 ℃ incubator for 12 h.

[0119] The size of the inhibition zone was measured with a vernier caliper and the antibacterial activity was evaluated.

[0120] 1.2.3 Determination of the antioxidant activity of the strain in vitro

[0121] Commercial kits were used to detect the total antioxidant capacity, anti-superoxide anion capacity, total superoxide dismutase activity, DPPH free radical scavenging capacity and hydroxyl free radical inhibition capacity in the fermentation supernatant of the strain.

[0122] 2 Results

[0123] 2.1 Determination of polyphenol oxidase activity produced by strains

[0124] The polyphenol oxidase activity of Enterococcus faecalis Z2MX, a strain with high polyphenol oxidase activity in the initial screening, and three strains of Enterococcus faecalis preserved in the strain resource bank were determined by fermentation supernatants of the fermentations for 48 h under the same conditions.

[0125] Table 1 Comparison of polyphenol oxidase activity (U / mL) produced by fermentation supernatants of different Enterococcus faecalis

[0126]

[0127] Note: Different lowercase letters in the same industry indicate significant differences ( P <0.05, the same or no letter shoulder indicates no significant difference ( P >0.05).

[0128] As shown in Table 1, no polyphenol oxidase activity was detected in the fermentation supernatant of BLCC2-0311. The polyphenol oxidase activity produced by Z2MX was the highest, at 15.00 U / mL fermentation supernatant, which was significantly higher than that of BLCC2-0323 and BLCC2-0591 ( P <0.05).

[0129] 2.2 Determination of antibacterial properties of strains

[0130] Table 2 Comparison of antibacterial properties of different Enterococcus faecalis fermentation supernatants

[0131]

[0132] As shown in Table 2, Z2MX fermentation broth has the broadest antibacterial spectrum and has antibacterial effects on all 8 tested indicator bacteria, and the diameters of the inhibition zones are larger than those of the other 3 strains of Enterococcus faecalis.

[0133] 2.3 Evaluation of the antioxidant activity of the strains in vitro

[0134] Table 3 Comparison of antioxidant capacity of fermentation supernatants of different Enterococcus faecalis

[0135]

[0136] Note: Different lowercase letters in the same industry indicate significant differences ( P <0.05, the same or no letter shoulder indicates no significant difference ( P >0.05).

[0137] As shown in Table 3, the Z2MX strain had the best in vitro antioxidant performance, with the highest total antioxidant capacity, total superoxide dismutase, anti-superoxide anion capacity, DPPH free radical scavenging capacity and hydroxyl free radical inhibition capacity. Among them, the total antioxidant capacity, anti-superoxide anion capacity and hydroxyl free radical inhibition capacity were significantly higher than those of the other three strains of Enterococcus faecalis ( P <0.05).

[0138] Example 3 Effects of Enterococcus faecalis Z2MX on the antioxidant capacity of mice

[0139] 1 Materials and methods

[0140] 1.1 Test materials

[0141] Test strains: Enterococcus faecalis Z2MX, BLCC2-0323 and BLCC2-0591.

[0142] 1.2 Experimental design and grouping

[0143] Fifty male Kunming mice were purchased and fed for 4 days. They were randomly divided into five groups according to the principle of no significant difference in body weight, with 10 mice in each group. The control group and the model group were gavaged with 0.2 mL of normal saline per mouse every day, and the probiotic group was gavaged with 0.2 mL of 10 7 CFU fermentation liquid / mouse. During the experimental period, except for the control group fed with the basal diet, the other experimental groups were fed with a high-iron diet containing 0.075% FeSO4. The experimental period was 21 days, and the specific experimental groups are shown in Table 4.

[0144] Table 4 Experimental groups and treatments

[0145]

[0146] The experiment examined the changes in body weight of each group of mice, the antioxidant capacity of mouse serum, and the antioxidant capacity of mouse colon.

[0147] 2 Results

[0148] 2.1 Effect of Enterococcus faecalis Z2MX on the body weight of mice

[0149] Table 5 Effect of Enterococcus faecalis Z2MX on the body weight of mice (g)

[0150]

[0151] Note: Different lowercase letters in the same industry indicate significant differences ( P <0.05, the same or no letter shoulder indicates no significant difference ( P >0.05). Same as the table below.

[0152] As shown in Table 5, the body weight of the model group was higher than that of the control group at 9 d, 12 d, 15 d and 18 d, but the differences were not significant ( P >0.05); at 21 days, the body weight of mice in the model group was significantly higher than that in the control group ( P <0.05), indicating that feeding a high-iron diet can cause obesity in mice; the weights of mice in each experimental group of Enterococcus faecalis were lower than those in the model group, which had a certain alleviating effect on the obesity of mice caused by a high-iron diet. Among them, the weight of the Enterococcus faecalis Z2MX group was not much different from that of the control group, which could alleviate the obesity of mice caused by a high-iron diet.

[0153] 2.2 Effects of Enterococcus faecalis Z2MX on serum antioxidant indices in mice

[0154] Table 6 Effects of Enterococcus faecalis Z2MX on serum antioxidant indexes in mice

[0155]

[0156] Note: Different lowercase letters in the same column indicate significant differences ( P <0.05, the same or no letter shoulder indicates no significant difference ( P >0.05). Same as the table below.

