CN115747092B - Two lactobacillus plantarum SR37-3 and SR61-2 with remarkable blood pressure reducing function and application - Google Patents

Abstract

The invention discloses two lactobacillus plantarum SR37-3 and SR61-2 with obvious blood pressure reducing function and application thereof. Lactobacillus plantarum (Lactobacillus plantarum) SR37-3, accession number is: GDMCC No:62390. lactobacillus plantarum (Lactobacillus plantarum) SR61-2, accession number is: GDMCC No:62389. two lactobacillus plantarum with the function of reducing blood pressure are obtained through screening, and are named lactobacillus plantarum (Lactobacillus plantarum) SR37-3 and lactobacillus plantarum (Lactobacillus plantarum) SR61-2. In vitro experiments prove that the two lactobacillus plantarum have stronger inhibition rate of angiotensin converting enzyme and have no hemolysis and drug resistance; in vivo experiments prove that both strains have better blood pressure reducing effect, and the functions of relieving kidney injury caused by hypertension, improving cardiovascular health and the like.

Description

Two lactobacillus plantarum SR37-3 and SR61-2 with remarkable blood pressure reducing function and application

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to two lactobacillus plantarum SR37-3 and SR61-2 with obvious blood pressure reducing function and application thereof.

Background

Cardiovascular disease has been a major public health problem in today's society, and hypertension is one of the major health risk factors leading to cardiovascular disease. The data show that the prevalence rate of adult hypertension in China reaches 27.9%, namely, 1 hypertension patient exists in every 4 adults, which seriously threatens the health of people.

For a long time, patients with hypertension often need to rely on drugs for a long time to control the progression of the disease. Currently available antihypertensive drugs are diuretics (thiazines), angiotensin converting enzyme inhibitors (Angiotensin converting enzyme inhibitor, ACEI), beta blockers, calcium channel blockers, angiotensin ii blockers. Among the drugs commonly used at present, ACEI drugs represented by captopril and enalapril have better clinical curative effects. ACEI drugs can inhibit the degradation of bradykinin for dilating blood vessel by inhibiting ACE activity, thereby reducing the generation of angiotensin II. Besides good antihypertensive effect, the ACEI drugs have few side effects, and clinical experiments prove that the drugs have 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 preparation of ACEI drugs at present mainly depends on chemical synthesis, and the synthesized drugs have adverse reactions, mainly including slight cough, hypotension, renal function reduction, hyperkalemia, angioneurotic edema and the like, and the toxic and side effects lead the application of the ACEI drugs to be greatly limited.

Therefore, it is a focus of research to develop new therapies and find safer antihypertensive products with less side effects. In recent years, sequencing technology has been continuously innovated, and microbial communities have been vigorously developed. This research is hot and has led us to reevaluate billions of microorganisms in the human gut. Intestinal microbiota has many protective and metabolic functions for host health, including functions of food fermentation degradation, digestion of polysaccharides and synthetic vitamins that are difficult for the host to digest, and the like. Especially, the deep research on probiotics is carried out, and the knowledge of the probiotics is not only remained on fermented foods such as pickle, yoghourt and the like. Probiotics are well developed in the fields of health care foods, specific foods and the like, and products such as probiotic electuary, capsules and the like appear successively. Studies have shown that some probiotics produce bioactive ingredients with prophylactic and regulatory effects on cardiovascular disease after fermentation of food. The bioactive factors are higher in safety and smaller in side effect, and can promote proliferation of beneficial microorganisms in intestinal tracts, regulate intestinal micro-ecology and further continuously and deeply influence host health.

The application of blood pressure lowering probiotics in food is mainly focused on dairy products. It is well recognized that probiotics such as lactic acid bacteria can hydrolyze proteins in dairy products to produce short peptides which have a blood pressure lowering effect. However, at present, the research on the blood pressure lowering fermented milk at home and abroad is not much. In 2002, seppo et al studied fermented milk produced by Evolus for lowering blood pressure. The blood pressure drop was more pronounced during the test than in the control group, but blood pressure was almost restored to the pre-test level after two weeks of cessation of Evolus fermented milk intake. In 2005, jauhiaain et al in a randomized, double-blind placebo controlled study 94 patients with hypertension ingested Lactobacillus helveticus fermented milk (with high concentration of VPP/IPP peptide) twice daily, and after 10 weeks of continuous ingestion, blood pressure was reduced compared to the control group (the difference in SBP/DBP compared to the control group was-4.1.+ -. 0.9/-1.8.+ -. 0.7mm Hg). The study of Ahren et al in 2015 found that after eating lactobacillus fermented milk product, the blood pressure of the hypertensive patient was significantly reduced without any side effects. At present, the countries such as Finland and Japan have blood pressure lowering products, and the countries still belong to the blank of blood pressure lowering probiotic products. The probiotic bacteria has excellent screening performance and obvious blood pressure reducing effect, can be used for producing functional foods, prevents and treats hypertension, regulates intestinal microecology and promotes cardiovascular health, and has great significance for implementing 'healthy Chinese' actions, promoting human health and improving human life quality.

Disclosure of Invention

The invention aims to provide two lactobacillus plantarum with the functions of reducing blood pressure, improving hypertension complications and promoting cardiovascular health and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: lactobacillus plantarum has effects of lowering blood pressure and promoting cardiovascular health, and is named Lactobacillus plantarum (Lactobacillus plantarum) SR37-3 and Lactobacillus plantarum (Lactobacillus plantarum) SR61-2.

Lactobacillus plantarum (Lactobacillus plantarum) SR37-3 was deposited at the Cantonese microorganism culture Collection (GDMCC) at 4-month 18 2022, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070, accession number is: GDMCC No:62390.

lactobacillus plantarum (Lactobacillus plantarum) SR61-2 was deposited at the Cantonese microorganism culture Collection (GDMCC) at 4-month 18 2022, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070, accession number is: GDMCC No:62389.

two lactobacillus plantarum with the function of reducing blood pressure are obtained through screening, and are named lactobacillus plantarum (Lactobacillus plantarum) SR37-3 and lactobacillus plantarum (Lactobacillus plantarum) SR61-2. In vitro experiments prove that the two lactobacillus plantarum have stronger inhibition rate of angiotensin converting enzyme and have no hemolysis and drug resistance; in vivo experiments prove that both strains have better blood pressure reducing effect, and the functions of relieving kidney injury caused by hypertension, improving cardiovascular health and the like.

