CN115044504A

CN115044504A - Enterococcus faecalis YZ-1 and probiotic application thereof

Info

Publication number: CN115044504A
Application number: CN202210625860.9A
Authority: CN
Inventors: 魏勇军; 阳雨蝶; 占闽宁; 朱丹丹
Original assignee: Henan Yuanzhi Biotechnology Co ltd; Zhengzhou University
Current assignee: Henan Yuanzhi Biotechnology Co ltd; Zhengzhou University
Priority date: 2022-06-02
Filing date: 2022-06-02
Publication date: 2022-09-13
Anticipated expiration: 2042-06-02
Also published as: CN115044504B

Abstract

The invention belongs to the field of microorganisms, relates to probiotics, and particularly relates to enterococcus faecalis YZ-1 and probiotic application thereof. The strain is separated from intestinal fecal samples of healthy young people, and has a preservation number of CCTCC M2022312, and a classification name of CCTCC M2022312Enterococcus faecalisYZ-1, the preservation unit is China center for type culture Collection, the preservation time is 2022 years, 3 months and 25 days, and the preservation address is university of Wuhan, China. The enterococcus faecalis YZ-1 isolate has the advantages of uncritical growth conditions, excellent stress resistance, no toxicity, harmlessness and better safety, and can survive in high-acid and high-bile salt environments; meanwhile, the strain can effectively inhibit the propagation of common intestinal pathogenic bacteria, has the capabilities of reducing cholesterol, removing free radicals, resisting cancer, resisting inflammation and the like, has the potential of treating liver inflammation, fibrosis and diabetes, and is a probiotic strain with development value.

Description

Enterococcus faecalis YZ-1 and probiotic application thereof

Technical Field

The invention belongs to the field of microorganisms, relates to probiotics, and particularly relates to enterococcus faecalis YZ-1 and probiotic application thereof.

Background

Healthy people have a large number of beneficial flora, and the flora mutually restrict and maintain the microecological balance in the body. While abuse of antibiotics can kill normal beneficial bacteria in the body, alter microflora composition, cause dysbacteriosis, increase the chance of infection, and even lead to more serious consequences. The use of antibiotics affects the activity of microbially regulated metabolites in the intestinal tract of a host, and the metabolites produced by the microbes protect the integrity of the intestinal epithelium and maintain the immune response of the gut. After the antibiotics are used, the change of microbial population diversity and community structure can cause the antibiotics to be easily attacked by pathogenic bacteria, so that the antibiotics are easily infected by the pathogenic bacteria such as clostridium difficile and the like, and further cause diarrhea or serious colitis, and the abuse of the antibiotics becomes an important cause for causing the imbalance of intestinal flora. The lactobacillus rhamnosus can inhibit the growth of pathogenic microorganisms by regulating the diversity and community constitution of intestinal microorganisms, thereby improving the flora structure and promoting the health of a host. In the research related to the improvement of human intestinal flora imbalance, people are prompted to pay more attention to the substitution or substitution method of antibiotics, such as probiotics, prebiotics, synbiotics and the like. Therefore, the method for exploring and researching the method for improving the intestinal flora imbalance has valuable economic value.

With the improvement of living standard and consumption standard, the concept of healthy diet of people is continuously strengthened. In order to enhance the autoimmunity, products such as probiotics are also well accepted. The definition of probiotics by the world health organization is: probiotics is a kind of active microorganisms beneficial to a host, and is a general term for active beneficial microorganisms which are planted in the intestinal tract and the reproductive system of a human body and can generate exact health efficacy so as to improve the microbial ecological balance of the host and exert beneficial effects on the intestinal tract; the probiotic preparation is usually a live bacterial preparation formed by compounding one or more lactic acid bacteria, and can play a plurality of roles of regulating intestinal flora, promoting intestinal health, enhancing immunity, inhibiting diarrhea and the like after being ingested.

Enterococcus faecalis: (A)Enterococcus faecalis) Is a gram-positive facultative anaerobe, is one of main flora in intestinal tracts of human beings and animals, is used as a probiotic and is widely applied to biomedicine. Animal experiments show that the microbial preparation prepared from enterococcus faecalis can improve the micro-ecological balance in intestinal tracts and prevent and treat the disorder of intestinal flora of animals; enterococcus faecalis can also enhance the activity of macrophages, promote the immune response of animals and improve the level of antibodies; enterococcus faecalis can form a biological film in the intestinal tract of animals and attach to the intestinal mucosa of animals to form a lactic acid bacteria barrier to resist the invasion of foreign pathogenic bacteria, viruses, mycotoxin and the like; enterococcus faecalis is also capable of producing various antibacterial substances and inhibiting common pathogenic bacteria in animals.

However, recent studies have shown that some enterococcus faecalis have virulence genes, and many drug-resistant enterococcus faecalis have emerged due to the long-term and largely irregular use of antibiotics; thus, a proportion of enterococcus faecalis strains may cause severe infections. Currently, there are fewer enterococcus faecalis with probiotic function and capable of being applied to human clinical treatment, and more strain isolation and functional validation experiments are required. The human probiotics has affinity to human bodies, is not easy to be rejected by immune systems of human bodies, is favorable for colonization in intestinal tracts and plays a probiotic function. In order to ensure that the probiotics can colonize in intestinal tracts and exert probiotic functions, the probiotics can still have a certain number of viable bacteria in gastrointestinal tracts by ensuring good gastrointestinal fluid tolerance capacity, and a large number of strain screening experiments are carried out in the subject group in order to find strains capable of fully exerting the functions of enterococcus faecalis in human bodies.

Disclosure of Invention

In order to realize the purpose, the invention discloses an enterococcus faecalis YZ-1 and probiotic application thereof.

The technical scheme of the invention is realized as follows:

according to the application, through carrying out isolated culture and screening on strains from human excrement, the adaptability of human gastrointestinal fluid, antibiotic resistance, human probiotic property and the like are detected, and through a series of in vivo and in vitro experiments, a probiotic strain enterococcus faecalis YZ-1 which can reduce cholesterol, eliminate free radicals, resist cancers and inflammation and has the potential of treating liver inflammation, fibrosis and diabetes is discovered.

