CN113913334B - Enterococcus faecalis EF-ZA1107-06 and application thereof - Google Patents
Enterococcus faecalis EF-ZA1107-06 and application thereof Download PDFInfo
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- CN113913334B CN113913334B CN202111222696.9A CN202111222696A CN113913334B CN 113913334 B CN113913334 B CN 113913334B CN 202111222696 A CN202111222696 A CN 202111222696A CN 113913334 B CN113913334 B CN 113913334B
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Abstract
The invention belongs to the technical field of microorganisms, and discloses enterococcus faecalis EF-ZA1107-06 and application thereof, wherein the strain is preserved in the microorganism strain preservation center (GDMCC) of Guangdong province at 2021, and the preservation number is GDMCCNo 61910. A series of safety experiments prove that the strain is safe and nontoxic, has strong gastrointestinal digestion resistance, high self-coagulation ability and strong antibacterial ability, has excellent cholesterol-lowering activity, antioxidant activity and anticancer activity, and can be applied to preparation of intestinal antibacterial drugs and feed additives.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to enterococcus faecalis EF-ZA1107-06 of an infant intestinal source and application thereof in preparation of medicines and feed additives for inhibiting intestinal pathogenic bacteria.
Background
Along with the improvement of living standard and consumption level, the healthy diet concept of people is continuously enhanced. Products such as probiotics have been more accepted as being more important for enhancing autoimmunity. The definition of probiotics by FAO/WHO is: probiotics are living microorganisms that when administered in sufficient amounts, provide health benefits to the host. 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 faecalis is a gram-positive facultative anaerobe, an intestinal primary resident bacterium, is often applied to various traditional fermentation products, and is a dominant microorganism population in traditional fermentation products. The enterococcus faecalis can produce bacteriocin, organic acid and other antibacterial substances in the growth process, and inhibit the growth of common pathogenic bacteria such as escherichia coli, staphylococcus aureus and the like. In addition, it also shows a certain probiotics in the aspects of reducing cholesterol, improving inflammatory reaction, regulating intestinal flora, enhancing immunity and the like, and is applied to the fields of medicine, feed, food and the like. At present, enterococcus faecalis is widely applied to feeds, and enterococcus faecalis is added into the feeds, so that the gastrointestinal microecological system of a host can be regulated, the immunity of livestock and poultry is enhanced, the disease resistance is improved, the production performance is improved, and the feed utilization rate is improved.
The human probiotics have affinity, are not easy to be rejected by the human immune system, are beneficial to colonise intestinal tracts and play a probiotic function. Therefore, the enterococcus faecalis is separated and screened from the intestinal tracts of infants, and the safety and the probiotics of the enterococcus faecalis are researched, so that the enterococcus faecalis has potential and value for development and utilization. Enterococci have a remarkable health-beneficial effect on the one hand, and some strains have a long history of safe use, and on the other hand, enterococci are a conditional pathogen, and there is a difference between strains, so that safety and nontoxicity are the primary important factors for screening of probiotic enterococcus faecalis. The strain has good gastrointestinal fluid tolerance, ensures that the strain still has a certain viable count in the gastrointestinal tract, enables the strain to be capable of colonising in the intestinal tract and plays a probiotic function.
At present, less enterococcus faecalis with cholesterol-reducing activity and anticancer activity is provided, and the enterococcus faecalis EF-ZA1107-06 with the advantages of safety and no toxicity, strong gastrointestinal fluid tolerance, high-efficiency cholesterol-reducing capability and anticancer activity of an infant intestinal source is provided, so that the enterococcus faecalis EF-ZA1107-06 is an excellent strain with cholesterol-reducing and anticancer effects. Meanwhile, the strain has good antibacterial capacity and antioxidant capacity, and can be applied to preparation of medicines and feed additives for inhibiting intestinal pathogenic bacteria.
Disclosure of Invention
The invention aims to provide a novel probiotic enterococcus faecalis EF-ZA1107-06 isolated from faeces of healthy 1 month old infants in Guangzhou city and application thereof.
In a first aspect of the invention, there is provided a strain of enterococcus faecalis EF-ZA1107-06.
In a second aspect of the invention, the safety evaluation of enterococcus faecalis EF-ZA1107-06 is provided, wherein the safety evaluation comprises nitroreductase experiments, amino decarboxylase experiments, indole experiments, hemolytic experiments, antibiotic susceptibility experiments, plasmid extraction experiments and animal acute oral toxicological experiments, the experiments prove that the enterococcus faecalis EF-ZA1107-06 nitroreductase experiments are negative, the amino decarboxylase experiments are negative, the indole experiments are negative and do not hemolyze, the strain is sensitive to ofloxacin, cefazolin, cefuroxime, tetracycline, penicillin, chloramphenicol and vancomycin, no acute toxic effect is caused on mice, and the strain is safe and nontoxic.
