CN115044504B

CN115044504B - Enterococcus faecalis YZ-1 and probiotic application thereof

Info

Publication number: CN115044504B
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: 2023-10-10
Anticipated expiration: 2042-06-02
Also published as: CN115044504A

Abstract

The application belongs to the field of microorganisms, and relates to probiotics, in particular to enterococcus faecalis YZ-1 and probiotic application thereof. The strain is separated from intestinal faeces sample of healthy young people, and has preservation number of CCTCC M2022312 and classification name of CCTCC M2022312Enterococcus faecalisYZ-1, the preservation unit is China center for type culture Collection, the preservation time is 2022, 3 and 25 days, and the preservation address is China university of Wuhan. The enterococcus faecalis YZ-1 isolate has the advantages of low growth condition, excellent stress resistance, capability of survival under high-acid and high-bile-salt environments, no toxicity, no harm and good safety; meanwhile, the strain can effectively inhibit the reproduction of common intestinal pathogenic bacteria, has the capabilities of reducing cholesterol, scavenging 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 application belongs to the field of microorganisms, and relates to probiotics, in particular to enterococcus faecalis YZ-1 and probiotic application thereof.

Background

A large number of beneficial flora exist in a healthy human body, and the flora are mutually restricted to jointly maintain the micro-ecological balance in the body. Whereas abuse of antibiotics kills normal beneficial bacteria in the body, alters the microbiota composition, causes dysbacteriosis, increases the chance of infection, and even leads to more serious consequences. The use of antibiotics affects the activity of metabolites regulated by microorganisms in the intestinal tract of the host, while metabolites produced by microorganisms can protect the integrity of intestinal epithelial cells and maintain the immune response of the intestinal tract. The diversity of microbial population and the change of community structure after the use of antibiotics can lead to the fact that the antibiotics are easy to be affected by pathogenic bacteria, so that pathogenic bacteria infection such as clostridium difficile is caused, diarrhea or severe colitis is further caused, and the abuse of antibiotics has become an important cause for causing intestinal flora imbalance. 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 host health. In research related to improving dysbacteriosis in the human intestinal tract, people are increasingly motivated to pay attention to alternatives or alternative methods of antibiotics, such as probiotics, prebiotics, synbiotics and the like. Therefore, the search and research of methods for improving intestinal dysbacteriosis is of valuable economic value.

Along with the improvement of living standard and consumption level, the healthy diet concept of people is continuously enhanced. Products such as probiotics have also gained more acceptance in order to enhance autoimmunity. The world health organization defines probiotics as follows: probiotics are active microorganisms beneficial to a host, are general names of active beneficial microorganisms which are fixedly planted in intestinal tracts and reproductive systems of human bodies and can generate definite health effects so as to improve the microecological balance of the host and exert beneficial effects on the intestinal tracts; the probiotic preparation is a living bacterial preparation which is usually formed by compounding one kind of lactobacillus or a plurality of kinds of lactobacillus, and can play a plurality of roles of regulating intestinal flora, promoting intestinal health, enhancing immunity, inhibiting diarrhea and the like after being taken.

Enterococcus faecalisEnterococcus faecalis) Is a gram-positive facultative anaerobe, one of the main flora in the human and animal intestinal tracts, and is widely used in biomedicine as a probiotic. Animal experiments show that the enterococcus faecalis prepared into the microbial preparation can improve the microecological balance in intestinal tracts and prevent and treat animal intestinal flora regional system disorder; enterococcus faecalis can also enhance the activity of macrophages, promote the immune response of animals and improve the antibody level; enterococcus faecalis can form a biological film in the intestinal tract of an animal to be adhered to the mucosa of the intestinal tract of the animal to form a lactobacillus barrier so as to resist invasion of external bacteria, viruses, mycotoxins and the like; enterococcus faecalis can also produce a plurality of antibacterial substances to inhibit common pathogenic bacteria in animals.

However, recent studies have shown that part of enterococcus faecalis has virulence genes, and many resistant enterococcus faecalis have emerged due to the long-term and large number of irregular uses of antibiotics; thus, a portion of the enterococcus faecalis strain may cause serious infections. Currently, fewer enterococcus faecalis strains with probiotic functions and capable of being applied to human clinical treatment require more strain isolation and function verification experiments. The human probiotics have more affinity to human body, are not easy to be rejected by the immune system of human body, are beneficial to colonise intestinal tracts and play a probiotic function. In order to ensure that probiotics can colonize the intestinal tract and exert the probiotics function, the probiotics need to have good gastrointestinal fluid tolerance capability to ensure that the probiotics still have a certain viable count in the gastrointestinal tract, and in order to find strains which can fully exert the functions of enterococcus faecalis in human bodies, a large number of strain screening experiments are carried out on the subject group.

