CN114717129B

CN114717129B - Lactobacillus rhamnosus and application thereof in preventing and relieving constipation symptoms

Info

Publication number: CN114717129B
Application number: CN202110966706.3A
Authority: CN
Inventors: 段治; 崔洪昌; 郭超群; 张景燕; 吴松洁; 李凯玲; 步心萍
Original assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Current assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2023-11-21
Anticipated expiration: 2041-08-23
Also published as: CN114717129A

Abstract

The invention provides lactobacillus rhamnosus, the preservation number of which is CCTCC NO: m2021904. The lactobacillus rhamnosus VHProbi M15 strain provided by the invention has strong tolerance to simulated artificial gastrointestinal fluid, and can successfully pass through the gastrointestinal tract to perform a probiotic function by colonic field planting. The strain is sensitive to common antibiotics such as ampicillin, tetracycline and the like, does not generate hemolysin, does not dissolve blood cells, and has good biological safety. Meanwhile, lactobacillus rhamnosus VHProbi M15 has a certain antioxidant function activity. The strain can also effectively degrade cholesterol and has the probiotic property of reducing serum cholesterol. The lactobacillus rhamnosus disclosed by the invention has no toxic or harmful effect on organisms, is expected to be used for developing foods, health-care products or medicines with the effect of preventing or relieving constipation, and has a wide application prospect.

Description

Lactobacillus rhamnosus and application thereof in preventing and relieving constipation symptoms

Technical Field

The invention belongs to the technical field of screening and application of probiotics, and particularly relates to lactobacillus rhamnosus and application thereof in preventing and relieving constipation symptoms.

Background

The term probiotics originates from greek, and along with the deep understanding of probiotics, keywords for regulating intestinal flora, home bacteria, living microorganisms, living bacteria number and the like are also integrated into the concept of probiotics. At present, probiotics are widely accepted by the definition of the national food and agricultural organization/world health organization, i.e. the active microorganisms that, when administered in sufficient quantity, exert a beneficial effect on the host. Probiotic bacteria and lysates thereof have also been shown in recent years to provide beneficial effects to humans.

The probiotics are helpful for improving intestinal microecology balance and can promote the intestinal canal of a host to maintain normal functions. The mechanism of improving intestinal dysregulation by probiotics may be achieved by means of immunomodulation, against bacteria with abnormal invasion in the intestinal tract, promotion of the production of short chain fatty acids by starch/non-starch polysaccharides, etc. The strain contained in the probiotics can be planted in the intestinal tract, and a biological film with a protective effect is formed on the surface of the intestinal mucosa. Since the niche on the intestinal epithelial cells is limited, it can also compete with bacterial pathogens for colonization sites, thereby achieving pathogen inhibition. Extracellular glycosidase produced by probiotics can degrade complex polysaccharide serving as a potential pathogenic bacteria and an endotoxin binding receptor thereof on intestinal mucosa epithelial cells, so that colonization and invasion of pathogenic bacteria can be prevented, translocation of intestinal bacteria is prevented, and inflammation is reduced by regulating cytokines. The probiotics can activate the related lymph and epithelial immune response of the intestines and increase the immunity of the organism; reducing peroxidized lipid in blood by scavenging free radicals, and delaying cell aging. Organic acid produced by metabolism of probiotics can reduce pH of intestinal environment, and is favorable for absorption of ferrous iron, calcium, phosphorus, cobalt and vitamin D. Thus, probiotics have been currently used in the treatment of a variety of gastrointestinal disorders, such as intestinal dysbacteriosis, antibiotic-associated bowel disease, intestinal infection, lactose intolerance, inflammatory bowel disease, colon cancer, and the like.

The occurrence and development of constipation are related to the imbalance of intestinal flora, and the supplementation of probiotics such as bifidobacteria, lactobacillus and the like for regulating intestinal microecology is one of effective measures for treating constipation. The probiotics applied to constipation at present can be divided into three types of single bacteria, mixed bacteria and synbiotics (single bacteria/mixed bacteria+prebiotics), the common probiotics comprise animal bifidobacteria, bifidobacterium longum, bifidobacterium bifidum, lactobacillus acidophilus, streptococcus, enterococcus and bacillus, and the common prebiotics in the synbiotics comprise fructo-oligosaccharides, xylo-oligosaccharides and the like.

In the study of constipation children, the lactobacillus reuteri DSM 17938 can obviously relieve constipation, the Bu and the like also find that Lactobacillus rhamnosusLcr has a certain treatment effect on constipation, and the side effect is small. In a clinical trial involving 56 children with constipation between 4 and 12 years old, researchers provided the constipation children with a mixture of lactulose and 7 probiotics each day, and found that the frequency of bowel movement was significantly improved in the constipation children after 4 weeks. In another study, colon transit time was significantly shortened in constipation patients after 2 weeks of yogurt containing polydextrose, lactobacillus acidophilus NCFM and bifidobacterium lactis HN 019. Wang et al studied the effect of Bifidobacterium adolescentis on relieving constipation by using a loperamide-induced BALB/c mouse constipation model, and various constipation symptoms of constipation mice after 17d of gastric lavage were significantly improved, and the abundance of Lactobacillus in constipation mice feces of treatment groups of Bifidobacterium adolescentis CCFM 669 and Bifidobacterium adolescentis CCFM 667 increased, and the abundance of Clostridium was reduced, indicating that probiotics improved constipation symptoms by affecting intestinal microbiome.

The pathogenesis of constipation is complex, and the probiotics characteristics of probiotics are different on the strain level, and the effects and the action mechanisms of the probiotics of different strains are different. Therefore, screening to obtain the probiotic bacterial strain with outstanding constipation relieving effect and clear action mechanism still remains the difficulty and hot spot of the current research.

Disclosure of Invention

The invention aims to provide lactobacillus rhamnosus (Lactobacillus rhamnosus) and application thereof in preventing and relieving constipation symptoms. The provided lactobacillus rhamnosus is separated from breast milk, and can effectively regulate gastrointestinal functions, and prevent and relieve constipation symptoms.

The lactobacillus rhamnosus provided by the invention is a lactobacillus rhamnosus (Lactobacillus rhamnosus) VHProbi M15 strain which is preserved in China center for type culture collection of university of Wuhan and Wuhan in China in 2021, 7 months and 19 days, and the preservation number is CCTCC NO: m2021904.

