CN117004538B

CN117004538B - Lactobacillus reuteri and application thereof

Info

Publication number: CN117004538B
Application number: CN202311091740.6A
Authority: CN
Inventors: 卓林双; 韩军涛; 金海利; 吕美博; 高梦; 赵珊; 梁伟
Original assignee: Dr Tong Health Industry Hebei Co ltd
Current assignee: Dr Tong Health Industry Hebei Co ltd
Priority date: 2023-08-28
Filing date: 2023-08-28
Publication date: 2024-03-26
Anticipated expiration: 2043-08-28
Also published as: CN117004538A

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus reuteri and application thereof. The preservation number of the lactobacillus reuteri (Limosifactobacillus reuteri) LR-T001 is CGMCC No.26792. The Lactobacillus reuteri LR-T001 provided by the invention has obvious growth inhibition effect on helicobacter pylori, can well grow in high-acid and high-bile salt concentration environments, can be well adapted to digestive tract environments, has good adhesion capability, can be rapidly planted in digestive tract systems to carry out rapid propagation, forms dominant bacterial colony, achieves the effect of rapidly and continuously inhibiting the growth of helicobacter pylori, effectively reduces the recurrence rate of helicobacter pylori infection, and has wide application prospects in the fields of clinical prevention, treatment and inhibition of helicobacter pylori infection related diseases and the like.

Description

Lactobacillus reuteri and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to lactobacillus reuteri and application thereof.

Background

Helicobacter pylori (Helicobacter pylori, HP) is a gram-negative bacillus that is S-shaped or curved. The cytotoxin-related gene (cagA) and the vacuolated toxin gene (vacA) are important pathogenic genes of helicobacter pylori, and are also main pathogenic factors causing chronic active gastritis, peptic ulcer, gastric MALT lymphoma and gastric cancer. Research shows that the incidence rate of atrophic gastritis and gastric cancer of the population with early age of primary infection of helicobacter pylori is relatively high, the helicobacter pylori infection and the death rate of gastric cancer are in parallel relation, and the world health organization already classifies the helicobacter pylori as a class I carcinogen. Thus, the control and treatment of helicobacter pylori is becoming more and more important.

At present, the conventional treatment of helicobacter pylori is mainly tetrad therapy (bismuth agent and proton pump inhibitor are combined with two antibiotics), and although the therapy can effectively eradicate helicobacter pylori in a short time, the helicobacter pylori is easy to be infected, even after healing, the helicobacter pylori can be infected again, the drug resistance of the helicobacter pylori continuously increases during the retreatment, and the eradication rate gradually decreases. Furthermore, the tetrad therapy causes the damage of normal flora of gastrointestinal tract and the colonization of external pathogenic bacteria, so that patients suffer from side effects of digestive tract such as abdominal pain, diarrhea, abdominal distention, constipation, nausea and the like. The research shows that some probiotics have the effect of resisting helicobacter pylori, and provide new thought for treating helicobacter pylori-caused diseases. However, the strains and the preparations thereof in the prior art have limited anti-helicobacter pylori effect, and still need to be matched with triple therapy or quadruple therapy to only play a role of auxiliary treatment. Although a small proportion of the probiotics may be used alone or in combination with other probiotics to treat helicobacter pylori, sufficient amounts are required to achieve a good therapeutic effect. Therefore, developing a method that can effectively inhibit helicobacter pylori infection is a problem to be solved.

Disclosure of Invention

Aiming at the problems of limited treatment effect, large dosage and the like of the existing probiotics for treating helicobacter pylori infection, the invention provides lactobacillus reuteri and application thereof.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

in a first aspect, the invention provides lactobacillus reuteri (Limosilactobacillus reuteri) LR-T001 with a preservation number of CGMCC No.26792.

Lactobacillus reuteri LR-T001 is selected from the feces of healthy human body, is classified and named as lactobacillus reuteri (Limosilactobacillus reuteri), and is preserved in China general microbiological culture collection center (CGMCC) for short in the year 03 and the month 10 of 2023, wherein the preservation number is CGMCC No.26792, and the preservation address is: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.

The biological characteristics of the lactobacillus reuteri LR-T001 provided by the invention are as follows: the MRS solid culture medium has the morphological characteristics of round, convex, milky, neat edge, poor transparency, small colony and short rod-shaped cell shape.