In a third aspect of the invention, the enterococcus faecalis EF-ZA1107-06 strain is provided with tolerance capability under the environment of artificial simulated gastrointestinal fluid. In vitro simulated digestion experiments are carried out, and after the simulated probiotics are ingested, the probiotics pass through the oral cavity and the gastrointestinal tract of a human body. The survival rate of the strain after being exposed to artificial simulated saliva for 10min is 97.3%, the survival rate after being continuously exposed to artificial simulated gastric fluid for 2h is 79.4%, and the survival rate after being continuously exposed to artificial simulated intestinal fluid for 3h is 67.2%. The strain of the invention has stronger tolerance to gastrointestinal fluids.
In a fourth aspect of the invention, there is provided the hydrophobicity and self-cohesion of the E.faecalis strain EF-ZA1107-06. The hydrophobicity of EF-ZA1107-06 is 17.74% when cultured for 1h, and the self-cohesive force reaches 96.0% when cultured for 24h.
In a fifth aspect of the invention, there is provided the inhibitory effect of the enterococcus faecalis EF-ZA1107-06 bacterial suspension or supernatant on pathogenic bacteria, wherein the inhibitory effect of the enterococcus faecalis EF-ZA1107-06 supernatant is significantly stronger than that of the bacterial suspension, the diameter (mm) of the supernatant on the inhibition zone of escherichia coli ATCC25922 is 20.5, the diameter (mm) of the inhibition zone of salmonella ATCC14028 is 16.7, the diameter (mm) of the inhibition zone of pseudomonas aeruginosa ATCC27853 is 15.0, the diameter (mm) of the inhibition zone of candida albicans SC5314 is 15.3, the diameter (mm) of the inhibition zone of listeria monocytogenes CMCC54002 is 17.3, and the diameter (mm) of the inhibition zone of the supernatant on staphylococcus aureus ATCC12592 is 16.7.
In a sixth aspect of the invention there is provided cholesterol lowering activity of said enterococcus faecalis EF-ZA1107-06, the cholesterol clearance of EF-ZA1107-06 being as high as 44.29%, i.e. 442.9. Mu.g/mL cholesterol in the culture medium being degraded.
In the seventh aspect of the invention, the antioxidant capacity of the supernatant fluid or the lysate or the bacterial suspension of the enterococcus faecalis EF-ZA1107-06 with different bacterial concentrations is provided, and the clearance rate of the supernatant fluid, the bacterial suspension and the lysate of the enterococcus faecalis EF-ZA1107-06 on DPPH free radicals is 79.38% -91.70%, 84.40% -93.16% and 52.51% -55.94% respectively.
In the eighth aspect of the invention, the inhibition effect of the supernatant fluid or the lysate or the inactivated thallus of the enterococcus faecalis EF-ZA1107-06 with different thallus concentrations on the proliferation of human breast cancer cells MDA-MB-231 is provided. With the increasing concentration of bacteria, the inhibition rate of each component EF-ZA1107-07 on cancer cells is gradually increased by 5 multiplied by 10 7 The inhibition rate of the CFU/mL inactivated thalli to the MDA-MB-231 of the human breast cancer cells is as high as 71.22%.
The strain EF-ZA1107-06 of the invention is derived from faeces of healthy infants in Guangzhou city of 1 month, and is obtained through a 16SrDNA sequencing identification result, and belongs to the enterococcus faecalis genus. A series of safety experiments prove that the strain is safe and nontoxic, has strong gastrointestinal digestion resistance, high self-coagulation ability and strong antibacterial ability, has excellent cholesterol-lowering activity, antioxidant activity and anticancer activity, and can be applied to preparation of medicines and feed additives for inhibiting intestinal pathogenic bacteria.
Preservation description
Strain name: enterococcus faecalis
Latin name: enterococcus faecalis
Strain number: EF-ZA1107-06.
Preservation mechanism: the collection of microorganism strains in Guangdong province.
Address: the university of first middle road 100 in Guangzhou city, building 59, academy of microorganisms of Guangdong province.
Preservation date: 2021, 9 and 1.
Accession numbers of the preservation center: GDMCC No. 61910.
Drawings
FIG. 1 example 1 colony morphology of E.faecalis EF-ZA1107-06 strain on MRS solid medium.
FIG. 2 example 1 gram of E.faecalis strain EF-ZA1107-06.
FIG. 3 is a phylogenetic tree diagram of E.faecalis strain EF-ZA1107-06.
FIG. 4 example 2A graph of the results of nitroreductase experiments on E.faecalis strain EF-ZA1107-06, with the left tube being a blank control, the middle tube being a control group to which E.coli ATCC25922 was added and the right tube being an experimental group to which E.faecalis strain EF-ZA1107-06 was added.
FIG. 5 is a graph showing the results of an amino acid decarboxylase experiment on E.faecalis strain EF-ZA 1107-06; lysine; ornithine; c: arginine.