The Riboprinter fingerprint of the lactobacillus rhamnosus VHProbi M15 is shown in figure 1, the RAPD fingerprint is shown in figure 2, and the rep-PCR fingerprint is shown in figure 3.

The 16s rDNA sequence of the lactobacillus rhamnosus VHProbi M15 provided by the invention is shown as SEQ ID NO. 1.

The invention also provides application of lactobacillus rhamnosus VHProbi M15 in preparation of a product for relieving constipation symptoms.

The invention also provides a product for relieving constipation symptoms, which comprises lactobacillus rhamnosus VHProbi M15 strain and/or lactobacillus rhamnosus VHProbi M15 fermentation product.

The product is a health product or a medicine.

The lactobacillus rhamnosus VHProbi M15 provided by the invention has strong tolerance to simulated artificial gastrointestinal fluid, has a survival rate as high as 99.8% after being digested in the artificial gastric fluid for 3 hours, and can successfully perform a probiotic function by being subjected to colonic field planting through the gastrointestinal tract. The strain is sensitive to common antibiotics such as ampicillin, tetracycline and the like, does not generate hemolysin, does not dissolve blood cells, and has good biological safety. Meanwhile, lactobacillus rhamnosus VHProbi M15 has a certain antioxidant function activity, the clearance rate of DPPH free radical reaches 46.19%, and the anti-lipid peroxidation inhibition rate of supernatant fluid reaches 43.37%. The strain can also effectively degrade cholesterol and has the probiotic property of reducing serum cholesterol.

Lactobacillus rhamnosus VHProbi M15 can significantly increase the water content of the mouse feces and the transport capacity of the constipation mouse small intestine, thereby being beneficial to promoting defecation and improving constipation symptoms.

Lactobacillus rhamnosus M15 can also significantly increase the content of acetic acid and total organic acids in the mouse faeces, acetic acid is a main product of colon fermentation, increasing the concentration of intestinal acetic acid can lead to an increase in intestinal osmotic pressure, and the water content of the intestinal contents is increased, so that the intestinal wall is stimulated, the intestinal peristalsis is increased, and constipation is relieved.

Lactobacillus rhamnosus VHProbi M15 can effectively regulate secretion of gastrointestinal regulatory peptides, and can reduce secretion of inhibitory transmitters endothelin, somatostatin-1 and vasoactive intestinal peptide by promoting secretion of excitatory transmitters motilin, gastrin and substance P, so as to effectively regulate gastrointestinal movement and gastric acid secretion and relieve constipation symptoms.

Lactobacillus rhamnosus VHProbi M15 can prevent and alleviate intestinal wall barrier damage caused by constipation, alleviate inflammatory reaction, and make intestinal mucosa barrier tend to be complete.

Lactobacillus rhamnosus VHProbi M15 can promote the abundance and uniformity of fecal flora in constipation mice, so that the abundance of dominant species at the portal and genus levels tends to the characteristics of the flora in mice in the blank group, the difference of the flora composition is reduced, the flora structure composition is similar to that of the blank group, and the abundance tends to the normal mouse level.

The lactobacillus rhamnosus VHProbi M15 has no toxic or harmful effect on organisms, is expected to be used for developing health-care products or medicines with the effect of relieving constipation, and has wide application prospect.

Drawings

FIG. 1 is a gram of M15 strain;

FIG. 2 is a chart of the Riboprinter fingerprint of the M15 strain;

FIG. 3 is a RAPD fingerprint of strain M15;

FIG. 4 is a rep-PCR fingerprint of M15 strain;

FIG. 5 is a graph showing the results of the fecal moisture content of each group of mice;

FIG. 6 is a graph of the intestinal thrust anatomic of each group of mice;

FIG. 7 is a graph showing results of small intestine thrust rate of mice in each group;

FIG. 8 is a graph showing the results of ileal pathological sections of mice in each group;

FIG. 9 is a graph showing results of gastrointestinal modulator peptides from each group of mice;

FIG. 10 is a graph showing the differential test between the various groups of alpha-diversity index sets of fecal samples from mice of each group;

FIG. 11 is a graph of a colony composition analysis cake at the portal level for each group of mouse fecal samples;

FIG. 12 is a bar graph of colony composition analysis at the genus level for each group of mouse fecal samples;

fig. 13 is a graph showing the results of PCoA analysis of fecal samples from mice in each group.

Detailed Description

The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The present invention will be described in detail with reference to specific examples.

EXAMPLE 1 isolation screening of Lactobacillus rhamnosus VHProbi M15

1. Primary screen

According to 2019 edition of human genetic resource coulomb regulation, after signing project commitment and informed consent with a sample provider, taking 1mL of fresh breast milk of a parturient in lactation period, which does not eat probiotic preparations within half a year, diluting the fresh breast milk with sterile normal saline, putting the diluted breast milk into a sterile sample bag, and beating and uniformly mixing the diluted breast milk with a homogenizer; and (3) taking 100 mu L of mixed solution for gradient dilution, coating the mixed solution on an MRS agar medium, and performing anaerobic culture at 37 ℃ for 48 hours, and performing microscopic examination on a single colony after the plate grows. According to the microscopic examination result, the applicant selects 20 potential lactobacillus strains, which are named as M01, M02, … … M19 and M20 respectively.

The preparation method of the MRS (Man Rogosa Sharpe) agar medium comprises the following steps:

1000mL of purified water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of Tween 80, 2.0g of citric acid diamine, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate, 15g of agar, pH adjustment of 6.2-6.5 and high-pressure sterilization at 121 ℃ for 15min.

2. Double screen

Preparing 1L of MRS liquid culture medium, sterilizing at 121deg.C for 15min, cooling, adding 3.2g of pig mucosa pepsin, shaking for dissolving, and placing in a 37 deg.C water bath shaking table for 1 hr to obtain acid-resistant culture medium.

The 20 strains of lactobacillus M01, M02, … … M19 and M20 obtained by screening are respectively inoculated into the acid-resistant culture medium according to the inoculum size of 6 percent, and are subjected to anaerobic static culture for 48 hours at 37 ℃, and the fermentation liquor is taken for bacterial count.