Disclosure of Invention

In order to achieve the aim, the application discloses enterococcus faecalis YZ-1 and a probiotic application thereof.

The technical scheme of the application is realized as follows:

the application detects the adaptability of human gastrointestinal fluid, the drug resistance of antibiotics, the probiotics of human body and the like by separating, culturing and screening strains from human body excrement, and discovers a probiotic strain enterococcus faecalis YZ-1 which can reduce cholesterol, remove free radicals, resist cancer and resist inflammation and has the potential of treating liver inflammation, fibrosis and diabetes through a series of in-vivo and in-vitro experiments.

Enterococcus faecalis strainEnterococcus faecalis) YZ-1 with preservation number of CCTCC M2022312 and classification name of CCTCC M2022312Enterococcus faecalisYZ-1, the preservation unit is China center for type culture Collection, the preservation time is 2022, 3 and 25 days, and the preservation address is China university of Wuhan.

And (5) identifying the biochemical characteristics of the strain. Specifically, the inventor finds that the bacteria are moderately sensitive to norfloxacin, vancomycin and macrolide antibiotics and are sensitive to common antibiotics (penicillin, cephalosporin, tetracycline, meropenem and the like); the enterococcus faecalis can resist the acidic environment of gastric juice (for example, the survival condition of the strain is good under the artificial gastric juice environment with the pH of 2.5-4.5); the enterococcus faecalis is also resistant to bile salt environments in the intestinal tract (e.g. at bile salt concentrations of 0.03-0.3%, the bacteria survive well).

It should be noted that "antibiotic sensitivity" in the present application means that the strain has weak resistance to the antibiotic, and the strain can be affected in normal growth under a trace administration condition. According to embodiments of the application, the enterococcus faecalis is sensitive to penicillins, cephalosporins, macrolides, tetracyclines, carbapenems, and chloramphenicol antibiotics.

According to an embodiment of the present application, the cephalosporins include at least one selected from the group consisting of cefoperazone, cefazolin, cefuroxime sodium; the quinolone is norfloxacin; the macrolide comprises at least one selected from erythromycin, clarithromycin and azithromycin; the beta-lactam is aztreonam; the tetracyclines are tetracyclines; the glycopeptides are vancomycin; the carbapenem is melopenem.

In addition, the enterococcus faecalis of the present application has high safety. The following aspects are specifically shown: there is no hemolysis; the transfer level of drug-resistant genes is low; does not affect the normal survival and weight gain of animals such as mice.

The enterococcus faecalis YZ-1 has an inhibitory effect on enteropathogenic bacteria, wherein the enteropathogenic bacteria are staphylococcus aureusStaphylococcus aureus) Listeria monocytogenesListeria monocytogenes) Coli @Escherichia coli) Salmonella (Salmonella)Salmonella) Pseudomonas aeruginosaPseudomonas aeruginosa) And candida albicansCandida albicans)。

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

The enterococcus faecalis YZ-1 can be used for preparing medicines for reducing cholesterol.

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

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

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

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

The application has the following beneficial effects:

1. the application newly discovers a human-derived strong acid-resistant enterococcus faecalis YZ-1, the enterococcus faecalis is treated for 3 hours under the condition of pH2.5, the viable count of the enterococcus faecalis is almost unchanged, and the viable count of the commercial enterococcus faecalis is reduced by about 8 times compared with the initial viable count after being treated for 3 hours under the condition of acidity, so that a new choice is provided for the treatment of related diseases.

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

3. The supernatant or lysate of enterococcus faecalis YZ-1 with different thallus concentration or inactivated thallus thereof has effect of inhibiting proliferation of human breast cancer cells MDA-MB-231. Along with the continuous increase of the concentration of bacteria, the inhibition rate of each component YZ-1 to cancer cells is gradually increased by 6 multiplied by 10 ⁷ The inhibition rate of the inactivated bacteria of CFU/mL to 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 application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the results of enterococcus faecalis YZ-1 exhibiting stability at pH 2.5-4.5.