FIG. 6 example 2 depicts the indole test results of E.faecalis strain EF-ZA1107-06, with the left test tube being the test group with E.faecalis strain EF-ZA1107-06, the middle test tube being the test group with the positive control of E.coli ATCC25922, and the right test tube being the blank control.
FIG. 7 is a graph showing the results of a hemolysis experiment of E.faecalis strain EF-ZA1107-06.
FIG. 8 shows the results of plasmid extraction experiments on E.faecalis strain EF-ZA1107-06.
FIG. 9 example 3 cholesterol standard curve.
FIG. 10 example 3 graphs of the results of experiments on total cholesterol removal capacity of enterococcus faecalis EF-ZA1107-06 and Lactobacillus rhamnosus standard strain ATCC 7469.
FIG. 11 shows the DPPH radical scavenging test results of E.faecalis EF-ZA1107-06 and Lactobacillus rhamnosus standard strain ATCC 7469; a) Supernatant fluid; b) Cracking liquid; c) Bacterial suspension.
FIG. 12 example 3 human normal hepatocytes LO treated with enterococcus faecalis EF-ZA1107-06 at various concentrations 2 Is a survival rate of (a).
FIG. 13 example 3 inhibition of MDA-MB-231 cell proliferation by enterococcus faecalis EF-A1107-06.
Note that: the different letters in figures 10, 11, 13 indicate that there is a significant difference (p < 0.05) between the groups.
Detailed Description
The invention provides a safe and nontoxic enterococcus faecalis EF-ZA1107-06 with probiotic activity, and particularly relates to an enterococcus faecalis which is of an infant intestinal source and resistant to gastrointestinal environment, has antibacterial capability, cholesterol-reducing activity, antioxidant activity and anticancer activity. The invention will be described and illustrated in detail below in connection with specific examples for a better understanding of the invention, but the scope of the invention is not limited to the examples shown below.
The following examples used the following media formulations:
MRS broth (available from Guangdong CycloKai microbiological technologies Co., ltd.):
taking 10g of casein enzyme digest, 10g of beef extract powder, 4g of yeast extract powder, 2g of tri-ammonium citrate, 5g of sodium acetate and magnesium sulfate (Mg SO) 4 ·7H 2 O) 0.2g, manganese sulfate (Mn SO) 4 ·4H 2 O) 0.05g, dipotassium hydrogen phosphate 2g, glucose 20g and Tween 80 1.08g, adding distilled water 1000mL, and adjusting pH to 5.7+ -0.2 after dissolution. Sterilizing at 121deg.C for 15min.
MRS solid Medium (available from Guangdong CycloKai microbiological technologies Co., ltd.):
taking 10g of peptone, 5g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of tri-ammonium citrate and 2g of magnesium sulfate (Mg SO 4 ·7H 2 O) 0.2g, manganese sulfate (Mn SO) 4 ·4H 2 O) 0.05g, agar 15g, adding distilled water 1000mL, dissolving, adjusting pH to 6.2+ -0.2, sterilizing at 121deg.C for 15min.
Nitroreductase assay medium:
taking 1g of bacteriological peptone, 0.1g of potassium nitrate, adding 100mL of distilled water, dissolving, adjusting the pH value of a culture medium to 7.4, subpackaging, and sterilizing at 121 ℃ for 15min.
Amino decarboxylase induction medium:
to 100mL of MRS broth medium, pyridoxal 5-phosphate was added in an amount of 0.005% by mass, mixed well, sub-packaged, and 0.1% by mass of lysine, ornithine and arginine were added, respectively, and autoclaved at 121℃for 15min.
Amino decarboxylase identification medium (BASM):
taking 5g of tryptone, 5g of yeast extract, 5g of beef extract, 2.5g of NaCl, 0.5g of glucose, 1g of Tween 80, 0.1g of calcium carbonate, 0.04g of ferrous sulfate, 0.2g of magnesium sulfate, 0.2g of manganese sulfate, 2g of ammonium citrate, 0.01g of thiamine, 2g of dipotassium hydrogen phosphate, 0.05g of pyridoxal 5-phosphate, 0.06g of bromocresol purple and 20g of agar, adding 1000mL of distilled water, after dissolving, adjusting the PH to 5.3, sterilizing for 10min at 121 ℃, subpackaging after slightly cooling, and respectively adding 1% of lysine, ornithine and arginine, shaking uniformly.
Peptone water medium:
taking 1g of bacteriological peptone and 0.5g of sodium chloride, adding 100mL of distilled water, adjusting the pH to 7.8 after dissolution, and sterilizing for 15min at 121 ℃.
LB agar medium:
taking 10g of peptone, 5g of sodium chloride, 1g of glucose, 5g of yeast extract powder, adding 1000mL of distilled water, dissolving, adjusting the pH to 7.0+/-0.2, and sterilizing at 121 ℃ for 15min.