The results show that in 20 lactobacillus fermentation broths obtained by preliminary screening, the logarithmic values of the viable bacteria amounts are 7.23, 6.94, 6.76, 7.56, 6.33, 5.39, 8.18, 6.35, 4.17, 5.28, 7.32, 7.35, 5.35, 8.18, 8.43, 4.99, 6.36, 7.41, 5.83 and 5.39Log CFU/mL respectively, wherein the M15 strain has the largest viable bacteria amount after being subjected to the rescreening by the acid-resistant culture medium, and the logarithmic value is as high as 8.43Log CFU/mL; the acid resistance of strain M15 was shown to be highest.

EXAMPLE 2 Strain identification bacteria and physicochemical Properties of M15 Strain

1. Colony morphology identification

After the M15 strain was inoculated on MRS agar medium and anaerobic cultured at 37℃for 24 hours, the colony morphology of M15 was observed, and the results are shown in FIG. 1. As can be seen, the single colony of M15 strain is beige, the diameter of the colony is about 0.5-1.5mm, the surface is moist, the colony is short rod-shaped under a microscope after gram staining, and some colonies are chain-shaped.

2. Identification of physiological and biochemical characteristics

Under the aseptic condition, a proper amount of fresh M15 bacterial liquid is taken, centrifuged for 5min at 5000rpm/min, washed for 2 times by PBS buffer, and then the bacterial body is diluted by 50 times after being resuspended by the same volume of PBS buffer, and the bacterial body is taken as an inoculation liquid.

1) Salinity tolerance test

Under aseptic conditions, 190. Mu.L of MRS liquid culture medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, respectively, 3 replicates of each salt concentration, and then 10. Mu.L of inoculation liquid was added, and wells without inoculation were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C, and observing whether the culture medium becomes turbid. The results showed that the M15 strain had a maximum tolerated salt concentration of 7%.

2) Carbon source metabolism test

The carbon source metabolism test was performed on the M15 strain using the API50CHL kit, and the experimental method was referred to the API50CHL kit instructions. The detection result shows that the M15 strain is lactobacillus rhamnosus, the% ID=99.6, the T value=0.78, and the comment is very good identification.

3) Glucose acidogenesis and gas production test

The medium formulation used in this example is as follows:

peptone 0.5g; 0.3g of yeast extract; tween 80.1 ml; salt solution a 0.5mL; salt solution B0.5 mL; 0.5g of sodium acetate; glucose 2.5g; 0.05mL of 2% bromocresol green (w/v); distilled water 100mL; the pH is 6.8-7.0.

The prepared culture medium was dispensed into large tubes containing inverted small tubes, 3 mL/tube, and autoclaved at 121℃for 15min.

Salt solution A: KH (KH) ₂ PO ₄ 10g、K ₂ HPO ₄ 1g, dissolved in distilled water, and the volume was set to 100mL.

Salt solution B: mgSO (MgSO) ₄ ·7H ₂ O 11.5g、MnSO ₄ ·2H ₂ O 2.4g、FeSO ₄ ·7H ₂ O0.68 g, dissolved in distilled water, was fixed to a volume of 100mL.

Under aseptic conditions, the inoculum was inoculated with 10% of the inoculum size, the medium without inoculation was used as a control, and then the top was capped with 2mL of sterile liquid paraffin and incubated at 37 ℃. The culture was continued for 6 days, and the presence or absence of change in the color of the medium was observed every day.

The results show that: after 6d of culture at 37 ℃, the culture medium turns yellow from green, and no gas exists in the small inverted tube, which indicates that the M15 strain ferments glucose to produce acid and does not produce gas.

4) Temperature tolerance test

Under aseptic conditions, the inoculation liquid is inoculated into 10mL of MRS liquid culture medium according to the inoculation amount of 10%, 10mL of MRS liquid culture medium without bacteria is used as a control, and the culture liquid is placed in a 15 ℃ constant temperature shaking incubator for 2 days, and is cultured for 7 days at 45 ℃ constant temperature shaking incubator, and whether the culture liquid becomes turbid is observed.

The results show that: culturing at 15 ℃ for 2 days, and then making the culture medium become turbid; after incubation at 45℃for 7 days, the medium remained clear. Thus, it was demonstrated that the M15 strain grew at 15℃and did not grow normally at 45 ℃.

3. Molecular biological identification

3.1 16s rDNA Gene sequence analysis

3.1.1 genomic DNA extraction

Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).

3.1.2, 16s rDNA Gene amplification

1) Primer sequence:

27F：AGAGTTTGATCCTGGCTCA；

1492R：GGTTACCTTGTTACGACTT。

2) Reaction system (50. Mu.L)

Table 1:16s rDNA PCR amplification System Table

By sequencing the PCR products, the 16s rDNA sequence SEQ ID NO. 1 of the M15 strain was obtained and the sequences were aligned in the NCBI database. The results showed that SEQ ID NO. 1 has the highest similarity to Lactobacillus rhamnosus (Lactobacillus rhamnosus), and therefore, M15 strain was initially identified as Lactobacillus rhamnosus (Lactobacillus rhamnosus).

3.2, riboprinter finger print

The purified single colony is dipped from an agar culture medium plate by a fungus taking rod, the single colony is placed into a sample tube with buffer solution, the single colony is stirred by a hand-held stirrer to be suspended in the buffer solution, then a sample frame is placed into a heater for inactivation and then placed into a Riboprinter system, and a bacterial identification result is obtained after DNA preparation, film transfer, imaging detection and data processing are carried out on the sample. The identification result shows that the M15 strain is lactobacillus rhamnosus, and the result of the Riboprinter fingerprint is shown in figure 2.

3.3, RAPD and rep-PCR fingerprint identification

3.3.1 RAPD finger print identification

1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');

2) RAPD reaction system

Table 2: RAPD reaction System Table

3) Electrophoresis

Preparing 1.5% agarose gel plates, using DL2000 DNA markers as result contrast, stabilizing the voltage for 100V electrophoresis for 80min, and finally detecting an electrophoresis pattern by using a gel imaging system; RAPD finger-prints of M15 strain are shown in FIG. 3.

3.4, rep-PCR finger print

1) rep-PCR primer

CTACGGCAAGGCGACGCTGACG。

2) reaction system of rep-PCR

Table 3: table of the reaction System of rep-PCR

3) Electrophoresis

DL2000 DNA Marker served as a result control. Detecting the amplification result by 100V voltage and 80min electrophoresis time. The rep-PCR fingerprint of M15 strain is shown in FIG. 4.

