CN114196575B - Panda source streptococcus paris and application thereof - Google Patents

Abstract

The invention provides a panda source streptococcus paris and application thereof, wherein the strain is named as: streptococcus lutetiensisS S7, deposited in Guangdong province culture Collection, with the deposit number GDMCC No: 61917. the strain, i.e., the gram-positive coccus, can grow at 40 ℃, has good acid resistance and cholate resistance, is high in safety, can regulate the expression of colon muc2 to promote mucus secretion, can regulate the relative expression amounts of colon IL-1 beta mRNA and TNF alpha mRNA of a DSS mouse model, and has good application prospects in treating colitis of pandas and improving immunity of the pandas.

Description

Panda-derived streptococcus paris and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to panda-derived streptococcus paris and application thereof.

Background

Pandas have long been plagued by intestinal disease, a major cause of death in pandas. Furthermore, in the pathogenicity, certain gut bacteria such as escherichia coli, klebsiella pneumoniae, and clostridium welchii have been shown to cause panda gut disease. These bacteria affect the balance and stability of intestinal micro-ecosystem to some extent, while the instability of intestinal microflora can further affect the digestion of bamboo by pandas and even the immune system of pandas.

Lactic acid producing bacteria such as streptococcus can regulate normal flora of gastrointestinal tract, maintain microecological balance, improve food digestibility, inhibit growth of intestinal putrefying bacteria, and improve host immunity. At present, most of the research on panda intestinal microorganisms mainly focuses on the cellulose degradation ability of the intestinal microorganisms, and little is known about the immune function of panda-related probiotics. S. paris S7 shows better tolerance and carries the gene sodA (encoding superoxide dismutase) which can be beneficial to the health of the host. This strain has good probiotic potential but needs further verification.

The DSS animal model was constructed based on the destruction of rodent epithelium by Dextran Sodium Sulfate (DSS), resulting in excessive tissue neutrophil and other damage and subsequent infiltration of acute immune cells, leading to colitis. Can take effect after being freely drunk for 7 days. The DSS model is morphologically and symptomatically very similar to ulcerative colitis, and the molding mechanism of DSS is associated with the direct toxic effects of DSS drugs on intestinal epithelium, which can cause erosion of intestinal epithelium, ultimately leading to the destruction of mucosal barrier integrity. DSS modeling has the advantages of simple method, easy repetition and easy acquisition and low price of DSS drugs, and is a chemical induction model used in experimental research. Therefore, DSS animal models were used in this study to evaluate the probiotic effect of the obtained strains.

Disclosure of Invention

Aiming at the blank of the immune function research of panda related probiotics in China at present, the invention provides a panda source streptococcus paris and application thereof, the strain can grow at a high temperature of 40 ℃, has good acid resistance and cholate resistance characteristics and high safety, can regulate the expression of colon muc2 to promote mucus secretion, and regulate the relative expression quantity of DSS mouse model colon IL-1 beta mRNA and TNF alpha mRNA, and has good application prospects in treating panda colitis and improving panda immunity. The technical scheme of the invention is as follows:

in a first aspect, the invention provides a panda-derived streptococcus paris strain, which is named as: streptococcus lutetiensis S7, deposited at 9/3/2021 at the Guangdong province collection of microorganisms, address: experiment building 5 of 100 # large institute in overseas junior middle school, mountain of Guangzhou, Guangdong province, with the preservation number GDMCC No: 61917.

The colony morphology of the streptococcus obtained by separation is small, is milk white and has a frankincense flavor. Gram-positive (bluish violet) cocci are found by gram-staining oil-microscopic examination (magnification: objective 100 Xeyepiece 16X), the cocci are arranged in a chain shape (figure 2), and the bacteria are identified as the streptococcus paris through sequencing.

Further, the panda-derived streptococcus paris could grow well at a temperature of 40 ℃ (fig. 3).

Furthermore, the panda-derived streptococcus Paris does not contain drug-resistant genes such as tetA, tetB, tetC, tetD, tetE, tetM, sul1, dfrA1, dfrA5 and intI 1.

Further, the panda source streptococcus paris has acid and bile salt resistance.

Further, the panda-derived streptococcus paris can regulate mouse colon muc2 expression and promote mucus secretion.

Further, the panda-derived streptococcus paris can regulate the expression of IL-1 β mRNA and TNF α mRNA in a DSS-induced enteritis model.

The construction of the DSS animal model is based on the destruction of rodent epithelium by Dextran Sodium Sulfate (DSS), resulting in excessive tissue neutrophil and other damage and subsequent infiltration of acute immune cells, leading to colitis. Usually, the effect can be obtained after free drinking for 7 days. The DSS model is morphologically and symptomatically very similar to ulcerative colitis, and the molding mechanism of DSS is associated with the direct toxic effects of DSS drugs on intestinal epithelium, which can cause erosion of intestinal epithelium, ultimately leading to the destruction of mucosal barrier integrity. DSS modeling has the advantages of simple method, easy repetition and easy acquisition and low price of DSS drugs, and is a chemical induction model used in experimental research. Therefore, the present invention employs DSS animal models to evaluate the prebiotic effect of panda-derived streptococcus paris.

In a second aspect, the invention provides an application of the panda-derived streptococcus paris in preparing a panda colitis medicament.

In a third aspect, the invention provides an application of the panda-derived streptococcus paris in preparing panda immunoregulation medicaments.

Compared with the prior art, the invention has the beneficial effects that:

the invention firstly carries out separation culture and biological characteristic research on the panda enteric streptococcus, and researches the growth characteristic, biochemical characteristic, system evolution relation of the strain and the like of the separated streptococcus. The panda source streptococcus paris of the invention can grow at the temperature of 40 ℃, has good acid and bile salt resistance, is safe and reliable, can regulate the expression of colon muc2, and enhances the intestinal mucosal immune barrier of a host. DSS induced enteritis animal models find that the streptococcus Paris S7 can reduce related intestinal inflammation indexes, generate immune factors with anti-inflammatory effects and relieve inflammatory reactions in the aspect of immunity. Has good application prospect in treating the colitis of the pandas and improving the immunity of the pandas.