The Lactobacillus reuteri LR-T001 provided by the invention has obvious growth inhibition effect on helicobacter pylori, grows well in environments with high acid and high bile salt concentration, has high acid resistance and bile salt resistance, can be well adapted to the digestive tract environment, has good adhesion capability, can be rapidly planted in the digestive tract system to perform rapid propagation to form dominant bacterial colony, achieves the effect of rapidly and continuously inhibiting the growth of helicobacter pylori, effectively reduces the recurrence rate of helicobacter pylori infection, and has wide application prospects in the fields of clinical prevention, treatment and inhibition of helicobacter pylori infection related diseases, microbial treatment of gastric diseases and the like; in addition, the lactobacillus reuteri LR-T001 also has good hydrogen peroxide tolerance, and has wide application prospect in the food industry and extremely high practical value.

In a second aspect, the invention also provides a probiotic composition comprising said lactobacillus reuteri LR-T001.

Further, the probiotic composition further comprises at least one of bacillus amyloliquefaciens, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum or bifidobacterium.

Lactobacillus reuteri LR-T001 of the present invention may be used in combination with probiotics having anti-helicobacter pylori efficacy or probiotics regulating gastrointestinal function, which are common in the art.

In a third aspect, the invention also provides the use of lactobacillus reuteri LR-T001 or a probiotic composition in the manufacture of a medicament for the treatment of helicobacter pylori infection.

In a fourth aspect, the invention also provides an anti-helicobacter pylori pharmaceutical formulation comprising said lactobacillus reuteri LR-T001 or said probiotic composition, and a pharmaceutically acceptable carrier.

Further, the dosage form of the helicobacter pylori resistant pharmaceutical preparation is one of powder, granule, emulsion, capsule, tablet, pill or oral liquid.

Further, the pharmaceutical preparation comprises not less than 1×10 ⁷ CFU/g L.reuteri LR-T001.

In a fifth aspect, the invention also provides application of the lactobacillus reuteri LR-T001 or the probiotic composition in preparing health-care food.

In a sixth aspect, the invention also provides a health food comprising the lactobacillus reuteri LR-T001 or the probiotic composition and a food additive.

Further, the health food is a probiotic solid beverage, fermented milk or fermented juice.

Further, the health food comprises not less than 1×10 ⁶ CFU/mL of Lactobacillus reuteri LR-T001.

The lactobacillus reuteri LR-T001 can be prepared into a pharmaceutical preparation with common pharmaceutical auxiliary materials or carriers in the field, and can also be prepared into health-care food with prebiotics, plant extracts or other solid components which can be mixed with probiotics for eating, so that the lactobacillus reuteri LR-T001 is convenient for various people to use.

For example, lactobacillus reuteri LR-T001 lyophilized powder can be mixed with common food additives such as inulin, galactooligosaccharide, fructooligosaccharide, fruit powder or citric acid to obtain solid beverage fermented juice, or the fermented liquid of lactobacillus reuteri LR-T001 can be mixed with common food additives such as fruit juice, fructooligosaccharide, etc. to obtain fermented juice; can also be fermented with milk, sweetener, emulsifier, etc. to obtain fermented milk.

In a seventh aspect, the invention also provides a metabolite prepared by liquid fermentation of lactobacillus reuteri LR-T001 as described above.

Further, the preparation method of the metabolite comprises the following steps:

s1, inoculating lactobacillus reuteri LR-T001 seed liquid into a culture medium, and standing for cultivation to obtain lactobacillus reuteri LR-T001 fermentation liquid;

s2, centrifuging the lactobacillus reuteri LR-T001 fermentation liquid, and taking supernatant to obtain a metabolite of lactobacillus reuteri LR-T001.

The lactobacillus reuteri LR-T001 provided by the invention has high safety and strong stability, can effectively inhibit the growth of helicobacter pylori, solves the problems of gastrointestinal dysfunction, gastrointestinal dysbacteriosis, increased pathogenic bacteria drug resistance and the like caused by unreasonable use of antibiotics for a long time in the process of preventing and treating helicobacter pylori infection, can be used as functional probiotics for preparing foods, health care products or medicines, and has extremely high development prospect.

Drawings

FIG. 1 shows colony morphology of Lactobacillus reuteri LR-T001 according to the present invention;

FIG. 2 shows the cell morphology of Lactobacillus reuteri LR-T001 according to the invention under gram staining;

FIG. 3 is a phylogenetic tree of Lactobacillus reuteri LR-T001 constructed based on sequence similarity of the 16S rRNA genes in example 1.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The medium used in the examples:

skim milk medium: 10% of skim milk powder, 2% of glucose, 88% of distilled water and 7min of sterilization at 121 ℃ for standby.