To sum up, the colony morphology and physiological and biochemical characteristic results of the M15 strain are uploaded to the http:// www.tgw1916.net/bacteria_log_desktop.htmL website, and the results published by De Clerck E, et al systems and applied microbiology,2004,27 (1) 50 are combined for comparison. From the comprehensive molecular biology identification results, it can be concluded that the M15 strain is a novel lactobacillus rhamnosus, which is named as lactobacillus rhamnosus VHProbi M15 and is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China at 7 months and 19 days in 2021: m2021904.

EXAMPLE 3 tolerance test of Lactobacillus rhamnosus VHProbi M15 against Artificial gastric juice and artificial intestinal juice

1. Preparation of artificial gastric juice

5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000mL of distilled water is added, pH is adjusted to 3.0 by dilute hydrochloric acid, and then sterilization is carried out for 20min at 115 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly shaken and dissolved, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1 hour so as to simulate the temperature of a human body.

2. Preparation of artificial intestinal juice

Respectively weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH ₂ PO ₄ 6.8g and 3.0g of ox gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then adding 1g of pancreatin before use, shaking to dissolve, and placing in a water bath shaker at 37 ℃ for warm water bath for 1h to simulate the temperature of human body.

3. Test method

1) Culturing Lactobacillus rhamnosus VHProbi M15 for 24 hr, collecting fresh bacterial liquid, centrifuging at 20000×g and 4deg.C for 10min, discarding supernatant, cleaning thallus once, and suspending with sterilized normal saline;

2) 1mL of the bacterial suspension is added into 50mL of artificial gastric juice, and the pH value is regulated to 3.0 by hydrochloric acid;

3) After digestion culture for 3 hours at 37 ℃, sampling and detecting the viable bacteria;

4) Taking 25ml of artificial gastric juice, digesting for 3 hours, centrifuging for 10 minutes at the temperature of 20000 Xg and 4 ℃, discarding the supernatant, cleaning the thalli once, and then suspending with sterilized normal saline; the collected bacterial suspension is added into 50ml of artificial intestinal juice again, and after digestion culture for 3 hours at 37 ℃, the bacterial suspension is sampled and detected for viable bacteria.

The viable bacteria counting method refers to national standard GB 4789.35-2016-food microorganism test lactobacillus test. The live bacterial amounts of lactobacillus rhamnosus VHProbi M15 strain after digestion with artificial gastric juice and artificial intestinal juice are shown in table 4.

Table 4: live bacteria scale after digestion of artificial gastrointestinal fluids (Log CFU/mL)

From Table 4, the lactobacillus rhamnosus VHProbi M15 screened by the method has strong tolerance to artificial gastric juice and artificial intestinal juice. After digestion for 3 hours in artificial gastric juice, the survival rate of the strain is up to 99.8%; after digestion for 3 hours in artificial intestinal juice, the survival rate of the strain can reach 31.4 percent. Therefore, the lactobacillus rhamnosus VHProbi M15 strain provided by the invention can reach the intestinal tract in a living way, and can realize effective field planting in the intestinal tract of a human body, thereby playing a probiotic role.

EXAMPLE 4 test of haemolytic and antibiotic resistance of Lactobacillus rhamnosus VHProbi M15

1. Hemolysis test

(1) Preparing an inoculation liquid: the frozen lactobacillus rhamnosus VHProbi M15 strain is streaked and inoculated in an MRS agar culture medium, cultured for 24-48 hours at the temperature of 37 ℃, subcultured for 1 time by the MRS liquid culture medium, and inoculated in a fresh MRS liquid culture medium at the temperature of 37 ℃ for 24-48 hours with the inoculum size of 5 percent to obtain fresh bacterial liquid serving as the inoculum.

(2) Preparation of blood cell culture medium: weighing the various components of TBS basic culture medium, dissolving, autoclaving at 121deg.C for 15min, cooling to 50deg.C, adding 5% sterilized defibrinated sheep blood, mixing, and plating.

(3) And (3) streaking culture: and streaking the test strain, inoculating the streaked strain to a prepared blood cell plate, culturing the strain in a 37 ℃ incubator, and observing whether the test strain has a hemolysis phenomenon or not in 24-48 hours.

The results showed that lactobacillus rhamnosus VHProbi M15 was unable to grow on the blood cell medium and the blood cell plate was unchanged, indicating that lactobacillus rhamnosus VHProbi M15 did not produce hemolysin and was unable to lyse blood cells.

2. Antibiotic resistance test

(1) Preparing antibiotics: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline and vancomycin are prepared into stock solution of 2048 mug/mL, and the stock solution is preserved at-20 ℃ for standby. When in use, the stock solution is serially diluted by 2 times by using BSM liquid culture medium to form use solution, and the gradient dilution concentration is 1-1024 mu g/mL and total 11 gradients.

(2) Preparing an inoculation liquid: taking a proper amount of fresh bacterial liquid (culturing for 24 hours at 37 ℃), centrifuging for 5 minutes at 5000rpm, washing once with sterile physiological saline, and diluting 50 times after re-suspending bacterial cells with the same volume of physiological saline to obtain an inoculation liquid.

(3) Determination of minimum inhibitory concentration MIC value of antibiotic for Lactobacillus rhamnosus VHProbi M15 by micro broth dilution method

an MRS liquid culture medium without antibiotics is added to the 1 st column of the 96-well plate as a negative control, 190 mu L of MRS liquid culture medium with antibiotics with different concentrations is sequentially added to the 2 nd to 12 th columns, 10 mu L of the inoculation liquid is inoculated respectively, 3 parallel wells are made, and 1 well of the non-added bacteria liquid is used as a blank.

b. 50. Mu.L of paraffin oil was added to cover the water and prevent evaporation.

c. The 96-well plate was incubated at 37℃for 24 hours, then removed, and OD was measured ₆₀₀ Values, MIC values of antibiotics against strains were counted with 24h results, and specific results are shown in table 5.

Table 5: antibiotic MIC value (μg/mL) of Lactobacillus rhamnosus VHProbi M15

From the results shown in Table 5, the lactobacillus rhamnosus VHProbi M15 provided by the invention is sensitive to common antibiotics such as ampicillin and tetracycline, and has good biological safety.