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

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

FIG. 4 shows the TGF-beta concentration (pg/mL) of human PBMCs after various treatments of culture filtrate, wherein normal control group represents cells without any treatment; the experimental group shows cells treated with 30g/L of culture filtrate of enterococcus faecalis YZ-1.

FIG. 5 is a standard graph of cholesterol used in the experiments.

FIG. 6 is the effect of enterococcus faecalis YZ-1 on fasting blood glucose in a high-fat model rat.

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

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

FIG. 9 is the effect of enterococcus faecalis YZ-1 on serum monocyte chemotactic protein-1 (MCP-1) of high-fat model rats.

FIG. 10 shows enterococcus faecalis YZ-1 at various concentrations against human normal hepatocytes LO ₂ Inhibition rate change plot.

FIG. 11 is a graph showing changes in 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 application will be clearly and completely described in conjunction with the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without any inventive effort, are intended to be within the scope of the application.

Example 1

Separation, purification and identification of enterococcus faecalis

(1) Sample collection and processing

The collected fresh feces of healthy young people are picked up for 5-10g, put into an EP pipe and transferred to a refrigerator at 4 ℃ for standby. In a sterile operating table, 1g of anaerobically preserved fecal sample is transferred to a sterile physiological saline solution containing 9mL of the sample until the sample is uniform, and 10-fold gradient dilution is sequentially performed to 1×10 ^-6 Pipetting 1X 10 with a pipette ^-4 、1×10 ^-5 、1×10 ^-6 A total of 3 gradients of 100. Mu.L of each dilution was plated in MRS agar medium, 3 plates per dilution.

MRS agar culture medium

10g/L of peptone, 5g/L of beef extract powder, 4g/L of yeast extract powder, 20g/L of glucose, tween-80, 1ml/L, 2g/L of dipotassium hydrogen 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 at 121deg.C with high pressure steam for 20min.

(2) Isolation and purification of strains

And (3) taking 100 mu L of the gradient diluted suspension, rapidly and uniformly coating the suspension into an MRS agar culture medium by using a coating rod, sealing the suspension by using a sealing film, placing the suspension into an anaerobic tank, and placing a bag of gas-producing anaerobic bag into the anaerobic tank to ensure an anaerobic environment. Culturing at 37 ℃ for 24 hours.

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

MRS liquid culture medium

10g/L of peptone, 5g/L of beef extract powder, 4g/L of yeast extract powder, 20g/L of glucose, 1mL of tween-80, 2g/L of dipotassium hydrogen 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 at 121deg.C with high pressure steam for 20min.

And (3) seed preservation is carried out on the bacterial liquid cultivated for the last time, 300 mu L of glycerol (50%, v/v) and 700 mu L of bacterial liquid are respectively taken, the bacterial liquid is filled into a seed preservation tube, numbered and frozen at the temperature of minus 80 ℃ for later experiments.

(3) Identification of Strain YZ-1

PCR amplification of 16S rRNA Gene

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

The PCR system was 50. Mu.L, wherein 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 primer used was 27F (5'-agagtttgatcctggctca-3'), SEQ ID NO:2 and 1492R (5'-ggttaccttgttacgactt-3'), SEQ ID NO:3. the amplified fragment was about 1500bp in length.

PCR conditions, pre-denaturation of DNA duplex at 95℃for 5 min; denaturation at 95℃for 30 s; annealing at 55 ℃ for 30 s; at 72℃for 70s, extension and 27 cycles, last at 72℃for 10min.

Agarose gel electrophoresis

0.8g agarose is weighed and dissolved in 80mL 1 xTAE solution, and after heating for 2min in a microwave oven, 8 mu L of nucleic acid dye is added; after the glue injection is solidified, 3-5 mu L of sample is added into each sample application hole; operating at 220V for 10min; the images were visualized by plate-gel under UV, and the clear-banded PCR products were selected and sequenced by Beijing qingke Biotech Co.

Analysis and identification of Gene sequence

Sequencing by Beijing Optimaceae Biotechnology Co., ltd. To obtain 16S rRNA gene sequence of strain YZ-1 shown in SEQ ID NO.1, and comparing with GenBank/EMBL/DDBJ database by BLAST analysis tool (BLAST. Ncbi. Lm. Nih. Gov), and analyzing and identifying that strain YZ-1 is enterococcus faecalisEnterococcus faecalis）。

Colony and fungus body shape

The colony is yellowish, round, and has neat edge and slightly raised edge. The bacterial form test results are gram positive, oval cocci, mostly in a double or short chain arrangement.

