Strain of <i>Lactobacillus rhamnosus </i>and application thereof in inhibiting <i>Helicobacter pylori </i>

Abstract

The disclosure discloses a strain of Lactobacillus rhamnosus and application thereof in inhibiting Helicobacter pylori, and belongs to the technical fields of microorganisms and medicine. The disclosure provides a strain of Lactobacillus rhamnosus CCFM1119. The Lactobacillus rhamnosus CCFM1119 can prevent and/or treat Helicobacter pylori infection, specifically embodied in that: (1) the Lactobacillus rhamnosus CCFM1119 can significantly relieve the gastrointestinal symptoms of patients with Helicobacter pylori infection; (2) the Lactobacillus rhamnosus CCFM1119 can significantly reduce the amount of colonization of Helicobacter pylori in patients with Helicobacter pylori infection; and (3) the Lactobacillus rhamnosus CCFM1119 can significantly increase the clearance rate of Helicobacter pylori in patients with Helicobacter pylori infection. Therefore, Lactobacillus rhamnosus CCFM1119 has great application prospects in the preparation of products (such as food or medicine) for preventing and/or treating Helicobacter pylori infection.

Claims

1. A Helicobacter pylori inhibitor, comprising Lactobacillus rhamnosus, wherein the Helicobacter pylori is Helicobacter pylori SS1; wherein the Lactobacillus rhamnosus is preserved in the Guangdong Microbial Culture Collection Center, under the preservation number GDMCC NO: 61013, on May 6, 2020; and wherein the inhibitor is a food or a medicine.

2. The Helicobacter pylori inhibitor according to claim 1, wherein a live count of the Lactobacillus rhamnosus is not less than 510.sup.9 CFU/mL or 510.sup.9 CFU/g.

3. The inhibitor of claim 1, wherein the inhibitor is the medicine, and wherein the medicine further comprises a drug carrier and/or a pharmaceutical excipient.

4. The inhibitor of claim 1, wherein the inhibitor is the medicine, and wherein the medicine is in a form of powders, granules, capsules, tablets, pills, or liquids.

5. The inhibitor according to claim 1, wherein the inhibitor is the food, and wherein the food further comprises dairy, bean, fruit, and/or vegetable.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 shows the adhesion rate of Helicobacter pylori to AGS cells in different groups.

(2) FIG. 2 shows changes in GSRS scores of Helicobacter pylori-positive patients in different groups.

(3) FIG. 3 shows changes in 14C-urea breath test values of Helicobacter pylori-positive patients in different groups.

(4) FIG. 4 shows the effect of storage time on the live count of Lactobacillus rhamnosus CCFM1119 bacterial powder.

DETAILED DESCRIPTION

(5) The Helicobacter pylori involved in the following examples is Helicobacter pylori SS1 from the National Type Culture Collection (NTCC). The Lactobacillus rhamnosus L. GG involved in the following examples is derived from the American Type Culture Collection (ATCC), and the preservation number is ATCC 53103. The F12 liquid medium and fetal calf serum involved in the following examples were purchased from Gibco, USA. The NaCl involved in the following examples was purchased from Sinopharm. The phenol red and urea involved in the following examples were purchased from Macklin. The Columbia medium involved in the following examples was purchased from OXOID, the United Kingdom. The sterile defibered sheep blood involved in the following examples was purchased from Hangzhou Sinry Bio-engineering Co., Ltd. The BHI liquid medium involved in the following examples was purchased from Qingdao Hope Bio-Technology Company.

Media Involved in the Following Examples are as Follows

(6) MRS solid medium (g/L): Peptone 10 g/L, beef extract 10 g/L, glucose 20 g/L, sodium acetate 2 g/L, yeast powder 5 g/L, diammonium hydrogen citrate 2 g/L, K.sub.2PO.sub.4.Math.3H.sub.2O 2.6 g/L, MgSO.sub.4.Math.7H.sub.2O 0.1 g/L, MnSO.sub.4 0.05 g/L, Tween 80 1 mL/L, and agar 20 g/L.