MRS liquid medium: 1.0g of soybean peptone, 0.2g of sodium citrate, 2.0g of glucose, 0.2g of dipotassium hydrogen phosphate, 0.5g of anhydrous sodium acetate, 0.0054g of manganese sulfate pentahydrate, 0.5g of yeast powder, 0.02g of magnesium sulfate heptahydrate, 0.1g of tween-80, 1.0g of beef extract and 100mL of distilled water, wherein the pH is adjusted to 6.5, and the mixture is sterilized at 121 ℃ for 15min for later use.

MRS solid medium: 1.0g of soybean peptone, 0.2g of sodium citrate, 2.0g of glucose, 0.2g of dipotassium hydrogen phosphate, 0.5g of anhydrous sodium acetate, 0.0054g of manganese sulfate pentahydrate, 0.5g of yeast powder, 0.02g of magnesium sulfate heptahydrate, 0.1g of tween-80, 1.0g of beef extract, 1.5g of agar, 100mL of distilled water, and sterilizing for 15min at 121 ℃ for later use.

Fermentation medium: 10g of soybean peptone, 5g of anhydrous sodium acetate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 0.04g of manganese sulfate pentahydrate, 5g of yeast extract powder, 0.2g of magnesium sulfate heptahydrate, 1g of tween-80, 10g of beef extract powder, 1000mL of purified water and sterilizing at 121 ℃ for 15min.

Example 1

1. Strain screening

Adding 1g of human body healthy excrement into 9mL of sterile PBS buffer solution, shaking and fully mixing, sequentially carrying out 10-time gradient dilution, and respectively taking 100 mu L of 10 ^-3 ～10 ^-5 The gradient dilution is evenly coated on an MRS solid culture medium, the culture medium is placed in an anaerobic incubator for culturing for 48 hours at 37 ℃, opaque milky white, round, glossy, regular in edge, raised on the surface and wet colonies are selected, repeated streak purification culture is carried out on the MRS solid culture medium, and the individual morphology is observed through an optical microscope, so that the lactobacillus strain is obtained.

2. Morphological observation and physiological biochemical identification

Bacterial strain LR-T001 is inoculated on MRS solid culture medium, and is put into a constant temperature incubator at 37 ℃ for anaerobic culture for about 48 hours, and the colony morphology, colony edge, color, transparency and the like on the culture medium are observed.

Strain LR-T001 was round, convex, milky white, well-defined and poorly transparent on MRS solid medium, with smaller colonies and short bars in cell shape (as shown in fig. 1-2).

Molecular biological identification

The total DNA of strain LR-T001 is used as template, and the 16S rRNA universal primer 27F (5'-AG AGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') are used for PCR amplification, and the amplified product is identified by 1.2% agarose gel electrophoresis and then sent to the biological engineering (Shanghai) Co., ltd for determining the nucleic acid sequence.

The PCR amplification used a 20. Mu.L reaction system comprising: 2X Taq PCR Master Mix. Mu.L, 0.5. Mu.L each of the upstream and downstream primers (10. Mu.M), 1.0. Mu.L (20 ng/. Mu.L) of the DNA template and ddH ₂ O 8.0μL。

PCR reaction conditions: firstly, pre-denaturing for 10min at 95 ℃; then denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, followed by 32 cycles in total and finally extension at 72℃for 10min.

The sequencing results of 16S rRNA were:

GGCTCAGGATGAACGCCGGCGGTGTGCCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCTTTATGGAGGG

comparing the 16S rRNA sequence of strain LR-T001 with the sequence in GenBank to obtain the 16S rRNA sequence of the similar lactobacillus reuteri standard strain, obtaining the evolutionary distance of the lactobacillus reuteri and related strains, and constructing a phylogenetic tree, as shown in figure 3. Based on the 16S rRNA sequence similarity analysis, strain LR-T001 was determined to be Lactobacillus reuteri (Limosilactobacillus reuteri).

The lactobacillus reuteri LR-T001 is preserved in China general microbiological culture Collection center (CGMCC) for a year of 2023 and a month of 03, and has a strain preservation number of CGMCC No.26792 and a preservation address of: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.

3. Activation and preservation of strains

First generation: the deposited strain was inoculated into 5ml of 10% skim milk medium, placed at 37℃for activation, and placed in a 4℃refrigerator for preservation until significant coagulation of skim milk in the medium was observed.

Second generation: the first-generation strain activated in advance is inoculated into 5mL MRS liquid culture medium according to the inoculation amount of 2%, and the strain is cultured for 12h at 37 ℃.