The physiological and biochemical reaction characteristics of the strain

Biochemical identification shows positive reactions of glucose, sucrose, maltose, lactose, trehalose, melezitose and sorbitol, and negative reactions of acacia, melibiose and catalyst.

Example 2

Acid and bile salt resistance study of strain

Culture of strains to be examined

MRS liquid medium was used for the cultivation of enterococcus faecalis isolate YZ-1 and commercial enterococcus faecalis strain. Accurately weighing according to the formula, adding distilled water, stirring, mixing, adjusting pH to 6.2, and sterilizing at 121deg.C for 20min. Enterococcus faecalis was inoculated into a sterilized MRS liquid medium at an inoculum size of 2%, and anaerobic culture was performed at 37 ℃.

Artificial gastric juice and intestinal juice preparation method

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

Artificial intestinal fluid, phosphate buffer (containing pancreatin, ph 6.8): taking 6.8g of monopotassium phosphate, adding 500mL of water to dissolve the monopotassium phosphate, and adjusting the pH to 6.8 by using 0.lmol/L sodium hydroxide solution; and (3) taking 10g of pancreatin, adding a proper amount of water to dissolve, mixing the two solutions, and adding water to dilute to 1000mL to obtain the pancreatic enzyme.

The specific experimental procedure is as follows:

gastrointestinal survival experiments

In artificial gastric juice and artificial intestinal juice, the effect of pH, bile salt concentration and treatment time on the survival rate of enterococcus faecalis was examined. Considering that gastric juice is in a state of about pH value of 2.5 in a normal state, the study simultaneously detects the survival condition of commercial enterococcus faecalis in a state of pH value of 2.5; considering that bile is in a state of about 0.2% in a usual state, the present study also 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: acid resistance test results

Remarks: enterococcus faecalis of the application: EF; commercial enterococcus faecalis: MEF.

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

TABLE 2 results of experiments on bile salt resistance

As shown in table 2 and the results of fig. 2, the concentration of bile salts in the artificial intestinal juice with concentration of 0.03% and 0.1% has little influence on the survival rate of enterococcus faecalis; when the concentration of bile salt is increased to 0.2%, the survival rate of thalli is slowly reduced along with the treatment time; at a bile salt concentration of 0.3%, enterococcus faecalis still has a survival rate of more than about 50% after 1 hour. These results all indicate that enterococcus faecalis has good cholate resistance. The viable count of the commercial enterococcus faecalis is obviously higher than the initial viable count in the trend of decreasing after being treated for 3 hours under the condition of 0.3 percent of bile salt. The enterococcus faecalis has higher bile salt resistance compared with the commercial enterococcus faecalis.

Example 3

Safety study of enterococcus faecalis strains

Drug resistance analysis

The preparation method is carried out by adopting a drug sensitive paper piece diffusion method. Diluting freshly cultured enterococcus faecalis with physiological saline to 10 ⁷ CFU/mL is evenly coated on MRS agar culture medium, drug sensitive paper sheets are placed after being placed for 3-5min at room temperature, the distance between the circle centers of the paper sheets is not less than 24mm, and the distance between the edge of the paper sheets and the edge of agar is not less than 15mm. The culture was inverted and incubated in an incubator at 37℃for 24 hours to measure the diameter of the inhibition ring. The enterococcus faecalis strain and its 30 th generation strain resistance were measured by a paper sheet diffusion method, and the results are shown in Table 3 below.

TABLE 3 results of enterococcus faecalis resistance

Note that: r = drug resistance; i = intermediary; s = sensitive; -: no bacteriostasis ring exists;

TABLE 4 results of drug resistance of enterococcus faecalis passaged 30 generation strains

as can be seen from the above-described resistance results, enterococcus faecalis and enterococcus faecalis-3 exhibit sensitivity and moderate sensitivity to most common almost all antibiotics. The drug resistance results of enterococcus faecalis and enterococcus faecalis-30 are consistent, which shows that the drug resistance of enterococcus faecalis is not affected in the passage process. The enterococcus faecalis antibiotics transfer risk is low, and the strain safety is high.