Enterococcus faecalis strainEnterococcus faecalis) YZ-1 with preservation number of CCTCC M2022312 and classification name ofEnterococcus faecalisYZ-1, the preservation unit is China center for type culture Collection, the preservation time is 2022 years, 3 months and 25 days, and the preservation address is university of Wuhan, China.

The result of the biochemical characteristic identification of the strain. Specifically, the inventor finds that the bacterium is moderately sensitive to norfloxacin, vancomycin and macrolide antibiotics and sensitive to common antibiotics (penicillins, cephalosporins, tetracyclines, meropenem and the like); the enterococcus faecalis can resist the acid environment of gastric juice (for example, the bacterial strain has good survival condition under the artificial gastric juice environment with the pH value of 2.5-4.5); the enterococcus faecalis can resist the bile salt environment in the intestinal tract (for example, the bacteria survive well under the condition of 0.03-0.3% of bile salt concentration).

The term "antibiotic-sensitive" as used herein refers to a strain that is weak against the antibiotic and can affect the normal growth of the strain under the condition of trace administration. According to an embodiment of the invention, the enterococcus faecalis is sensitive to penicillin, cephalosporin, macrolide, tetracycline, carbapenem and chloramphenicol antibiotics.

According to an embodiment of the present invention, the cephalosporins include at least one selected from the group consisting of cefoperazone, cefazolin, cefuroxime sodium; the quinolone is norfloxacin; the macrolides include at least one selected from the group consisting of erythromycin, clarithromycin, azithromycin; the beta-lactam is aztreonam; the tetracycline is tetracycline; the glycopeptide is vancomycin; the carbapenems are meropenem.

In addition, the enterococcus faecalis of the present invention is highly safe. The concrete aspects are as follows: is not hemolytic; the transfer level of the drug-resistant gene is low; does not affect the normal survival and weight gain of animals such as mice.

The enterococcus faecalis YZ-1 has an inhibiting effect on enteropathogenic bacteria, wherein the enteropathogenic bacteria are staphylococcus aureus (Staphylococcus aureus: (Staphylococcus aureus))Staphylococcus aureus) Listeria monocytogenes (I), (II)Listeria monocytogenes) Escherichia coli (E.coli)Escherichia coli) Salmonella bacteria (A), (B)Salmonella) Pseudomonas aeruginosaPseudomonas aeruginosa) And Candida albicans (C.), (Candida albicans)。

Further, the liquid culture of enterococcus faecalis YZ-1 according to the present application is used for preparing an anti-inflammatory drug. The liquid culture is essentially a secondary metabolite of the strain; the medicament is in a dosage form for oral administration or in a dosage form for topical administration.

The application of enterococcus faecalis YZ-1 in preparing medicines for reducing cholesterol is provided.

The enterococcus faecalis YZ-1 is applied to the preparation of medicines for reducing blood sugar.

The application of the enterococcus faecalis YZ-1 in preparing medicines for reducing inflammatory factors related to liver inflammation and/or fibrosis.

The enterococcus faecalis YZ-1 is applied to the preparation of the medicine for efficiently removing the free radicals.

The enterococcus faecalis YZ-1 is applied to the preparation of the medicine for resisting human breast cancer cell MDA-MB-231.

The invention has the following beneficial effects:

1. the application newly discovers a humanized strong acid resistant enterococcus faecalis YZ-1, the viable count of the enterococcus faecalis is hardly changed after the enterococcus faecalis is treated for 3 hours under the acidic condition under the condition of pH2.5, and after the commercially available enterococcus faecalis is treated for 3 hours under the acidic condition, the viable count is reduced by about 8 times compared with the initial viable count, so that a new choice is provided for treating related diseases.

2. Compared with commercially available enterococcus faecalis, the viable count of the enterococcus faecalis is high, the enterococcus faecalis has high bile salt resistance, the cholesterol clearance rate is up to 65.36 +/-0.25%, and namely 653.6 mu g/mL cholesterol in a culture medium is degraded. The clearance rates of the supernatant, the bacterial suspension and the lysate of the enterococcus faecalis YZ-1 to DPPH free radical are 82.19-94.37%, 86.72-95.38% and 56.73-58.32% respectively, and the clearance rates are at the highest concentration (2 x 10) ⁸ CFU/mL), the clearance rate of the YZ-1 bacterial suspension on DPPH is as high as 95.38%.

3. The supernatant or lysate of enterococcus faecalis YZ-1 with different thallus concentrations or the inactivated thallus thereof has the function of inhibiting the proliferation of human breast cancer cells MDA-MB-231. The inhibition rate of each component YZ-1 on cancer cells is gradually increased with the increasing of the bacteria concentration, and 6 multiplied by 10 ⁷ The inhibition rate of CFU/mL inactivated bacteria on human breast cancer cells MDA-MB-231 is up to 82.32 +/-0.25%.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph showing the results that enterococcus faecalis YZ-1 was stable at pH2.5 to 4.5.

FIG. 2 is a graph showing the results of the stabilization of enterococcus faecalis YZ-1 in artificial intestinal fluids with bile salt concentration of 0.03% -0.3%.

FIG. 3 is a graph showing the IL-10 concentration (pg/mL) measured after culture filtrates of human PBMCs cells treated differently, wherein a normal control group represents cells without any treatment; the positive control group represents cells treated with Phytohemagglutinin (PHA) at 200 ng/mL; the experimental group shows cells treated with culture filtrate of enterococcus faecalis YZ-1 at 30 g/L.

FIG. 4 is a graph showing the TGF- β concentrations (pg/mL) measured after differently treated culture filtrates of human PBMCs cells, wherein a normal control group represents cells without any treatment; the experimental group shows cells treated with culture filtrate of enterococcus faecalis YZ-1 at 30 g/L.

FIG. 5 is a standard curve of cholesterol used in the experiment.

