CN112410250A - Enterococcus faecalis and application thereof - Google Patents
Enterococcus faecalis and application thereof Download PDFInfo
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- CN112410250A CN112410250A CN202011265258.6A CN202011265258A CN112410250A CN 112410250 A CN112410250 A CN 112410250A CN 202011265258 A CN202011265258 A CN 202011265258A CN 112410250 A CN112410250 A CN 112410250A
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- enterococcus faecalis
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- enterococcus
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Abstract
The invention relates to the technical field of microorganisms, and particularly discloses enterococcus faecalis with a high-efficiency blood pressure lowering effect and application thereof, wherein the enterococcus faecalis comprises a 16s rRNA sequence which is at least 95% identical to SEQ ID No.10, the enterococcus faecalis is enterococcus faecalis 1004-9 or enterococcus faecalis 1004-10 or enterococcus faecalis 1028-1, the enterococcus faecalis 1004-9, the enterococcus faecalis 1004-10 and the enterococcus faecalis 1028-1 are all preserved in Guangdong province microorganism strain preservation center, and the preservation number of the enterococcus faecalis 1004-9 is GDMCC NO: 60992, the enterococcus faecalis 1004-10 has a collection number of GDMCC NO: 60993, the collection number of enterococcus faecalis 1028-1 is GDMCC NO: 60994. the enterococcus faecalis obtained by screening has a strong angiotensin converting enzyme inhibition rate, can efficiently reduce blood pressure, has strong gastrointestinal digestion resistance and is free of hemolysis.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to enterococcus faecalis with a high-efficiency blood pressure lowering effect and application thereof.
Background
Cardiovascular disease is already a major public health problem in today's society, and hypertension is the most major risk factor for cardiovascular and cerebrovascular diseases. The latest data show that the prevalence rate of adult hypertension in China reaches 27.9%, that is, 1 hypertension patient exists in every 4 adults, and the health of people is seriously threatened.
The existing commonly used blood pressure lowering drugs comprise diuretics (thiazines), Angiotensin Converting Enzyme Inhibitors (ACEIs), beta-receptor blockers, calcium channel blockers and Angiotensin II receptor blockers, and in addition, some traditional Chinese medicines also have certain effect on blood pressure regulation. Among the currently used drugs, the ACEI drugs represented by captopril and enalapril have better clinical curative effects. The ACEI drug can inhibit ACE activity to reduce angiotensin II, and inhibit bradykinin degradation for dilating blood vessel to achieve effects of dilating blood vessel and lowering blood pressure. The ACEI medicine has good antihypertensive effect and fewer side effects, and clinical tests prove that the medicine has good safety for patients with hypertension complicated with coronary heart disease, heart failure, cardiac hypertrophy, cardiomyopathy, diabetes, mild proteinuria, mild renal insufficiency and the like. However, the manufacturing of ACEI drugs at present mainly depends on chemical synthesis, while synthetic drugs have some adverse reactions, mainly including slight cough, hypovolemia, renal function reduction, hyperkalemia, angioneurotic edema and the like, and the application of ACEI drugs is greatly limited due to the toxic and side effects.
Therefore, finding a blood pressure lowering functional product with higher safety and less side effects is a research focus of people. Research shows that some bioactive components in food have better preventing and regulating effect on some cardiovascular diseases. The bioactive factors can be obtained from daily diet, have higher safety and less side effect, and are the best substitute for the medicine for preventing and treating hypertension. In recent years, the functional food is more and more concerned by people, and the eating of the functional food not only can obtain basic nutrient substances required by organisms in daily life, but also has a promotion effect on human health, so the appearance of the functional food develops a new field of food industry.
The functionality of probiotic fermented milk products is currently a hot spot of research at home and abroad, and the function of lowering blood pressure of fermented milk products gradually draws attention, but at present, domestic research on blood pressure-lowering fermented milk is less. In 1900 Masayoshi Furushiro et al, the effect of oral Lactobacillus casei (Lactobacillus casei) autolysed extract on systolic blood pressure was studied with Spontaneous Hypertensive Rats (SHR). The results show that lactobacillus casei can significantly reduce the blood pressure of SHR and has no influence on the blood pressure of normal rats. The blood pressure reduction is characterized in that the blood pressure regulation still has a certain effect within a period of time after the medicine is stopped taking. In 2000, Kawase found that fermented milk by using Streptococcus thermophilus (Streptococcus thermophilus) TMC1543 and Lactobacillus casei TMC0409 not only can reduce cholesterol, but also has an obvious blood pressure reducing effect. A plurality of ACE inhibitory peptides can be obtained by mixing and fermenting Gobbetti with Lactococcus lactis FT4 and Lactobacillus bulgaricus subsp SS 1. In 2006, Muguerza studied the blood pressure lowering activity of fermented milk of Enterococcus faecalis (Enterococcus faecalis), and found that the systolic blood pressure of SHR can be lowered by more than 30mmHg by using fermented milk of Enterococcus faecalis. At present, blood pressure lowering products are already made available in Finland, Japan and other countries, the research and development of blood pressure lowering probiotics in China still remain blank, and a great deal of work is still needed in the aspect of screening and culturing good probiotics with the blood pressure lowering characteristic. The health food with the function of reducing blood pressure is prepared by improving diet to prevent and treat hypertension and screening probiotics to ferment skim milk, and has important significance for developing new probiotics related functional food and promoting human health.