Example 1: screening and identification of strains
Feces from healthy infants within one month of the month center of the loved children in Guangzhou city were collected with a sterile disposable feces collection cup, and the samples were diluted in a gradient of 10 in normal saline over 4 hours 7 The sample dilutions were plated in MRS solid medium and incubated anaerobically for 48h at 37 ℃. Selecting single bacterial colony according to the size, shape and other characteristics of bacterial colony, culturing on MRS solid culture medium for more than 3 times by streaking, obtaining a separated and purified bacterial strain, and naming the bacterial strain as EF-ZA1107-06. The bacterium isThe colony morphology of the strain is shown in figure 1, the colony morphology is characterized by a middle bulge, is white or milky white and opaque, has smooth and glossy surface and tidy edge, and has a colony diameter of about 0.5-1.0 mm.
In order to identify the strain, the bacterial form after gram staining is observed by a microscope, and the result is shown in figure 2, and the strain can be primarily judged to be enterococcus faecalis by combining physiological and biochemical reactions such as catalase, contact enzyme, gas production, sugar fermentation and the like. Molecular biology identification is carried out by 16S rDNA sequencing, and the 16S rDNA sequencing result shows that the EF-ZA1107-06 strain is enterococcus faecalis. The 16S rDNA sequence of the strain is shown in SEQ NO.1. The separated and purified enterococcus faecalis EF-ZA1107-06 strain is stored in 30% glycerol and frozen at-20 ℃.
Example 2: safety evaluation of enterococcus faecalis EF-ZA1107-06
Inoculating the frozen strain in a refrigerator at-20 ℃ into an MRS broth culture medium, activating for 2 times continuously to obtain seed fermentation liquor, inoculating the seed fermentation liquor into the MRS broth culture medium in a volume ratio of 3:100, and culturing overnight at 37 ℃ to obtain the strain fermentation liquor.
2.1 nitroreductase Activity assay
Enterococcus faecalis EF-ZA1107-06 and a positive control strain E.coli ATCC25922 which are cultured overnight by using an MRS broth culture medium are respectively inoculated into a nitroreductase detection culture medium according to an inoculum size of 3% (v/v), the nitroreductase detection culture medium without inoculated strain is used as a blank control, the culture medium is incubated for 48-96 hours at a constant temperature of 37 ℃, alpha-naphthylamine and sulfanilic acid solution are sequentially added into the culture medium, the mixture is uniformly mixed, and the color change of the culture medium is observed. The red color of the culture medium represents that the detection result of nitroreductase is positive, otherwise, the detection result of nitroreductase is negative. As shown in FIG. 4, the result of the enterococcus faecalis EF-ZA1107-06 nitroreductase is negative, namely the enterococcus faecalis EF-ZA1107-06 does not produce active nitroreductase in the proliferation metabolic process, and nitrate is not reduced to nitrite.
2.2 detection of Aminodecarboxylase Activity
The method comprises the steps of inoculating an overnight cultured enterococcus faecalis EF-ZA1107-06 fermentation liquor into an induction culture medium according to an inoculum size of 1% (v/v), simultaneously taking escherichia coli ATCC25922 as a positive control strain, taking lactobacillus rhamnosus ATCC7469 (LGG) as a negative control strain, culturing for 20-24 hours at 37 ℃, transferring for 6 times according to the same method, uniformly mixing the overnight cultured strain fermentation liquor, respectively sucking 100 mu L of bacterial liquor, uniformly coating the bacterial liquor on a BASM culture medium, culturing for 4 days at 37 ℃, and observing the color of the culture medium. The identification medium was yellow negative and purple positive. As shown in FIG. 5, enterococcus faecalis EF-ZA1107-06 amino-decarboxylase gave a negative result, i.e., the strain did not produce amino-decarboxylase.
2.3 indole experiments
The fermentation liquid of the enterococcus faecalis EF-ZA1107-06 cultured overnight and the liquid of the positive control strain escherichia coli ATCC25922 are inoculated into peptone water culture medium according to the inoculum size of 3% (v/v), a blank control is arranged at the same time, the blank control is cultivated for 72 hours at the constant temperature of 37 ℃, indole reagent is added for 8-10 drops, and the color change of the liquid level is observed. As shown in FIG. 6, the liquid surface of the culture medium Escherichia coli ATCC and 25922 was rose-red, the result was positive, while the liquid surface of the culture medium EF-ZA1007-06 was yellow, and the result was negative, which indicated that the culture medium EF-ZA1107-06 did not produce tryptophan enzyme during the proliferation process.
2.4 hemolysis experiments
Under the aseptic condition, enterococcus faecalis EF-ZA1107-06 is picked, streaked on a Columbia blood agar plate, cultured for 48 hours at the constant temperature of 37 ℃, and whether obvious hemolysis rings exist around the colony is observed. As shown in FIG. 7, there was no obvious hemolytic ring around enterococcus faecalis EF-ZA1107-06, i.e. the strain did not cause hemolytic injury to human body.