The invention also claims the application of the lactobacillus plantarum SR37-3 or lactobacillus plantarum SR61-2 in preparing products for reducing blood pressure, slowing down the production of inflammatory substances by serum and improving kidney injury and promoting cardiovascular health.

The invention also provides a product for reducing blood pressure, slowing down the production of inflammatory substances by serum and improving kidney injury and promoting cardiovascular health, which contains lactobacillus plantarum SR37-3 or lactobacillus plantarum SR61-2 as an active ingredient.

The invention also provides a method for culturing the lactobacillus plantarum SR37-3 or the lactobacillus plantarum SR61-2, and a proper amount of the lactobacillus plantarum SR37-3 or the lactobacillus plantarum SR61-2 is inoculated into a culture medium for culturing.

As a preferred embodiment of the present invention, the medium is MRS medium.

The invention also claims a specific nucleotide sequence of lactobacillus plantarum SR37-3 or SR61-2, wherein the specific nucleotide sequence of lactobacillus plantarum SR37-3 is shown as SEQ ID NO.1, and the specific nucleotide sequence of lactobacillus plantarum SR61-2 is shown as SEQ ID NO. 2.

Furthermore, the invention also claims a corresponding PCR primer group, wherein the primer group is designed according to the nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.

Each primer set includes a forward primer and a reverse primer, and a nucleotide sequence is detected by designing the corresponding primer set for PCR based on the corresponding nucleotide sequence.

As a preferred embodiment of the invention, the primer set for detecting lactobacillus plantarum SR37-3 comprises nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO. 4, and the nucleotide sequence for detecting lactobacillus plantarum SR61-2 is shown in SEQ ID NO.5 and SEQ ID NO. 6.

The invention also provides a detection method for detecting the lactobacillus plantarum SR37-3 or the lactobacillus plantarum SR61-2, which comprises the following steps:

s1: performing PCR amplification using the primer set;

s2: detecting the amplified product by gel electrophoresis;

s3: and observing whether the amplified product meets the expectations.

As a preferred embodiment of the present invention, the PCR amplification system in S1 comprises PCR Mix, template DNA, primer set and sterilized double distilled water.

As a preferred embodiment of the present invention, the PCR amplification system was 2X San Taq PCR Mix 10. Mu.L, 1.0. Mu.L of template DNA, 1. Mu.L of primers each, and the volume was made up to 20. Mu.L by sterilizing double distilled water.

As a preferred embodiment of the present invention, the PCR amplification procedure in S1 is as follows: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s; annealing at 70 ℃ for 30s; extending at 72 ℃ for 60s; carrying out denaturation, annealing and extension for 35 cycles; finally, the extension is carried out for 5min at 72 ℃. Pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s; annealing at 70 ℃ for 30s; extending at 72 ℃ for 25s; carrying out denaturation, annealing and extension for 37 cycles; finally, the extension is carried out for 5min at 72 ℃.

The beneficial effects of the invention are as follows:

1. the probiotics in the present invention are excellent strains isolated from fermented foods.

2. Compared with the traditional antihypertensive chemical drugs, the invention has no toxic or side effect and residue risk to the ecological environment, is safer to human bodies and is more economical.

3. The invention has good effects of reducing blood pressure and promoting cardiovascular health, and is specifically embodied as follows:

a. the lactobacillus plantarum provided by the invention has higher inhibition rate of angiotensin converting enzyme after fermenting skim milk.

b. The strain shows better safety in-vitro and in-vivo safety experiments.

c. The composition has certain regulation effects on aspects of hypertension characterization, enzyme, hormone, inflammatory factors and the like in serum in vivo tests, and has the effects of protecting kidney injury caused by hypertension and improving cardiovascular health.

4. The invention has proved that the invention has no potential harm to human body from genome and phenotype detection, animal test and other multi-angle.

5. The functional material basis and metabolic pathways are initially mined by metabonomics means.

Lactobacillus plantarum (Lactobacillus plantarum) SR37-3 was deposited at the Cantonese microorganism culture Collection (GDMCC) at 4-month 18 2022, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070, accession number is: GDMCC No:62390.

lactobacillus plantarum (Lactobacillus plantarum) SR61-2 was deposited at the Cantonese microorganism culture Collection (GDMCC) at 4-month 18 2022, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070, accession number is: GDMCC No:62389.

drawings

FIG. 1 shows the antihypertensive effect of two Lactobacillus plantarum strains in vivo, the data being expressed in mean+ -SE.

FIG. 2 shows the results of in vivo serum ELISA assays.

FIG. 3 is a graph of a UPLC-MS/MS data multivariate statistical analysis of LC-MS analysis of hypertensive rat serum (a-f) samples; PCA score map distribution for the W, LN, LN+SR37-3 and SR61-2 groups in positive model (a) and negative model (b); an OPLS-DA model arrangement test pattern between metabonomics data set W and LN, positive pattern (r2= (0,0.989), a PCA score pattern distribution of the group of q2= (0, -0.00648)) (c) and negative pattern ((r2= (0,0.876), q2= (0, -0.458)) (d), an OPLS-DA score pattern of metabonomics data set, positive pattern (r2y=0.997; q2y=0.575) (e), negative pattern (r2y=0.981; q2y=0.732) (f), LC-MS analysis of hypertensive rat stool (g-l) sample, an arrangement pattern of W, LN, ln+sr37-3 and SR61-2 groups in positive pattern (g) and negative pattern (h), an OPLS-DA model arrangement pattern between metabonomics data set W and LN, positive pattern (r2= (0,0.597), q2= (0, -0.52) (i), negative pattern (r2= (r2=0. 0,0.759), q2y=0.732) (e), positive pattern (g2y=0.994).

FIG. 4 is a differential metabolite pathway enrichment, a and e being layered aggregation heatmaps between W and LN groups constructed from important metabolites of serum and cecal samples, respectively, showing the relative increase/decrease in metabolite content and their similarity between individual samples; columns correspond to different groups and rows correspond to altered metabolites; color represents metabolite expression values; green: least, red: the highest; b. c and d are metabonomic views of serum samples in the path analysis using metabolic analysis between the W and LN groups, between the LN and LN+SR37-3 groups, and between the LN and LN+SR61-2 groups, respectively, while f, g, and h are metabonomic views of cecal content samples.