Disclosure of Invention
The enterococcus faecalis screened by the invention has high angiotensin converting enzyme inhibition rate, can reduce blood pressure efficiently, has strong gastrointestinal digestion resistance and is free from hemolysis.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides an enterococcus faecalis comprising a 16s rRNA sequence having at least 95% identity to SEQ ID No. 10.
In a preferred embodiment of the Enterococcus faecalis according to the present invention, the Enterococcus faecalis is named as Enterococcus faecalis (Enterococcus faecalis)1004-9, or named as Enterococcus faecalis (Enterococcus faecalis)1004-10, or named as Enterococcus faecalis (Enterococcus faecalis)1028-1, and the Enterococcus faecalis (Enterococcus faecalis)1004-9, Enterococcus faecalis (Enterococcus faecalis)1004-10, and Enterococcus faecalis (Enterococcus faecalis)1028-1 are all preserved in Guangdong provincial culture collection center at 4-3 days in 2020, addresses: the microbial research institute of Guangdong province No. 100 of the Mieli Zhonglu city, Guangzhou, China, the preservation number of the Enterococcus faecalis (Enterococcus faecalis)1004-9 is GDMCC NO: 60992, the Enterococcus faecalis (Enterococcus faecalis)1004-10 has a deposit number of GDMCC NO: 60993, the Enterococcus faecalis (Enterococcus faecalis)1028-1 has a deposit number GDMCC NO: 60994.
the inventor obtains three Enterococcus faecalis strains with the function of reducing blood pressure through a large number of researches and screening for the first time, and the three Enterococcus faecalis strains are named as Enterococcus faecalis (Enterococcus faecalis)1004-9, Enterococcus faecalis (Enterococcus faecalis)1004-10 and Enterococcus faecalis (Enterococcus faecalis) 1028-1. The test proves that the three Enterococcus faecalis have stronger angiotensin converting enzyme inhibition rate, can reduce blood pressure, has stronger gastrointestinal tract digestion resistance, does not have hemolytic activity and has better safety, in addition, the three Enterococcus faecalis obtained by screening the invention is sensitive to penicillin, ampicillin and chloramphenicol, shows a medium for erythromycin and shows drug resistance to vancomycin, norfloxacin and rifampicin, wherein the Enterococcus faecalis (Enterococcus faecalis)1004-9 and Enterococcus faecalis (Enterococcus faecalis)1004-10 are sensitive to tetracycline, and the Enterococcus faecalis (Enterococcus faecalis)1028-1 shows drug resistance to tetracycline.
As a preferred embodiment of the Enterococcus faecalis according to the present invention, the Enterococcus faecalis (Enterococcus faecalis)1004-9 comprises the specific molecular target shown in SEQ ID NO.1, the Enterococcus faecalis (Enterococcus faecalis)1004-10 comprises the specific molecular target shown in SEQ ID NO.2, and the Enterococcus faecalis (Enterococcus faecalis)1028-1 comprises the specific molecular target shown in SEQ ID NO. 3.
In a second aspect, the invention provides a primer for detecting Enterococcus faecalis, wherein primers corresponding to Enterococcus faecalis (Enterococcus faecalis)1004-9 are SEQ ID No.4 and SEQ ID No. 5; the primers corresponding to the Enterococcus faecalis (Enterococcus faecalis)1004-10 are SEQ ID NO.6 and SEQ ID NO.7, and the primers corresponding to the Enterococcus faecalis (Enterococcus faecalis)1028-1 are SEQ ID NO.8 and SEQ ID NO. 9.
In a third aspect, the invention provides an application of the enterococcus faecalis in preparing a microbial preparation with a blood pressure reducing effect.
In a fourth aspect, the invention provides the application of the enterococcus faecalis in preparing foods and medicines with the effect of reducing blood pressure.
As a preferred embodiment of the use according to the invention, the food product is a functional food product.
As a preferred embodiment of the use according to the invention, the functional food is a fermented food.
In a fifth aspect, the invention provides a food or a medicine with a function of reducing blood pressure, which comprises the enterococcus faecalis.
As a preferred embodiment of the food or the drug of the present invention, the food or the drug further comprises a pharmaceutical adjuvant, the adjuvant may be corn flour, glucose or sucrose, etc., and the adjuvant may improve the performance of the microbial preparation, wherein the adjuvant and the carrier are not limited to the above-mentioned materials, but may be other materials conventional in the art.
The three-strain enterococcus faecalis provided by the invention has higher angiotensin converting enzyme inhibition rate; the enterococcus faecalis obtained by screening has good capability of simulating gastrointestinal digestion, enriches a strain resource library, and has great application potential and value in the aspect of developing functional foods for preventing and treating hypertension.
Test results show that the angiotensin converting enzyme inhibition rate of the probiotic fermented blood pressure lowering product Evolus is 85% -90%, and the angiotensin converting enzyme inhibition rate of the screened three-strain enterococcus faecalis is higher than that of Evolus, so that the enterococcus faecalis has a better blood pressure lowering effect.
Compared with the prior art, the invention has the following beneficial effects:
the three Enterococcus faecalis strains with the function of reducing blood pressure are obtained by screening for the first time and are respectively named as Enterococcus faecalis (Enterococcus faecalis)1004-9, Enterococcus faecalis (Enterococcus faecalis)1004-10 and Enterococcus faecalis (Enterococcus faecalis) 1028-1.