2.5 antibiotic susceptibility experiments
Taking fermentation broth of enterococcus faecalis EF-ZA1107-06 cultured overnight, and regulating concentration to about 2×10 8 CFU/mL is taken, 100 mu L of CFU/mL is evenly coated on MRS solid culture medium, after bacterial liquid is fully absorbed, antibiotic drug sensitive tablets are placed, the mixture is placed at room temperature for 20min, and the mixture is cultivated for 24h at the constant temperature of 37 ℃. The drug sensitive tablets comprise ofloxacin, cefazolin, cefuroxime, tetracycline, chloramphenicol, penicillin and vancomycin, and 3 drug sensitive sheets are parallel. The diameter of the inhibition zone was measured with a ruler. And simultaneously, staphylococcus aureus CMCC (B) 26003 is selected as a quality control strain for a drug sensitivity experiment.The sensitivity of the strain to various antibiotics was judged according to the relevant standards established by the american society for clinical and laboratory standardization (Clinical and Laboratory Standards Institute, CLSI) (see table 1). The experimental results of the drug sensitive paper sheets are shown in Table 2, and the sensitivity of enterococcus faecalis EF-ZA1107-06 to seven antibiotics is very high.
TABLE 1 drug sensitive paper content and drug resistance criterion
TABLE 2 results of drug sensitive paper experiments
2.6 plasmid extraction experiments
1.5mL of an overnight cultured enterococcus faecalis EF-ZA1107-06 fermentation broth was taken, bacterial plasmids were extracted according to the instructions of the Biyundian plasmid miniprep kit, and the obtained liquid was subjected to agarose gel electrophoresis with E.coli ATCC25922 as a positive control. Taking 5 mu L of liquid extracted from the kit, adding 1 mu L of 6×loading Buffer, blowing and sucking uniformly, and spotting on agarose gel. The running solution was 1 XTBE running buffer, the voltage was set to 150V, and the working time was 40min. After electrophoresis is finished, the migration distance on the agarose gel is analyzed by using an image recording analysis system. The agarose gel is prepared by taking 2g agarose, adding 100mL of 1 XTBE electrophoresis buffer, heating and boiling until the liquid is transparent, adding 5 mu LGoldhiew nucleic acid dye, cooling to 60 ℃, pouring into an electrophoresis tank, and cooling and solidifying. As shown in FIG. 8, the electrophoresis result shows that the lanes of the enterococcus faecalis EF-ZA1107-06 are not provided with bands, which indicates that the enterococcus faecalis EF-ZA1107-06 does not contain plasmids and the drug resistant genes cannot be transferred to the human genome.
2.7 acute oral toxicity test in mice
20 SPF-grade KM mice were taken, and the weight of the mice was 18-22 g. The experiment uses a limiting method, and only one dosage group of 10.0g/kg body weight is set. Animals fasted for 6 hours before the experiment, and the drinking water is not limited. Fermenting enterococcus faecalis EF-ZA1107-06 in logarithmic growth phaseThe concentration of the liquid is adjusted to 3X 10 by using normal saline 8 CFU/mL, stomach is filled 1 time on an empty stomach, the stomach filling capacity is 20.0mL/kg body weight, the feeding is continued for 1 hour after the stomach filling, the body weight, poisoning performance, death number and death time of the animal are observed and recorded, and the observation period is 14 days. The experimental results are shown in Table 3, and the results show that the oral liquid containing enterococcus faecalis EF-ZA1107-06 has acute oral toxicity LD50 of more than 10.0g/kg body weight for male and female KM mice, belongs to actual non-toxic grade, and has no acute toxicity effect on mice.
TABLE 3 test results of acute oral toxicity test of test substances on mice
Example 3: study of the Probiotics of enterococcus faecalis EF-ZA1107-06
Inoculating the frozen strain in a refrigerator at-20deg.C into MRS broth culture medium, and activating for 2 times to obtain seed fermentation broth. 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.