Virulence factor analysis

The haemolysin and the biofilm formation of enterococcus faecalis are measured by a literature report method, and the in vivo safety evaluation of animals is carried out according to the current edition pharmacopoeia method.

(1) By extracting plasmids from the strain, the strain does not contain plasmids, which shows that the transfer level of drug resistance genes is low and the safety of the strain is high.

(2) Hemolysin detection

Bacterial secreted hemolysin can cause erythrocyte lysis or other tissue damage. The production of hemolysin is mainly related to virulence genes such as cylA, cylB and the like. The application has no hemolytic ring around the enterococcus faecalis colony, the control strain has slight hemolytic ring, and the staphylococcus aureus has obvious hemolytic ring around. The results show that the enterococcus faecalis strain has the advantages of negative hemolysis experiment, no hemolysis of the strain and high safety.

(3) Evaluation of in vivo animal safety

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

Are all normal. The body weight of the mice before and after gastric lavage was recorded as shown in Table 5.

TABLE 5 weight changes in mice before and after gastric lavage

As a result, the weight of each group of mice increased, and the difference in the weight change between the two groups was not large (the difference between the two groups was not statistically significant, and p > 0.05).

Example 4

Probiotic study of enterococcus faecalis YZ-1

The strain frozen in the refrigerator at the temperature of minus 20 ℃ is inoculated in MRS agar culture medium and is activated for 2 times continuously, thus obtaining seed fermentation liquor. Inoculating the seed fermentation broth into MRS liquid culture medium at a volume ratio of 3:100, and culturing overnight at 37 ℃ to obtain strain fermentation broth.

(1) Experiment for inhibiting pathogenic bacteria

Gram-positive bacteria (staphylococcus aureus ATCC12592, listeria monocytogenes CMCC 54002), gram-negative bacteria (escherichia coli ATCC25922, salmonella ATCC14028, pseudomonas aeruginosa ATCC 27853) and fungi (candida albicans SC 5314) were used as indicator strains. Proper amount of indicator fungus (about 10) ⁸ CFU/mL) was added to LB agar medium to give a final concentration of about 10 ⁶ CFU/mL, shaking, pouring the flat plate, punching after the culture medium is solidified, respectively injecting 150 mu L of enterococcus faecalis YZ-1 supernatant and bacterial suspension, drying the liquid in the holes by air, culturing for 24 hours at 37 ℃, and measuring the diameter of a bacteriostasis zone. The supernatant was obtained by centrifuging fermentation broth of enterococcus faecalis YZ-1 cultured overnight (8000 r/min,5 min), and filtering with 0.22 μm filter membrane. The bacterial suspension is the fermentation broth of enterococcus faecalis YZ-1 cultured overnight, centrifugated (8000 r/min,5 min), and the bacterial suspension is taken and resuspended 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 capacities (mm) of enterococcus faecalis YZ-1 and LGG on six indicator bacteria

Note that: the differences between the letters in the same row represent significant differences (p < 0.05).

As shown in Table 6, the enterococcus faecalis YZ-1 and the lactobacillus rhamnosus LGG both have good antibacterial effect, and the inhibitory effect of YZ-1 supernatant on the escherichia coli is stronger than that of the LGG supernatant.

(2) In vitro anti-inflammatory Activity test of culture filtrate

Preparation of human PBMCs cells

Healthy human volunteers from 20 to 25 years old are students at school from the university of Zhengzhou college of medicine.

Healthy human volunteers were subjected to blood drawing and a solution of citric acid glucose (ACD) as an anticoagulant. Then added to a centrifuge tube and centrifuged at 720g rpm-gradient for 30 minutes at 4℃and then the lymphocyte layer is collected. 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 thus obtained were conditioned to a cell concentration of 4X 10 with RPMI 1640 medium containing 10% FBS ⁶ cell/mL was kept ready.

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. The inoculum of the strain was then inoculated into MRS liquid medium supplemented with a mixture of 5-30% milk, 1-10% soybean meal and 3% glucose-maltodextrin, respectively, in an inoculum amount of about 1-3% (v/v), and cultured at 37℃for 24 hours. After that, the cells were precipitated by centrifugation at about 3000-6000rpm for 60 minutes, and then the supernatant was collected and subjected to heat sterilization treatment, thereby obtaining a liquid culture of enterococcus faecalis YZ-1.