Drawings
FIG. 1 is a graph showing the results of the angiotensin-converting enzyme inhibitory ratio assay of different strains in example 3;
FIG. 2 is a schematic diagram of the phylogenetic tree of 16S rDNA of Enterococcus faecalis (Enterococcus faecalis) 1004-9;
FIG. 3 is a schematic diagram of the phylogenetic tree of 16S rDNA of Enterococcus faecalis (Enterococcus faecalis) 1004-10;
FIG. 4 is a schematic diagram of the 16S rDNA phylogenetic clade of Enterococcus faecalis (Enterococcus faecalis) 1028-1;
FIG. 5 is a schematic diagram showing the results of a GI tract simulation experiment using Enterococcus faecalis (Enterococcus faecalis)1004-9, Enterococcus faecalis (Enterococcus faecalis)1004-10 and Enterococcus faecalis (Enterococcus faecalis) 1028-1;
FIG. 6 is a graph showing the results of measuring the hydrophobicity of Enterococcus faecalis (Enterococcus faecalis)1004-9, Enterococcus faecalis (Enterococcus faecalis)1004-10, and Enterococcus faecalis (Enterococcus faecalis) 1028-1;
FIG. 7 is a schematic diagram I showing the result of electrophoresis in the PCR detection specificity evaluation of Enterococcus faecalis (Enterococcus faecalis)1004-9 in example 5;
FIG. 8 is a schematic diagram II showing the result of electrophoresis in the PCR detection specificity evaluation of Enterococcus faecalis (Enterococcus faecalis)1004-9 in example 5;
FIG. 9 is a schematic diagram I showing the result of electrophoresis in the PCR detection specificity evaluation of Enterococcus faecalis (Enterococcus faecalis)1004-10 in example 5;
FIG. 10 is a schematic diagram II showing the result of electrophoresis in the PCR detection specificity evaluation of example 5, Enterococcus faecalis (Enterococcus faecalis) 1004-10;
FIG. 11 is a schematic diagram I showing the result of electrophoresis in the PCR detection specificity evaluation of example 5 Enterococcus faecalis (Enterococcus faecalis) 1028-1;
FIG. 12 is a schematic diagram II showing the result of electrophoresis in the PCR detection specificity evaluation of example 5 Enterococcus faecalis (Enterococcus faecalis) 1028-1;
FIG. 13 is a schematic diagram showing the results of a hemolysis experiment of Enterococcus faecalis (Enterococcus faecalis) 1004-9;
FIG. 14 is a schematic diagram showing the results of a hemolysis experiment of Enterococcus faecalis (Enterococcus faecalis) 1004-10;
FIG. 15 is a schematic diagram showing the results of a hemolysis experiment of Enterococcus faecalis (Enterococcus faecalis) 1028-1.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1 preservation information of enterococcus faecalis
The invention provides Enterococcus faecalis, which is named as Enterococcus faecalis (Enterococcus faecalis)1004-9, or named as Enterococcus faecalis (Enterococcus faecalis)1004-10, or named as Enterococcus faecalis (Enterococcus faecalis)1028-1, 1004-9, 1004-10 and 1028-1, and is preserved in Guangdong province microbial culture collection center 4-3 days in 2020, addresses: the collection number of Enterococcus faecalis (Enterococcus faecalis)1004-9 is GDMCC NO: 60992, Enterococcus faecalis (Enterococcus faecalis)1004-10 with deposit number GDMCC NO: 60993, Enterococcus faecalis (Enterococcus faecalis)1028-1 with deposit number GDMCC NO: 60994. enterococcus faecalis (Enterococcus faecalis)1004-9, Enterococcus faecalis (Enterococcus faecalis)1004-10 and Enterococcus faecalis (Enterococcus faecalis)1028-1 comprise a 16s rRNA sequence having at least 95% identity to SEQ ID No. 10.
Example 2 isolation and identification of lactic acid bacteria
Collecting human feces with normal and healthy blood pressure in the world long-life county-Banana Ling region as a sample, adding about 1g of the feces sample into 10mL of TPY liquid culture medium in an aseptic environment, shaking, mixing uniformly, culturing for 24h at 37 ℃ under an anaerobic condition to prepare a stock solution, and sucking 0.5mL of bacterial solution for gradient dilution. Adding physiological saline to obtain 10-1、10-2、10-3、10-4、10-5Diluting the gradiental bacteria suspension, selecting 10-3、10-4、10-5And (3) sucking 200 mu L of three gradient bacterium suspensions to TPY solid culture medium and modified TPY solid culture medium respectively, smearing the three gradient bacterium suspensions to be uniform by using a disposable sterile coating rod, and culturing for 48h under the anaerobic condition at 37 ℃. Typical colonies on the plate were picked, streaked for purification to give pure colonies, the seeds were preserved and extracted for bacterial DNA, PCR amplification primer selection 27F: 5'-AGA GTT TGA TCC TGG CTC AG-3', respectively; 1492R: 5'-CTAC GGC TAC CTT GTT ACG A-3' are provided. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 35 cycles of 95 ℃ for 15s, 56 ℃ for 30s and 72 ℃ for 45s, and annealing and extending for 10min at 72 ℃. And then performing first-generation sequencing, comparing the sequence with an NCBI database, performing homology analysis, and selecting lactic acid bacteria for experiment.