(7) MRS liquid medium (g/L): Peptone 10 g/L, beef extract 10 g/L, glucose 20 g/L, sodium acetate 2 g/L, yeast powder 5 g/L, diammonium hydrogen citrate 2 g/L, K.sub.2PO.sub.4.Math.3H.sub.2O 2.6 g/L, MgSO.sub.4.Math.7H.sub.2O 0.1 g/L, MnSO.sub.4 0.05 g/L, and Tween 80 1 mL/L.

Detection Methods Involved in the Following Examples are as Follows

(8) Detection method of live count: National standard GB 4789.35-2016 National Food Safety Standard, Food Microbiology Detection, Lactic Acid Bacteria Detection.

(9) A Preparation Method of Helicobacter pylori Cells Involved in the Following Examples is as Follows:

(10) Helicobacter pylori is streaked on a Columbia blood agar medium, and cultured in a three-gas incubator (85% N.sub.2, 10% CO.sub.2 and 5% O.sub.2) at 37 C. for 3 days to obtain a single colony. The single colony is picked and inoculated in BHI medium containing 5% (v/v) fetal calf serum, and cultured in a three-gas incubator (85% N.sub.2, 10% CO.sub.2 and 5% O.sub.2) at 37 C. for 4 days to obtain a seed solution. The seed solution is inoculated in BHI liquid medium at an inoculation amount of 2% (v/v), and the seed solution is cultured in a three-gas incubator (85% N.sub.2, 10% CO.sub.2 and 5% O.sub.2) at 37 C. for 4 days to obtain a Helicobacter pylori bacterial solution. The Helicobacter pylori bacterial solution is centrifuged at 8,000 g for 10 min and filtered to obtain Helicobacter pylori bacterial cells.

(11) The Columbia blood agar medium is prepared as follows: 39 g of Columbia medium solid powder is dissolved in 1 L of water. The solution is sterilized at 121 C. for 15 min. After cooling to 55 C. to 60 C., 7.5% (v/v) sterile defibered sheep blood is added, and the solution is mixed uniformly and poured into a plate.

(12) A Preparation Method of Lactobacillus rhamnosus Cells Involved in the Following Examples is as Follows:

(13) Lactobacillus rhamnosus is streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony is picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in MRS liquid medium at an inoculation amount of 2% (v/v) and cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution is centrifuged at 8,000 g for 10 min and filtered to obtain Lactobacillus rhamnosus bacterial cells.

Example 1: Screening and Identification of Lactobacillus rhamnosus

(14) 1. Screening

(15) Fresh feces of healthy humans from Kunshan, Jiangsu was taken as a sample. The sample was pretreated, and the pretreated sample was stored in a refrigerator at 80 C. in about 30% glycerol. After the sample was taken out and thawed, the sample was mixed uniformly. 0.5 mL of the sample was pipetted and added to 4.5 mL of 0.9% physiological saline and subjected to gradient dilution. An appropriate gradient dilution was selected and spread on an MRS solid medium. The dilution was cultured at 37 C. for 48 h. Typical colonies were picked and streaked on an MRS plate for performing purification. A single colony was picked and transferred to an MRS liquid medium for performing enrichment to obtain the strain CCFM1119 (the original number of the strain is JS-SZ-2-1), and the strain was preserved with 30% glycerol in a tube.

(16) 2. Identification

(17) The genome of the CCFM1119 was extracted, and the 16S rDNA of the CCFM1119 was amplified and sequenced (completed by Sangon Biotech (Shanghai) Co., Ltd.). By sequencing analysis, the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1. The sequence was compared in GenBank, and the result showed that the strain was Lactobacillus rhamnosus, named Lactobacillus rhamnosus CCFM1119.