Drawings

FIG. 1 is a gram stain microscopic examination (1000X) of Streptococcus S7 isolated in example 1

FIG. 2 is a graph of the growth curves of S7S in example 2 at different temperatures.

FIG. 3 is a bar graph showing the change in body weight and food intake of mice in example 3 of the present invention, wherein FIG. 3-1 shows the change in body weight of mice in the acute toxicity test; FIG. 3-2 shows the body weight change of mice in chronic toxicity test; FIG. 3-3 shows the change in food intake of mice in acute toxicity test; FIGS. 3-4 show the change in food intake of chronic toxicity test mice.

FIG. 4 is a genus in which t-test is significantly different between the experimental group and the control group in example 4 of the present invention, wherein FIG. 4-1 is the result for 14 days and FIG. 4-2 is the result for 28 days.

FIG. 5 shows the real-time fluorescent quantitative PCR detection of mouse colon muc2 mRNA expression level in example 5 of the present invention.

Fig. 6 is a statistical analysis of mucosal thickness in example 5 of the present invention.

FIG. 7 shows the colon mucosa thickness (28d) of each group of mice detected by AB-PAS staining in example 5 of the present invention, wherein FIG. 7-1 shows the results of 28 days in the control group, and FIG. 7-2 shows the results of 28 days in the treatment group.

FIG. 8 shows the construction of a DSS animal model in example 6 of the present invention.

FIG. 9 is a graph showing the change in body weight of mice of each group during the molding period in example 6 of the present invention.

FIG. 10 shows the DAI index records of each group in example 6 of the present invention.

FIG. 11 is a histogram of colon length of each group of mice in example 6 of the present invention.

FIG. 12 is a histogram of spleen weights of groups of mice in example 6 of the present invention.

FIG. 13 is a bar graph of MPO enzyme activity in colon tissue of each group of mice in example 6 of the present invention.

FIG. 14 is a histogram of the mRNA expression levels of IL-6, IL-1. beta. and TNF. alpha. genes in the colon of each group of mice in example 6 of the present invention, wherein FIG. 14-1 is the mRNA expression level of the IL-6 gene, FIG. 14-2 is the mRNA expression level of the IL-1. beta. gene, and FIG. 14-3 is the mRNA expression level of the TNF. alpha. gene.

FIG. 15 is a staining pattern of HE of a partial colon tissue of each group of mice in example 6 of the present invention, FIGS. 15-1 and 15-2 are staining patterns of 200 μm and 50 μm, respectively, in a control group, FIGS. 15-3 and 15-4 are staining patterns of 200 μm and 50 μm, respectively, in a DSS group, and FIGS. 15-5 and 15-6 are staining patterns of 200 μm and 50 μm, respectively, in a treatment group.

FIG. 16 is a histogram of histological score of the groups of mice in example 6 of the present invention.

Detailed Description

In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.

Example 1

Separation, identification and activation of panda-derived streptococcus paris S7

1. Isolation and identification of panda source streptococcus paris S7

And (3) diluting the collected panda excrement sample in a gradient manner by using PBS (phosphate buffer solution), coating the diluted panda excrement sample on an MRS (methicillin resistant Staphylococcus aureus) solid culture medium, and culturing the sample in a constant-temperature incubator at 37 ℃ for 18 h. And selecting the suspicious colonies for separation and purification again, wherein the colony morphology of the streptococcus obtained by separation is small, the streptococcus is milk white, and the streptococcus gives off a frankincense taste. The strain was found to be gram-positive (bluish violet) cocci by gram-stain oil-microscopic examination (magnification: objective 100 Xeyepiece 16X), and the cocci were arranged in a chain form (FIG. 1). Single colonies were picked and cultured in MRS liquid medium for 18h (37 ℃). Extracting DNA according to the steps of the hectacke bacterial DNA extraction kit, amplifying the 16S rRNA gene by using bacterial DNA as a template and 27F and 1492R as primers, and performing 16S rRNA marker gene sequencing identification.

2. Activation and culture of strains

Taking out the bacteria-protecting tube from a refrigerator at minus 80 ℃, streaking and reviving streptococcus Paris S7 on an MRS solid plate, culturing for 18h at 37 ℃, selecting a single colony to inoculate in an MRS liquid culture medium, culturing for 18h at 37 ℃, then inoculating in the MRS liquid culture medium with the inoculation amount of 2%, culturing for 18h at 37 ℃, continuously activating for three generations, then inoculating in the MRS liquid culture medium with the inoculation amount of 2% to perform amplification culture, and culturing for 18h at 37 ℃. The pellet was centrifuged at 8000 Xg for 10min, washed 3 times with PBS and used in the experiments of the subsequent examples.

Example 2

Evaluation of temperature tolerance, antibiotic tolerance, acid and bile salt resistance of panda-derived Streptococcus Paris S7

1. Temperature tolerance assay of strains

The method comprises the following specific steps:

(1) bacterial liquid culture: a clitocybe is selected from a slant culture of a panda source streptococcus paris S7 strain, inoculated in a meat soup culture medium (5 g of beef extract, 10g of peptone and 5g of sodium chloride, and water is added to 1000 mL), and kept stand for culture at 37 ℃ for about 12 hours, and the bacterial liquid is a seed culture solution.

(2) Subpackaging the culture solution and correcting the zero point: 25mL of the medium was pipetted with a sterile pipette into a Erlenmeyer flask with a sidearm tube, the uninoculated medium was poured into its sidearm tube, and the zero point was adjusted on the photoelectric colorimeter, i.e., the OD600 value on the photoelectric colorimeter was at zero point (three parallel replicates were set for each strain).

(3) Inoculation and zero time measurement: 2.5mL of seed culture was pipetted into the inoculation flask and shaken well. The freshly inoculated culture broth was poured into a side arm tube and the OD600 value of the photoelectric colorimeter was determined, which was the zero time reading in the post-inoculation strain growth curve.