77 lactic acid bacteria were obtained by co-screening, and the results of the comparison are shown in Table 1.
TABLE 1 lactic acid bacteria identification results
Example 3 screening of blood pressure lowering enterococcus faecalis
Inoculating the activated strain into 11% (w/v) skim milk sterilized at 105 deg.C for 15min at 4%, culturing at 37 deg.C for 24 hr, activating for 3 times, inoculating into skim milk at 4%, and culturing at 37 deg.C until the skim milk coagulates. Centrifuging the prepared fermented milk at 4 deg.C for 10min at 7000 Xg, collecting supernatant, adjusting pH to 7.5 with 5mol/L NaOH solution, centrifuging at 4 deg.C for 3min at 11000 Xg, collecting supernatant, filtering with 0.45 μm filter membrane, and storing the obtained whey at 4 deg.C for use. 10 μ L of 0.25U ACE, 10 μ L whey samples were added to 4 wells of a 96-well plate, respectively; 10 μ L whey sample, 10 μ L50 mmol/L Tris-HCl; 10 μ L of 0.25U ACE, 10 μ L of 50mmol/L Tris-HCl; 20 μ L50 mmol/L Tris-HCl. 150 μ L of 0.88mmol/L FAPGG in 37 ℃ water bath for 15min was added at the fastest rate. Immediately after the sample addition, the initial absorbance of each sample well was measured at 37 ℃ at 340nm with a microplate reader after shaking for 30s, and each absorbance was designated as a1，b1，c1，d1After incubation at 37 ℃ for 15min, the absorbance of each well was measured again under the same conditions, and each well was designated as a2，b2，c2，d2The absorbance decrease value of each sample well is A ═ a1-a2、B＝b1-b2、C＝c1-c2、D＝d1-d2. And calculating the ACE inhibition ratio of the fermented milk according to a formula. Obtaining the strain with higher ACE inhibition rate after calculation
ACE inhibition (%) - (C-D) - (a-B) ]/(C-D) × 100%
The ACE inhibition rate of the separated lactic acid bacteria is measured, the measurement result is shown in figure 1, 3 strains of lactic acid bacteria are found to have higher ACE inhibition rates, wherein the inhibition rates of 1004-9, 1004-10 and 1028-1 are 97.10%, 96.89% and 92.23% respectively, and are enterococcus faecalis, and the 16S rDNA phylogenetic clade of the three strains is shown in figure 2-4. According to the relevant research results, the ACE inhibition rate of the probiotic fermentation blood pressure lowering product Evolus is 85% -90%, and the ACE inhibition rate of three strains of enterococcus faecalis screened by the research is higher than that of Evolus.
Example 4 biological Properties of enterococcus faecalis
(1) Test for tolerance to artificial gastrointestinal fluids
Tolerance to artificial gastric juice test: PBS (pH7.4) buffer solution was adjusted to pH 3.0 with 0.1mol/L HCl, pepsin (3g/L) was added, and after dissolution, the mixture was filtered through a 0.22 μm sterile filter membrane and ready to use. Activating the selected strain, inoculating the prepared bacterial liquid into 10mL of TPY liquid culture medium at an inoculum size of 2%, standing and culturing at 37 deg.C under anaerobic condition for 24h, centrifuging at 4000 Xg for 10min, collecting thallus, washing thallus with PBS (pH7.4) for 3 times, and adjusting the bacterial liquid concentration to 10 with PBS9CFU/mL (reference OD600 of 1.0), ready for use. Adding 1mL of PBS resuspended lactobacillus bacterial liquid into 5mL of simulated gastric juice, adding 1.5mL of 0.5% (w/v) NaCl, mixing uniformly, quickly placing into a 37 ℃ incubator, culturing for 0h and 3h, diluting with sterile physiological saline according to a gradient of 1:10, sucking 100 mu L of diluent, uniformly coating the diluent on a TPY solid culture medium by using a disposable coating rod, culturing for 48h at 37 ℃ under an anaerobic condition, counting live lactobacillus bacteria, and calculating the survival rates of different lactobacillus strains under the condition of pH 3.0 for 0h and 3h according to the following formula.
Survival rate (%) ═ 3h survival number of lactic acid bacteria/0 h survival number of lactic acid bacteria × 100%;
tolerance to artificial intestinal juice experiment: PBS buffer was adjusted to pH 8.0 with 0.1mol/L NaOH, and 1mg/mL trypsin and 0.3% bovine bile salt were added. Adding 1mL of resuspended lactobacillus thallus (bacterial suspension is prepared into the same tolerance artificial gastric juice) into 5mL of simulated intestinal fluid, quickly and uniformly mixing, putting into an incubator at 37 ℃, diluting with sterile physiological saline according to a gradient of 1:10 after 0h and 4h, sucking 100 mu L of diluent, uniformly coating the diluent on a TPY solid culture medium by using a disposable coating rod, culturing at 37 ℃ under an anaerobic condition, counting live lactobacillus, and calculating the survival rate of different lactobacillus strains for 4h according to the following formula.