FIG. 5 is a graph showing the results of morphological observation of kidney tissue of a rat in example 3, wherein a is W (control group), b is LN (model group), c is LN+SR37-3, and d is LN+SR61-2.

FIG. 6 is a diagram showing a strain hemolysis experiment, a is Lactobacillus plantarum SR37-3, b is Lactobacillus plantarum SR61-2, c is a positive control

FIG. 7 is a standard curve, a standard curve (a) and an original curve (b) of a fluorescent quantitative PCR detection of the high-efficiency depressurization strain SR 37-3.

FIG. 8 is a standard curve, a standard curve (a) and an original curve (b) of a fluorescent quantitative PCR detection of the high-efficiency depressurization strain SR61-2.

Detailed Description

In order that the invention may be more readily understood, a further description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings.

EXAMPLE 1 isolation and characterization of Lactobacillus plantarum SR37-3 and SR61-2

1.1 isolation of probiotics and preservation of the Strain library

And (3) taking the Spirac fermented food as a sample, taking 0.1g of salted fish sample, adding 10ml of MRS liquid culture medium into the sample in a sterile environment, vibrating and uniformly mixing, carrying out enrichment culture for 24 hours under the anaerobic condition at 37 ℃, and sucking 0.5ml of bacterial liquid for gradient dilution. Adding physiological saline to make into 10 ^-1 To 10 ^-5 Diluting gradient bacterial suspension, selecting 10 ^-3 、10 ^-4 、10 ^-5 Three gradient bacterial suspensions were aspirated 100 μl to MRS agar medium, smeared uniformly with a spreading bar, and cultured at 37deg.C under anaerobic conditions for 48h. Picking a typical colony on a flat plate to an MRS agar culture medium for streak purification, picking a single colony after purification, inoculating the single colony into the MRS liquid culture medium, performing anaerobic culture at 37 ℃ for 48 hours, and preserving 30% glycerol in an ultralow temperature refrigerator at-80 ℃.

1.2 identification of Lactobacillus

Bacterial DNA extraction was performed using bacterial DNA extraction kit (Mabio, CHINA) followed by PCR amplification using 2 XPCR mix (Dongshengmbio, CHINA). The PCR amplification primer adopts a 16S rRNA gene universal primer, and the upstream primer sequence is 27F:5'-AGA GTT TGA TCC TGG CTC AG-3'; the downstream primer sequence is 1492R:5'-CTAC GGC TAC CTT GTT ACG A-3'. The PCR reaction conditions were: pre-denaturation at 95℃for 5min; for 35 cycles of 95℃30s,56℃30s,72℃1min 30s, and annealing extension at 72℃for 10min. The PCR products were cut to recover and then sequenced for the first generation (done by Souzhou Jin Weizhi Biotechnology Co.). The obtained 16S rRNA gene sequence is compared with NCBI database (https:// blast. NCBI. Lm. Nih. Gov), and the strain with Identity and Coverage similarity of more than 99% with the known lactobacillus plantarum in the comparison result can be determined to be lactobacillus plantarum.

Wherein the sequence of the 16S rRNA gene of the strain claimed in the patent is shown in SEQ ID No. 7-8. The sequence was aligned to NCBI database (https:// blast. NCBI. Lm. Nih. Gov) and the result suggested that it had the highest homology to Lactobacillus plantarum, designated Lactobacillus plantarum (Lactobacillus fermentum) SR37-3 (16S rRNA corresponding to SEQ ID No. 7) and SR61-2 (16S rRNA corresponding to SEQ ID No. 8).

EXAMPLE 2 Whole genome sequencing of two Lactobacillus plantarum strains

a determination of genomic DNA concentration

1) Bacterial DNA extraction kit is used for extracting bacterial DNA, and the bacterial DNA is extracteddsDNAHS reagent->The dsDNAHS Buffer was diluted 200-fold and the final volume of assay solution per tube was 200. Mu.L.

2) 190. Mu.L of working solution is added to a standard substance measuring tube, 10. Mu.L of standard substance is added, and the mixture is mixed for 2 to 3 seconds. 199. Mu.L of working solution was added to each sample measurement tube, 1. Mu.L of the sample was added, and after the preparation, the tube wall was flicked with a finger for two to three seconds to mix well. After 2min of dark place, the Qubit samples were assayed.

b fragmentation of genomic DNA

100ng of DNA was added to the sample tube and adjusted to 79. Mu.L with sterile water. DNA was interrupted by Covaris M220, 500bp in size

c end repair and purification of fragmented DNA

Transfer sample from sample tube to 1.5mL centrifuge tube and add according to Table 1

Table 1 DNA repair reagents

After incubation for 20min at room temperature 180. Mu.L of magnetic beads were added to the centrifuge tube. The DNA was thoroughly mixed with the magnetic beads by pipetting 5 times, after brief centrifugation, incubated for 5min at room temperature. The sample was placed on a magnetic rack until the solution became clear, and the supernatant was carefully aspirated with a pipette to avoid damage to the magnetic bead layer. 500. Mu.L of freshly prepared 70% ethanol was added to the centrifuge tube and incubated for 30s, and the tube was turned in place for 2-3 rounds. After the solution became clear, the supernatant was carefully aspirated with a pipette to avoid damage to the magnetic bead layer. This procedure was repeated once. And taking out the sample from the magnetic rack, centrifuging briefly, putting back the sample on the magnetic rack, and sucking and discarding the residual ethanol to avoid damaging the magnetic bead layer. And opening the tube cover on the magnetic frame to naturally dry the magnetic bead layer at room temperature, wherein the operation process is not suitable for more than 5min. The sample was removed from the magnet holder, 25 μl of sterile water was added, and the mixture was blown 5 times with a pipette, vortexed for 10s, and thoroughly mixed. After brief centrifugation, the magnet rack was returned until the solution became clear, and the supernatant was collected and transferred to a new 0.2mL PCR tube.

d linker ligation, gap repair and library purification

The corresponding reagents were added to the above sample tubes as in table 2 and centrifuged briefly. The sample tubes were then placed in the PCR instrument with the procedure shown in table 3 and after the reaction was completed, the samples were transferred to a new 1.5mL centrifuge tube in preparation for the next purification.