Example 5 hydrophobic cell surface test of lactobacillus rhamnosus VHProbi M15

1. Preparation of bacterial liquid to be tested

The purified lactobacillus rhamnosus VHProbi M15 colony is picked and inoculated in a newly prepared MRS liquid culture medium, and is cultured for 24-48 hours at 37 ℃. Inoculating 1% (V/V) of the strain into MRS liquid culture medium, continuously culturing at 37deg.C for 24-48 hr, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile physiological saline for 2 times, and sterilizing with 0.1M KNO ₃ The bacterial cells were resuspended in 1mL of the solution and used as the bacterial liquid to be tested.

2. Surface hydrophobicity determination

mu.L of the above bacterial suspension was pipetted into 2450. Mu.L of 0.1M KNO ₃ And record OD ₆₀₀ Is A ₀ 1.5mL of the bacterial suspension was mixed with 500. Mu.L of xylene, and the mixture was allowed to stand at room temperature for 10 minutes (a two-phase system was formed). Vortex oscillating the two-phase system for 2min, standing for 20min, and reforming into water phase and organic phase. Carefully aspirate the aqueous phase (not the organic phase), OD ₆₀₀ Absorbance A was measured at ₁ 。

Hydrophobicity% = (a ₀ -A ₁ )/A ₁ X% calculation, measurement of the average of three experiments.

The results show that: the hydrophobicity of the surface of the lactobacillus rhamnosus VHProbi M15 cell provided by the invention is 14.39%, and the standard deviation is 0.11%.

Example 6 Lactobacillus rhamnosus VHProbi M15 antioxidant function assay

1. Determination of DPPH-clearing Capacity of Strain

1) Preparation of PBS bacterial suspension

Single colony with excellent growth state is inoculated into 3mL of MRS liquid culture medium, and is cultured for 24h at 37 ℃, the culture solution is taken as an inoculating solution, and is inoculated into 50mL of MRS liquid culture medium according to the inoculating amount of 2 percent, and the culture solution of the strain is obtained by standing and culturing for 24 h. After 1mL of bacterial liquid is sucked up and bacterial cells are collected, the bacterial cells are washed by 1mL of buffer solution for 2 times, and then 2mL of buffer solution is added to resuspend the bacterial cells for standby.

2) Determination of the ability of the Strain to scavenge DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical

Taking 1mL of PBS bacterial suspension of the strain to be detected, adding 1mL of 0.4mM of the ready-prepared DPPH free radical solution, uniformly mixing, then placing the mixture at room temperature for shading reaction for 30min, and then measuring the absorbance A of the sample at the wavelength of 517nm _{Sample of} 3 replicates were measured. The control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution and with equal volumes of PBS and ethanol mixed solution. The clearance is calculated according to the following formula: clearance% = [1- (a) _{Sample of} -A _{Blank space} )/A _Control ]X 100%. The results are shown in Table 6.

Table 6: DPPH free radical scavenging rate table

From the data in Table 6, the lactobacillus rhamnosus VHProbi M15 provided by the invention can effectively remove DPPH free radicals, and the removal rate reaches 46.19%.

2. Bacterial strain anti-lipid peroxidation experiment

1) Preparation of culture and fermentation supernatant, cell and intracellular extract of lactic acid bacteria:

culturing lactobacillus in MRS liquid culture medium at 37deg.C for 24 hr, transferring for 3 generations, centrifuging at 6000rpm/min at 4deg.C for 10min, and collecting supernatant to obtain fermentation supernatant. The collected cells were centrifuged at 6000r/min for 10min with PBS buffer (pH 7.4), and washed 3 times. The bacterial cells were resuspended in PBS buffer to a bacterial cell concentration of 1.0X10 ⁹ cells/mL to obtain a bacterial suspension.

2) Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.

3) To 0.5mL of PBS solution (pH 7.4) was added 1mL of linoleic acid emulsion, 1mL of LFASO ₄ (1%) and 0.5mL of the sample was added, the mixture was added in a 37℃water bath for 1.5h, and 0.2m of the mixture was addedL TCA (4%), 2mL TBA (0.8%), water bath at 100deg.C for 30min, rapid cooling, centrifuging at 4000rpm/min for 15min, collecting supernatant, and measuring absorbance at 532nm to obtain A; the control group was A with 0.5mL distilled water instead of the sample ₀ 。

Inhibition rate/% = (a ₀ －A)/A ₀ ×100％。

Note that: a is absorbance of a sample group; a is that ₀ Absorbance was used as control. The results are shown in Table 7.

TABLE 7 Table 7 inhibition of lipid peroxidation

From the data in Table 7, it can be seen that the supernatant of Lactobacillus rhamnosus VHProbi M15 provided by the invention has an anti-lipid peroxidation inhibition rate of 43.37%.

Example 7 Lactobacillus rhamnosus VHProbi M15 in vitro cholesterol degradation experiments

1. Preparing a cholesterol micelle solution: 1g of cholesterol was accurately weighed, dissolved in absolute ethanol, and fixed to 100mL, and sterilized by filtration through a 0.22 μm microporous filter under aseptic conditions.

2. 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of Tween 80, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt and 1000mL of distilled water are weighed, the pH is 7.3, sterilization is carried out at 115 ℃ for 30min, and then a cholesterol solution is added to make the final concentration of cholesterol be 0.1%.

Inoculating fresh lactobacillus rhamnosus VHProbi M15 bacterial liquid according to an inoculum size of 0.1%, standing at 37 ℃ for 48 hours, taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10 minutes, centrifuging at 3000 revolutions for 5 minutes, and taking supernatant for measuring cholesterol content. Cholesterol measurement method reference GB/T5009.128-2003 < measurement of cholesterol in food >.

The results show that: the degradation rate of the lactobacillus rhamnosus VHProbi M15 provided by the invention on cholesterol reaches 12.27%.

Example 8 use of Lactobacillus rhamnosus VHProbi M15 for alleviating constipation in mice

1 Experimental animal treatment and model construction

1.1 Experimental consumables

Table 8: experimental consumable information table

1.2 treatment of laboratory animals

SPF-class BALB/c mice, namely male and 24 mice, are randomly divided into 4 groups, and 6 mice in each group are respectively a blank group, a building block group, a positive group and a probiotic group. Wherein, the mice in the blank group do not do any treatment and are filled with normal saline in the whole process; the constipation model is built by the building module, the positive group and the probiotic group mice by adopting sucralfate, and after the building module and the positive group mice are modeled, equal amounts of physiological saline and phenolphthalein (constipation treating drugs) are respectively infused into the stomach, and before and after the modeling of the probiotic group mice, the stomach is infused with probiotic liquid. Mice in each group were free to drink water. The specific treatment method is as follows:

(1) Blank group: after the adaptation is finished, 1mL of physiological saline is infused every day;

(2) Building a module: 1mL of physiological saline is infused into the stomach each time until the test is finished;

(3) Positive group: 1mL of phenolphthalein solution is infused every time to 14 days, 1mL of physiological saline is infused every day from 15 days to the end of the experiment, and the dosage of the phenolphthalein is infused according to the standard of 70mg/Kg according to the weight of the mice;

(4) Probiotic group: after the adaptive feeding is finished, the gastric lavage of probiotics is started, and the gastric lavage is continued until the test is finished, wherein the gastric lavage is performed for 1 multiplied by 10 per day ⁹ CFU/mL bacterial liquid 1mL.