Survival rate (%) ═ 4h survival number of lactic acid bacteria/0 h survival number of lactic acid bacteria × 100%;
the results of artificial gastrointestinal fluid simulation experiments on the screened strains are shown in figure 5, and the gastric juice-resistant survival rates of enterococcus faecalis 1004-9, enterococcus faecalis 1004-10 and enterococcus faecalis 1028-1 are respectively 99.50%, 54.50% and 63.77%; the intestinal juice resistant survival rates of enterococcus faecalis 1004-9, enterococcus faecalis 1004-10 and enterococcus faecalis 1028-1 are 140.95%, 65.51% and 41.18%, respectively.
(2) Determination of hydrophobic rate of enterococcus faecalis
The activated bacterial liquid is centrifuged at 4000 Xg for 10min to collect thalli, the thalli is washed by 5mL PBS (50mmol/L, pH 6.5) buffer solution, centrifuged at 4000 Xg for 10min, the supernatant is poured off, and the washing is repeated twice. Adjusting the concentration of lactobacillus with buffer solution as blank control at 560nm wavelength0The value is about 1.00. And (3) adding 0.8mL of dimethylbenzene into 4mL of bacteria liquid after turbidity adjustment, oscillating for 30s, stopping for 10s, oscillating for 30s, standing for 7min for layering, taking the lower-layer water phase, taking a buffer solution as a blank control, measuring and recording the value A at the wavelength of 560 nm. The calculation of the surface hydrophobicity of the lactic acid bacteria cell is shown in the following formula. The test was carried out with the probiotic Lactobacillus johnsonii MH-68 added to the product as a control strain.
The hydrophobic rate H ═ A ═0-A)/A0]×100％；
The hydrophobic rate of the selected strains was measured, and the results are shown in FIG. 6, in which the hydrophobic rates of enterococcus faecalis 1004-9, enterococcus faecalis 1004-10 and enterococcus faecalis 1028-1 were 13.4%, 19.7% and 5.5%, respectively, while the hydrophobic rate of control strain MH-68 was 19.2%.
Example 5 detection of specific molecular targets of enterococcus faecalis
(1) Excavation of specific novel molecular targets
Performing bioinformatics analysis according to a GenBank database and a whole genome DNA sequence self-tested by the team; screening to obtain a specific gene segment of 3 strains of enterococcus faecalis, wherein the nucleotide sequence of the gene segment is shown in SEQ ID NO. 1-SEQ ID NO. 3.
Wherein, the sequence SEQ ID NO.1 is a specific gene segment of enterococcus faecalis 1004-9 strain, SEQ ID NO.2 is a specific gene segment of enterococcus faecalis 1004-10 strain, and SEQ ID NO.3 is a specific gene segment of enterococcus faecalis 1028-1 strain.
(2) Primer validity detection
Specific PCR amplification primer sets (comprising forward primers and reverse primers) are designed according to the sequences SEQ ID NO. 1-NO. 3 in the step (1), and the sequences of the primer sets are shown in the following table 2.
TABLE 2 specific PCR detection primer set
Preparation of S1 DNA template: respectively culturing three strains of enterococcus faecalis in a TPY liquid culture medium in an enrichment manner, and respectively extracting bacterial genome DNA of the three strains of enterococcus faecalis by using a bacterial genome DNA extraction kit to serve as templates to be detected;
s2 PCR amplification:
the PCR detection system is as follows:
wherein:
when the template DNA is enterococcus faecalis 1004-9, the primer is the primer in the primer group 1;
when the template DNA is enterococcus faecalis 1004-10, the primer is the primer in the primer group 2;
when the template DNA is enterococcus faecalis 1028-1, the primer is the primer in the primer group 3;
PCR amplification procedure:
wherein, when the primer group 1 is used, the annealing temperature is 65.5 ℃, and the extension time is 20 s; when the primer set 2 is used, the annealing temperature is 68 ℃, and the extension time is 20 s; when primer set 3 was used, the annealing temperature was 68 ℃ and the extension was 30 seconds.
S3: and (3) carrying out gel electrophoresis on the PCR amplification products, and observing whether a single amplification band exists in the position of the size of the corresponding product of each primer group. If the amplification single band does not appear in other strain templates, the corresponding target is a strain-specific molecular target.
45 strains of enterococcus faecalis, 45 strains of non-target enterococcus and 90 strains of non-enterococcus were subjected to PCR detection according to the above method. Wherein, S1 and the DNA template are prepared by respectively extracting the genome DNA of each bacterium; s2, in PCR amplification, the primers used are the primers in the primer set. A blank was set, the template of which was an aqueous solution without genome.
The strains of the respective bacteria used and the results of the detection are shown in Table 3 below, in which "-" in the column of the results of the detection indicates negative. The electrophoresis results of the PCR products are shown in FIGS. 7-12, and + is a positive control; m is 2000Maker, C is blank.
TABLE 3 test results of the test for the detection specificity evaluation of enterococcus faecalis according to the present invention
As can be seen from FIGS. 7-12, only the target strain shows amplified bands, and neither the non-target Enterococcus nor the non-target Enterococcus has the target bands, which indicates that only the target strain in the method contains the specific molecular targets, i.e., Enterococcus faecalis (Enterococcus faecalis)1004-9 strain, Enterococcus faecalis (Enterococcus faecalis)1004-10 strain, and Enterococcus faecalis (Enterococcus faecalis)1028-1 strain contain the specific molecular targets.