TABLE 2PCR reaction System

TABLE 3 PCR procedure

100. Mu.L of magnetic beads were added to the tube. The DNA was thoroughly mixed with the magnetic beads by pipetting, and after brief centrifugation, incubated for 5min at room temperature. Placing the sample on a magnetic rack, standing for 3min or after the solution becomes clear, carefully sucking the supernatant with a pipette to avoid damaging the magnetic bead layer. 500. Mu.L of freshly prepared 70% ethanol was added directly to the centrifuge tube, incubated for 30s and the tube was turned in place for 2-3 rounds. After the solution became clear, the supernatant was aspirated with a pipette to avoid damaging the magnetic bead layer. And taking out the sample from the magnetic rack, centrifuging briefly, putting back the sample on the magnetic rack, and sucking and removing residual ethanol by using a pipetting gun to avoid damaging the magnetic bead layer. And opening a tube cover of the centrifuge tube on the magnetic frame, and naturally airing the magnetic bead layer for 5min at room temperature. Taking the sample from the magnetic rack, adding 20 mu L of sterile water, blowing with a pipetting gun for 5 times, carrying out vortex oscillation for 10s, uniformly mixing, centrifuging for a short time, placing the sample back to the magnetic rack until the solution becomes clear, collecting the supernatant, and transferring the supernatant into a new 0.2mL PCR tube.

Size screening of e linker-containing libraries

HandleSizeSelect ^TM Two combs on 2% agarose Gel were pulled off and inserted into iBase from left to right ^TM Is a kind of medium. mu.L of the prepared DNA sample was added to the spotted well (upper row), 10. Mu.L of 50 bpDNALader was added to the M well, 25. Mu.L of sterile Water was added to the spotted well (lower row), and 10. Mu.L of nucleic-free Water was added to the M well. Covering the transilluminator, inserting instrument power supply, and selecting SizeSelect in mode ^TM 2% procedure, set time to 20min. When the indicator tape on the Ladder matching the size of the target recovery strip runs to the reference line, the instrument is suspended, about 10. Mu.L of nucleic-free Water is added to the sample collection well, the instrument is started, the corresponding strip in the Ladder is waited for entering the sample collection well, the instrument is stopped, and the sample is carefully sucked into a new 0.2mL PCR tube by using a pipette.

f library amplification and purification

The corresponding reagents were added to the sample tubes as shown in Table 4. After fully mixing, the sample tube is put into a PCR instrument

TABLE 4 PCR reaction procedure

The procedure was set according to the conditions in table 5. After the reaction was completed, the PCR product was transferred to a new 1.5mL centrifuge tube.

TABLE 5 PCR reaction procedure

130. Mu.L of magnetic beads were added to the sample tube. The DNA was thoroughly mixed with the magnetic beads, centrifuged briefly and incubated at room temperature for 5min. The sample was placed on a magnetic rack and after the solution became clear, the supernatant was aspirated off. 500. Mu.L of freshly prepared 70% ethanol was added to the centrifuge tube and incubated for 30s, and the tube was turned in place for 2-3 rounds. After the solution becomes clear, the supernatant is sucked and removed, the sample is taken out from the magnetic rack, centrifuged briefly and then put back on the magnetic rack, the residual ethanol is sucked and removed by a 20 mu L pipetting gun to avoid damaging the magnetic bead layer, the tube cover of the centrifuge tube is opened, the magnetic bead layer is naturally dried for 5min at room temperature, the sample is taken off from the magnetic rack, 20 mu L of sterile water is added, and vortex oscillation is carried out for 10s, so that the mixture is thoroughly mixed. After brief centrifugation, the magnet rack was returned until the solution became clear, and the supernatant was collected and transferred to a new 0.2mL PCR tube.

g determination of library concentration after amplification

Library concentration determination after amplification and a genomic DNA concentration determination

h, performing on-machine test

i after sequencing, the whole genome sequences of the lactobacillus plantarum SR37-3 and SR61-2 are obtained. And the virulence or drug resistance genes were annotated using Abricate software alignment VFDB (Virulence Factor Data base), ARG-Annot (Antibiotic Resistance Gene-ANNOTION), CARD (the Comprehensive Antibiotic Research Database), resfinder databases, with the results shown in tables 6-7 below. Both strains do not contain virulence genes, and the drug-resistant genes are generally considered to have coverage rate more than or equal to 95% and identification rate more than or equal to 90%, and are determined to exist. The safety of the strain is fully proved by carrying out drug resistance and virulence gene analysis on the genome level.

TABLE 6 Whole genome virulence and resistance Gene analysis of Strain SR37-3 ("/" indicates no)

TABLE 7 Whole genome virulence and resistance Gene analysis of Strain SR61-2 ("/" indicates no)

Example 3 determination of angiotensin converting enzyme inhibition ratio

The activated strain was inoculated in an inoculum size of 4% by volume into 11% (w/v) skim milk sterilized at 105℃for 15min, and cultured at 37℃until skim milk coagulated. Centrifuging the prepared fermented milk at 4deg.C and 7000 Xg for 10min, collecting supernatant, adjusting pH to 7.5,4 with 5mol/L NaOH solution, centrifuging at 11000 Xg for 3min, collecting supernatant, filtering with 0.45 μm filter membrane, and storing the obtained whey at 4deg.C for use. 10. Mu.L of 0.25U ACE and 10. Mu.L of whey samples were added to 4 wells of a 96-well plate, respectively; 10. Mu.L of whey sample, 10. Mu.L of 50mmol/L Tris-HCl; 10. Mu.L of 0.25U ACE, 10. Mu.L of 50mmol/L Tris-HCl;20 μL of 50mmol/L Tris-HCl. 150. Mu.L of FAPGG at 0.88mmol/L in a 37℃water bath for 15min were each added at the fastest rate. Measuring initial absorbance of each sample well at 37deg.C and 340nm immediately after shaking for 30s with enzyme-labeled instrument, respectively designated as a ₁ ，b ₁ ，c ₁ ，d ₁ After incubation at 37℃for 15min, the absorbance of each sample well was again measured under the same conditions and was designated as a, respectively ₂ ，b ₂ ，c ₂ ，d ₂ The absorbance decrease value of each sample well was a=a, respectively ₁ -a ₂ 、B＝b ₁ -b ₂ 、C＝c ₁ -c ₂ 、D＝d ₁ -d ₂ . And calculating the ACE inhibition rate of the fermented milk according to a formula. After calculation, the strain with higher ACE inhibition rate is obtained.