[0157] As shown in Table 6, compared with the control group, the serum polyphenol oxidase activity of mice in the model group was significantly reduced ( P <0.05), the malondialdehyde content increased significantly ( P <0.05), indicating that feeding a high-iron diet could significantly reduce the antioxidant capacity of mice.

[0158] There was no significant difference in the polyphenol oxidase activity between the group fed with Enterococcus faecalis Z2MX and the control group ( P >0.05), but significantly higher than the model group, oral administration of Enterococcus faecalis BLCC2-0323 and BLCC2-0591 groups ( P <0.05); the malondialdehyde content was the lowest in the group with oral administration of Enterococcus faecalis Z2MX, and there was no significant difference between the control group and the group with oral administration of Enterococcus faecalis BLCC2-0323 ( P >0.05), but significantly lower than the model group and the group gavage with Enterococcus faecalis BLCC2-0591 ( P <0.05).

[0159] 2.3 Effects of Enterococcus faecalis Z2MX on antioxidant indices in mouse colon tissue

[0160] Table 7 Effects of Enterococcus faecalis Z2MX on antioxidant indices in mouse colon tissue

[0161]

[0162] Note: Different lowercase letters in the same industry indicate significant differences ( P <0.05, the same or no letter shoulder indicates no significant difference ( P >0.05). Same as the table below.

[0163] As shown in Table 7, compared with the control group, the polyphenol oxidase activity (PPO), total antioxidant capacity, glutathione peroxidase activity and catalase activity in the model group were significantly reduced ( P <0.05), the malondialdehyde content increased significantly ( P <0.05). This indicates that feeding a high-iron diet significantly reduced the antioxidant activity of mice.

[0164] Compared with the control group, the activity of polyphenol oxidase (PPO) and glutathione peroxidase in the group given oral administration of Enterococcus faecalis Z2MX were not significantly different from those in the control group ( P >0.05), but were significantly higher than those of the BLCC2-0323 and BLCC2-0591 groups ( P <0.05); the total antioxidant capacity and catalase activity of the group gavage with Enterococcus faecalis Z2MX were significantly higher than those of the model group ( P <0.05, but there was no significant difference with the other experimental groups ( P >0.05). The malondialdehyde content of the group fed with Enterococcus faecalis Z2MX was significantly lower than that of the groups fed with Enterococcus faecalis BLCC2-0323 and BLCC2-0591 ( P <0.05, but there was no significant difference compared with the control group ( P >0.05).

[0165] 2.4 Effects of Enterococcus faecalis Z2MX on mouse colon tissue

[0166] The paraffin sections and staining results of the colon tissues of mice in each group are shown in Figure 3 As shown. In the control group, the goblet cells of the colon tissue were intact and arranged regularly and neatly, the villi were arranged neatly, tightly and with clear boundaries, and there was no inflammatory cell infiltration; in the model group, the colon tissue was scattered, the crypt structure partially disappeared or deformed, and there was a large number of inflammatory cell infiltration (as marked by the red box), indicating that the high-iron diet caused colon inflammation; compared with the control group, the morphological structure of the colon tissue in each E. faecalis treatment group was basically the same, the colon mucosal epithelial tissue was relatively intact, the crypt glands were relatively intact, the goblet cells were relatively abundant, and they were arranged regularly and neatly. The results showed that after gavage for 10 7The Enterococcus faecalis at a rate of CFU / mouse has a certain alleviating and protective effect on the colon oxidative damage induced by high-iron diet in mice.

[0167] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Although the present application is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Enterococcus faecalis, which was named Enterococcus faecalis ( Enterococcus faecalis )Z2MX, and was deposited in the China Center for Type Culture Collection on May 14, 2024, with the deposit number being CCTCC NO: M 2024920.

2. A composition comprising the Enterococcus faecalis according to claim 1.

3. A product comprising the Enterococcus faecalis according to claim 1, and at least one auxiliary material; wherein: The product described is a pharmaceutical product.

4. A product comprising the Enterococcus faecalis according to claim 1, and at least one auxiliary material; wherein: The product is a bacteriostatic agent.

5. A product comprising the Enterococcus faecalis according to claim 1, and at least one auxiliary material; wherein: The product described is an antioxidant.

6. A product comprising the Enterococcus faecalis according to claim 1, and at least one auxiliary material; wherein: The product is a feed additive.

7. A product comprising the Enterococcus faecalis according to claim 1, and at least one auxiliary material; wherein: The product is a microecological preparation.

8. Use of the Enterococcus faecalis according to claim 1 in producing polyphenol oxidase.

9. Use of the Enterococcus faecalis according to claim 1 in preparing a product for improving intestinal oxidative damage.

10. The use according to claim 9, characterized in that: The product described is a pharmaceutical product.

11. The use according to claim 9, characterized in that: The product is a feed additive.

12. The use according to claim 9, characterized in that: The product is a microecological preparation.

13. A method for producing polyphenol oxidase, comprising: The Enterococcus faecalis of claim 1 is fermented and cultured to produce polyphenol oxidase, and the polyphenol oxidase is isolated and obtained.

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