Test example and evaluation of safety of enterococcus faecalis
(1) Antibiotic sensitivity test
Selecting 8 common antibiotics of penicillin, ampicillin, erythromycin, tetracycline, vancomycin, norfloxacin, chloramphenicol and rifampicin. Centrifuging the prepared bacterial liquid at 4000 Xg for 10min, washing with sterile PBS solution for 3 times, and diluting the bacterial liquid with PBS to 108CFU/mL or so. The bacteria solution was picked up with a sterile cotton swab and evenly spread over the entire plate surface. After the drug sensitive paper is pasted, the drug sensitive paper is lightly pressed by using sterile forceps to be firmly pasted with the surface of the culture medium. Each flat plate with the diameter of 90mm is stuck with 4 drug sensitive paper sheets, and the distance from the center of each paper sheet to the center is not less than 24 mm. Marking the name of the antibiotic, measuring the diameter of the inhibition zone from the back of the plate by a caliper after culturing for 18h at 37 ℃, and recording the result. Staphylococcus aureus ATCC25923 was used as a quality control strain. The results obtained are shown in Table 4 below (note: R is drug resistant, I is intermediate, S is sensitive).
TABLE 4 enterococcus faecalis antibiotic susceptibility
As can be seen from Table 4, enterococcus faecalis 1004-9, enterococcus faecalis 1004-10 and enterococcus faecalis 1028-1 were shown to be sensitive to penicillin, ampicillin and chloramphenicol, exhibited a mediator for erythromycin, and showed resistance to vancomycin, norfloxacin and rifampicin, enterococcus faecalis 1004-9 and enterococcus faecalis 1004-10 were shown to be sensitive to tetracycline, and enterococcus faecalis 1028-1 was shown to be resistant to tetracycline.
(2) Hemolysis test
Under aseptic conditions, the activated bacterial liquid is streaked on a blood plate by using a disposable aseptic inoculating loop, cultured for 48 hours at 37 ℃, and the hemolysis phenomenon is observed. Hemolysis can develop three features on blood plates: (1) α hemolysis: grass green hemolysis rings appear around colonies, and this bacterium is generally opportunistic. (2) Beta hemolysis: a broad, transparent hemolytic ring appears around the colony, and is generally highly pathogenic. (3) Gamma hemolysis: no hemolytic rings appeared around the colonies, and the strains were generally nonpathogenic.
As can be seen from FIGS. 13 to 15, no hemolytic ring appears around each colony, and therefore, neither Enterococcus faecalis (Enterococcus faecalis)1004-9, Enterococcus faecalis (Enterococcus faecalis)1004-10, nor Enterococcus faecalis (Enterococcus faecalis)1028-1 has hemolytic activity, and has good safety.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> institute of microorganisms of Guangdong province (center for analysis and detection of microorganisms of Guangdong province), Guangdong Huanji Biotech Co., Ltd
<120> enterococcus faecalis and application thereof
<130> 2020.11.06
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 525
<212> DNA
<213> specific molecular target of Enterococcus faecalis (Enterococcus faecalis)1004-9
<400> 1
ttgatatcta aaaatagtga ttgtgaagat aaatatatga gctatgatgg tattgataaa 60
aaagctactg tagaaatttc atttatttca ggtggtacat tatttacatc taatgggcct 120
caaatactta agcctaactt ttatgcaaaa gcaagtatga aagctaatga tgatagtgct 180
gaatttaata taaacctaaa tccagctatt tctccatatg taaagaagat agaaacgaga 240
tggcggcatc cactttttcc tccatataat gaaaaaaccc aaacaagttt tactcctaga 300
ataattatag aattaaaata tagtaaaata tggcatagta ttgacaagtt atttacttca 360
gataaatata cagaattatt gcatgatgtt acaaataaaa aaattcaaga gattaagcaa 420
caagttttga gtatgccgaa taatcaagat gctttagaaa tgattaaatt aattactaaa 480
gctgaacaat tatgggagaa aaagagacag ctgttacaaa agtaa 525
<210> 2
<211> 345
<212> DNA
<213> specific molecular target of Enterococcus faecalis (Enterococcus faecalis)1004-10
<400> 2
atggtagatc gaatttctac aactgacact tcactttcag ttcatttagg aaaagttact 60
ccagatgatg catatgtaat cacttatgga ttaaatgtga ctcctggaat tacaccacaa 120
gactttggca cacgctataa caacgctcgt atgacttctg gaaatattgt gaaggaaagc 180
aatgttccag taattctaaa aaaaaaatta gaaaatgatg cttctgtttt agaaaaaagt 240
gtagataaaa cacagcttgc gacaaattcc gcaagtctag aatatcgttt aaaaacagaa 300
tctaataaaa gaaaaaagtc accaaacaaa tcatttgatg actag 345
<210> 3
<211> 2295
<212> DNA
<213> specific molecular target of Enterococcus faecalis (Enterococcus faecalis)1028-1
<400> 3
ttggatacta aaataactgt aagtggtagt attgttaaag agttgtctga aaaaattcct 60
aataatatta ttgcattaaa tgaattagtg aaaaatgcat atgatgctgg ctctaaacaa 120
gttgatataa acatttctac tggtgaaaaa aaattatcaa taaaagatta tgggattgga 180