FIG. 6 shows the effect of enterococcus faecalis YZ-1 on fasting plasma glucose in rat model with high fat.

FIG. 7 shows the effect of enterococcus faecalis YZ-1 on fasting serum insulin in a rat in a hyperlipidemic model.

FIG. 8 shows the effect of enterococcus faecalis YZ-1 on serum interleukin 1-beta (IL-1 beta) in rat model with high fat.

FIG. 9 shows the effect of enterococcus faecalis YZ-1 on serum monocyte chemoattractant protein-1 (MCP-1) in rat hyperlipidaemia model.

FIG. 10 shows that different concentrations of enterococcus faecalis YZ-1 are in normal hepatocyte LO ₂ Inhibition rate change graph.

FIG. 11 is a graph showing the change of the inhibition rate of enterococcus faecalis YZ-1 at different concentrations on human breast cancer cells MDA-MB-231.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Separation, purification and identification of enterococcus faecalis

(1) Sample collection and processing

5-10g of collected fresh excrement of healthy young people is picked, put into an EP (EP) tube and transferred into a refrigerator at 4 ℃ for standby. In a sterile operating platform, 1g of anaerobically preserved fecal sample is transferred to sterile saline containing 9mL until the mixture is uniform, and gradient dilution is sequentially performed by 10 times until the concentration is 1X 10 ^-6 Sucking 1X 10 by using a pipette ^-4 、1×10 ^-5 、1×10 ^-6 A total of 3 dilutions were made in 100. mu.L each in MRS agar medium, 3 plates per dilution.

MRS agar medium

10g/L of peptone, 5g/L of beef extract powder, 4g/L of yeast extract powder, 20g/L of glucose, 80 g/L of Tween, 1mL/L of beef extract powder, 2g/L of dipotassium phosphate, 5g/L of anhydrous sodium acetate, 2g/L of ammonium citrate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate and 20g/L of agar powder, adding ultrapure water to 1L, and adjusting the pH to 6.0-6.4. Sterilizing with high pressure steam at 121 deg.C for 20 min.

(2) Strain isolation and purification

And (3) quickly and uniformly coating 100 mu L of the suspension after gradient dilution into an MRS agar culture medium by using a coating rod, sealing by using a sealing film, putting into an anaerobic tank, and putting into an aerogenic anaerobic bag to ensure an anaerobic environment. Culturing at 37 deg.C for 24 h.

Typical colonies on the above agar medium were picked, inoculated into an anaerobic tube containing 5mL of MRS liquid medium, and cultured at 37 ℃ for 24 hours. Solid and liquid MRS media were inoculated 5 times repeatedly.

MRS liquid medium

10g/L of peptone, 5g/L of beef extract powder, 4g/L of yeast extract powder, 20g/L of glucose, 801 mL/L of tween-801, 2g/L of dipotassium phosphate, 5g/L of anhydrous sodium acetate, 2g/L of ammonium citrate, 0.2g/L of magnesium sulfate and 0.05g/L of manganese sulfate, adding ultrapure water to 1L, and adjusting the pH to 6.0-6.4. Sterilizing with high pressure steam at 121 deg.C for 20 min.

And (3) preserving the strains of the last culture, respectively taking 300 mu L of glycerol (50%, v/v) and 700 mu L of strains, loading the strains into a seed preservation tube, numbering, and placing the seeds at-80 ℃ for freezing and storing for subsequent experiments.

(3) Identification of Strain YZ-1

PCR amplification of 16S rRNA Gene

And obtaining an amplification template. And (3) putting 1mL of the bacterial liquid of the bacterial strain obtained by the last culture in a 1.5mL EP tube, centrifuging at the rotating speed of 10000 g for 1min, discarding the culture medium, adding 1mL of sterilized ultrapure water, resuspending, centrifuging at the rotating speed of 10000 g for 1min again, discarding the supernatant, repeating for 3 times, finally adding 1mL of 0.2mmol/L lysozyme, resuspending, and oscillating on an oscillator for 20min to obtain the template for PCR amplification.

The PCR system was 50. mu.L, where Mix was 25. mu.L, template was 1. mu.L, primer 27F was 1. mu.L (10. mu.M), primer 1492R was 1. mu.L (10. mu.M), ddH ₂ O was 22. mu.L.

The primers used were 27F (5'-agagtttgatcctggctca-3'), SEQ ID NO: 2 and 1492R (5'-ggttaccttgttacgactt-3'), SEQ ID NO: 3. the length of the amplified fragment is about 1500 bp.

PCR conditions, pre-denaturation of DNA double strands at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; 72 ℃, 70s, extension, and cycling 27 times, finally holding at 72 ℃ for 10 min.

Agarose gel electrophoresis

Weighing 0.8g of agarose, dissolving in 80mL of 1 × TAE solution, heating in a microwave oven for 2min for dissolving, and adding 8 μ L of nucleic acid dye; after glue injection and solidification, adding 3-5 mu L of sample into each sample hole; operating at 220V for 10 min; the images of the rubber plate are observed under UV, and the PCR product with clear strips is selected and sent to Beijing Optimala Biotechnology Limited company for sequencing.

Analysis and identification of gene sequence

Sequencing by Beijing Ongji science and technology Limited to obtain the 16S rRNA gene sequence of the strain YZ-1 as shown in SEQ ID NO.1, comparing with GenBank/EMBL/DDBJ database by BLAST analysis tool (BLAST. ncbi. nlm. nih. gov), and analyzing and identifying that the strain YZ-1 is enterococcus faecalis ((R))Enterococcus faecalis）。

Bacterial colony and bacterial morphology

The colony is light yellow, round, neat in edge, and slightly raised. The results of the mycosis examination are gram-positive, oval cocci, mostly arranged in double or short chain.

The physiological and biochemical reaction characteristics of the strain

And carrying out biochemical identification to obtain a positive reaction of glucose, sucrose, maltose, lactose, trehalose, melezitose and sorbitol and a negative reaction of Arabic gum, melibiose and a catalyst.