(4) Culturing and measuring the growth amount: placing the triangular flask after the measurement at zero time on a constant-temperature water bath shaking table for shake culture, wherein the culture temperature is 37 ℃, and the frequency of the shaking table is about 100 Xg/min. During the culture, the Erlenmeyer flask should be removed from the shaker every 30min, the broth should be poured into a side-arm test tube, and OD600 values should be read on a photoelectric colorimeter (Shanghai apparatus electric instruments Co., Ltd., model 721G), data measured each time are recorded, and the zero point of the photoelectric colorimeter should be corrected with the culture broth of a blank control tube at each measurement.

(5) Drawing a growth curve: and (3) taking the measured time as an abscissa and the logarithm of the number of bacteria (OD600 value) as an ordinate, and drawing points on semi-logarithmic coordinate paper to obtain a curve, namely the growth curve of the measured strain under the experimental conditions. Setting temperature gradient (20 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 50 deg.C and 60 deg.C), measuring optimum growth temperature, measuring thallus concentration by turbidimetry, and drawing growth curve of the strain at different temperatures (t), as shown in FIG. 2, Streptococcus Paris S7 grows well at 40 deg.C.

2. Antibiotic resistance test

Selecting a plurality of single panda source streptococcus paris S7 bacterial colonies, directly inoculating 4.5% normal saline to prepare bacterial suspension, and adjusting the turbidity of the suspension to 0.5 McLee unit by using a turbidity comparator; after the turbidity of the suspension is adjusted, the suspension is immersed in a sterile cotton swab, and the swab is rotated above the liquid tightly against the inner wall of the test tube several times to remove the excess liquid on the cotton swab, but the amount should be appropriate. Streaking the entire agar surface with a swab, spreading evenly from the top to the bottom of the plate; the antibacterial paper sheets are stuck to the surfaces of the agar inoculated with bacteria, each paper sheet must be pressed down to ensure that the paper sheet is completely contacted with the surface of the agar, no matter the paper sheets are placed independently or paper sheet distribution equipment is arranged, the paper sheets must be distributed uniformly, the distance between the circle centers of the two paper sheets is not less than 24mm, and the distance between the edges of the paper sheets and the edges of the agar is not less than 15 mm; after placing the paper sheets, within 15min, the MH agar was placed upside down in a constant temperature incubator and incubated for 16-18 h. After incubation, the MH plate with the paper attached was placed on a black non-reflective background and the diameter of the zone of inhibition (diameter of the zone of complete inhibition) was measured using a vernier caliper. The strain sensitivity interpretation refers to CLSI standard, and reports the sensitivity, intermedium and drug resistance of the tested bacteria to the tested drugs according to the diameter value of the antibacterial ring. The detection of antibiotics mainly comprises the following steps: tetracycline, doxycycline, sulfamethoxazole, azithromycin, vancomycin, rifampin, clindamycin, meropenem, levofloxacin, clarithromycin, ofloxacin, chloramphenicol, penicillin, ceftriaxone, erythromycin, and ampicillin. The PCR is used for detecting the drug resistance genes, and the detected drug resistance genes are as follows: tetA, tetB, tetC, tetD, tetE, tetM, sul1, dfRA1, dfRA5 and intI1, and the test result shows that no drug resistance gene was detected.

3. Determination of acid-resistant and cholate-resistant characteristics of target strain

Pepsin was resuspended in sterilized 0.5% (W/V) physiological saline to a concentration of 3g/L, and pH was adjusted to 2.0 to obtain simulated gastric fluid. The pancreatin was resuspended in sterilized 0.5% (W/V) physiological saline to a concentration of 1g/L, 0.3% bile salt was added and the pH was adjusted to 8.0 to obtain a simulated intestinal fluid. Taking 3 parts of 1mL of panda-derived streptococcus paris S7 bacterial liquid, centrifuging, washing, respectively suspending in 1mL of 0.5% (W/V) sterile saline, 1mL of simulated gastric fluid and 1mL of simulated intestinal fluid, mixing uniformly, and placing in a constant-temperature incubator at 37 ℃ for culture. 1mL of simulated gastric juice, taking 100 mu L of bacterial liquid respectively at 1 hour and 3 hours, counting viable bacteria, calculating the survival rate, and determining the acid resistance. 1mL of simulated intestinal fluid, taking 100 mu L of bacterial liquid respectively at 2h and 4h for viable count, calculating the survival rate, and determining the cholate resistance. Wherein the survival rate (%) ═ P₁/P₀×100，P₁For the number of viable bacteria after stress treatment, P₀Viable count before stress treatment.

As shown in Table 1, the survival rate of S7 in gastric juice was 1.3% + -0.5% (9.4X 10) at 1h⁷CFU/mL), the survival rate of the strain in gastric juice at 3h is 0.1% + -0.1% (9.8X 10)⁶CFU/mL); the survival rate of the streptococcus Paris S7 in pancreatic juice at 2h is 28.0% + -5.3% (2.1X 10)⁸CFU/mL), the survival rate of the streptococcus Paris S7 in pancreatic juice is 3.2% + -0.5% (2.5X 10) at 4h⁷CFU/mL). The streptococcus paris S7 has relatively good acid-resistant and bile-salt-resistant properties.

TABLE 1 results of acid and bile salt resistance characteristics of Strain S7

According to the determination of the temperature tolerance, antibiotic tolerance, acid resistance and bile salt resistance of the strain, the strain has the potential of becoming probiotics.

Example 3

Acute and chronic toxicity animal experiment

3.1 Experimental animals

40 mice of 4-week-old SPF grade adult C57BL/6 mice are selected respectively and strictly controlled in temperature and humidity. The experiment was started 7 days after the mice were acclimated and relevant experimental records were made according to the experimental requirements.