In 15d, except for the blank group, the building module, the positive group and the probiotics group respectively administer sucralfate with the concentration of 50% according to 1 mL/intragastric administration, and the continuous intragastric administration is carried out for 2 days; fasted overnight after the 2 nd day of gastric lavage, free drinking; 50% sucralfate was administered at 0.5 mL/intragastrically on day 3, followed by 5 to 21 days of continuous intragastrical administration.

2 index detection

2.1 fecal moisture content

After 24h of final gastric lavage (day 22), wet feces from mice were collected (8:00 a.m. to 8:00 a.m.), wet feces weight was weighed (only/day), feces were freeze-dried, feces dry weight was recorded, and water content was calculated.

2.2 small intestine Propulsion Rate

After 24 hours of last gastric lavage of sucralfate, each group of mice was respectively given 3% inactivated charcoal by gastric lavage, CO after 1 hour ₂ And (3) stopping asphyxia, rapidly separating and taking the small intestine, drawing the small intestine into a straight line, placing the small intestine on a sterile cushion sheet, measuring the length of an intestine tube (from the pylorus to the ileocecum) as the total length of the small intestine, taking the distance from the front edge of the carbon powder to the pylorus as the propelling distance of the carbon powder in the small intestine, and calculating the propelling rate and the propelling inhibition rate of the carbon powder.

Carbon powder advance rate (%) =carbon powder advance distance in intestine (cm)/small intestine total length (cm) ×100%.

2.3 organic acid content in feces

All groups were tested for acetic acid, propionic acid, butyric acid and total organic acid content by taking the feces on days 7, 14, 21.

2.4 neurotransmitter content in serum

After 24h of last gastric administration of sucralfate, the orbit was bled, centrifuged, serum was taken, and the Elisa method was used to detect the levels of 6 neurotransmitters, motilin (MTL), gastrin (GAS), substance P (SP), endothelin (SS), somatostatin-1 (ET-1) and Vasoactive Intestinal Peptide (VIP) in the serum.

2.5 histopathological examination

After 24h of final gastric lavage of sucralfate, mice were sacrificed, ileal tissues of the mice were harvested, dehydrated, embedded, sectioned, HE stained, and observed for histopathological changes.

2.6 fecal flora detection

Feces were taken on all groups, 14 and 21, frozen at-80℃and DNA was extracted for analysis of the structure of intestinal flora by 16S rRNA high-throughput sequencing.

3 data processing

All experimental data are expressed as mean ± standard deviation, data statistics and mapping are performed by GraphPad Prism 7.0 software, single factor analysis of variance is adopted for comparison among multiple groups of data, t-test is adopted for comparison among two groups of data, and significant difference is judged by P < 0.05.

4 experimental results

4.1 fecal moisture content

The water content of the excrement is related to the hardness of the excrement, and the excrement is dried when the water content is low, so that the excrement is difficult to discharge, and the water content of the excrement is one of common indexes for treating constipation. The comparison of the fecal moisture content changes for each group of mice is shown in fig. 5 and table 9.

Compared with the blank group, the fecal water content of the model building mice was reduced and had significant differences (P < 0.05), indicating successful constipation modeling.

Compared with the modeling group, the water content of the feces of the mice in the probiotics group is obviously increased, and the mice in the probiotics group are recovered to normal level, which is equivalent to that in the blank group and is slightly lower than that in the positive group. Therefore, the lactobacillus rhamnosus VHProbi M15 provided by the invention can effectively soften the mouse feces, and is beneficial to improving constipation.

Table 9: comparison table of fecal moisture content for each group of mice (n=6,)

4.2 small intestine Propulsion Rate

Small intestine transit time is one of the indicators measuring the overall intestinal transit capacity. The applicant adopts a carbon powder propulsion experiment to observe the small intestine propulsion rate. The results are shown in FIGS. 6 and 7.

The modeled group showed a decrease in small intestine turnover rate compared to the blank group, and the difference was significant (P < 0.05). The small intestine turnover rate of the probiotic group was significantly increased compared to the modeled group, comparable to the blank and positive groups. Therefore, the lactobacillus rhamnosus VHProbi M15 provided by the invention can obviously improve the transportation capacity of the small intestine of a constipation mouse, so that the constipation mouse is beneficial to promoting defecation and improving constipation symptoms.

4.3 short chain fatty acid content in feces

Probiotics need to be subjected to colonization and metabolism in intestinal tracts to have beneficial effects on hosts, and metabolic products Short Chain Fatty Acids (SCFAs) including acetic acid, propionic acid, butyric acid and the like are important products of fermentation of intestinal flora of mammals, can participate in important physiological metabolic processes in vivo, such as providing capability for colonic mucosa cells, promoting absorption of water and electrolyte, promoting proliferation of epithelial cells, and affecting gastrointestinal motility and other physiological effects. Therefore, measuring SCFAs content in the intestinal tract has become an important means for evaluating whether a strain can relieve constipation. The effect of different treatments on the intestinal microenvironment of constipation mice was assessed by measuring the concentration of SCFAs in the feces. The specific results are shown in Table 10.

The results show that the acetic acid, propionic acid, butyric acid and total organic acid content in the feces of mice in the blank group, the building block group and the positive group are all stable before modeling (the first 14 days); however, the acetic acid content in the feces of the mice in the probiotics group is obviously increased (P is less than 0.05), 19.8% is increased on the 14 th day compared with the blank group, but the propionic acid, butyric acid and total organic acid contents are not obviously changed.