atggacgaag aagatgtgaa aaaattattt catatatctt caagtgtaaa aaaatatgga 240
atgatcacta atgtaaatgg actggaaagg attatgcaag gttcaaaggg actaggtttt 300
ttgtcagttt tcaaatttgg gaatagagtg agatggaaaa catcgaaagg aaagacttta 360
gaattttttg ctgattatag tgatattctt agtaaagagg atattagtga atataatgtt 420
tttattaatg aattagatga agaatttatt ggaacagaga taacgataga tttaaatgag 480
tataatactg attcattgtc taattatttt agtgaagaaa aaaacaggga aaagctattg 540
aattccttca ttttttttaa agaaaataat accattaaac atgattctaa ttttgttatt 600
aagttagaaa tagatgccga atcatatcaa actgatttaa acttatcatt acaaagtgaa 660
tctcccgatc agcagcttgt tagagtaaag tatgatagta ataccaaaac aataagttat 720
tattcaaata aaaacgtaag tgttccattg tacaatgagc caatggattt tgattctgaa 780
gaatactcag tagttttaga tttacaaaca tttttactaa aacctcgtgg caaagaaaaa 840
attagtaagt tcttttggca cccagtatca ggcgagctaa caccgttagt gtacgtaaat 900
aataatttgt ttaataacta cgaattgttt gatactaatc taatgaggtc aaaaaaatac 960
agcaatgtta tgtctcaaat gataggttat gtatctatca ttagtagtag cacctctata 1020
gattttaatt cagatagaac gaggtttgtt caaaatgaat taacagataa tatttctgga 1080
tttttagaaa agttaaatga aaggatacaa accactagtt ctgaattgaa aaacgaactt 1140
ggtggtaaag ttagattttt aagaacaaat gagatatctc aagaaatggt aacagaagac 1200
tttgattatt tatctttagt aaaagaaaat tttaaagtta aatcaaatat aacctatgag 1260
aataagggtg atataattgt atacaattta ttcgatatag atgaaaaagt attgattaaa 1320
cagaaggaaa aggaacaagt aatatcaaat gttgaatttt attatttgca attatctgaa 1380
gaggaaaaaa aaggattact ttctgagtta tctagcttca atacaataaa attcgaaggt 1440
gatattgttg attcatttga tcttttaaaa gatggtaaat gggaattttt caaagaagat 1500
gatagccatg tttttataaa aaatgttcaa atattaagcc ctcaacagcc taaaataata 1560
caaaaaacaa aagttgttga gttgcataaa gaatatagct acgatgactt atttgatttt 1620
gaaaatagtt tttatgagaa ggataaagga gtttttcctg acttaaagac agaaacaata 1680
aaatatattc ataatgataa gaaaaaagga attatttctt ttggaatgga acaagaatta 1740
tcttttcccg tatgtttaag agataaaaaa actagtaaaa cgcatgagat agaagccgtt 1800
tttagggtaa tatctaaatc tcatgatatc tctcaaacga aaaaaggaat agactttata 1860
aagatgccta tcagtaatgg tgctgatctg ccaggaaata taataggatt tatagcagaa 1920
ttaaatctcc taaataataa gaatgaattt tcctattcgt tcgtatcttc ttttcgaaca 1980
ctggtggagc tatgtgttct cgatatattg aataaaaaag gaataggcaa agataagagc 2040
ttggcaaata attataaaga agtactaaag ttatatccag attatattga caatatacat 2100
gatgaaaaag atcagcagat gattctaaat ctttttaatg caatagaagc accttctgag 2160
aggaaagcgt atatagcttt cttaaatcta tgtactcatg gtagtagtac aatgatttca 2220
aaaaaagaaa tagaatacaa aacaaaagag cttagcttac ttttagaata tttaaatttt 2280
ttaaataagg aataa 2295
<210> 4
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 4
<210> 5
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 5
cagctgtctc tttttctccc a 21
<210> 6
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 6
<210> 7
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 7
<210> 8
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 8
tgggattgga atggacgaag a 21
<210> 9
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 9
gctgctgatc gggagattca 20
<210> 10
<211> 1422
<212> DNA
<213> Artificial Synthesis
<400> 10
gctggctcca aaaggttacc tcaccgactt cgggtgttac aaactctcgt ggtgtgacgg 60
gcggtgtgta caaggcccgg gaacgtattc accgcggcgt gctgatccgc gattactagc 120
gattccggct tcatgcaggc gagttgcagc ctgcaatccg aactgagaga agctttaaga 180
gatttgcatg acctcgcggt ctagcgactc gttgtacttc ccattgtagc acgtgtgtag 240
cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg gtttgtcacc 300
ggcagtctcg ctagagtgcc caactgaatg atggcaacta acaataaggg ttgcgctcgt 360
tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtca 420
ctttgtcccc gaagggaaag ctctatctct agagtggtca aaggatgtca agacctggta 480
aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540
aattcctttg agtttcaacc ttgcggtcgt actccccagg cggagtgctt aatgcgtttg 600
ctgcagcact gaagggcgga aaccctccaa cacttagcac tcatcgttta cggcgtggac 660
taccagggta tctaatcctg tttgctcccc acgctttcga gcctcagcgt cagttacaga 720
ccagagagcc gccttcgcca ctggtgttcc tccatatatc tacgcatttc accgctacac 780
atggaattcc actctcctct tctgcactca agtctcccag tttccaatga ccctccccgg 840
ttgagccggg ggctttcaca tcagacttaa gaaaccgcct gcgctcgctt tacgcccaat 900
aaatccggac aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt 960
ggctttctgg ttagataccg tcaggggacg ttcagttact aacgtccttg ttcttctcta 1020
acaacagagt tttacgatcc gaaaaccttc ttcactcacg cggcgttgct cggtcagact 1080
ttcgtccatt gccgaagatt ccctactgct gcctcccgta ggagtctggg ccgtgtctca 1140
gtcccagtgt ggccgatcac cctctcaggt cggctatgca tcgtggcctt ggtgagccgt 1200
tacctcacca actagctaat gcaccgcggg tccatccatc agcgacaccc gaaagcgcct 1260
ttcactctta tgccatgcgg cataaactgt tatgcggtat tagcacctgt ttccaagtgt 1320
tatccccctc tgatgggtag gttacccacg tgttactcac ccgtccgcca ctcctctttc 1380
caattgagtg caagcactcg ggaggaaaga agcgttcgac tt 1422
Claims (10)