3.2 acute toxicity animal experiments

The experiment is carried out according to the requirements of national standard GB15193.3-2014 national food safety Standard acute oral toxicity experiment, male and female mice are randomly selected after the adaptation period is ended and are divided into 2 groups, namely a blank group and a formula group, wherein each group comprises 10 male and female mice, the formula group comprises 10 enema panda source streptococcus paris S7 bacterial suspension, and the amount of the enema bacteria reaches 10⁹CFU/mouse, blank group gavage PBS. The preparation is administered by intragastric administration at one time, and the dosage is 0.2 mL/tube. After gavage, mice were observed and recorded for behavioral characteristics as well as death and toxicity. The body weight and food intake were recorded by weighing at 0, 3, 7 and 14 days before the experiment, and on 14 days, the mice were subjected to orbital bleeding for blood routine and blood biochemistry, roughly dissected, and the relevant organs were weighed, and if there was a change in volume, color, etc. of the organs, pathological examination of the organs was performed. The experimental data are all expressed as mean ± standard error of mean, data statistics are analyzed by one-way ANOVA, and Tukey's multiple test, and the data difference is considered significant when the p value is less than 0.05.

3.3 Chronic toxicity test

Experiments are carried out according to the requirements of national standards GB 15193.13-2015 national food safety standard 30-day feeding test and GB15193.22-2014 national food safety standard 28-day import toxicity test, male and female mice are randomly selected after the adaptation period is finished and divided into 2 groups, the groups are respectively a blank group and a formula group, 10 males and females in each group, the blank group is perfused with gastric PBS, the formula group is perfused with gastric panda source streptococcus Paris S7 bacterial suspension, and the amount of the perfusing bacteria reaches 10⁹Continuous daily gavage and observation for CFU/patientMice were observed to record behavioral characteristics as well as mortality and toxicity. Body weight and food intake were recorded at 0, 3, 7, 14, 21, 28 days of the experiment, and on day 28, mice were bled orbitally to determine blood routine and blood biochemistry. Mice are dissected and relevant organs are weighed, and if there is a change in volume, color, etc. of an organ, pathological examination of the organ should be performed. The experimental data are all expressed as mean ± standard error of mean, data statistics are analyzed by one-way ANOVA, and Tukey's multiple test, and the data difference is considered significant when the p value is less than 0.05.

3.4 detection of physiological indices of mouse blood

The mouse blood physiological indexes are detected by a full-automatic blood cell analyzer, and the detection indexes comprise White Blood Cells (WBC), Red Blood Cells (RBC), Hemoglobin (HGB) and Platelets (PLT). The liver and kidney function detection indexes comprise glutamic-pyruvic transaminase (ALT) by a dry chemical method, detailed steps refer to an application instruction of a glutamic-pyruvic transaminase test strip, an alkaline phosphatase (ALP) by a disodium phenylphosphate colorimetric method, and detailed steps refer to an alkaline phosphatase determination kit (purchased from Nanjing institute of bioengineering). The liver and kidney function index detection method adopts a picric acid method for creatinine (Cr), and the detailed steps refer to a creatinine determination kit. Urea (Urea) in the blood of the mice was detected by the urease indigo method, and the detailed procedure was referred to the measurement kit. All experimental data were subjected to factor analysis of variance and correlation analysis using SPSS software. The experimental data are expressed as mean values plus or minus standard error of mean, data statistics are analyzed by one-way ANOVA and Tukey's multiple tests, and the data difference is considered to be significant when the p value is less than 0.05.

3.5 analysis of the results

In the observation period of 14 days of acute toxicity animal experiment and 28 days of chronic toxicity animal experiment, all groups of mice normally move, have good spirit and normal drinking water, and have no poisoning and death. No obvious pathological changes of mouse organs are found after the mouse is dissected. In acute and chronic toxicity animal experiments, there was no significant difference in organ coefficients (p >0.05) between mice in the control and treated groups measured after dissection (tables 2 and 3). The change of the body weight and food intake of the mice in each experimental group within 14 days of the acute toxicity animal experiment and 28 days of the chronic toxicity animal experiment is shown in fig. 3, and the body weight and food intake of the mice in the control group and the treated group are not obviously abnormal (p > 0.05). In addition, in acute and chronic toxicity animal experiments, the blood index of each group of mice was normal, and there was no significant difference (p >0.05) between the blood index of the control group and the treated group (as shown in tables 2 to 9).

TABLE 2 organ coefficient index of mice fed in acute toxicity experiment

Note: heart: heart, lever: liver, spleens: spleen and lung: lung, kidney, stomach: stomach, colon: colon, denum: duodenum, cecum: the cecum. There was no significant difference p >0.05 for each group.

TABLE 3 index of organ coefficient of mice fed in chronic toxicity experiment

Note: heart: heart, liver: liver, spleens: spleen and lung: lung, kidney, stomach: stomach, colon: colon, denum: duodenum, cecum: the cecum. There was no significant difference p >0.05 for each group.

TABLE 4(a) blood indices of mice fed in acute toxicity test

Note: WBC (10^ 9/L): number of leukocytes; neu (%): the percentage of neutrophils; lym (%): percentage of lymphocytes; mon (%): percent monocytes; eos (%): percentage of eosinophils; bas (%): percentage of basophils; neu # (10^ 9/L): the number of neutrophils; lym # (10^ 9/L): the number of lymphocytes; mon # (10^ 9/L): the number of monocytes; bas # (10^ 9/L): basophil number. There was no significant difference p >0.05 for each group.

TABLE 5(b) blood indices of mice fed in acute toxicity test

Note: RBC (10^ 12/L): the number of red blood cells; HGB (g/L): the concentration of hemoglobin; HCT (%): hematocrit; mcv (fl): mean hematocrit; mch (pg): mean corpuscular hemoglobin content; MCHC (g/L): mean corpuscular hemoglobin concentration; RDW _ CV (%): coefficient of variation of distribution width of erythrocytes; RDW _ sd (fl): standard deviation of distribution width of red blood cells. There was no significant difference p >0.05 for each group.