After modeling (days 15-21)), the acetic acid, propionic acid, butyric acid and total organic acid content in the stool of the model mice was significantly reduced (P < 0.05) compared to the blank group. Compared with a modeling group, the acetic acid content in the feces of the mice in the probiotics group is obviously improved, and the level of the mice is close to that of a blank group and slightly lower than that of a positive group; the total organic acid content is also obviously improved, which is equivalent to that of a blank group and a positive group; but the propionic acid and butyric acid contents were not significantly different from the modeled group. Therefore, the lactobacillus rhamnosus M15 provided by the invention can obviously improve the content of acetic acid and total organic acid in the mouse faeces, wherein the acetic acid is a main product of colon fermentation, and increasing the concentration of acetic acid in the intestinal tract can lead to increasing the osmotic pressure of the intestinal tract, so that the water content in the intestinal content is increased, thereby stimulating the intestinal wall, increasing the intestinal peristalsis and further relieving constipation.

Table 10: comparison table of short chain fatty acid content in feces of mice of each group

The a-f on different row numbers represent significant differences between groups (P < 0.05)

4.4 changes in the content of MTL, gas, ET-1, SS, SP, VIP gastrointestinal Conditioning peptides in serum

Constipation-related gastrointestinal regulatory peptides are capable of exerting important regulatory actions on gastrointestinal motility, motilin (MTL), gastrin (GAS) and Substance P (SP) being excitatory transmitters, endothelin (SS), somatostatin-1 (ET-1) and Vasoactive Intestinal Peptide (VIP) being inhibitory transmitters. Motilin affects water and electrolyte transport, promotes gastric contractions and segmental motion of the small intestine, accelerates intestinal transit time, and increases colonic motion. Gastrin stimulates secretion of gastric acid and pepsin, promotes growth of the mucosal epithelium of the digestive tract, promotes contraction of the smooth muscle of the gastrointestinal tract and relaxation of the pyloric sphincter. Substance P mainly regulates the contraction of the gastrointestinal tract, intestinal peristalsis and gastric acid secretion. Endothelin-1, a multifunctional peptide, plays an important role in cardiovascular, neuroendocrine and gastrointestinal functions. Somatostatin inhibits the release of gastrointestinal hormones. The vasoactive peptide can relax the sphincter of the gastrointestinal tract.

The invention adopts an Elisa method to detect the change of 6 gastrointestinal regulating peptides of motilin, gastrin, somatostatin-1, endothelin, substance P and vasoactive peptide in the serum of mice. The results are shown in FIG. 9.

The results showed that excitatory transmitter motilin, gastrin and substance P were significantly reduced (P < 0.01) and inhibitory transmitter endothelin, somatostatin-1 and vasoactive intestinal peptide were significantly increased (P < 0.01) in the serum of the modular mice compared to the blank group; whereas the excitatory transmitter motilin, gastrin and substance P were significantly elevated (P < 0.01) and the inhibitory transmitter endothelin, somatostatin-1, vasoactive intestinal peptide were significantly reduced (P < 0.01) in the probiotic and positive groups compared to the modeled group. Therefore, the lactobacillus rhamnosus VHProbi M15 provided by the invention can increase small intestine peristalsis and gastric acid secretion by regulating the secretion of gastrointestinal regulatory peptides.

4.5 ileal histopathological observations in mice

The results of the ileal pathology section of each group of mice observed under the light microscope are shown in fig. 8.

(1) Blank group: the three-layer structure of the intestinal wall of the mouse is complete, the mucosal surface is covered with a single ciliated columnar epithelium, the epithelium and the inherent layer below the epithelium form intestinal villi protruding into the intestinal cavity, and the epithelium is sunk into the inherent layer to form intestinal glands;

(2) Building a module: inflammatory cell infiltration occurs in intestinal mucosa tissue of mice, intestinal lesions are necrotic detachment of intestinal villi, and mucosal ulcer occurs;

(3) Probiotic group: the intestinal inflammation degree of the mice is reduced, the intestinal mucosa is basically normal, intestinal villi is edematous, and a small amount of intestinal villi is necrotized;

(4) Positive group: the intestinal mucosa structure of the mice is basically normal, and small amount of inflammatory cell infiltration occurs in the intestinal mucosa.

The results show that the lactobacillus rhamnosus M15 provided by the invention can effectively prevent and relieve intestinal wall barrier damage caused by constipation, reduce inflammatory reaction and enable intestinal mucosa barrier to be complete.

4.6 group differences in fecal flora in mice

To investigate the effect of lactobacillus rhamnosus VHProbi M15 on constipation mice intestinal flora, the collected groups of mouse faeces were subjected to a flora structure analysis, 48 samples yielded a total of 2350178 effective sequence numbers of 16srdna V4 regions, with an average sequence length of 252bp.

4.6.1 analysis of flora diversity

The diversity of the different groups of mouse fecal sample populations was reflected by an alpha diversity analysis, the richness of the different sample populations was reflected by the Sobs index, and the diversity of the sample populations was reflected by the Shannon index. The results are shown in FIG. 10.

(1) Compared with the blank group, the Sobs index and Shannon index of the modeling group are both reduced, and the significant difference exists, which indicates that the abundance and uniformity of species in the feces of the mice of the modeling group are significantly reduced.

(2) Compared to the modeled group, the Sobs index of the positive and probiotic groups increased with significant differences (positive group, P <0.01; probiotic group, P < 0.05), shannon index increased without significant differences. Therefore, the species richness in the feces of the mice in the positive group and the probiotics group is obviously increased, and the flora diversity is improved.

4.6.2 analysis of the composition of the flora

The mouse fecal flora at the phylum level is mainly composed of bacteroides, firmicutes, proteus and verrucomicrobia. The composition of species at the portal level in the faeces of each group of mice is shown in figure 11.

(1) Compared with the blank group, the bacteroides in the mouse feces of the modeling group are reduced, while the firmicutes, the warts and the Proteus are increased more, and the actinomycetes are also increased.

(2) Compared with the modeling group, the thick-walled fungus door and the Proteus door in the feces of the mice in the positive group are obviously reduced, and the wart fungus door is obviously increased.

(3) Compared with the modeling group, the probiotics group mice have increased bacteroides in feces, the firmicutes, the warts and the proteobacteria are reduced, and the species composition at the portal level is equivalent to that of a blank group and is close to the normal level.

The species composition at the genus level for each group of mice is shown in figure 12.