Example 2

Research on acid resistance and bile salt resistance of strain

Cultivation of the Strain to be tested

The MRS liquid culture medium is used for culturing an enterococcus faecalis isolated strain YZ-1 and a commercially available enterococcus faecalis strain. Weighing according to the formula, adding distilled water, stirring, adjusting pH to 6.2, and sterilizing at 121 deg.C for 20 min. Inoculating enterococcus faecalis to a sterilized MRS liquid culture medium in an inoculation amount of 2%, and performing anaerobic culture at 37 ℃.

Method for preparing artificial gastric juice and intestinal juice

Artificial gastric juice: taking 16.4mL of dilute hydrochloric acid, adding about 800mL of water and 10g of pepsin, shaking up, and adding water to dilute into 1000mL to obtain the finished product.

Artificial intestinal juice, i.e. phosphate buffer (containing pancreatin, ph 6.8): taking 6.8g of monopotassium phosphate, adding 500mL of water for dissolving, and adjusting the pH value to 6.8 by using 0.lmol/L sodium hydroxide solution; dissolving pancreatin 10g in water, mixing the two solutions, and diluting to 1000 mL.

The specific experimental procedures are as follows:

gastrointestinal survival assay

The effect of pH, bile salt concentration and treatment time on the survival rate of enterococcus faecalis was examined in artificial gastric and intestinal fluids. Considering that gastric juice is at about pH2.5 in the usual state, the present study simultaneously examined the survival of commercially available enterococcus faecalis at pH 2.5; considering that bile is in a state of about 0.2% in a usual state, the present study simultaneously examined the survival of commercially available enterococcus faecalis in a state of about 0.3% in an extreme bile concentration. The experimental results are as follows:

table 1: results of acid resistance experiments

Remarking: the enterococcus faecalis of the invention: EF; commercially available enterococcus faecalis: and (4) MEF.

As shown in table 1 and fig. 1, enterococcus faecalis exhibited good simulated gastric juice tolerance. The enterococcus faecalis of the present invention showed stable activity at pH2.5-4.5 within 3 h. Under the condition of pH2.5, the viable count of the enterococcus faecalis treated for 3 hours under the acidic condition is almost unchanged, and after the commercially available enterococcus faecalis is treated for 3 hours under the acidic condition, the viable count is reduced by about 8 times compared with the initial viable count. Therefore, the enterococcus faecalis has higher viable count compared with the commercially available enterococcus faecalis, and has higher acid resistance.

TABLE 2 results of bile salt resistance experiment

Remarking: the enterococcus faecalis of the invention: EF; commercially available enterococcus faecalis: MEF.

As shown in table 2 and fig. 2, the survival rate of enterococcus faecalis was not significantly affected by the artificial intestinal juice with bile salt concentrations of 0.03% and 0.1%; when the concentration of bile salts is increased to 0.2%, the survival rate of thalli is slowly reduced along with the processing time; when the concentration of bile salt is 0.3%, the survival rate of the enterococcus faecalis still above about 50% after 1 h. These results all indicate that enterococcus faecalis has good bile salt resistance. After the viable count of the commercially available enterococcus faecalis is treated for 3 hours under the condition of 0.3 percent of bile salt, the decline trend of the viable count compared with the initial viable count is obviously higher than that of the enterococcus faecalis. Therefore, the enterococcus faecalis has higher viable count compared with the commercially available enterococcus faecalis, and has higher bile salt resistance.

Example 3

Enterococcus faecalis strain safety study

Drug resistance analysis

The method is carried out by adopting a drug sensitive paper diffusion method. Will be freshCultured enterococcus faecalis diluted to 10% with physiological saline ⁷ And uniformly coating CFU/mL on an MRS agar culture medium, placing at room temperature for 3-5min, and placing drug sensitive paper sheets with circle center spacing not less than 24mm and paper sheet edge distance not less than 15mm from agar edge. The cells were cultured in an inverted state in an incubator at 37 ℃ for 24 hours to measure the diameter of the zone of inhibition. The resistance of enterococcus faecalis strains and their 30 th generation strains was determined by the paper diffusion method, and the results are shown in table 3 below.

TABLE 3 enterococcus faecalis resistance results

Note: r ═ drug resistance; i is intermediary; s is sensitive; -: no bacteriostatic zone is formed;

TABLE 4 enterococcus faecalis passage 30 strain resistance results

as can be seen from the above resistance results, enterococcus faecalis and enterococcus faecalis-3 showed sensitivity and moderate sensitivity to almost all common antibiotics. The results of the drug resistance of the enterococcus faecalis and the enterococcus faecalis-30 are consistent, which shows that the drug resistance of the enterococcus faecalis is not influenced in the process of passage. Therefore, the enterococcus faecalis antibiotic disclosed by the invention is low in transfer risk and high in strain safety.

Virulence factor analysis

Hemolysin and biofilm formation of enterococcus faecalis are measured by adopting a literature report method, and safety evaluation in animal bodies is carried out according to the method of the current pharmacopoeia.

(1) The strain does not contain plasmids by plasmid extraction, which shows that the transfer level of drug-resistant genes is low and the strain has high safety.

(2) Hemolysin detection

Hemolysin secreted by bacteria can cause erythrolysis or other tissue damage. The production of hemolysin is mainly related to virulence genes such as cylA, cylB and the like. The enterococcus faecalis provided by the invention has no hemolytic ring around the bacterial colony, the control strain has slight hemolytic ring, and the staphylococcus aureus has obvious hemolytic ring around the bacterial colony. The result shows that the enterococcus faecalis strain disclosed by the invention is negative in hemolysis experiment, has no hemolysis and is high in safety.

(3) Evaluation of safety in animals

After the mice are gavaged, the health condition is good, compared with the normal saline group, the activity condition of the mice is not different, and the hair state of the mice is not different

All are normal. The body weights of the mice before and after gastric lavage were recorded as shown in Table 5.