Example 2: Inhibition Effect of Lactobacillus rhamnosus on Helicobacter pylori Growth

(18) An MRS liquid medium was used as a negative control. Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in MRS liquid medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min and filtered with a 0.22 m sterile filter membrane to obtain supernatant. The diameter of the inhibition zone of the supernatant of Lactobacillus rhamnosus CCFM1119 on Helicobacter pylori was measured by the Oxford cup method to indicate the effect of inhibiting the growth of Helicobacter pylori. The measurement results are shown in Table 1. (For details of the Oxford cup method, please refer to the literature: Zhang Tingting, Zhai Qixiao, Jin Xing, et. al. Screening and characterization of lactic acid bacteria with antagonistic activities against Campylobacter jejuni from chicken manure. Microbiology China, 2017, (44): 118-125).

(19) It can be seen from Table 1 that the MRS liquid medium has no inhibition zone on Helicobacter pylori, while the diameter of the inhibition zone of the Lactobacillus rhamnosus CCFM1119 supernatant on Helicobacter pylori can reach 12.92 mm, indicating that the Lactobacillus rhamnosus CCFM1119 can inhibit the growth of Helicobacter pylori.

(20) TABLE-US-00001 TABLE 1 The diameter of the inhibition zone of Lactobacillus rhamnosus CCFM1119 on Helicobacter pylori Diametere of inhibition Group pH zone (mm) Negative control 6.20 0 CCFM1119 3.54 12.92 0.19

Example 3: Effect of Lactobacillus rhamnosus on Adhesion of Helicobacter pylori

(21) Specific Steps are as Follows:

(22) (1) Preparation of Resuspension

(23) Helicobacter pylori cells were resuspended in an F12 medium to a concentration of 110.sup.7 CFU/mL to obtain a Helicobacter pylori resuspension. Lactobacillus rhamnosus L. GG cells were resuspended in an F12 medium to a concentration of 110.sup.7 CFU/mL to obtain a Lactobacillus rhamnosus L. GG resuspension. Lactobacillus rhamnosus CCFM1119 cells were resuspended in an F12 medium to a concentration of 110.sup.7 CFU/mL to obtain a Lactobacillus rhamnosus CCFM1119 resuspension.

(24) (2) Preparation of Helicobacter pylori-Infected AGS Cells

(25) AGS cells were resuspended in an F12 medium containing 5% (v/v) fetal calf serum, then added to a 96-well plate (210.sup.4 cells/well), and cultured at 37 C. in 5% CO.sub.2 for 12-16 h. Until the AGS cells were in an adherent state, the AGS cells were washed 3 times with PBS to remove dead cells. The Helicobacter pylori resuspension was added to the washed AGS cells, and cultured in an incubator at 37 C. in 5% CO.sub.2 for 2 h. The AGS cells were washed with a PBS solution 3 times to remove unabsorbed Helicobacter pylori and obtain Helicobacter pylori-infected AGS cells.

(26) (3) AGS cells not infected with Helicobacter pylori and not treated with Lactobacillus rhamnosus L. GG or Lactobacillus rhamnosus CCFM1119 were a blank group.

(27) Helicobacter pylori-infected AGS cells not treated with Lactobacillus rhamnosus L. GG or Lactobacillus rhamnosus CCFM1119 were a model group (Hp group).

(28) Helicobacter pylori-infected AGS cells treated with Lactobacillus rhamnosus L. GG and Helicobacter pylori-infected AGS cells treated with Lactobacillus rhamnosus CCFM1119 were experimental groups, named an Hp+LGG group and an Hp+CCFM1119 group respectively.

(29) 0.2 mL of Lactobacillus rhamnosus L. GG resuspension or Lactobacillus rhamnosus CCFM1119 resuspension was added to Helicobacter pylori-infected AGS cells respectively, and the cells were cultured in an incubator at 37 C. in 5% CO.sub.2 for 2 h to obtain Helicobacter pylori-infected AGS cells treated with the Lactobacillus rhamnosus L. GG and Helicobacter pylori-infected AGS cells treated with the Lactobacillus rhamnosus CCFM1119. After the Helicobacter pylori-infected AGS cells treated with the Lactobacillus rhamnosus L. GG and the Helicobacter pylori-infected AGS cells treated with the Lactobacillus rhamnosus CCFM1119 were washed with a PBS solution 5 times, 200 L of urease reagent (9 g/L NaCl, 14 g/mL phenol red, 20 mM urea, pH 6.8) was added to the Helicobacter pylori-infected AGS cells treated with the Lactobacillus rhamnosus L. GG and the Helicobacter pylori-infected AGS cells treated with the Lactobacillus rhamnosus CCFM1119 respectively, and the cells were cultured in an incubator at 37 C. in 5% CO.sub.2 for 2 h to obtain culture solutions.