The culture filtrate of enterococcus faecalis YZ-1 was spray-dried before the following experiments, and then dissolved in an appropriate amount of RPMI 1640 medium and adjusted to a concentration of 30g/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 each 1X 10 ⁵ Cell/well number cultureIn 96-well plates containing 0.2. 0.2 mL RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin) and in an incubator (37 ℃,5% CO) ₂ ) Is cultured 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 supplementation.

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

Method for measuring concentration of IL-10

The IL-10 concentration determination of the supernatants of the normal control group, the positive control group and the experimental group was performed using an IL-10ELISA kit. The absorbance values measured for each group were each converted to their concentration (pg/mL) according to a standard curve previously made with IL-10 standards having different known concentrations relative to their own absorbance values.

Method for measuring concentration of TGF-beta

TGF-beta concentration assays of supernatants of normal control and experimental groups were performed using TGF-beta ELISA kits. The absorbance values measured for each group were each converted to their concentrations (pg/mL) according to a standard curve previously made with TGF-beta standards having different known concentrations relative to their own absorbance values.

Results of concentration measurement of IL-10

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

Concentration measurement of TGF-beta

TGF-beta concentrations were 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-beta concentration of the experimental group was significantly increased compared to the normal control group, and the culture filtrate was able to effectively activate human PBMCs cells to stimulate secretion of TGF-beta.

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

(3) Total cholesterol removal assay

The total cholesterol standard curve was determined using a total cholesterol test kit. And (3) respectively diluting cholesterol standard solution (2 mg/mL) to 2, 1.6, 1.2, 0.8, 0.4 and 0.2mg/mL by distilled water, accurately sucking 2.5 mu L of diluent with each concentration, adding into a 96-well plate, adding 250 mu L of working solution, uniformly mixing, placing in a 37 ℃ incubator for incubation for 10min, and measuring absorbance value at the wavelength of 510nm, wherein each concentration is in parallel for 3 times. Cholesterol standard curves were plotted with software Excel (fig. 5). YZ-1 fermentation broth cultured overnight was inoculated into 5mL of 1mg/mL cholesterol medium at an inoculum size of 5% (v/v), shaken well, and incubated at 37℃for 24 hours while a blank group (cholesterol medium without bacteria) was set. Centrifuging (8000 r/min,5 min) to obtain 2.5 μl of supernatant, adding into 96-well plate, adding 250 μl of working solution, mixing, incubating in 37 deg.C incubator for 10min, measuring absorbance at 510nm, taking cholesterol standard curve equation, and calculating cholesterol concentration (C) of supernatant. Total cholesterol clearance = [ (C-null-C-like)/C-null) ]x100%. The cholesterol medium is: dissolving 0.05g cholesterol with a small amount of absolute ethanol under heating, adding into 50mL MRS liquid culture medium, shaking, and sterilizing at 121deg.C for 15min.

Total cholesterol removal by 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 of YZ-1 is as high as (65.36 + -0.25)%, i.e., 653.6 μg/mL cholesterol in the medium is degraded, comparable to LGG. At low bacterial concentrations YZ-1 has a greater cholesterol lowering capacity than LGG. Indicating that enterococcus faecalis YZ-1 has high activityHas cholesterol reducing effect. The cholesterol concentration in serum is reduced, cardiovascular diseases such as arteriosclerosis, hypertension and the like are prevented, and the strain can be applied to research and development of functional foods with cholesterol reducing effect.

(4) In vivo evaluation test for blood glucose reducing function

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

Enterococcus faecalis YZ-1 has the function of reducing fasting blood glucose and blood insulin of rats. FIG. 6 shows the effect of enterococcus faecalis YZ-1 on fasting blood glucose in a high-fat model rat. The average blood sugar content of enterococcus faecalis YZ-1 intervention group (YZ-1 intervention group) and simvastatin intervention group (drug intervention group) is 2.80+/-0.56 mmol/L and 2.71+/-0.45 mmol/L, which are obviously lower than that of the high-fat model group of 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 high-fat model rats (figure 7). Serum insulin average contents of enterococcus faecalis YZ-1 (YZ-1 intervention group) and simvastatin intervention group (drug intervention group) are 28.23+/-2.02 mU/L and 38.25+/-1.98 mU/L, which are obviously lower than that of a high-fat model group 48.25+/-1.78 mU/L; the average serum insulin content of the high-fat model group is 48.25+/-1.78 mU/L, which is obviously higher than that of the normal control group of 23.12+/-2.86 mU/L.