Third generation: the second-generation strain was inoculated into 5mL of MRS liquid medium at an inoculum size of 2%, and cultured at 37℃for 12 hours.

Fourth generation: the third generation strain was inoculated into 1mL of MRS liquid medium at an inoculum size of 5%, and cultured at 37℃for 12 hours.

The fourth generation strain is liquid strain for fermentation, a fermentation culture medium is adopted, the strain is inoculated according to 10% of the liquid in a fermentation tank, the pH value of a fermentation liquid is regulated to be constant 6.0 by 2mol/L sodium hydroxide solution during fermentation, the fermentation temperature is 37 ℃, the fermentation time is 7-8h, and the OD of the fermentation liquid is to be obtained ₆₀₀ Stopping fermentation when the value is stable or no longer increases, reducing the temperature to 10-15 ℃, standing, centrifuging at 14000rpm for 1.5-2h, taking bacterial sludge, and adding the bacterial sludge according to the ratio of 1:0.32Adding a protective agent, wherein the protective agent comprises 12% of skim milk powder, 10% of trehalose, 0.5% of sodium glutamate, 1% of glycerol and the balance of sterile water, and after emulsification, putting into a vacuum freeze dryer for freeze drying, taking out, crushing, and detecting parameters such as viable count, physicochemical index and the like.

Example 2

Lactobacillus reuteri LR-T001 acid and bile salt resistance test:

lactobacillus reuteri LR-T001 fermentation broth in example 1 was centrifuged at 4000r/min at 4 ℃ for 3min, the supernatant was discarded, the cells were washed 2 times with PBS buffer, inoculated into modified MRS liquid media having pH values of 2.0, 2.5, 3.0, 3.5, 4.0, respectively, at 1% inoculum size, anaerobically cultured at 37 ℃, sampled at 0h, 2h, 4h, respectively, and viable count was performed. And cultured in a conventional MRS liquid medium as a control group. The results are shown in Table 1.

TABLE 1 viable count of bacterial powders of Lactobacillus reuteri LR-T001 at different pH values (10 ¹¹ CFU/g)

Time	pH2.0	pH2.5	pH3.0	pH3.5	pH4.0	Control group
							0h	3.8	4.9	3.9	4.2	4.2	4.0
2h	3.1	3.3	3.5	3.2	3.5	3.8
							4h	2.6	3.7	3.1	2.8	3.9	3.6

Similarly, the strain was inoculated into an improved MRS liquid medium having a concentration of 0.1%, 0.3% and 0.6% by mass of bovine bile salt at an inoculum size of 1%, anaerobically cultured at 37℃and sampled at 0h, 2h, 4h and 6h, respectively, to count the number of viable bacteria. And cultured in a conventional MRS liquid medium as a control group. The results are shown in Table 2.

TABLE 2 viable count of bacterial powders of Lactobacillus reuteri LR-T001 at various pig bile salt concentrations (10) ¹¹ CFU/g)

Time	0.1％	0.3％	0.5％	Control group
					0h	4.3	2.8	1.9	4.4
2h	3.9	2.2	1.2	4.4
					4h	3.7	1.7	0.91	4.2
6h	3.4	1.2	0.73	3.8

The results prove that the lactobacillus reuteri LR-T001 has higher acid resistance and bile salt resistance.

Example 3

Lactobacillus reuteri LR-T00 tolerates the ability to mimic the gut environment:

preparation of simulated gastric fluid:

accurately measuring 16.4mL of hydrochloric acid with the mass concentration of 100g/L, adding distilled water for dilution, adjusting the pH value to be 3.0, then adding pepsin, calculating the addition according to the mass concentration ratio of 1g/100mL, and filtering and sterilizing by using a microporous filter membrane after full dissolution to prepare simulated gastric juice for later use.

Preparation of artificial intestinal juice

Weighing KH ₂ PO ₄ 3.4g, adding 250mL of distilled water for dissolution, adjusting the pH value to 6.8 by using 0.4g/100mL of NaOH solution, adding water for dilution to 500mL, then adding trypsin according to the mass concentration ratio of 1g/100mL, and filtering and sterilizing by using a microporous filter membrane after full dissolution to prepare simulated intestinal juice for later use.