3.1 artificial simulation of gastrointestinal tolerability experiments
Taking fermentation broth of enterococcus faecalis EF-ZA1107-06 cultured overnight, centrifuging at 8000r/min for 5min, washing with sterile PBS buffer solution (pH 7.4) for 3 times, and re-suspending with sterile physiological saline. Inoculating the bacterial suspension into artificial simulated saliva according to an inoculum size of 10% (v/v), culturing at 37 ℃ for 10min, sampling during culturing for 0min and 10min, and counting the number of viable bacteria. And centrifuging (8000 r/min,5 min) the residual culture solution after sampling, re-suspending with sterile PBS buffer solution (pH 7.4) and centrifuging (8000 r/min,5 min), repeating for 2 times, re-suspending the bacterial sludge in artificial simulated gastric fluid, culturing at 37 ℃ for 2h, sampling, and counting the number of viable bacteria. The remaining culture solution after sampling was centrifuged (8000 r/min,5 min), resuspended in sterile PBS buffer solution (pH 7.4) and centrifuged, and the procedure was repeated 2 times, bacterial sludge was taken, resuspended in artificial simulated intestinal fluid, sampled at 3h, and the viable count was counted. LGG was used as a control strain. Survival= [ logCFU (N) t )/logCFU(N 0 )]X 100%, where N 0 For exposure to artificial simulated saliva for 0minNumber of viable bacteria at the time, N t The number of viable bacteria when the strain was exposed to artificial simulated saliva for 10min, to artificial simulated gastric fluid for 2h and to artificial simulated intestinal fluid for 3 h. Wherein, the preparation of the artificial simulated saliva is as follows: 0.071g of disodium hydrogen phosphate, 0.006g of monopotassium phosphate and 0.24g of sodium chloride are weighed, a small amount of distilled water is added for complete dissolution, the pH of the solution is regulated to 6.75 by 1mol/L HCl and 1mol/L NaOH, 0.027g of alpha-amylase is added, distilled water is used for constant volume to 30mL, and a 0.22 mu m filter membrane is used for sterilization for standby. The preparation of the artificial simulated gastric juice comprises the following steps: weighing 0.88g of sodium chloride, dissolving with a small amount of distilled water, regulating the pH of the solution to 3 with 1mol/L HCl and 1mol/L NaOH, adding pepsin according to the concentration of 5g/L, fixing the volume to 30mL with distilled water, and sterilizing with a 0.22 mu m filter membrane for later use. The preparation of the artificial simulated intestinal juice comprises the following steps: weighing 0.27g of monopotassium phosphate, weighing a small amount of distilled water for dissolution, adjusting the pH of the solution to 6.8 by using 1mol/L HCl and 1mol/L NaOH, adding trypsin according to the proportion of 0.1g/L, fixing the volume to 30mL by using distilled water, and sterilizing by using a 0.22 mu m filter membrane for later use. As shown in Table 4, after the enterococcus faecalis EF-ZA1107-06 and LGG are exposed to artificial saliva, the survival rate of the enterococcus faecalis EF-ZA1107-06 is kept to be more than 90%, and the survival rate of the enterococcus faecalis EF-ZA1107-06 is obviously higher than that of the LGG (p is smaller than 0.05), and after the enterococcus faecalis EF-ZA 11006 and LGG are continuously exposed to artificial saliva for 10min, artificial gastric juice for 2h and artificial intestinal juice for 3h, the survival rate of the enterococcus faecalis EF-ZA 11006 disclosed by the invention is still more than 65%, so that the enterococcus faecalis EF-ZA 11006 disclosed by the invention has better tolerance to the artificial gastric juice and can reach the intestinal tract to exert the probiotic effect.
TABLE 4 survival of enterococcus faecalis EF-ZA1107-06 and LGG in artificial simulated gastrointestinal fluids (%)
Note that: the differences between the letters in the same column represent significant differences (p < 0.05).
3.2 test of the hydrophobic Properties of enterococcus faecalis EF-ZA1107-06
Taking an overnight cultured enterococcus faecalis fermentation liquor, centrifuging (8000 r/min,10 min), re-suspending with a sterile PBS buffer solution (pH 7.4), centrifuging, repeating for 2 times, taking bacterial mud, re-suspending with the PBS solution, and adjusting the OD600 value of the bacterial suspension to be 0.5+/-0.02. Take 3mLMixing the bacterial suspension with 1mL of dimethylbenzene, vortex oscillating for 2min, standing at room temperature for 0.5h and 1h respectively, taking water phase, measuring OD600 value, and A 0 The OD600 before mixing was determined. Hydrophobicity= [ (a) 0 -A t )/A 0 ]X 100%. The hydrophobicity of lactobacillus rhamnosus standard strain ATCC7469 (LGG) was determined by the same method as described above. As shown in Table 5, the E.faecalis EF-ZA1107-06 was more hydrophobic than LGG when left to stand for 0.5 h.
TABLE 5 hydrophobicity of enterococcus faecalis EF-ZA1107-06 and LGG (%)
Note that: the differences between the letters in the same row represent significant differences (p < 0.05).
3.3 test of self-coagulation ability of enterococcus faecalis EF-ZA1107-06
Centrifuging (8000 r/min,10 min) fermentation broth of enterococcus faecalis cultured overnight, re-suspending with sterile PBS buffer solution (pH 7.4), centrifuging, repeating for 2 times, re-suspending bacterial mud with PBS solution, vortex oscillating, standing at room temperature, standing for 0, 2, 6, 20, 24 hr respectively, and sucking supernatant to determine OD600 value, self-coagulation ability = [ (A) 0 -A t )/A 0 ]X 100%. Wherein A is t To OD600 value after placement t h, A 0 OD600 was set for 0 h. The self-clotting ability of lactobacillus rhamnosus standard strain ATCC7469 (LGG) was determined in the same manner as described above. As shown in Table 6, the self-coagulation ability of enterococcus faecalis EF-ZA1107-06 can reach (96.0+ -1.7)% when the product is left stand for 24 hours, and the product has good self-coagulation ability.