The results show that enterococcus faecalis YZ-1 strain has the effect of remarkably reducing blood sugar and serum insulin and has similar blood sugar reducing effect with simvastatin. Lipid metabolism disorders such as hypertriglyceridemia and hyperfree fatty acidemia are closely related to insulin resistance. Insulin resistance reduces the efficiency of insulin in promoting glucose uptake and utilization, and the body's compensatory hypersecretion of insulin produces hyperinsulinemia to maintain blood glucose stability. Insulin resistance is prone to metabolic syndrome and type 2 diabetes. Enterococcus faecalis YZ-1 strain has blood lipid reducing effect, so as to relieve insulin resistance, and reduce blood sugar and serum insulin.

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

Effect of enterococcus faecalis YZ-1 on serum interleukin 1-beta (IL-1 beta) in high-fat model rats (figure 8). The average content of serum IL-1 beta in enterococcus faecalis YZ-1 intervention group and simvastatin intervention group is 14.32+/-2.08 pg/mL and 16.76+/-1.25 pg/mL, which are obviously lower than 25.32+/-2.12 pg/mL of the high-fat model group; the average content of serum IL-1 beta in the high-fat model group is 25.32+/-2.12 pg/mL, which is obviously higher than that in the normal control group of 18.23+/-2.56 pg/mL.

Effect of enterococcus faecalis YZ-1 on serum monocyte chemotactic protein-1 (MCP-1) of high-fat model rats (fig. 9). The average content of serum MCP-1 of enterococcus faecalis YZ-1 intervention group is 489.98 +/-98.23 pg/mL, which is obviously lower than that of high-fat model group 860.56 +/-65.22 pg/mL; the average content of serum MCP-1 in the simvastatin intervention group is 712.59 +/-48.34 pg/mL, and has no obvious difference with that in the high-fat model group 860.56 +/-65.22 pg/mL; the average serum MCP-1 content of the high-fat model group is 860.56 +/-65.22 pg/mL, which is obviously higher than that of the normal control group 486.12 +/-56.23 pg/mL (p < 0.001).

The results show that the enterococcus faecalis YZ-1 strain has the effect of obviously reducing serum IL-1 beta and MCP-1 inflammatory factors, and has stronger effect of reducing serum inflammatory factors than simvastatin. Liver fibrosis is the final outcome of the continued development of liver injury from a variety of causes, and MCP-l is an important macrophage chemokine in tissue injury repair, which plays an important role in liver injury and repair. IL-1β is another important inflammatory cell that causes fibrosis, is the primary inducer of the pro-inflammatory response, and can enhance the extent of the inflammatory response by coordinating and promoting the expression of other inflammatory factors, such as TNF- α. In summary, MCP-l and IL-1β are important inflammatory factors in liver fibrosis. Enterococcus faecalis YZ-1 significantly reduces the content of serum IL-1 beta and MCP-1 inflammatory factors, thereby reducing the occurrence of liver inflammation and liver fibrosis.

(6) DPPH free radical scavenging experiments

Diluting the overnight cultured enterococcus faecalis YZ-1 fermentation broth with sterile physiological saline to 5×10 respectively ⁷ 、1×10 ⁷ 、5×10 ⁶ 、1×10 ⁶ CFU/mL, DPPH radical scavenging ability of the 4 dilutions of cell lysates, supernatants and bacterial suspensions were measured separately. Taking 2mL of a sample to be detected and 2mL of an ethanol solution of LDPPH, uniformly mixing, carrying out dark reaction for 40min, setting a control group (absolute ethyl alcohol is used for replacing the sample solution) and a blank group (absolute ethyl alcohol is used for replacing the DPPH solution), and measuring the absorbance at 517 and nm wavelength. Free radical clearance= [1- (sample-a air/a pair)]X 100%. The DPPH value was 0.2mmol/L, and 0.0078g of DPPH was taken and 100mL of the solution was taken out with absolute ethanol. The supernatant is obtained by centrifuging (8000 r/min,5 min) the fermentation broth under the concentration, and filtering with 0.22 μm filter membrane. The bacterial suspension is the fermentation broth with the concentration, centrifugated (8000 r/min,5 min) and the bacterial is taken to be suspended in sterile physiological saline. The cell lysate is obtained by treating the bacterial liquid at the concentration with a cell ultrasonic breaker for 30min (800W, 15 s), centrifuging (8000 r/min,5 min), collecting supernatant, and filtering with 0.22 μm filter membrane. The DPPH radical scavenging ability of Lactobacillus rhamnosus LGG was determined in the same manner as described above. The results show that the clearance rates of the supernatant, the bacterial suspension and the lysate of enterococcus faecalis YZ-1 on DPPH free radicals are 82.19% -94.37%, 86.72% -95.38% and 56.73% -58.32%, respectively, and the clearance capacities of the bacterial suspension and the lysate of YZ-1 on DPPH free radicals are higher than that of LGG under the same concentration. The higher the cell concentration, the higher the DPPH clearance, and the higher the concentration (5X 10) ⁷ CFU/mL), the clearance rate of YZ-1 bacterial suspension to DPPH is up to 95.38%.