ACE inhibition ratio (%) = [ (C-D) - (a-B) ]/(C-D) ×100%

The ACE inhibition rates of the two strains were measured, and the experimental results show that the inhibition rates of the fermented whey of the lactobacillus plantarum SR37-3 and SR61-2 are 70.5% and 68.9% respectively.

Example 4 two Lactobacillus plantarum in vivo antihypertensive effects and related mechanisms

S1 experiment design

Wistar rats (8 weeks old) were randomized after 7 days of adaptive feeding and treated as follows:

1. normal group (W): the stomach is irrigated with 10ml/Kg physiological saline water daily, and the water is freely drunk. 2. Model set (LN): 400mg/L N of the water is added into the water ^G -nitro-L-arginine methyl ester hydrochloride (L-NAME), 10ml/Kg physiological saline per day. SR37-3 intervention group (LN+SR37-3): 400mg/L L-NAME was added to the drinking water and 10ml/Kg SR37-3 fermented milk was added to the water daily (the preparation method was the same as in example 2). Sr61-2 intervention group (ln+sr61-2): 400mg/L L-NAME was added to the drinking water, and 10ml/Kg SR61-2 fermented milk was added to the drinking water per day (the preparation method was the same as in example 3). The experiment was approved by the ethical committee, the gastric lavage cycle was 4 weeks, the initial systolic and diastolic pressures were measured, and thereafter, the systolic and diastolic pressures were measured every two weeks, and the blood pressure results are shown in fig. 1. The blood pressure rise is inhibited by the intervention of the fermented milk of the strains SR37-3 and SR61-2. At the end of 4 weeks, extended intake of probiotic fermented milk inhibited SBP (170.22 + -8.40 and 133.28+ -6.09 LN and LN+SR37-3, respectively) and p compared to model group<0.01 (In) and DBP (LN and LN+SR37-3 were 133.83 + -5.91 and 103.00 + -6.41, respectively), LN+SR37-3 compared to model group, p<0.01 A) rise in the pressure. After two weeks of L-NAME intake in drinking water, blood pressure (SBP/DBP) increased significantly from 125.67 + -4.26/89.83+ -6.01 mmHg to 157.89 + -6.69/114.22 + -2.55 mmHg. Our results have advantages compared to TSAI et al, which reduce SBP 19mmHg after gastric lavage of fermented whey SHR rats. Overall our results have certain advantages, later validated using different models.

Detection of related substances in S2 serum

The animals were fasted for 16h and were collected and serum was isolated and tested for the following indicators using the kit. The detection results are shown in FIG. 2. The detection indexes are as follows:

1. is related to blood pressure regulating mechanism (NE, ET-1, ang I, ang II, ALD)

2. Is related to oxidative stress and inflammation (IFN-beta, TNF-alpha, IL-6, IL-1 beta)

The results show that: the SR37-3 and SR61-2 fermented milk can reduce factors related to regulation of hypertension mechanism, such as NE, ET-1, ang I, ang II, ALD and the like, and can reduce inflammatory factors, such as IFN-beta, TNF-alpha, IL-6 and IL-1 beta, in serum of hypertensive rats. This suggests that SR61-2 and SR37-3 may exert hypotensive effects through the angiotensin system and reduce inflammatory responses.

S3 serum and fecal non-targeted metabonomics

Serum sample pretreatment

200ul of serum is taken, 1ml of mixed solution of methanol and acetonitrile (methanol: acetonitrile=1:1v/v) is added, the mixture is uniformly mixed, the mixture is placed into ice water for freezing at the temperature of minus 20 ℃ for 1h after ultrasonic treatment for 20min, supernatant is centrifugally taken (4 ℃ and 13000r/min and 15 min), concentrated by a vacuum drying box, 50% acetonitrile is added for redissolution, and 150ul is centrifugally taken for loading. And 10ul of the mix was taken from each tube as QC samples.

Fecal sample pretreatment

Weighing (50+/-5) mg of solid fecal sample, and adding precooled methanol: acetonitrile: 250ul of water (4:4:2, V:V) mixed solution, homogenizing twice, performing ultrasonic treatment in water bath for 10min, standing at-20deg.C for 1 hr, supernatant was collected by centrifugation (12000 r/min,10min,4 ℃ C.), dried in a vacuum oven at room temperature and then dried with acetonitrile: 150ul of the water (1:1, V: V) mixture was subjected to redissolution vortex for 30s, and the supernatant was sampled.

LC-MS on-line analysis

The column was ACQUITY UPLC HSS T (2.1X100, 1.8 um). Elution was performed in a gradient at a flow rate of 0.3ml/min and a column temperature of 40 ℃. And a heating electrospray ion source (HESI) is adopted, the spray voltage is 3500V at the positive electrode and 2000V at the negative electrode. The capillary temperature was 320 ℃. The sheath gas flow rate was 45arb and the assist gas flow rate was 8arb (-) or 10arb (+ or-). Adopting positive and negative ion switching collection mode, wherein the scanning mode is Full Scan/dd-MS2; full MS resolution was set to MS Full Scan70000FWHM and MS/MS17500FWHM.

LC-MS data processing, namely importing the acquired LC-MS/MS data raw file into Compound Discoverer 2.0.0 software for data preprocessing.

Multivariate statistical analysis: the data obtained above were imported into Simca 14.1 software for analysis. The change of metabolic profiles of normal and model groups was investigated using orthogonal partial least squares discriminant analysis (OPLS-DA), and differential metabolites were found in combination with VIP >1 (serum) and VIP >2 (stool) and t-test (p < 0.05) in the figures. The reliability of the data model is described using the R2 and Q2 values. Statistical analysis: the data are all expressed in mean+ -SD, the analysis is performed by adopting sigma plot14.0 statistical software, independent sample t test is adopted for comparison between two groups, single factor variance analysis is adopted for comparison between multiple groups, and the statistical difference is considered to be present by p < 0.05. Metabolic profile analysis: the complex data obtained from the four groups of serum and stool samples were subjected to dimension reduction using PCA-X and OPLS-DA.