TABLE 6 self-coagulant forces of enterococcus faecalis EF-ZA1107-06 and LGG (%)
Note that: the difference between the different representatives of the letters in the same row is remarkable (p < 0.05)
3.4 experiments to inhibit pathogenic bacteria
Gram positive bacteria (Staphylococcus aureus ATCC12592, prunus salicina)Stonecrop CMCC 54002), gram-negative bacteria (e.g., 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 EF-ZA1107-06 supernatant and bacterial suspension, air-drying liquid in the holes, culturing at 37 ℃ for 24 hours, and measuring the diameter of a bacteriostasis ring. The supernatant was obtained by centrifuging fermentation broth of enterococcus faecalis EF-ZA1107-06 cultured overnight (8000 r/min,5 min), and filtering with 0.22 μm filter membrane. The bacterial suspension is fermentation broth of enterococcus faecalis EF-ZA1107-06 cultured overnight, centrifuging (8000 r/min,5 min), and taking the bacterial suspension to be resuspended in sterile physiological saline. The bacteriostatic ability of lactobacillus rhamnosus standard strain ATCC7469 (LGG) was determined in the same manner as described above. As shown in Table 7, the results show that the enterococcus faecalis EF-ZA1107-06 and the lactobacillus rhamnosus LGG have good antibacterial effects, and the effect of the EF-ZA1107-06 supernatant on inhibiting the escherichia coli is stronger than that of the LGG supernatant. Enterococcus faecalis EF-ZA1107-06 can be applied to feed additives, inhibit the reproduction of pathogenic bacteria in animal intestinal tracts, improve the disease resistance of animals, and contribute to the increase of the economic benefit of livestock and poultry animal cultivation.
TABLE 7 inhibition by enterococcus faecalis EF-ZA1107-06 and LGG against six indicator bacteria (mm)
Note that: the differences between the letters in the same row represent significant differences (p < 0.05).
3.5 Total Cholesterol removal experiment
The total cholesterol standard curve was determined using a total cholesterol test kit. Diluting cholesterol standard solution (2 mg/mL) with distilled water to 2, 1.6, 1.2, 0.8, 0.4, 0.2mg/mL, accurately sucking 2.5 μl of each concentration of the diluted solution, adding into 96-well plate, addingAdding 250 μl of working solution, mixing, incubating in a 37 ℃ incubator for 10min, and measuring absorbance at 510nm wavelength, wherein each concentration is performed in parallel for 3 times. Cholesterol standard curves were plotted with software Origin 8.0, see figure 9. The EF-ZA1107-06 fermentation broth cultured overnight was inoculated into 5mL of 1mg/mL cholesterol medium at an inoculum size of 5% (v/v), shaken and simultaneously a blank group (cholesterol medium without bacteria) was set for culturing at 37℃for 24 hours. 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) Empty space -C Sample )/C Empty space )]X 100%. The cholesterol medium is as follows: 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. The total cholesterol removal capacity of lactobacillus rhamnosus standard strain ATCC7469 (LGG) was determined in the same manner as described above. As a result, as shown in FIG. 10, at the highest concentration (2X 10 8 CFU/mL), the clearance rate of the cholesterol of EF-ZA1107-06 is as high as (44.29 +/-0.30)%, namely 442.9 mug/mL of cholesterol in the culture medium is degraded, which is equivalent to LGG. At low bacterial concentration, EF-ZA1107-06 has stronger cholesterol-lowering ability than LGG. Indicating that enterococcus faecalis EF-ZA1107-06 has high cholesterol reducing capability. Reducing cholesterol concentration in serum is beneficial for preventing cardiovascular diseases such as arteriosclerosis and hypertension.
3.6DPPH free radical scavenging experiments
Diluting the fermentation broth of enterococcus faecalis EF-ZA1107-06 with sterile physiological saline to 2×10 8 、1×10 8 、5×10 7 、2×10 7 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 DPPH ethanol solution, 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 nm. Radical scavenging rate= [1- (a) Sample -A Empty space /A For a pair of )]X 100%. The DPPH value was 0.2mmol/L, and 0.0078g of DPPH was taken and the volume was adjusted to 100mL 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. As shown in FIG. 11, the clearance rates of the supernatant, bacterial suspension and lysate of enterococcus faecalis EF-ZA1107-06 on DPPH free radicals are 79.38% -91.70%, 84.40% -93.16% and 52.51% -55.94%, respectively, and under the same concentration, the clearance capacities of the bacterial suspension and lysate of EF-ZA1107-06 on DPPH free radicals are higher than that of LGG. The higher the cell concentration, the higher the DPPH clearance, and the higher the concentration (2X 10) 8 CFU/mL), the clearance rate of EF-ZA1107-06 bacterial suspension to DPPH is up to 93.16%.