TABLE 6(c) blood indices of mice fed in acute toxicity test

Note: PLT (10^ 9/L): number of platelets; MPV (fL): mean platelet volume; PDW: width of platelet distribution; PCT (%): thrombocyte hematocrit; ALT (U/L): alanine aminotransferase; cre (μmol/L): creatinine. There was no significant difference p >0.05 for each group.

TABLE 7(a) blood indices of mice fed in chronic toxicity test

Note: WBC (10^ 9/L): number of leukocytes; neu (%): the percentage of neutrophils; lym (%): percentage of lymphocytes; mon (%): percent monocytes; eos (%): percentage of eosinophils; bas (%): percentage of basophils; neu # (10^ 9/L): the number of neutrophils; lym # (10^ 9/L): the number of lymphocytes; mon # (10^ 9/L): the number of monocytes; bas # (10^ 9/L): basophil number. There was no significant difference p >0.05 for each group.

TABLE 8(b) blood indices of mice fed in chronic toxicity test

Note: RBC (10^ 12/L): the number of red blood cells; HGB (g/L): the concentration of hemoglobin; HCT (%): hematocrit; mcv (fl): mean hematocrit; mch (pg): mean red blood cell hemoglobin content; MCHC (g/L): mean corpuscular hemoglobin concentration; RDW _ CV (%): coefficient of variation of distribution width of erythrocytes; RDW _ sd (fl): standard deviation of distribution width of red blood cells. There was no significant difference p >0.05 for each group.

TABLE 9(c) blood indices of mice fed in chronic toxicity test

Note: PLT (10^ 9/L): number of platelets; MPV (fL): mean platelet volume; PDW: width of platelet distribution; PCT (%): platelet aggregation; ALT (U/L): alanine aminotransferase; cre (μmol/L): creatinine. There was no significant difference p >0.05 for each group.

In a word, the panda source streptococcus paris provided by the invention is expected to be developed into panda colitis medicines and panda immunoregulation medicines.

Example 4

High-throughput sequencing of mouse feces 16S rRNA

4.1 sample Collection

During the course of the chronic experiment in mice of example 3, mouse feces were collected under sterile conditions in the middle (day 14) and end (day 28) of the experiment for 16S rRNA high-throughput sequencing.

4.2 Total DNA extraction and 16S rRNA high throughput sequencing

Mobio Power Fecal was used according to the manufacturer's instructions^TMThe DNA kit extracts total genomic DNA from collected mouse feces, PCR-amplifies the hypervariable region V4(83bp) of the 16S rRNA gene using universal primers 520F (5 '-barcode + AYTGGGYDTAAAGNG-3') and 802R (5 '-TACNVGGGTATCTAATCC-3'), and then submits the PCR product to Shannocao biotechnology Co., Ltd for sequencing based on Illumina HiSeq 2500 platform.

4.3 quality control and statistical analysis of data

And (3) performing quality control on the sequenced off-line data to obtain clean data, analyzing the flora structure according to out, and calculating the different species between the control group and the experimental treatment group by T-test. As shown in fig. 4, at day 14, the abundance of streptococci in the experimental treatment group was significantly higher than that in the control group (p ═ 0.003< 0.01). On day 28, the abundance of streptococci in the experimental treatment group was significantly higher than that in the control group (p-0.002 < 0.01). It can be seen that the streptococcus has reached a stable enrichment state in the intestinal tract of mice, and it is noted that the experimental group also has a higher Leuconostoc (Leuconostoc) and Lactococcus (Lactococcus) than the control group (p < 0.01).

Example 5

Study on intestinal mucosa of mice

5.1 extraction of mouse Colon tissue RNA and quantitative real-time PCR amplification of muc2 Gene

Based on the chronic toxicity test in mice in example 3, colon tissue from mice was obtained on day 30, and total RNA was extracted from the colon tissue from mice using an RNA extraction kit according to the manufacturer's instructions. Then using RT Easy^TMII kit reverse transcription of total RNA into cDNA, detection by SYBRGreen Master Mix kit real-time quantitative PCR (qPCR)mRNA expression of gene muc 2. Specific primers (muc2) used were: an upstream primer: 5 '-ATGC-CCACCTCCTCAAAGAC-3', downstream primer: 5'-GTAGTTTCCGTTGGAACAGTGAA-3', GAPDH is used as internal reference for expression of related genes 2^-ΔΔCtAccording to the method, the GAPDH upstream primer is 5'-TGTGTCCGTCGTGGATCTGA-3', and the downstream primer is 5 '-CCTGCTTCACCACCTTCTT-GA-3'. The experimental data are expressed as mean values plus or minus standard error of mean, data statistics are analyzed by one-way ANOVA and Tukey's multiple tests, and the data difference is considered to be significant when the p value is less than 0.05.

5.2 pathological observation of mouse AB-PAS

Taking the colon of the mouse under the aseptic condition, and taking the colon tissue of the mouse by 1cm³The fixed tissue specimens were fixed in 4% paraformaldehyde (CAS: 30525-89-4, Kyoto Chemicals, Inc.) for 48h and then sectioned with reference to the HE staining method. Dyeing by using AB-PAS, slicing and dewaxing to water, dip-dyeing or drop-dyeing by using Alisin blue dye liquor for 5-10min, slightly washing by using water, oxidizing by using 0.5% periodic acid water solution for 5-10min, washing by using running water for several minutes, changing and washing by using distilled water twice, dip-dyeing in a dark place by using a Schiff reagent for 30min, washing by using running water for 5-10min, and observing the dye slice by using an optical microscope.