From the PCA plot scores, we can observe that in the positive ion mode, the W, LN, ln+sr37-3 and ln+61-2 groups are located at different positions (fig. 3a, g), whereas in the negative ion mode there is no significant separation between the groups (fig. 3b, h). The OPLS-DA scores in both positive and negative ion modes were significantly separated for the W and LN groups, both serum and cecal content, as shown in figures 3e, f, k and l, respectively. From the distribution trend we can further confirm that the hypertension model was successfully built using L-NAME. Chemical identification of these characteristic metabolites is achieved by local and on-line databases based on mass fragmentation patterns and isotope peak ratios, including HMDB @www.HMDB.ca). Biomarker analysis: serum sample results showed that 30 metabolites between W and LN, 21 metabolites between LN and ln+sr37-3, and 28 metabolites between LN and ln+sr61-2 were identified, as shown in table 8, including amino acids, fatty acids, amide derivatives, and choline derivatives, and cecum content samples showed that 27 metabolites between W and LN, 31 metabolites between LN and ln+sr37-3, and 24 metabolites between LN and ln+sr61-2 were identified, as shown in table 9, including amino acids, fatty acids, amide derivatives, bile derivatives, pyrimidine, and choline derivatives, and we performed a pathway analysis on the characteristic metabolites using metaanalysis 4.0 (www.MetaboAnalyst.ca) for a more comprehensive understanding of the mechanism of hypertension. Metabolic pathway analysis: differential metabolites were introduced into MetPA (Metabo analysis 3.0, http:// www.Metaboanalyst.ca) for pathway enrichment analysis. In the serum samples, since there were significant changes in 31 metabolites between W and LN, there were no changes in the results7 metabolic pathways significantly affected by hypertension were found (fig. 4 b), of which 3 were amino acid metabolisms, similar to the results in the cecal content samples, of which the first five metabolic pathways were amino acid metabolisms (fig. 4 f). Based on 22 significantly altered metabolites between LN and LN+SR37-3, 5 metabolic pathways were found that were significantly affected by the fermented milk of Lactobacillus plantarum strain SR37-3 (FIG. 4 c), of which 3 were amino acid metabolism and mainly involved in histidine and pyrimidine metabolism in the faecal samples (FIG. 4 g). In view of the significant variation in 29 metabolites between LN and LN+SR61-2, 5 metabolic pathways significantly affected by the fermented milk of Lactobacillus plantarum strain SR61-2 were found (FIG. 4 d), mainly involving fatty acid and amino acid metabolism, but in fecal samples, mainly involved in primary bile acid biosynthesis (FIG. 4 h). The heat map shows that there is a significant separation between LN and W groups in the serum (fig. 4 a) and cecal content (fig. 4 e) samples. However, in the control and model groups, most serum and cecal content metabolites have the same trend. Metabolites are divided into two large clusters (up and down). The metabolites of the upward cluster were mainly enriched in the control group, while the metabolites of the downward cluster were mainly enriched in the disease group. In particular, we have found that L-phenylalanine, L- (-) -methionine and L-valine are three different metabolites involved in both intestinal and blood metabolism. Phenylalanine, tyrosine and tryptophan biosynthesis and phenylalanine metabolism are common metabolic pathways of serum and cecal contents, and may be related to the pathogenesis of the disease. In the blood metabolome, the SR37-3 fermented milk can increase the content of nicotinamide in the body, and the nicotinamide has a series of functions of resisting aging, improving hypercholesterolemia, protecting endothelial cells of patients with coronary heart disease, reducing secretion and release of ET-1, and the like. SR61-2 fermented milk can increase betaine and arachidonic acid content in vivo. Betaine has certain curative effects on coronary heart disease, liver diseases, cardiovascular diseases and nerve diseases, and has the functions of regulating and controlling human metabolic balance and physiological balance, and the mechanism is that the betaine participates in methionine and homocysteine reaction. Arachidonic acid has effects of lowering blood sugar, reducing blood lipid, and lowering cholesterol. Studies show that arachidonic acid can significantly reduce low-density lipoprotein in bloodWhite (LDL-C) and very low density lipoprotein (VLDL-C), elevated high density lipoprotein (HDL-C) are important for cardiovascular health. Arachidonic acid also has a series of biological effects of dilating blood vessels, participating in immunomodulation, regulating neuroendocrine, promoting cell division, etc.

TABLE 8 UPLC-MS/MS technology based blank and model group serum differential metabolite assignment Table

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TABLE 9 UPLC-MS/MS technology based blank and model group cecal content differential metabolite identification Table

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R.t.: retention time (minutes), W: animals not treated with L-NAME received standard chow, LN L-NAME treated rats received standard chow, LN+SR37-3:L-NAME treated rats received Lactobacillus plantarum strain SR37-3 fermented milk lavage, LN+SR61-2:L-NAME treated rats received Lactobacillus plantarum strain SR61-2 fermented milk lavage. HMDB: human metabolome database.

S4 renal morphology observations

The rat in example 3 was dissected and kidney tissue was harvested and HE stained to observe its tissue morphology and pathological changes, and the specific procedure was performed as material-fixation-trimming-dehydration-transparency-wax-embedding-slicing-flaking. The results are shown in FIG. 5.

From fig. 5, it can be seen that the kidney tissue of the control group has uniform glomerulus distribution in cortex, uniform cell number and matrix in glomerulus, round and full tubular epithelial cells, regular brush-like edges, and no obvious abnormality in medulla; connective tissue between urinary tubules is renal interstitium, and the interstitium has no obvious hyperplasia; no apparent inflammatory cell infiltration was seen. The kidney tissue of the model group was denatured by small arterial smooth muscle cell vacuoles, and small vacuoles were seen in the cytoplasm (black arrows) compared to the control group. While SR37-3 intervention group showed no obvious abnormality in kidney, SR61-2 intervention group showed little increase in renal tubule nuclei and vacuolation (black arrow), and no other obvious abnormality. It shows that long-time hypertension can cause chronic damage to the kidney of the rat, and the probiotic can have a certain relieving effect after intervention.