3.7 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 EF-ZA1107-06 sample solution: taking 3 parts of EF-ZA1107-06 fermentation liquor, placing one part of the fermentation liquor at 121 ℃ for high temperature treatment for 15min, centrifuging (8000 r/min,5 min), and re-suspending the fermentation liquor by using sterile PBS to prepare an inactivated thallus 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 percent 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, and 100 mu LEF is added into each holeZA1107-06 sample solution was incubated in a 5% CO2 incubator at 37℃for 24h. 3 parallel controls were set for each concentration, with DMEM complete medium instead of sample as blank. After the culture, the cell viability was examined by MTT method, and the result is shown in FIG. 12, where EF-ZA1107-06 vs. LO 2 The safe concentration of cells was 2X 10 4 ～5×10 7 CFU/mL. Thus, the concentration was selected to measure the inhibition of the proliferation of the cancer cells by EF-ZA1107-06.
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 each well is added with 2 multiplied by 10 4 ～5×10 7 CFU/mL EF-ZA1107-06 sample solution 100 μl 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 37deg.C for 48h. The survival rate of cancer cells was determined by MTT method, and the inhibition rate of EF-ZA1107-06 on cancer cells was calculated. Cancer cell inhibition rate= [1- (a) Treatment group /A Blank group )]X 100%. As a result, as shown in FIG. 13, 5X 10 7 The inhibition rate of the inactivated bacteria of CFU/mL to human breast cancer cells MDA-MB-231 is as high as (71.22+/-0.72)%, which is obviously higher than that of 5-FU. The result shows that the enterococcus faecalis EF-ZA1107-06 has better anticancer activity and larger potential application value.
Sequence listing
<110> university of North China
<120> enterococcus faecalis EF-ZA1107-06 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1417
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
catgcagtcg aacgcttctt tcctcccgag tgcttgcact caattggaaa gaggagtggc 60
ggacgggtga gtaacacgtg ggtaacctac ccatcagagg gggataacac ttggaaacag 120
gtgctaatac cgcataacag tttatgccgc atggcataag agtgaaaggc gctttcgggt 180
gtcgctgatg gatggacccg cggtgcatta gctagttggt gaggtaacgg ctcaccaagg 240
ccacgatgca tagccgacct gagagggtga tcggccacac tgggactgag acacggccca 300
gactcctacg ggaggcagca gtagggaatc ttcggcaatg gacgaaagtc tgaccgagca 360
acgccgcgtg agtgaagaag gttttcggat cgtaaaactc tgttgttaga gaagaacaag 420
gacgttagta actgaacgtc ccctgacggt atctaaccag aaagccacgg ctaactacgt 480
gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc 540
gagcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat 600
tggaaactgg gagacttgag tgcagaagag gagagtggaa ttccatgtgt agcggtgaaa 660
tgcgtagata tatggaggaa caccagtggc gaaggcggct ctctggtctg taactgacgc 720
tgaggctcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 780
cgatgagtgc taagtgttgg agggtttccg cccttcagtg ctgcagcaaa cgcattaagc 840
actccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca 900
caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 960
atcctttgac cactctagag atagagcttt cccttcgggg acaaagtgac aggtggtgca 1020
tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080
tattgttagt tgccatcatt tagttgggca ctctagcgag actgccggtg acaaaccgga 1140
ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1200
caatgggaag tacaacgagt cgctagaccg cgaggtcatg caaatctctt aaagcttctc 1260
tcagttcgga ttgcaggctg caactcgcct gcatgaagcc ggaatcgcta gtaatcgcgg 1320
atcagcacgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 1380
gagtttgtaa cacccgaagt cggtgaggta acctttt 1417
Claims (3)
1. Enterococcus faecalis strainEnterococcus faecalis) EF-ZA1107-06, wherein said strain was deposited at the Guangdong province microorganism strain deposit center (GDMCC) on 1/9/2021 under accession number GDMCC No. 61910.
2. The use of enterococcus faecalis as claimed in claim 1 in the manufacture of a medicament for inhibiting enteropathogenic bacteria, said enteropathogenic bacteria being staphylococcus aureus @Staphylococcus aureus) Listeria monocytogenesListeria monocytogenes) Coli @Escherichia coli) Salmonella (Salmonella)Salmonella) ATCC14028 and Pseudomonas aeruginosaPseudomonas aeruginosa) And candida albicansCandida albicans)。
3. Use of enterococcus faecalis according to claim 1 for the preparation of a feed additive.
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