5.3 analysis of results

To evaluate whether S7, Streptococcus Paris, stimulates the expression of mouse colon muc2, the present study first examined the mRNA expression level of mouse colon muc2 gene by qPCR, and showed that 14 days after inoculation of the strain, the colon muc2 mRNA expression of the treated mice was increased by about 1.6. + -. 0.5 times (p < 0.05); 28 days after inoculation with the strain, colon muc2 mRNA expression was increased by about 1.9. + -. 0.3 fold (p <0.01) in the treated mice compared to the control group (FIG. 5). In addition, AB-PAS staining showed (as in FIGS. 6 and 7), a significant increase in colonic mucosal thickness (p 0.02 ≦ 0.05) in the treated mice at 28 d. These results indicate that streptococcus paris S7 may regulate colon muc2 expression, promoting mucus secretion.

Example 6

Construction of DSS enteritis disease model

The mice used in the experiment were healthy C57BL/6 female mice purchased from Sichuan Daoshuo Biotechnology Co., Ltd, the age of the mice was 7 weeks, the body weight was 18. + -.1 g, and all the procedures were performed according to the animal welfare regulations. According to the mature DSS animal prevention experimental model (fig. 8), the experimental mice were randomly divided into three groups of 5 mice, the experiment was performed for a total of 30 days, the prevention period was defined from day 1 to day 21, the modeling period was defined from day 22 to day 28, the mice were sacrificed on day 30, colon tissues were dissected and spleens were taken and weighed.

During the experiment, mice in each group were treated differently (table 10), (1) control group (control): throughout the experimental period, mice were fed with feed and normal drinking water while gavage with 200 μ L of sterile PBS daily. (2) DSS group (DSS): in the prevention period, the feed and the normal drinking water are supplied to the mice, in the molding period, the feed and the drinking water are changed into 2.5 percent DSS aqueous solution, and 200 mu L of sterile PBS is applied to the stomach every day. (3) Treatment group (treat): in the prevention period, the feed and the normal drinking water are supplied to the mice, the treatment group normally supplies the feed in the molding period, the drinking water is 2.5 percent DSS water solution, and the stomach is irrigated by 10 percent every day in the whole experiment period¹⁰CFU/mL of concentrated bacterial solution of Streptococcus Paris S7 (200. mu.L). During the experiment, the mice were observed daily for changes in signs while relevant data were recorded.

TABLE 10 Experimental design content

Experimental process and result analysis:

6.1 DSS animal models weight Change

The body weights of the mice in each group were maintained in a substantially slow growth state during the prevention period, and there was no significant difference between the body weights of the mice in each group. After entering the molding period, the control group of mice continuously keeps the original slow growth speed and the state is relatively stable; from the trend of weight change, the weight loss of the mice in the DSS group is obvious; the weight of mice in the treatment group also decreased, but the decrease curve was slightly slower than in the DSS group. The weights of the groups at the last day of the molding session were all separated by large differences from each other. As can be seen from fig. 9, there was a very significant difference in body weight between the treated and DSS group mice compared to the control group at day 7 of the molding period (p < 0.001); meanwhile, there was also a significant difference in the body weight change of mice in the treatment group and DSS group (p < 0.05).

6.2 disease Activity index score

Disease Activity Index (DAI) includes body weight change, hematochezia status and stool characteristics, and specific scoring criteria are shown in table 11. During the modeling period, the body weight of the mouse is measured every day, and the hematochezia condition and the stool character of the mouse are detected. Scoring was performed according to table 11, DAI ═ weight change score + hematochezia score + stool trait score/3. Fecal occult blood is measured by a fecal occult blood test by a benzidine method, and if reddish brown or bright red blood can be seen by naked eyes in the feces, the feces are naked-eye bloody stool. The stool characters are divided into normal, loose and loose stool, the normal stool of the mouse is shaped and granular, if the stool is increased in viscosity and easy to scatter, but the stool is loose if the stool is not adhered to the anus, and if the stool is not shaped or is in a thin water shape, the stool is adhered to the anus and is in a thin stool.

TABLE 11 DAI scoring criteria

Note: percent change in body weight, "(X body weight-initial body weight)/initial body weight" × 100%

The DSS-and treatment-group mice were induced to develop experimental colitis models by 2.5% DSS, except for the control group of mice. In the control group, all mice had normal diet and drinking, and normal stool color and shape; in the DSS group, no death of the mice occurred during the molding period, the mice had a severe weight loss from day 3, and had hematochezia symptoms beginning on day 4 with loose stools; in the treatment group, mice did not die throughout the experiment, body weight declined from day 4 and hematochezia appeared at day 5, as well as loose stools and unformed. As can also be seen from the DAI index (fig. 10), the DAI index was 0 throughout the control group. The DAI indices of the DSS group (6.89 + -0.89) and the treatment group (6.53 + -0.45) were separated by a large difference (p <0.001) from the control group at the last day of the molding period. No significant difference in DAI index occurred between DSS and treatment groups (p > 0.05).

6.3 animal model Colon Length and spleen weight determination

Animal models colon length (cm) and spleen weight (g) were measured after dissection in each experimental group of mice.

On day 30 of the end of the experiment, the colon of the mouse was dissected out and the length of the colon (from cecum to anus) was recorded. As shown in FIG. 11, the colon length of the control mice was 8.34. + -. 0.11cm on average; the colon length of the mice in the DSS group is averagely 7.28 +/-0.15 cm; the colon length of the treated mice is 7.54 +/-0.11 cm on average. The differences in colon length between the DSS group and the mice in the treated group and the control group were very significant (p <0.001) by statistical t-test (anova) analysis. Meanwhile, the DSS group differed significantly from the treatment group (p < 0.05).

After each group of mice was dissected, spleen weights of each group of mice were measured. The spleen weight of the control group mice is 0.066 +/-0.011 g, the spleen weight of the DSS group mice is 0.096 +/-0.011 g, and the spleen weight of the treatment group mice is 0.088 +/-0.008 g. Through statistical analysis, the spleen weight of the mice in the DSS group is remarkably different from that of the control group (p <0.01), and the spleen weight of the mice in the treatment group is also remarkably different from that of the control group (p < 0.05); however, there was no significant difference between the DSS group and the treatment group (fig. 12).