(7) Anticancer Activity cell experiment

Bovine serum from foetus: double antibody: DMEM medium = 1:0.1:9 ratio DMEM complete medium was formulated. For human normal liver cell LO ₂ And human breast cancer cells MDA-MB-231 for resuscitation and passaging.

Preparation of YZ-1 sample solution: taking 3 parts of YZ-1 fermentation broth, placing one part of the fermentation broth at 121 ℃ for high temperature treatment for 15min, centrifuging (8000 r/min,5 min), and re-suspending the fermentation broth with sterile PBS to obtain an inactivated bacterial group; crushing one part by ultrasonic for 15min (800W, 9 s), centrifuging, and filtering the supernatant by a 0.22 mu m filter membrane to obtain a lysate group; one portion was centrifuged (8000 r/min,5 min) and the supernatant was filtered through a 0.22 μm filter to give a supernatant set. Each set of samples was diluted to different concentrations with DMEM complete medium.

Cultured LO ₂ Cells were digested, centrifuged, and the cell density was adjusted to 80000 cells/mL, and applied to 96-well plates at 100. Mu.L per well. Placing in 37 ℃ and 5% CO ₂ Culturing in an incubator for 24 hours. After the cell coverage area reaches about 85%, the old culture medium is sucked and removed, 100 mu L of YZ-1 sample solution is added into each hole, and 37 ℃ and 5% CO are added ₂ Culturing in an incubator for 24 hours. 3 parallel controls were set for each concentration, with DMEM complete medium instead of sample as blank. After the completion of the culture, the cell viability was examined by MTT method, and the results (FIG. 10) showed that YZ-1 vs. LO ₂ The safe concentration of cells was 3X 10 ⁴ ～6×10 ⁷ CFU/mL. Thus, the concentration was selected to determine the inhibition of cancer cell proliferation by YZ-1. MDA-MB-231 cells were digested with 0.25% pancreatin, centrifuged (1200 r/min,5 min), the cells were diluted to 80000/mL, 100. Mu.L per well was added to 96-well plates, and placed at 37℃with 5% CO ₂ Culturing in an incubator for 24 hours. When the cell coverage area reaches about 85%, the old culture medium is sucked and removed, the cells are washed 3 times by PBS, and 3X 10 cells are added into each well ⁴ ～6×10 ⁷ CFU/mL YZ-1 sample solution 100. Mu.L was used as the treatment group, DMEM complete medium was used as the blank control group instead of the sample, 5-fluorouracil (5-FU) was used as the positive control group instead of the sample, and the culture was performed at 37℃for 48 hours. The survival rate of cancer cells was determined by MTT method, and the inhibition rate of YZ-1 against cancer cells was calculated. Cancer cell inhibition = [1- (treatment group a/blank group a)]X 100%. The results (FIG. 11) show that 6X 10 ⁷ The inhibition rate of the inactivated thalli of CFU/mL to human breast cancer cells MDA-MB-231 is up to 82.32 +/-0.25%, which is obviously higher than that of 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 effect.

The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the application.

<110> university of Zhengzhou

Henan Yuanzhi biotechnology Co., ltd

<120> an 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 of CCTCC M2022312Enterococcus faecalisYZ-1, the preservation unit is China center for type culture Collection, the preservation time is 2022, 3 and 25 days, and the preservation address is China university of Wuhan.

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

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

4. Use of enterococcus faecalis YZ-1 according to claim 1 for the manufacture of a medicament for reducing liver inflammation and/or fibrosis related inflammatory factors.

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