Example 5 evaluation of safety of two Lactobacillus plantarum strains in vitro

(1) Antibiotic susceptibility test

6 common antibiotics were selected for drug susceptibility testing. The selected antibiotics are as follows: chloramphenicol, tetracycline, erythromycin, gentamicin, amoxicillin, and ampicillin. Centrifuging 4000 Xg of the prepared bacterial liquid for 10min, washing with sterile PBS solution for 3 times, diluting the bacterial liquid, then soaking the bacterial liquid with a sterile cotton swab, and uniformly coating the whole surface of the flat plate. After the drug sensitive paper is attached, the drug sensitive paper is lightly pressed by sterile forceps to be firmly attached to the surface of the culture medium. The flat plate with the diameter of 90mm is respectively attached with 4 drug sensitive paper sheets, and the center-to-center distance between the paper sheets is not less than 24mm. The name of the antibiotic is marked, after the antibiotic is cultured for 18 hours at 37 ℃, the diameter of the bacteriostasis ring is measured from the back of the plate by a caliper, and the result is recorded. Staphylococcus aureus ATCC25923 is used as a quality control strain, and a patent drug resistant strain 833-1 is used as a control. The results obtained are shown in Table 10 below (note: R is drug resistant, I is intermediate, S is sensitive).

Resistance of probiotics to antibiotics can limit the use of probiotics. Bacterial resistance results can be judged as three forms of resistance (R), mediation (I) and sensitivity (S) according to CLSI 2015 antimicrobial drug susceptibility test criteria. As can be seen from Table 10, SR37-3 and SR61-2 exhibited sensitivity to various antibiotics, which fully demonstrated the safety characteristics of both strains.

Table 10 antibiotic susceptibility of two Lactobacillus plantarum strains

(2) Hemolysis experiment

Under aseptic condition, the activated bacterial liquid is streaked on a blood plate by a disposable aseptic inoculation loop, and is cultured for 48 hours at 37 ℃ to observe the hemolysis phenomenon. Hemolysis can form three features on the blood platelets: (1) alpha hemolysis: grass green hemolytic rings appear around colonies, which are generally opportunistic. (2) beta hemolysis: a broad transparent hemolytic ring appears around the colony, and is generally highly pathogenic. (3) gamma hemolysis: there were no hemolytic rings around the colonies, and in general the strains were nonpathogenic.

As shown in FIG. 6, no hemolytic ring appeared around each colony, so that both Lactobacillus plantarum (SR 37-3 and SR 61-2) have no hemolysis and have good safety.

Example 6 evaluation of in vivo safety of two Lactobacillus plantarum strains

S1 experiment design

Wistar male and female rats (190-210 g) were subjected to adaptive feeding for 7 days for testing, and acute toxicity experiments were performed using the maximum tolerance method. The people can fasted for 16 hours before the experiment, and the drinking water is not limited. Normal group: the food is eaten and drunk freely without treatment. The treatment group used the maximum lavage method. The grouping is as follows:

normal group 1: the male mice were free to eat and drink water without treatment.

Normal group 2: the female mice were free to eat and drink water without treatment.

Treatment group 3: by 1X 10 ¹⁵ CFU/Kg.bw live bacteria (SR 37-3) were lavaged once in male mice.

Treatment group 4: by 1X 10 ¹⁵ CFU/Kg.bw live bacteria (SR 37-3) were lavaged once to the female mice.

Treatment group 5: by 1X 10 ¹⁵ CFU/Kg.bw live bacteria (SR 61-2) were lavaged once in male mice.

Treatment group 6: by 1X 10 ¹⁵ CFU/Kg.bw live bacteria (SR 61-2) were lavaged once to the female mice.

After gastric lavage, the patients were observed for 14 days continuously, the poisoning manifestations and death were recorded, and initial and final body weights were weighed. And (5) after the experiment is expired, taking the liver and spleen for weighing after blood sampling and sacrifice, and calculating the liver-body ratio and spleen-body ratio.

Organ index, data were analyzed using sigma plot14.0 software, and mean±sd values were used to represent each set of values, and One Way ANOVA was used to check their significance. Labeling was used when there was a significant difference between each male and female treated group and the male and female normal group, respectively. The experimental results (table 11) show that there was no significant difference in organ index between each dosing group and the normal group.

TABLE 11 liver and spleen organ coefficients for animals of each group

Blood routine and blood biochemical detection after S2 toxicity experiment

The method for detecting blood routine items by using the full-automatic blood analyzer comprises the following steps: white blood cell count, basophil count, neutrophil count, eosinophil count, lymphocyte count, monocyte count, basophil proportion, neutrophil proportion, eosinophil proportion, lymphocyte proportion, monocyte proportion, red blood cell count, hemoglobin content, mean red blood cell volume, mean red blood cell hemoglobin content, mean red blood cell hemoglobin concentration, coefficient of variation in distribution width of red blood cells, standard deviation of distribution width of red blood cells, hematocrit, platelet count, mean platelet volume, distribution width of platelets, platelet hematocrit, 22 items.

Serum is taken and used for detecting blood biochemical projects by a full-automatic biochemical analyzer. Comprising the following steps: glutamic pyruvic transaminase (ALT), total Protein (TP), albumin (ALB), globulin (GLOB), bilirubin (TBIL), alkaline phosphatase (ALP), glutamyl transpeptidase (γ -GT), urea nitrogen (BUN), serum Creatinine (CRE), uric Acid (UA), blood Glucose (GLU), total Cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (LDL-C), low density lipoprotein cholesterol (LDL-C) 15 total. The above two detection results are shown in the accompanying tables 12, 13, 14 and 16. In the table, SR37-3 represents the treatment of the lavage of the SR37-3 strain, and SR61-2 represents the treatment of the lavage of the SR61-2 strain. The data were analyzed using sigma plot14.0 software and each set of values was expressed as mean±sd values, and their significance was checked using One Way ANOVA. Labeling was used when there was a significant difference between each male and female treated group and the male and female normal group, respectively. Experimental results showed no significant differences in organ index between each dosing group and the normal group.