The fermentation broth of Lactobacillus reuteri LR-T001 in example 1 was centrifuged at 8000r/min at 4℃for 4min, the supernatant was discarded, the cells were washed 2 times with PBS buffer, resuspended in a mixed solution of 1.9mL of simulated gastric fluid and 0.1mL of 10% skim milk solution, and after aerobic culture at 37℃for 2h, 100. Mu.L of the bacterial broth was taken for viable count.

Similarly, the fermentation broth of Lactobacillus reuteri LR-T001 in example 1 was centrifuged at 8000r/min at 4℃for 4min, the supernatant was discarded, the cells were washed with PBS buffer 2 times, resuspended in 1.9. Mu.L of simulated duodenal fluid, and after anaerobic culture at 37℃for 2h, 100. Mu.L of the cells were counted for viable bacteria. The results are shown in Table 3.

TABLE 3 resistance to simulated digestive tract environmental competence

The result shows that the lactobacillus reuteri LR-T001 has better capability of tolerating the simulated digestive tract and has higher application potential in the development of anti-helicobacter pylori medicines and health-care foods.

Lactobacillus reuteri LR-T001 tolerance to hydrogen peroxide:

the fermentation broth of Lactobacillus reuteri LR-T001 in example 1 was centrifuged at 4000r/min at 4℃for 3min, the supernatant was discarded, the cells were washed 2 times with PBS buffer, inoculated in a modified MRS liquid medium having hydrogen peroxide concentrations of 0.5mmol/L and 1.0mmol/L, respectively, at 1% inoculum size, and sampled at 0h, 3h, and 6h, respectively, for viable count. And a conventional MRS liquid medium was inoculated as a control group. The results are shown in Table 4.

TABLE 4 hydrogen peroxide resistance (10 ¹¹ CFU/mL)

Time	0.5mmol/L	1.0mmol/L	Control group
				0h	3.6	1.7	4.3
3h	3.1	1.3	4.2
				6h	2.6	0.6	4.0

Example 4

Adhesion capability test on human gastric mucosal cells GES-1:

regulating GES-1 cell concentration to 1×10 ⁴ /mL，Added to a 96-well plate at 37℃with 5% CO ₂ After the GES-1 cells are in an adherent state, washing the GES-1 cells for 3 times by using PBS buffer solution to obtain washed GES-1 cells (blank group).

Diluting helicobacter pylori suspension with sterile physiological saline to concentration of 2×10 ⁷ CFU/mL, 1mL was added to the washed GES-1 cells at 37℃with 5% CO ₂ After 2h incubation in the incubator (II), the unadsorbed H.pylori was washed 3 times with PBS buffer to clean the H.pylori infected GES-1 cells (HP group).

Into helicobacter pylori infected GES-1 cells, 10. Mu.L of Lactobacillus reuteri LR-T001 suspension (1.0X10. Mu.L of fermentation broth was centrifuged and resuspended in PBS) ⁸ CFU/mL), at 37℃with 5% CO ₂ Is cultured for 2 hours to obtain the helicobacter pylori infected GES-1 cells (treatment group) treated with the Lactobacillus reuteri LR-T00.

After washing each well 5 times with PBS, 200. Mu.L of urease reagent was added to each cell group, and the mixture was washed with 5% CO at 37℃C ₂ Culturing for 2 hours in an incubator of (2) to obtain culture solutions, and measuring the absorbance of the culture solutions of different groups at the wavelength of 550nm by using an enzyme-labeled instrument, wherein each group is subjected to 3 parallel experiments. The results are shown in Table 5.

Adhesion = (OD value of treatment group blank)/(OD value of HP group OD value of blank) ×100%

The adhesion of HP group is defined as 100%, the adhesion rate of treatment group is 21.2%, which shows that Lactobacillus reuteri LR-T001 can effectively reduce the colonization ability of helicobacter pylori to gastric mucosa cells, and is better used for eliminating helicobacter pylori after infection.

Example 5

Self-agglomerating ability detection

Centrifuging the lactobacillus reuteri LR-T001 fermentation broth of example 1 at 4000r/min and 4deg.C for 3min, discarding supernatant, washing thallus with PBS buffer for 2 times, and re-suspending in PBS buffer to obtain thallus concentration of 1×10 ⁸ Bacterial suspension of CFU/mL is subjected to stationary culture at 37 ℃, supernatant liquid is taken at 0h, 2h, 4h, 6h, 12h and 24h,determination of OD ₆₀₀ Values, 3 parallel experiments were set and self-coagulation rates were calculated. The results are shown in Table 5.

Self-aggregation ratio (%) = (a) ₀ -A _t )/A ₀ ×100％

Wherein A0 is an OD of 0h ₆₀₀ Value, at is OD At different sampling time points ₆₀₀ Values.