(30) The absorbance of the culture solutions of different groups was measured at a wavelength of 550 nm by using a microplate reader. The adhesion rate determined by subtracting the absorbance of the blank group from the absorbance of the model group is 100%. The relative adhesion rate was the value obtained by the absorbance of the remaining groups minus the absorbance of the blank group versus the value of obtained by the absorbance of the model group minus the absorbance of the blank group. The measurement results are shown in FIG. 1.

(31) It can be seen from FIG. 1 that after treatment with Lactobacillus rhamnosus CCFM1119, the adhesion rate of Helicobacter pylori to the AGS cells decreased significantly, from 100% in the model group (Hp group) to about 70%. However, Lactobacillus rhamnosus L. GG did not significantly reduce the adhesion rate of Helicobacter pylori to the AGS cells, and the adhesion rate of Helicobacter pylori hardly changed. The result shows that Lactobacillus rhamnosus CCFM1119 can effectively reduce the adhesion of Helicobacter pylori to the AGS cells.

Example 4: Effect of Lactobacillus rhamnosus on Gastrointestinal Symptoms in Helicobacter pylori-Positive Patients

(32) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(33) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(34) The components of the protectant include: 130 g/L skimmed milk powder.

(35) 26 Helicobacter pylori-positive infected patients (Table 2 shows the population distribution of recruited patients, and the difference in baseline conditions between the two groups of people is of no statistical significance) were recruited. The 26 Helicobacter pylori-positive infected patients were randomly divided into 2 groups, including 13 in a placebo group (Placebo) and 13 in a Lactobacillus rhamnosus CCFM1119 group (CCFM1119).

(36) The placebo group (Placebo) took placebo twice a day, and the Lactobacillus rhamnosus CCFM1119 group took the bacterial powder twice a day. The whole experiment period is 1 month (the placebo and the Lactobacillus rhamnosus bacterial powder contain different components, but the appearances and packaging of the products are the same without significant difference). The two groups of patients filled out the Gastrointestinal Symptom Rating Scale (GSRS) before and after the experiment (Table 3 shows the Gastrointestinal Symptom Rating Scale). The average value of each group was calculated to characterize the gastrointestinal health status of each group, and the improvement of gastrointestinal symptoms of patients after the experiment was evaluated. The measurement results are shown in FIG. 2.

(37) It can be seen from FIG. 2 that before and after the experiment, the Helicobacter pylori-positive infected patients in the placebo group (Placebo) had a GSRS score 5 or above. However, the Helicobacter pylori-positive infected patients in the Lactobacillus rhamnosus CCFM1119 group (CCFM1119) had a GSRS score of about 6 before the start of the experiment, and dropped to about 2 after the end of the experiment. The result shows that Lactobacillus rhamnosus CCFM1119 can significantly relieve the gastrointestinal symptoms of Helicobacter pylori-infected patients.

(38) TABLE-US-00002 TABLE 2 Population distribution of recruited Helicobacter pylori-positive infected patients Number Drinker/ Smoker/ of people Male/ non- non- Group (N) Age female drinker smoker Placebo 13 48.15 3.70 2/11 1/12 0/13 CCFM1119 13 51.67 4.26 4/9 2/11 0/13