(1) Compared with the blank group, the abundance of Muribaculaceae (color block 1 from bottom to top) and Lactobacillus (color block 3 from bottom to top) in the building block is obviously reduced, while the abundance of Achroman (color block 2 from bottom to top) and Streptococcus (color block 6 from bottom to top) is obviously increased;

(2) The abundance of Muribaculaceae is reduced in the positive group compared to the modeled group, and the abundance of Acremonium and Alloprvotella (color lump 5 from bottom to top) is significantly increased.

(3) Compared to the modeled group, the abundance of the musicualace genus, lactobacillus genus and Alloprevotella genus of the probiotic group increased, while the abundance of the ackermannium genus and streptococcus genus decreased, and the species composition at the genus level tended to be in the community structure of the blank group.

The abundance of dominant species at the genus level is different for the positive group and the probiotic group, indicating that the drugs used in the positive group are different from those used in the probiotic group in terms of their modulating effect on the intestinal flora.

4.6.3 flora composition differential analysis

The variability of the composition of the fecal sample flora of the mice of the different groups was studied using beta diversity, as shown in figure 13.

PCoA principal coordinate analysis shows that the structure of the mice flora in the blank group is different from that of the mice flora in the modeling group, the structure of the mice flora in the positive group is different from that of the mice flora in the probiotic group, and the structure of the mice flora in the blank group is similar to that of the mice flora in the probiotic group.

The results show that the lactobacillus rhamnosus VHProbi M15 provided by the invention can improve the richness and uniformity of fecal flora of constipation mice, so that the abundance of dominant species on the levels of doors and genus tends to the characteristics of the flora of mice in a blank group, the difference of flora composition is reduced, the composition of the flora structure is similar to that of the blank group, and the flora structure tends to the level of normal mice.

In conclusion, the lactobacillus rhamnosus VHProbi M15 provided by the invention has strong tolerance to simulated artificial gastrointestinal fluids, which lays a foundation for the probiotic strains to successfully pass through the gastrointestinal tract to perform the probiotic function by colonic colonisation. The hemolysis test proves that lactobacillus rhamnosus VHProbi M15 does not generate hemolysin, does not dissolve blood cells, and has good biological safety. Meanwhile, lactobacillus rhamnosus VHProbi M15 can remove DPPH free radicals, inhibit lipid peroxidation, has a certain antioxidant function activity, can degrade cholesterol, and has the probiotic property of reducing serum cholesterol. Animal experiments prove that the lactobacillus rhamnosus VHProbi M15 can remarkably improve the water content of the mouse excrement and the running capability of the mouse intestine, and can also improve the content of acetic acid and total organic acid in the mouse excrement. Lactobacillus rhamnosus VHProbi M15 can effectively prevent and relieve constipation symptoms of mice by regulating secretion of gastrointestinal regulatory peptides, increasing intestinal peristaltic ability, regulating intestinal flora and other ways, and is hopeful to be developed into health care products or medicines with constipation relieving effects.

Sequence listing

<110> Qingdao blue organism Co., ltd

<120> Lactobacillus rhamnosus and its use in preventing and alleviating constipation symptoms

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1431

<212> DNA

<213> Lactobacillus rhamnosus (Lactobacillus rhamnosus)

<400> 1

gctcgctccc taaaagggtt acgccaccgg cttcgggtgt tacaaactct catggtgtga 60

cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg cgtgctgatc cgcgattact 120

agcgattccg acttcgtgta ggcgagttgc agcctacagt ccgaactgag aatggcttta 180

agagattagc ttgacctcgc ggtctcgcaa ctcgttgtac catccattgt agcacgtgtg 240

tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc 300

accggcagtc ttactagagt gcccaactaa atgctggcaa ctagtcataa gggttgcgct 360

cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg 420

tcattttgcc cccgaagggg aaacctgatc tctcaggtga tcaaaagatg tcaagacctg 480

gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 540

gtcaattcct ttgagtttca accttgcggt cgtactcccc aggcggaatg cttaatgcgt 600

tagctgcggc actgaagggc ggaaaccctc caacacctag cattcatcgt ttacggcatg 660

gactaccagg gtatctaatc ctgttcgcta cccatgcttt cgagcctcag cgtcagttac 720

agaccagaca gccgccttcg ccactggtgt tcttccatat atctacgcat ttcaccgcta 780

cacatggagt tccactgtcc tcttctgcac tcaagtttcc cagtttccga tgcacttcct 840

cggttaagcc gagggctttc acatcagact taaaaaaccg cctgcgctcg ctttacgccc 900

aataaatccg gataacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 960

cgtggctttc tggttggata ccgtcacgcc gacaacagtt actctgccga ccattcttct 1020

ccaacaacag agttttacga cccgaaagcc ttcttcactc acgcggcgtt gctccatcag 1080

acttgcgtcc attgtggaag attccctact gctgcctccc gtaggagttt gggccgtgtc 1140

tcagtcccaa tgtggccgat caacctctca gttcggctac gtatcattgc cttggtgagc 1200

cgttacctca ccaactagct aatacgccgc gggtccatcc aaaagcgata gcttacgcca 1260

tctttcagcc aagaaccatg cggttcttgg atttatgcgg tattagcatc tgtttccaaa 1320

tgttatcccc cacttaaggg caggttaccc acgtgttact cacccgtccg ccactcgttc 1380

aaaatcaaat caagatgcaa gcacctttca ataatcagaa ctcgttcgac c 1431

Claims

1. Lactobacillus rhamnosus, characterized in that, lactobacillus rhamnosus (Lactobacillus rhamnosus) have a preservation number of cctccc NO: m2021904.

2. The lactobacillus rhamnosus of claim 1 wherein the lactobacillus rhamnosus has a Riboprinter fingerprint as shown in figure 1.

3. The lactobacillus rhamnosus according to claim 1, wherein the RAPD fingerprint of lactobacillus rhamnosus is shown in figure 2.

4. The lactobacillus rhamnosus according to claim 1, wherein the rep-PCR fingerprint of lactobacillus rhamnosus is shown in figure 3.

5. The lactobacillus rhamnosus according to claim 1, wherein the 16s rDNA sequence of lactobacillus rhamnosus is SEQ ID NO. 1.

6. The use of lactobacillus rhamnosus as claimed in claim 1 for the manufacture of a health product or medicament for alleviating constipation symptoms.

7. A health product or medicine for relieving constipation symptoms, which is characterized in that the health product or medicine contains lactobacillus rhamnosus as defined in claim 1.