TABLE 5 weight changes of mice before and after gastric lavage

As a result, the body weight of mice in each group was increased, and the body weight changes in the two groups were not very different (the difference between the two groups was not statistically significant, and p > 0.05).

Example 4

Probiotic activity study of enterococcus faecalis YZ-1

Inoculating the strain frozen in a refrigerator at the temperature of 20 ℃ below zero into an MRS agar culture medium, and continuously activating for 2 times to obtain seed fermentation liquor. Inoculating the seed fermentation liquor into an MRS liquid culture medium according to the volume ratio of 3:100, and carrying out overnight culture at 37 ℃ to obtain the strain fermentation liquor.

(1) Experiment for inhibiting pathogenic bacteria

Gram-positive bacteria (staphylococcus aureus ATCC12592, listeria monocytogenes CMCC54002), gram-negative bacteria (escherichia coli ATCC25922, salmonella ATCC14028, pseudomonas aeruginosa ATCC27853) and fungi (candida albicans SC5314) were used as indicator strains. Taking appropriate amount of indicator bacteria (about 10) ⁸ CFU/mL) was added to LB agar medium to give a final concentration of about 10 ⁶ CFU/mL, shaking up, pouring the plate, solidifying the culture medium, punching, injecting 150 mu L of supernatant YZ-1 of enterococcus faecalis and bacterial suspension respectively, air-drying the liquid in the hole, culturing at 37 ℃ for 24h, and measuring the diameter of the inhibition zone. The supernatant is fermentation broth of overnight cultured enterococcus faecalis YZ-1Centrifuging (8000r/min,5min), collecting supernatant, and filtering with 0.22 μm filter membrane. The bacterial suspension is obtained by centrifuging fermentation liquor of overnight cultured enterococcus faecalis YZ-1 (8000r/min,5min), and suspending thallus in sterile physiological saline. The bacteriostatic ability of Lactobacillus rhamnosus standard strain ATCC7469(LGG) was determined in the same manner as described above.

Table 6 shows the inhibitory potency (mm) of enterococcus faecalis YZ-1 and LGG on six indicator bacteria

Note: the difference between letters in the same row represents significant difference (p < 0.05).

As shown in Table 6, both enterococcus faecalis YZ-1 and Lactobacillus rhamnosus LGG had good inhibitory effects, and the inhibitory effect of the supernatant YZ-1 on Escherichia coli was greater than that of the supernatant LGG.

(2) In vitro anti-inflammatory Activity assay of culture filtrates

Preparation of human PBMCs cells

Healthy human volunteers between 20 and 25 years of age were college students from the Zhengzhou university college of pharmacy.

Blood was drawn from healthy human volunteers and citrate dextrose (ACD) solution was used as an anticoagulant. Then added to a centrifuge tube and centrifuged at 720g rpm density-gradient for 30 minutes at 4 ℃ before collecting lymphocyte layer. The red blood cells were then lysed with a red blood cell lysis buffer and centrifuged at 3000rpm for 10 minutes at 4 ℃ to remove residual red blood cells. The human PBMCs cells thus obtained were adjusted to 4X 10 cell concentration in RPMI 1640 medium containing 10% FBS ⁶ cells/mL are ready for use.

Preparation of enterococcus faecalis YZ-1 liquid culture

Enterococcus faecalis YZ-1 was first inoculated into MRS liquid medium and cultured at 37 ℃ for about 24 hours, and the resulting culture was used as an inoculum. Then, the inoculum sources of the strains were inoculated in about 1-3% (v/v) of the inoculum size, respectively, to MRS liquid medium supplemented with a mixture of 5-30% milk, 1-10% soybean powder, and 3% glucose-maltodextrin, and cultured at 37 ℃ for 24 hours. Thereafter, the cells were centrifuged at about 3000-6000rpm for 60 minutes to precipitate the cells, and then the supernatant was collected and subjected to heat sterilization treatment, thereby obtaining a liquid culture of enterococcus faecalis YZ-1.

Before the following experiment, the culture filtrate of enterococcus faecalis YZ-1 was spray-dried, and then dissolved in an appropriate amount of RPMI 1640 medium to adjust the concentration to 30 g/L.

Treatment of YZ-1 culture filtrate on human PBMCs cells

The human PBMCs cells obtained above were first divided into 3 groups including 1 normal control group, 1 positive control group and 1 experimental group. The cells of each group were treated at 1X 10 ⁵ Cell/well number cultured in 96-well culture plates containing 0.2 mL of RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin) and in an incubator (37 ℃, 5% CO) ₂ ) And culturing for 48 hours.

The culture medium of the experimental group was then replaced with RPMI 1640 medium supplemented with 30g/L of enterococcus faecalis YZ-1 culture filtrate. The medium of the positive control group was replaced with RPMI 1640 medium supplemented with 200ng/mL phytohemagglutinin, while the medium of the normal control group was replaced with RPMI 1640 medium without addition.

Each group of cells was cultured in an incubator (37 ℃, 5% CO) ₂ ) After 48 hours of medium culture, the resulting culture was centrifuged at 3000rpm for 10 minutes, and the supernatant was taken for the following analysis.

Method for measuring IL-10 concentration

The determination of IL-10 concentration in the supernatant of the normal control group, the positive control group and the experimental group was carried out using an IL-10ELISA kit. The absorbance values measured for each group were converted to their concentrations (pg/mL) based on a standard curve previously prepared with IL-10 standards of different known concentrations relative to their own absorbance values.

Method for measuring TGF-beta concentration

TGF-. beta.concentration determination of the supernatants for the normal control group and the experimental group was performed using a TGF-. beta.ELISA kit. The absorbance values measured for each group were converted to their concentrations (pg/mL) based on a standard curve previously made with TGF- β standards having different known concentrations relative to their own absorbance values, respectively.

Measurement results of IL-10 concentration

Human PBMCs cells were treated with culture filtrate of enterococcus faecalis YZ-1 and the IL-10 concentration measured (FIG. 3). As can be seen from FIG. 3, the IL-10 concentration in the experimental group was significantly increased and significantly higher than that in the positive control group, compared to the normal control group, indicating that the culture filtrate was able to effectively activate human PBMCs cells to stimulate IL-10 secretion.