(39) TABLE-US-00003 TABLE 3 Gastrointestinal Symptom Rating Scale Item Manifestations Score Abdominal pain (Physical discomfort, subjective No or transient pain 0 point feeling of pain. The type of pain can be based on Occasional pain, affecting some 1 point the patient's description and the nature of the pain. social activities For example, upper abdominal pain, according to its Prolonged pain, requiring treatment, 2 points typical location, can be considered as an acid- and affecting many social activities related symptom, just like the characteristics of Severe pain, affecting all social 3 points eating and antacid relief. Hernia pain is usually activities severe and is located in the lower abdomen. Persistent dull pain usually lasts for a few hours and is of moderate severity. Graded according to the degree, frequency, duration, relieving factors and social activity impacts.) Heartburn (Manifested as discomfort or burning No or transient heartburn 0 point sensation behind the breastbone. Graded according Occasional short-time heartburn 1 point to the degree, frequency, duration, relieving factors Frequent and prolonged discomfort, 2 points and social activity impacts.) requiring treatment to relieve Persistent discomfort, which can 3 points only be temporarily relieved by antacids Acid reflux (Manifested as a sudden occurrence of No or short reflux 0 point acid reflux. Graded according to the degree, Occasional offensive reflux 1 point frequency, duration, relieving factors and social Reflux once or twice a day, requiring 2 points activity impacts.) treatment to relieve Reflux several times a day, antacid 3 points treatment for which can only provide short-time and insignificant relief Upper abdomen tightness (Manifested as upper No or transient tightness sensation 0 point abdomen tightness that can be relieved by eating Occasional short-time discomfort, 1 point or antacids. Without eating or taking medicine, the requiring no food or antacids tightness sensation progresses to pain. Graded between meals according to the degree, frequency, duration, Discomfort with prolonged time and 2 points relieving factors and social activity impacts.) increased frequency, requiring food or antacids between meals to relieve Persistent discomfort, frequently 3 points requiring for food or antacids Nausea and vomiting (Representing nausea and No nausea 0 point vomiting worsened by nausea. Graded according to Occasional transient discomfort 1 point the degree, frequency, duration, relieving factors Frequent and prolonged nausea, no 2 points and social activity impacts.) vomiting Persistent nausea, frequent vomiting 3 points Borborygmus (Manifested as a rumbling in the No or transient borborygmus 0 point abdomen. Graded according to the degree, Short-time and occasional 1 point frequency, duration, relieving factors and social borborygmus and discomfort activity impacts.) Frequent and prolonged 2 points borborygmus, which can be controlled by activities without affecting social activities Persistent borborygmus, seriously 3 points affecting social activities Abdominal distension (Manifested as gas swelling in No or transient abdominal distension 0 point the abdomen. Graded according to the degree, Short-time occasional abdominal 1 point frequency, duration, relieving factors and social distention activity impacts.) Frequent and long-time abdominal 2 points distension, which can be controlled by adjusting the dress Persistent abdominal distension, 3 points seriously affecting social activities Belching (Graded according to the degree, No or transient belching 0 point frequency, duration, relieving factors and social Occasional offensive belching 1 point activity impacts.) Frequent belching, affecting some 2 points social activities Frequent belching, seriously 3 points affecting social activities Increased flatus (Graded according to the degree, No increase in flatus 0 point frequency, duration, relieving factors and social Short-time occasional discomfort 1 point activity impacts.) Frequent and prolonged discomfort, 2 points affecting some social activities Increased number of attacks, 3 points seriously affecting social activities Decreased defecation (Graded according to the Once a day 0 point degree, frequency, duration, relieving factors and Once every three days 1 point social activity impacts.) Once every five days 2 points Once every seven days or less 3 points Increased defecation (Graded according to the Once a day 0 point degree, frequency, duration, relieving factors and Three times a day 1 point social activity impacts.) Five times a day 2 points Seven times a day or more 3 points Loose stools (Graded according to the degree, Standard consistency 0 point frequency, duration, relieving factors and social Slightly loose 1 point activity impacts.) Mushy 2 points Watery 3 points Hard feces (Graded according to the degree, frequency, Standard consistency 0 point duration, relieving factors and social Slightly hard 1 point activity impacts.) Hard 2 points Hard and segmented 3 points A sense of urgency to defecate (Manifested as a Normal control 0 point sense of urgency to defecate, the feeling of inability Occasional sense of urgency to 1 point to control defecation. Graded according to the defecate degree, frequency, duration, relieving factors and Frequent sense of urgency to 2 points social activity impacts.) defecate and the sudden need to go to the toilet, affecting social activities Fecal incontinence 3 points Feeling of incomplete defecation No feeling of incomplete defecation 0 point and effortless defecation Occasional difficulty in defecation; 1 point occasional feeling of incomplete defecation Definite difficulty in defecation, 2 points usually accompanied by a feeling of incomplete defecation Extreme difficulty in defecation; 3 points routine feeling of incomplete defecation