TABLE 12 routine analysis of blood from female groups

TABLE 13 routine analysis of blood from individual male groups

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TABLE 14 Biochemical analysis of blood from female animals

TABLE 15 Biochemical analysis of blood from Male animals

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EXAMPLE 7 specific Gene fragment of Lactobacillus plantarum SR61-2

(1) Excavating lactobacillus plantarum SR61-2 different species specific molecular targets

Performing bioinformatics analysis according to the GenBank database and the genome-wide DNA sequence self-tested by the team; screening to obtain specific gene fragments of lactobacillus plantarum SR37-3 and SR61-2, wherein the nucleotide sequences of the gene fragments are shown as SEQ ID NO. 1-2. Wherein the sequence SEQ ID NO.1 is a SR37-3 strain-specific gene fragment, and the sequence SEQ ID NO.2 is a SR61-2 strain-specific gene fragment

(2) Primer availability detection

Specific PCR amplification primer sets (including forward and reverse primers) were designed according to the sequence SEQ ID NO.1 as described in (1), and the primer set sequences are shown in Table 16 below.

TABLE 16 specific PCR detection primer set

S1, preparation of a DNA template: culturing SR37-3 and SR61-2 in MRS liquid culture medium, extracting their DNA respectively by using bacterial genome DNA extraction kit as template to be detected;

s2 PCR amplification:

the PCR detection system is as follows:

primer set 1PCR amplification procedure:

primer set 2PCR amplification procedure:

s3: and (3) taking PCR amplified products to carry out gel electrophoresis, and observing whether the positions of the primer groups corresponding to the sizes of the products exist in the target positions. If other strain templates do not have bands at the target location, the corresponding targets are strain-specific molecular targets.

PCR was performed as in example 6. Wherein, the S1 DNA template is prepared to extract genome DNA of each bacterium respectively; in the S2 PCR amplification, the primer used is the primer in the primer group. A blank is set, and the template of the blank is an aqueous solution without genome. The strains of each bacterium used and the results of the tests are shown in tables 17 to 18, in which "-" in the column of the results of the tests indicates negative.

TABLE 17 evaluation test results of the detection specificity of Lactobacillus plantarum SR37-3 of the invention

TABLE 18 evaluation test results of the detection specificity of Lactobacillus plantarum SR61-2 of the invention

As can be seen from the two tables, only the target strain can generate an amplified band at the target fragment position and has high brightness, which indicates that only the target strain contains a specific molecular target in the method.

EXAMPLE 7 establishment of methods for fluorescent quantitative PCR detection of Lactobacillus plantarum SR37-3 and SR61-2

Bacterial DNA was extracted using a genomic DNA extraction kit (Magen Biotech, china) and its concentration was measured. The concentration is changed into the copy number, and the conversion formula between the DNA concentration and the copy number is as follows:

wherein A-copy number per μL of DNA; C-DNA mass concentration in ng/. Mu.L; the length of the L-DNA fragment is in bp. Gradient dilution of DNA to a copy number of 10 ² ～10 ¹⁰ Between them. And (3) performing fluorescent quantitative PCR by taking the products after gradient dilution as templates. Fluorescent quantitative PCR system (20 μl): 2 XSYBR PCR premix 10. Mu.L, upstream primer 0.6. Mu.L, downstream primer 0.6. Mu.L, template 1. Mu.L, 50 XSORO X Reference Dye 0.4. Mu.L, ddH ₂ O7.4. Mu.L. PCR procedure 1: pre-denaturation at 95 ℃ for 15min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, and 2-4 steps for 40 times. Standard curves were made with copy number and Ct value of the corresponding sample. The results are shown in fig. 7-8, where Ct values are linear with lg copy number, giving an SR37-3 standard curve of y= 44.089-2.8593X. X represents lg copy number and Y represents Ct value. R2=0.991. The R61-2 standard curve is y= 45.7602-3.5494X. X represents lg copy number and Y represents Ct value. R2= 0.9956.

Claims (8)

1. Lactobacillus plantarum（Lactobacillus plantarum）SR37-3, deposit number is: GDMCC No:62390.

2. lactobacillus plantarum（Lactobacillus plantarum）SR61-2, accession number is: GDMCC No:62389.

3. use of lactobacillus plantarum SR37-3 according to claim 1 or lactobacillus plantarum SR61-2 according to claim 2 for the preparation of a product for reducing blood pressure, slowing down the production of inflammatory substances in the serum of patients suffering from hypertension, improving kidney damage caused by hypertension.

4. The use according to claim 3, wherein the product is a medicament.

5. A product for reducing blood pressure, reducing inflammatory substances produced in serum of patients with hypertension, and improving kidney damage caused by hypertension, which comprises lactobacillus plantarum SR37-3 of claim 1 or lactobacillus plantarum SR61-2 of claim 2 as an active ingredient.

6. The use of a primer set for amplifying a specific nucleotide sequence of lactobacillus plantarum SR37-3 as defined in claim 1 in identifying lactobacillus plantarum SR37-3 or the use of a primer set for amplifying a specific nucleotide sequence of lactobacillus plantarum SR61-2 as defined in claim 2 in identifying lactobacillus plantarum SR61-2, wherein the specific nucleotide sequence of lactobacillus plantarum SR37-3 is shown as SEQ ID NO.1, and the specific nucleotide sequence of lactobacillus plantarum SR61-2 is shown as SEQ ID NO. 2.

7. The primer set for detecting the lactobacillus plantarum SR37-3 of claim 1 or the lactobacillus plantarum SR61-2 of claim 2, wherein the nucleotide sequences of the primers in the primer set for detecting the lactobacillus plantarum SR37-3 are shown in SEQ ID NO.3 and SEQ ID NO. 4 respectively, and the nucleotide sequences of the primers in the primer set for detecting the lactobacillus plantarum SR61-2 are shown in SEQ ID NO.5 and SEQ ID NO. 6 respectively.

8. A detection method for detecting lactobacillus plantarum SR37-3 according to claim 1 or lactobacillus plantarum SR61-2 according to claim 2, characterized by comprising the steps of:

s1: performing PCR amplification using the primer set of claim 7;

s2: detecting the amplified product by gel electrophoresis;

s3: and observing whether the amplified product meets the expectations.