Measurement results of TGF-. beta.concentration

The TGF-. beta.concentration measured after treatment of human PBMCs cells with culture filtrate of enterococcus faecalis YZ-1 (FIG. 4). As can be seen from FIG. 4, the TGF- β concentration of the experimental group was significantly increased compared to the normal control group, and the culture filtrate was able to effectively activate the human PBMCs cells to stimulate them to secrete TGF- β.

The culture filtrate can effectively stimulate the human PBMCs cells to secrete IL-10 and TGF-beta, and further achieve excellent anti-inflammatory effect through immune regulation. Therefore, the culture filtrate of enterococcus faecalis YZ-1 is considered to have a high potential for developing an anti-inflammatory drug and is expected to be available for the treatment of inflammatory disorders.

(3) Total Cholesterol removal test

The standard curve of cholesterol was determined using a total cholesterol test kit. Diluting cholesterol standard solution (2mg/mL) with distilled water to 2, 1.6, 1.2, 0.8, 0.4, 0.2mg/mL respectively, accurately sucking 2.5 μ L of each concentration of the diluted solution, adding into a 96-well plate, adding 250 μ L of the working solution, mixing, placing in an incubator at 37 ℃ for incubation for 10min, measuring absorbance value at 510nm wavelength, and making 3 times of parallels for each concentration. The cholesterol standard curve was plotted using software Excel (fig. 5). The fermentation broth YZ-1 cultured overnight was inoculated into 5mL of 1mg/mL cholesterol medium in an amount of 5% (v/v), shaken, and simultaneously set as a blank (cholesterol medium without inoculum), and cultured at 37 ℃ for 24 hours. Centrifuging (8000r/min,5min), adding 2.5 μ L of supernatant into 96-well plate, adding 250 μ L of working solution, mixing, incubating at 37 deg.C for 10min, measuring absorbance at 510nm, introducing into standard curve equation of cholesterol, and calculating cholesterol concentration (C) of supernatant. Total cholesterol clearance ═ [ (crom-C)/crom) ] × 100%. The cholesterol culture medium is as follows: dissolving 0.05g cholesterol with small amount of anhydrous ethanol under heating, adding into 50mL MRS liquid culture medium, shaking to obtain cholesterol culture medium with final concentration of 1mg/mL, and autoclaving at 121 deg.C for 15 min.

The total cholesterol-removing ability of Lactobacillus rhamnosus standard strain ATCC7469(LGG) was determined in the same manner as described above at the highest concentration (2X 10) ⁸ CFU/mL), the cholesterol clearance rate of YZ-1 is as high as (65.36 +/-0.25)%, namely 653.6 mu g/mL of cholesterol in the culture medium is degraded, and the cholesterol is equivalent to LGG. The cholesterol-lowering ability of YZ-1 is stronger than that of LGG under the condition of low bacteria liquid concentration. Therefore, the enterococcus faecalis YZ-1 has high cholesterol-reducing capability. The reduction of cholesterol concentration in serum is beneficial to preventing cardiovascular diseases such as arteriosclerosis, hypertension and the like, and the bacterial strain can be applied to the research and development of functional foods with the function of reducing cholesterol.

(4) In vivo evaluation test for hypoglycemic function

32 female SD rats of about 200 g body weight were randomly divided into four groups, one group was given normal maintenance diet, and three groups were given high fat diet (cholesterol 1%, lard 10%, 0.2% cholate, 10% egg yolk powder). Three groups of high fat diet were orally administered 10 every other day after one week of feeding ⁹ CFU enterococcus faecalis YZ-1 bacterial liquid, one group of the bacterial liquid is given with physiological saline every other day, the other group of the bacterial liquid is given with simvastatin with the weight of 20mg/kg every other day three weeks before the termination of the experiment, the experiment is terminated for five weeks, and the blood sugar and the insulin level of the fasting serum of the rat are detected to evaluate the prevention and intervention effect of the enterococcus faecalis YZ-1 on the metabolic disorder model.

Enterococcus faecalis YZ-1 has effects of reducing fasting blood glucose and blood insulin of rat. FIG. 6 shows the effect of enterococcus faecalis YZ-1 on fasting plasma glucose in a rat with high fat. The average blood sugar content of enterococcus faecalis YZ-1 dry pretreatment group (YZ-1 dry pretreatment group) and simvastatin dry pretreatment group (drug dry pretreatment group) is 2.80 +/-0.56 mmol/L and 2.71 +/-0.45 mmol/L, which are both obviously lower than that of the high-fat model group by 4.89 +/-0.32 mmol/L; the average blood sugar content of the high-fat model group is 4.89 +/-0.32 mmol/L, which is obviously higher than that of the normal control group by 2.56 +/-0.27 mmol/L.

Effect of enterococcus faecalis YZ-1 on fasting serum insulin in rat hyperlipidic model (fig. 7). The average content of serum insulin of a enterococcus faecalis YZ-1 group (YZ-1 dry pre-group) and a simvastatin dry pre-group (drug dry pre-group) is 28.23 +/-2.02 mU/L and 38.25 +/-1.98 mU/L, which are both obviously lower than that of a high-fat model group 48.25 +/-1.78 mU/L; the mean content of serum insulin in the high-fat model group is 48.25 +/-1.78 mU/L, which is obviously higher than that in the normal control group by 23.12 +/-2.86 mU/L.

The results show that the enterococcus faecalis YZ-1 strain has the function of obviously reducing blood sugar and serum insulin, and has the similar blood sugar reducing effect with simvastatin. Lipid metabolism disorders such as hypertriglyceridemia and high free fatty acidemia are closely related to insulin resistance. Insulin resistance decreases the efficiency of insulin in promoting glucose uptake and utilization, and the body produces hyperinsulinemia by compensatory hypersecretion of insulin to maintain the stability of blood glucose. Insulin resistance is predisposed to metabolic syndrome and type 2 diabetes. The enterococcus faecalis YZ-1 strain has the function of reducing blood fat, thereby reducing insulin resistance and causing the reduction of blood sugar and serum insulin.