Example 5: Effect of Lactobacillus rhamnosus on Colonization Amount and Clearance Rate of Helicobacter pylori in Helicobacter pylori-Positive Patients

(40) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline for 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(41) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(42) The components of the protectant include: 130 g/L skimmed milk powder.

(43) 26 Helicobacter pylori-positive infected patients (Table 2 shows the population distribution of recruited patients, and the difference in baseline conditions between the two groups of people is of no statistical significance) were recruited. The 26 Helicobacter pylori-positive infected patients were randomly divided into 2 groups, including 13 in a placebo group (Placebo) and 13 in a Lactobacillus rhamnosus CCFM1119 group (CCFM1119).

(44) The placebo group (Placebo) took placebo twice a day, and the Lactobacillus rhamnosus CCFM1119 group took the bacterial powder twice a day. The whole experiment period is 1 month (the placebo and the Lactobacillus rhamnosus bacterial powder contain different components, but the appearances and packaging of the products are the same without significant difference). The 14C-urea breath test values of the Helicobacter pylori-positive infected patients in the placebo group and the Lactobacillus rhamnosus CCFM1119 group were measured by a 14C-urea breath test reagent bag and a tester before and after the experiment respectively, to evaluate the amount of colonization and clearance rate of Helicobacter pylori in the patients. The measurement results are shown in FIG. 3 and Table 4.

(45) The evaluation criterion for the amount of colonization of Helicobacter pylori is: The decrease in the 14C-urea breath test values of Helicobacter pylori-positive infected patients after the end of the experiment compared with the 14C-urea breath test values of the Helicobacter pylori-positive infected patients before the start of the experiment.

(46) The evaluation criterion for the clearance rate of Helicobacter pylori is as follows: The threshold of the clinical 14C-urea breath test value is 100, that is, if the 14C-urea breath test value is greater than or equal to 100, the infection of Helicobacter pylori is positive, and if the 14C-urea breath test value is lower than 100, the infection of Helicobacter pylori is negative. After the end of the experiment, whether the Helicobacter pylori-positive infected patient becomes negative is used to evaluate the increase of the clearance rate of Helicobacter pylori-positive infected patients.

(47) It can be seen from FIG. 3 that after the end of the experiment, the 14C-urea breath test values of the Helicobacter pylori-positive infected patients in the placebo group (Placebo) were almost the same as before the start of the experiment. However, the 14C-urea breath test values of the Helicobacter pylori-positive infected patients in the Lactobacillus rhamnosus CCFM1119 group (CCFM1119) decreased by about 100 compared with that before the start of the experiment, and the two groups have a significant difference. The result indicates that the Lactobacillus rhamnosus CCFM1119 can significantly reduce the amount of colonization of Helicobacter pylori in Helicobacter pylori-infected patients.

(48) It can be seen from Table 4 that after the end of the experiment, 2 out of 13 people in the placebo group (Placebo) became Helicobacter pylori negative, and the negative rate was 15.38%. Of the 13 people in the Lactobacillus rhamnosus CCFM1119 group, 8 people became Helicobacter pylori negative, and the negative rate was as high as 61.54%, which was significantly higher than that of the placebo group. The result indicates that the Lactobacillus rhamnosus CCFM1119 can significantly improve the clearance rate of Helicobacter pylori in the Helicobacter pylori-infected patients.

(49) TABLE-US-00004 TABLE 4 Clearance rate of Helicobacter pylori-positive patients in different groups Number Negative of people Positive Negative rate Group (N) n n (%) Placebo 13 11 2 15.38 CCFM1119 13 5 8 61.54* Note: *indicates a significant difference compared with the placebo group (p < 0.05).