1. An enterococcus faecalis comprising a 16s rRNA sequence having at least 95% identity to SEQ ID No. 10.
2. The Enterococcus faecalis according to claim 1, wherein said Enterococcus faecalis is Enterococcus faecalis (Enterococcus faecalis)1004-9, or Enterococcus faecalis (Enterococcus faecalis)1004-10, or Enterococcus faecalis (Enterococcus faecalis)1028-1, said Enterococcus faecalis (Enterococcus faecalis)1004-9, Enterococcus faecalis (Enterococcus faecalis)1004-10, and Enterococcus faecalis (Enterococcus faecalis)1028-1 are all preserved in Guangdong provincial culture collection, and said Enterococcus faecalis (Enterococcus faecalis)1004-9 has a collection number of GDMCC NO: 60992, the Enterococcus faecalis (Enterococcus faecalis)1004-10 has a deposit number of GDMCC NO: 60993, the Enterococcus faecalis (Enterococcus faecalis)1028-1 has a deposit number GDMCC NO: 60994.
3. the Enterococcus faecalis according to claim 2, wherein said Enterococcus faecalis (Enterococcus faecalis)1004-9 comprises the specific molecular target shown in SEQ ID No.1, said Enterococcus faecalis (Enterococcus faecalis)1004-10 comprises the specific molecular target shown in SEQ ID No.2, and said Enterococcus faecalis (Enterococcus faecalis)1028-1 comprises the specific molecular target shown in SEQ ID No. 3.
4. A primer for detecting Enterococcus faecalis according to claim 2, wherein the primers corresponding to Enterococcus faecalis (Enterococcus faecalis)1004-9 are SEQ ID No.4 and SEQ ID No. 5; the primers corresponding to the Enterococcus faecalis (Enterococcus faecalis)1004-10 are SEQ ID NO.6 and SEQ ID NO.7, and the primers corresponding to the Enterococcus faecalis (Enterococcus faecalis)1028-1 are SEQ ID NO.8 and SEQ ID NO. 9.
5. Use of enterococcus faecalis according to claim 1 or 2 for the preparation of a microbial preparation having a blood pressure lowering effect.
6. Use of enterococcus faecalis according to claim 1 or 2 for the preparation of food and pharmaceutical products having a blood pressure lowering effect.
7. Use according to claim 6, wherein the food product is a functional food product.
8. Use according to claim 7, wherein the functional food is a fermented food.
9. A food or pharmaceutical product having a blood pressure lowering function, comprising the enterococcus faecalis according to claim 1.
10. The food or pharmaceutical product of claim 9, further comprising a pharmaceutical excipient.
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Citations (5)
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|---|---|---|---|---|
| JPH05201871A (en) * | 1992-01-27 | 1993-08-10 | Nichinichi Seiyaku Kk | Agent for inhibiting rise of blood pressure and for preventing cardiomegaly |
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| EP1640447A1 (en) * | 2003-05-21 | 2006-03-29 | Grupo Leche Pascual S.A. | BIOACTIVE PEPTIDE-PRODUCING STRAINS OF i ENTEROCOCCUS FAECALIS /i , BIOACTIVE PEPTIDES AND APPLICATIONS THEREOF |
| KR100628539B1 (en) * | 2005-05-17 | 2006-09-26 | 남양유업 주식회사 | Yogurt composition for the control of a blood pressure |
| CN110499266A (en) * | 2019-08-05 | 2019-11-26 | 昆明理工大学 | One plant of enterococcus faecalis and its application |
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2020
- 2020-11-12 CN CN202011265258.6A patent/CN112410250A/en active Pending
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|---|---|---|---|---|
| JPH05201871A (en) * | 1992-01-27 | 1993-08-10 | Nichinichi Seiyaku Kk | Agent for inhibiting rise of blood pressure and for preventing cardiomegaly |
| JPH07304677A (en) * | 1994-05-11 | 1995-11-21 | Nichinichi Seiyaku Kk | Antihypertensive agent |
| EP1640447A1 (en) * | 2003-05-21 | 2006-03-29 | Grupo Leche Pascual S.A. | BIOACTIVE PEPTIDE-PRODUCING STRAINS OF i ENTEROCOCCUS FAECALIS /i , BIOACTIVE PEPTIDES AND APPLICATIONS THEREOF |
| KR100628539B1 (en) * | 2005-05-17 | 2006-09-26 | 남양유업 주식회사 | Yogurt composition for the control of a blood pressure |
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| ANA QUIRÓS 等: "Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis", 《INTERNATIONAL DAIRY JOURNAL》 * |
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