TABLE 5 self-agglomeration rate

Hydrophobic ability detection

Centrifuging the lactobacillus reuteri LR-T001 fermentation broth of example 1 at 4000r/min and 4deg.C for 3min, discarding supernatant, washing thallus with PBS buffer for 2 times, re-suspending in PBS buffer, and adjusting bacterial suspension OD ₆₀₀ Taking 2mL of the bacterial suspension and 2mL of dimethylbenzene in a test tube, vortex oscillating for 2min, standing at room temperature for 20min, vortex mixing, standing at room temperature for 20min for layering, and measuring absorbance value A at 600nm by water phase ₁ 3 replicates were measured in each group using PBS buffer as a blank.

Hydrophobicity= (1-A1/A0) ×100%

Wherein A0 is the initial absorbance value of the bacterial suspension at 600nm, and A1 is the absorbance value of the aqueous phase of the stationary phase layering.

The calculated hydrophobicity of lactobacillus reuteri LR-T001 was 89.5%.

The result proves that the lactobacillus reuteri LR-T001 has higher self-coagulation capability, can reach a plateau stage rapidly in 6 hours, is about 85.87 percent, has stronger hydrophobicity, can form a barrier rapidly on gastric mucosa, and prevents the colonization and infection of harmful bacteria.

Example 6

In vitro helicobacter pylori inhibition capability detection

Accurately measuring 16.4mL of hydrochloric acid with the mass concentration of 100g/L, adding distilled water for dilution, adjusting the pH value to 3.0, then adding pepsin, calculating the addition according to the mass concentration ratio of 1g/100mL, and filtering and sterilizing by using a microporous filter membrane after full dissolution to prepare simulated gastric fluid for later use.

Diluting helicobacter pylori suspension to a concentration of 1×10 ⁸ CFU/mL, 2mL was added to 20mL simulated gastric fluid, and then 2mL of Lactobacillus reuteri LR-T001 suspension (fermentation broth was centrifuged and resuspended in PBS solution, 1.0X10) ⁷ CFU/mL) is placed on a micro-aerobic environment shaking table at 37 ℃ for culture, and the viable count is sampled and detected at 0h, 0.5h, 1h, 1.5h and 2h respectively, so as to calculate the inactivation rate. The blank was physiological saline at pH 7.0.

Inactivation ratio (%) = (number of viable bacteria detected at different times—number of initial viable bacteria)/number of initial viable bacteria×100%

The test result proves that the inactivation rate of the helicobacter pylori can reach more than 90% after 0.5h of culture, and the helicobacter pylori can be basically inactivated in 1.5h, so that the Lactobacillus reuteri LR-T001 can obviously inhibit the growth of the helicobacter pylori in the stomach environment.

In conclusion, the lactobacillus reuteri LR-T001 with the preservation number of CGMCC NO.26792 has strong self-condensation capability and high hydrophobicity, can quickly form a protective barrier on gastric mucosa, has high capability of adhering helicobacter pylori, can prevent the colonization of helicobacter pylori, effectively prevent and eliminate helicobacter pylori invaded in vivo, and has wide application prospect.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.

Claims

1. Lactobacillus reuteri @ and its preparing processLimosilactobacillus reuteri) LR-T001, which is characterized in that the preservation number is CGMCC No.26792.

2. A probiotic composition comprising lactobacillus reuteri LR-T001 according to claim 1.

3. The probiotic composition of claim 2, further comprising at least one of bacillus amyloliquefaciens, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, or bifidobacterium.

4. Use of lactobacillus reuteri LR-T001 according to claim 1 or a probiotic composition according to claim 2 for the manufacture of a medicament against helicobacter pylori infection.

5. An anti-helicobacter pylori pharmaceutical formulation comprising lactobacillus reuteri LR-T001 according to claim 1 or a probiotic composition according to claim 2, and a pharmaceutically acceptable carrier.

6. The helicobacter pylori resistant pharmaceutical preparation according to claim 5, wherein the dosage form is one of powder, granule, emulsion, capsule, tablet, pill or oral liquid.

7. Use of lactobacillus reuteri LR-T001 according to claim 1 or of a probiotic composition according to claim 2 in the preparation of a health food.

8. A health food comprising lactobacillus reuteri LR-T001 according to claim 1 or the probiotic composition according to claim 2, and a food additive.

9. The health food of claim 8, wherein the health food is a probiotic solid beverage, fermented milk or fermented juice.