(5) In vivo evaluation of reducing inflammatory factors associated with liver inflammation and liver fibrosis

Effect of enterococcus faecalis YZ-1 on serum Interleukin 1-beta (IL-1. beta.) in rat model with high fat (FIG. 8). The average content of IL-1 beta in serum of an enterococcus faecalis YZ-1 dry pre-group and a simvastatin dry pre-group is 14.32 +/-2.08 pg/mL and 16.76 +/-1.25 pg/mL, and both are obviously lower than that of a high-fat model group by 25.32 +/-2.12 pg/mL; the average content of IL-1 beta in serum of the high-fat model group is 25.32 +/-2.12 pg/mL, which is obviously higher than that of the normal control group by 18.23 +/-2.56 pg/mL.

Effect of enterococcus faecalis YZ-1 on serum monocyte chemotactic protein-1 (MCP-1) of model rat with hyperlipidemia (FIG. 9). The average content of MCP-1 in serum of a enterococcus faecalis YZ-1 dry preparation group is 489.98 +/-98.23 pg/mL, and is obviously lower than 860.56 +/-65.22 pg/mL of a high-fat model group; the average content of MCP-1 in serum of a simvastatin dry pre-group is 712.59 +/-48.34 pg/mL, and the difference with 860.56 +/-65.22 pg/mL in a high-fat model group is not significant; the mean content of MCP-1 in serum of the high-fat model group is 860.56 +/-65.22 pg/mL, which is remarkably higher than that of 486.12 +/-56.23 pg/mL (p is less than 0.001) of a normal control group.

The results show that the enterococcus faecalis YZ-1 strain has the function of remarkably reducing serum IL-1 beta and MCP-1 inflammatory factors, and has stronger effect of reducing the serum inflammatory factors than simvastatin. Liver fibrosis is the final outcome of the continuous development of liver injury caused by various reasons, and MCP-l is an important macrophage chemotactic factor in tissue injury repair and plays an important role in liver injury and repair. IL-1 β is another important inflammatory cell that causes fibrosis, is a major inducer of the pro-inflammatory response, and enhances the extent of the inflammatory response by synergizing with and promoting the expression of other inflammatory factors, such as TNF- α. In conclusion, MCP-l and IL-1 β are important inflammatory factors for liver fibrosis. Enterococcus faecalis YZ-1 significantly reduces the levels of serum IL-1 beta and MCP-1 inflammatory factors, thereby reducing the incidence of liver inflammation and liver fibrosis.

(6) DPPH free radical scavenging experiment

Respectively diluting overnight cultured enterococcus faecalis YZ-1 fermentation broth to 5 × 10 with sterile normal saline ⁷ 、1×10 ⁷ 、5×10 ⁶ 、1×10 ⁶ CFU/mL, the DPPH free radical scavenging capacity of the 4 dilutions of cell lysate, supernatant and suspension was determined separately. Taking 2mL of a sample to be detected and 2mL of an ethanol solution of LDPPH, mixing uniformly, carrying out dark reaction for 40min, setting a control group (replacing the sample solution with absolute ethanol) and a blank group (replacing the DPPH solution with absolute ethanol), and measuring the light absorption value at the wavelength of 517 nm. Free radical clearance ═ 1- (a-like-a empty/a pair)]X 100%. The above DPPH was 0.2mmol/L, and 0.0078g of DPPH was taken and the volume of 100mL was determined with absolute ethanol. The supernatant is obtained by centrifuging the fermentation liquid at the above concentration (8000r/min,5min), and filtering with 0.22 μm filter membrane. The bacterial suspension is obtained by centrifuging fermentation liquor at the concentration (8000r/min,5min), and taking thallus for resuspending in sterile physiological saline. The cell lysate is obtained by treating the bacterial liquid with the concentration for 30min (800W, 15s, 15s) with a cell ultrasonication instrument, centrifuging (8000r/min,5min), collecting the supernatant, and filtering with a 0.22 μm filter membrane. The DPPH radical scavenging ability of Lactobacillus rhamnosus LGG was measured in the same manner as described above. The results show that enterococcus faecalis YZ-1 supernatant and bacterial suspensionAnd the clearance rate of the lysate to DPPH free radicals is 82.19-94.37%, 86.72-95.38% and 56.73-58.32%, and under the same concentration, the clearance capacity of the bacterial suspension and the lysate of YZ-1 to DPPH free radicals is stronger than that of LGG. The higher the cell concentration, the higher the DPPH clearance, at the highest concentration (5X 10) ⁷ CFU/mL), the clearance rate of the YZ-1 bacterial suspension on DPPH is as high as 95.38%.

(7) Cell experiments for anticancer Activity

With fetal bovine serum: double resistance: DMEM complete medium was prepared in a ratio of 1:0.1: 9. Human normal liver cell LO ₂ And human breast cancer cell MDA-MB-231 for recovery and passage.

Preparation of YZ-1 sample solution: taking 3 parts of YZ-1 fermentation liquor, placing one part of the fermentation liquor at 121 ℃ for high-temperature treatment for 15min, centrifuging (8000r/min,5min), and carrying out heavy suspension by using sterile PBS to prepare an inactivated thallus group; one part is subjected to ultrasonic disruption for 15min (800W, 9s, 9s), centrifuged, and the supernatant is filtered by a 0.22 mu m filter membrane to prepare a lysate group; one part is centrifuged (8000r/min,5min) and the supernatant is filtered through a 0.22 μm filter membrane to obtain a supernatant group. Each set of samples was diluted to different concentrations with DMEM complete medium.