6.4 determination of Colon tissue Myeloperoxidase (MPO)

The colon tissue was taken out, washed clean with PBS, ground, and the supernatant was examined according to the procedure of myeloperoxidase examination kit (Bio-engineering, Inc.). Adding 3mL of distilled water, 0.2mL of sample and 0.2mL of reagent IV into a tube, mixing uniformly, performing water bath at 37 ℃ for 30min, adding 0.05mL of reagent IV, mixing uniformly, performing water bath at 60 ℃ for 10min, taking out, and then performing ultrasonic wave treatment at 460nm (OD)₄₆₀) The absorbance was measured. The calculation formula is as follows: MPO enzyme activity (U/g tissue wet weight) ═ (measured OD value-control OD value)/11.3 × sample size (g). Definition of enzyme activity unit: per gram of tissue wet sheet in the reaction system at 37 DEG C₂O₂Decomposed 1 mu mol to 1 enzyme activity unit (U).

MPO is a mark of activation of neutrophil functions and represents the infiltration degree of inflammatory cells, and the higher the MPO enzyme activity is, the deeper the infiltration degree of inflammatory cells is. As can be seen from fig. 13: after the induction of DSS, MPO in colon tissues of mice in the DSS group (9.03 +/-1.56U/g) and the treated group (6.89 +/-1.19U/g) is greatly increased, and the difference with the control group (3.96 +/-0.86U/g) is very obvious (p is less than 0.001). By feeding the streptococcus Paris S7, compared with the DSS group, the MPO enzyme activity caused by DSS induction is reduced in the treatment group (p is less than 0.05).

6.5 colonic IL-6, IL-1 beta and TNF alpha fluorescence quantification

6.5.1 colonic tissue RNA extraction and reverse transcription

Adding 450 mu.L of Buffer Rlysine-AG into a centrifugal tube of 1.5mL of RNase-free for later use; grinding colon tissue of mice of 20-50mg control group, DSS group and treatment group into powder with liquid nitrogen, adding into the above 1.5mL centrifuge tube, immediately shaking and mixing, shaking for 2min, and standing at room temperature for 3 min; centrifuging at 12,000 Xg and 4 deg.C for 3min, and transferring the supernatant into a 1.5mL RNase-free centrifuge tube; adding 1/2 volume of absolute ethyl alcohol, and fully and uniformly mixing; putting the adsorption column into a collection tube, transferring all the solution into the adsorption column by using a liquid transfer device, standing for 1min, centrifuging for 1min at room temperature of 12,000 Xg, and pouring off waste liquid in the collection tube; placing the adsorption column back into the collection tube, adding 500 μ L GT Solution, standing for 1min, centrifuging at room temperature of 10,000 × g for 1min, and pouring off the waste liquid in the collection tube; putting the adsorption column back into the collecting pipe, adding 500. mu.L of NT Solution, standing for 2min, centrifuging at room temperature of 10,000 Xg for 1min, and pouring out waste liquid in the collecting pipe; placing the adsorption column back into the collection tube, and centrifuging at room temperature of 12,000 Xg for 2 min; placing the adsorption column into a 1.5mL centrifuge tube of RNase-free, and adding 30-50 μ L DEPC-treated ddH into the center of the adsorption membrane₂O, standing for 2min, centrifuging at room temperature for 2min at 12,000 Xg, and storing the obtained RNA solution at-70 ℃ or using the RNA solution for subsequent tests. Using RT Easy^TMII kit (WUDUF Biotech Co., Ltd.) Total RNA was reverse transcribed into cDNA, step kit instructions. In the Real-time quantitative PCR reaction, the reverse transcription product needs to be diluted to make the CT value of the internal reference gene between 16 and 18, and 1 mu L of diluted cDNA is taken for reaction.

6.5.2 fluorescent quantitative PCR (Real-time PCR)

The primers used were as follows: TNF α upstream primer: 5'-CCAAAGGGATGAGAAGTTCC-3', downstream primer: 5'-CTCCACTTGGTGGTTTGCTA-3', respectively;

IL-1 β upstream primer: 5 '-TTCAGGCAGGCA-GTATCA-3', downstream primer: 5'-GTCACAACCAGCAGGTTA-3', respectively;

IL-6 upstream primer: 5 '-C-CCGGAGGAGACTTCAG-3', downstream primer: 5'-CAGATTGCCATTGCACAAC-3', respectively;

GAPDH upstream primer: 5'-TGTGTCCGTCGTGGATCTGA-3', downstream primer: 5 '-CCTGCTT-CACCACCTTCTTGA-3'.

The reaction system is as follows: 0.5. mu.L of forward primer (10. mu.M), 0.5. mu.L of reverse primer (10. mu.M), 10. mu.L of SYBR Green Master Mix, and 1. mu.L of template cDNA using ddH₂O make up to 20. mu.L. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, and annealing/extension at 60 ℃ for 30s, the reaction amounting to 40 cycles. By comparing Ct (2)^-△△Ct) Relative mRNA expression was calculated using GAPDH as a reference gene.

6.5.3 analysis of results

The expression of colon-associated inflammatory factor mRNA was examined by qPCR for each group of mice (fig. 14). The relative expression quantity of the IL-6mRNA in the colon of the mice in the DSS group is 3 +/-0.99 times of the IL-6 gene; the relative expression amount of IL-6mRNA in colon of mice in the treated group is 3 +/-1.02 times. Among them, the relative expression amount of the colon IL-6mRNA of the mice in the DSS group is not obviously different from that of the mice in the treatment group. As for IL-1 beta gene, the relative expression quantity of IL-1 beta mRNA of the colon of the mice in the DSS group is 7 +/-1.79 times; the relative expression amount of IL-1. beta. mRNA in the colon of the treated mice was 5. + -. 0.94-fold. Among them, the relative expression of colon IL-1 beta mRNA of mice in the DSS group is remarkably different from that of the mice in the treatment group (p < 0.05). For TNF alpha, the relative expression quantity of colon TNF alpha mRNA of DSS group mice is 7 +/-0.69 times; the relative expression amount of colon TNF alpha mRNA of the treated mice is 5 +/-1.45 times. Among them, the relative expression amount of colon TNF α mRNA of mice in the DSS group was significantly different from that in the treatment group (p < 0.01).

6.6 pathological Observation of HE staining

Taking control under sterile conditionsColon 1-2cm in group, DSS group, treatment group³After fixation in 10% neutral formalin for 48h, the tissue blocks were trimmed to a thickness of 5 mm; area is less than 0.7cm²Then washing with water; ethanol dehydration in the upper row (low to high) 50% (2h) → 70% (overnight) → 80% (2h) → 90% (2h) → 95% (2h) → 100% (2 times, 1 h/times); then, the mixture is transparent by dimethylbenzene (alcohol is replaced) for 20 min/time; waxing the tissue block, wherein the melting point is 56-58 ℃, the waxing time is 1.5h, the waxing time is divided into three times, and the operation is carried out in a constant temperature box for 0.5 h/time; embedding the tissue in paraffin and trimming the paraffin blocks, using 2cm blocks of wood³Stick to the wax block (up without wax); slicing: using a microtome, a microtome knife → sharpen (mirror, oilstone, knife edge back) to cut the tissue 5-10 μm thin; and spreading the slices in water bath at 40 ℃; sheet sticking: mixing glue and protein glycerol according to the ratio of 1: 1, preparing; slicing for 24h and naturally drying; dewaxing the xylene for 2 times and 3 min/time; alcohol (downstream) removal of xylene: 100% (2min) → 100% (2min) → 90% (2min) → 80% (2min) → 70% (2min) → 50% (2min) → water (2 min); staining C nucleus with hematoxylin for 30 min; separating with 0.5% hydrochloric acid water solution or 0.1% hydrochloric acid alcohol (10%) solution for 50 s; flushing with running water for 1h or rapid bluing (one beaker of water +10 drops of ammonia for 1min, followed by washing) eosin stain C: 50% alcohol (2min) → 70% (2min) → 80% (2min) → eosin (1-3 min) → 90% (2min) → 100% (2min) → 100% (2 min); the xylene is transparent again for 2 times and 3 min/time; gum seal (solvent: xylene). The sections were observed under an optical microscope imaging system. Histological scoring was performed using the Fedorak histological integration standard (table 12).

TABLE 12 histological lesion scoring criteria

Note that: (a) number of ulcer formations: none-0, 1 ulcer-1, 2 ulcer-2, 3 ulcer-3, > 3 ulcer-4; (b) epithelial cell changes: normal-0, goblet cell deletion-1, large area deletion of goblet cells-2, crypt deletion-3, large area deletion of crypts or polypoid regeneration-4; (c) inflammatory infiltration: no-0, infiltration-1 around crypts, infiltration-2 of mucosal muscularis, general infiltration of mucosal muscularis, thickening of mucosa-3, and infiltration-4 of submucosa; (d) lymph node formation: none-0, 1 lymph node-1, 2 lymph nodes-2, 3 lymph nodes-3, > 3 ulcers-4.

The experimental results are as follows: the colon epithelial cells of the control group mice are complete and have no obvious lesion; the colon epithelial cells of the mice in the DSS group are seriously damaged, crypt-shaped structures and goblet cells cannot be observed, and a large amount of inflammatory cell infiltration can be clearly observed; the colonic epithelial cells in the treated group were slightly necrotic, crypt structures were also not visible, and in addition, there was only a small number of goblet cells and a mild inflammatory cell infiltrate (fig. 15). From the histological score (fig. 16), the colon histological score of DSS group mice was significantly increased (p <0.001) compared to the control group, and the DSS-induced histological score was decreased by the treatment group (p < 0.05).

In conclusion, the biological characteristics of the streptococcus paris in the panda intestinal tract are researched, and the probiotic function of the streptococcus paris is evaluated. The Latin of the panda-derived Streptococcus Paris is Streptococcus lutetiensis, can tolerate related acid, alkali and bile salts, is safe and reliable, can be used for up-regulating the expression of mouse colon muc2 gene when fed to the Streptococcus Paris, can enhance the intestinal mucosal immune barrier of a host, and can be used for discovering that the Streptococcus Paris S7 has the effect of relieving intestinal inflammation by a DSS-induced enteritis animal model. Therefore, the panda-derived streptococcus paris provided by the invention is expected to be developed into panda colitis medicaments and panda immunoregulation medicaments.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. A panda source streptococcus paris strain is characterized in that: the panda source streptococcus paris is named as: streptococcus lutetiensis S7, deposited in Guangdong province microorganism culture collection center with the collection number GDMCC No: 61917.

2. the strain of panda-derived streptococcus paris according to claim 1, wherein: the strain is gram-positive coccus.

3. The strain of panda-derived streptococcus paris according to claim 1, wherein: the panda-derived streptococcus Paris grows at a temperature below 40 ℃.

4. The strain of panda-derived streptococcus paris according to claim 1, wherein: the panda derived streptococcus Paris does not contain tetA, tetB, tetC, tetD, tetE, tetM, sul1, dfrA1, dfrA5 and intI1 drug resistance genes.

5. The strain of panda-derived streptococcus paris according to claim 1, wherein: the panda source streptococcus paris has the characteristics of acid resistance and bile salt resistance.

6. The strain of panda-derived streptococcus paris according to claim 1, wherein: the panda-derived streptococcus paris can regulate mouse colon muc2 expression and promote mucus secretion.

7. The strain of panda-derived streptococcus paris according to claim 1, wherein: the panda source streptococcus paris can regulate the expression of IL-1 beta mRNA and TNF alpha mRNA of a DSS induced enteritis model.

8. Use of the panda-derived streptococcus paris as claimed in any of claims 1 to 7 in the manufacture of a medicament for the treatment of panda colitis.

9. Use of Streptococcus paris from panda according to any one of claims 1 to 7 in the preparation of a panda immunomodulatory medicament.

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