Culturing the cultured LO ₂ Cells were digested, centrifuged, cell density adjusted to 80000 cells/mL, and added to a 96-well plate at 100. mu.L per well. Adding 5% CO at 37 deg.C ₂ Culturing in an incubator for 24 h. After the cell coverage area reaches about 85%, the old culture medium is discarded, 100. mu.L of YZ-1 sample solution is added into each well, and the mixture is placed at 37 ℃ and 5% CO ₂ Culturing in an incubator for 24 h. 3 replicates were set for each concentration, and DMEM complete medium was used as a blank instead of the sample. After completion of the culture, the cell viability was examined by MTT method, and the results (FIG. 10) show that YZ-1 is responsible for LO ₂ The safe concentration of cells was 3X 10 ⁴ ～6×10 ⁷ CFU/mL. Therefore, the concentration of YZ-1 was selected to measure the inhibitory effect on the proliferation of cancer cells. MDA-MB-231 cells were digested with 0.25% pancreatin, centrifuged (1200r/min, 5min), diluted to 80000 cells/mL, added to a 96-well plate at 100. mu.L per well, and placed at 37 ℃ with 5% CO ₂ Culturing in an incubator for 24 h. When the cell coverage area reaches about 85%, the old medium is discarded, washed 3 times with PBS, and 3 × 10 cells are added to each well ⁴ ～6×10 ⁷ CFU/mL of YZ-1 sample solution (100. mu.L) was used as a treatment group, DMEM complete medium was used as a blank control group instead of the sample, and 5-fluorouracil (5-FU) was used as a positive control group instead of the sample, and the mixture was incubated at 37 ℃ for 48 hours. The survival rate of cancer cells was measured by MTT method, and the inhibition rate of YZ-1 on cancer cells was calculated. Cancer cell inhibition rate ═ 1- (a treatment group/a blank group)]X100%. The results (FIG. 11) show that 6X 10 ⁷ The inhibition rate of CFU/mL inactivated bacteria on human breast cancer cell MDA-MB-231 is up to 82.32 +/-0.25%, which is obviously higher than 5-FU. The result shows that enterococcus faecalis YZ-1 has better anticancer activity and has larger potential application value in producing functional food with anticancer function.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

<110> Zhengzhou university

Henan Yuan Zhi Biotech Co., Ltd

<120> enterococcus faecalis YZ-1 and probiotic application thereof

<141> 2022-06-02

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1460

<212> DNA

<213> Enterococcus faecalis (Enterococcus faecalis)

<400> 1

gtggcgggtg ctataatgca gtcgaacgct tctttcctcc cgagtgcttg cactcaattg 60

gaaagaggag tggcggacgg gtgagtaaca cgtgggtaac ctacccatca gagggggata 120

acacttggaa acaggtgcta ataccgcata acagtttatg ccgcatggca taagagtgaa 180

aggcgctttc gggtgtcgct gatggatgga cccgcggtgc attagctagt tggtgaggta 240

acggctcacc aaggccacga tgcatagccg acctgagagg gtgatcggcc acactgggac 300

tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa 360

agtctgaccg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt 420

tagagaagaa caaggacgtt agtaactgaa cgtcccctga cggtatctaa ccagaaagcc 480

acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 540

attgggcgta aagcgagcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa 600

ccggggaggg tcattggaaa ctgggagact tgagtgcaga agaggagagt ggaattccat 660

gtgtagcggt gaaatgcgta gatatatgga ggaacaccag tggcgaaggc ggctctctgg 720

tctgtaactg acgctgaggc tcgaaagcgt ggggagcaaa caggattaga taccctggta 780

gtccacgccg taaacgatga gtgctaagtg ttggagggtt tccgcccttc agtgctgcag 840

caaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 900

gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 960

taccaggtct tgacatcctt tgaccactct agagatagag ctttcccttc ggggacaaag 1020

tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080

acgagcgcaa cccttattgt tagttgccat catttagttg ggcactctag cgagactgcc 1140

ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1200

ctacacacgt gctacaatgg gaagtacaac gagtcgctag accgcgaggt catgcaaatc 1260

tcttaaagct tctctcagtt cggattgcag gctgcaactc gcctgcatga agccggaatc 1320

gctagtaatc gcggatcagc acgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380

ccgtcacacc acgagagttt gtaacacccg aagtcggtga ggtaaccttt tggagccagc 1440

cgcctaaggt gattgatttc 1460

<210> 2

<211> 19

<212> DNA

<213> Unknown (Unknown)

<400> 2

agagtttgat cctggctca 19

<210> 3

<211> 19

<212> DNA

<213> Unknown (Unknown)

<400> 3

ggttaccttg ttacgactt 19

Claims

1. Enterococcus faecalis strainEnterococcus faecalis) YZ-1 with preservation number of CCTCC M2022312 and classification name ofEnterococcus faecalisYZ-1, the preservation unit is China center for type culture Collection, the preservation time is 2022 years, 3 months and 25 days, and the preservation address is university of Wuhan, China.

2. Use of enterococcus faecalis YZ-1 according to claim 1 for the preparation of a medicament for inhibiting enteropathogenic bacteria.

3. Use according to claim 2, characterized in that: the enteropathogenic bacteria are one or more of staphylococcus aureus, listeria monocytogenes, escherichia coli, salmonella, pseudomonas aeruginosa or candida albicans.

4. Use of enterococcus faecalis YZ-1 according to claim 1 for the preparation of an anti-inflammatory medicament.

5. Use according to claim 4, characterized in that: the anti-inflammatory drug is culture filtrate of enterococcus faecalis YZ-1.

6. Use of enterococcus faecalis YZ-1 according to claim 1 for the preparation of a cholesterol lowering medicament.

7. Use of enterococcus faecalis YZ-1 according to claim 1 for the preparation of a medicament for lowering blood glucose.

8. Use of enterococcus faecalis YZ-1 according to claim 1 for the preparation of a medicament for reducing inflammatory factors associated with liver inflammation and/or fibrosis.

9. Use of enterococcus faecalis YZ-1 according to claim 1 for the preparation of a medicament for efficient scavenging of free radicals.

10. Use of enterococcus faecalis YZ-1 according to claim 1 for the preparation of a medicament against human breast cancer cells MDA-MB-231.