Example 6: Application of Lactobacillus rhamnosus

(50) Lactobacillus rhamnosus CCFM1119 can be used to prepare bacterial powder. The specific preparation process of the bacterial powder is as follows:

(51) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline for 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(52) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(53) The components of the protectant include: 130 g/L skimmed milk powder.

(54) 2 g of Lactobacillus rhamnosus CCFM1119 bacterial powder was accurately weighed and dissolved in 10 mL of sterile physiological saline to obtain an original bacterial suspension. 0.5 mL of the original bacterial suspension was taken and added to 4.5 mL of sterile physiological saline, and the solution was uniformly mixed. At this time, the original bacterial suspension is diluted 10 times and recorded as n=10. 0.5 mL of the diluted bacterial suspension was taken and added to 4.5 mL of sterile physiological saline. At this time, the original bacterial suspension is diluted 100 times and recorded as n=10.sup.2. By analogy, the original bacterial suspension was diluted 1.010.sup.8 times. 0.1 mL of the bacterial suspensions with dilution multiples of 1.010.sup.6 (n=10.sup.6), 1.010.sup.7 (n=10.sup.7) and 1.010.sup.8 (n=10.sup.8) were taken and inoculated into MRS solid medium, and put the medium upside down in an anaerobic box. The bacterial suspensions were cultured in an anaerobic box at 37 C. for 2 d to 3 d, and the live bacteria were counted. The measurement was performed once a week for one month to determine the storage stability of the Lactobacillus rhamnosus CCFM1119 bacterial powder. The measurement results are shown in FIG. 4.

(55) It can be seen from FIG. 4 that the initial live count of the Lactobacillus rhamnosus CCFM1119 bacterial powder was higher than 10.sup.10 CFU/bag, and meets the product specifications. During storage for one month, the live count of the Lactobacillus rhamnosus CCFM1119 bacterial powder did not decrease significantly compared with the initial period, and the live count was always higher than 10.sup.10 CFU/bag, indicating that the properties of the Lactobacillus rhamnosus CCFM1119 bacterial powder are relatively stable during the short-term storage of one month.

Example 7: Application of Lactobacillus rhamnosus

(56) Lactobacillus rhamnosus CCFM1119 can be used to prepare a capsule product. The specific preparation process of the capsule product is as follows:

(57) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline for 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was added to a sodium alginate solution with a concentration of 30 g/L to a concentration of 210.sup.9 CFU/mL, and then the solution was fully stirred to make the cells of Lactobacillus rhamnosus CCFM1119 evenly dispersed in the sodium alginate solution to obtain a mixed solution. The mixed solution was squeezed into a calcium chloride solution with a concentration of 20 g/L to form colloidal particles. After the formed colloidal particles were statically solidified for 30 min, the colloidal particles were filtered and collected. The collected colloidal particles were freeze-dried for 48 h to obtain a powder. Medicinal capsules were filled with the powder to obtain a capsule product.

(58) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

Example 8: Application of Lactobacillus rhamnosus

(59) Lactobacillus rhamnosus CCFM1119 can be used to prepare tablets. The specific preparation process of the tablets is as follows:

(60) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(61) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(62) The components of the protectant include: 130 g/L skimmed milk powder.

(63) 25.7 parts by weight of the Lactobacillus rhamnosus CCFM1119 bacterial powder, 55.0 parts by weight of starch, 4.5 parts by weight of a cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 parts by weight of talc, 1.0 part by weight of sucrose, and 1.0 part by weight of water were weighed to obtain raw materials. The raw materials were mixed to obtain wet granules. The wet granules were compressed using a tablet press of Zhongnan Pharmaceutical Machinery Factory, and then the tablets were dried using a small medicine dryer of Qingzhou Yikang Traditional Chinese Medicine Machinery Co., Ltd. to obtain the tablets.

Example 9: Application of Lactobacillus rhamnosus

(64) Lactobacillus rhamnosus CCFM1119 can be used to prepare fermented milk. The specific preparation process of the fermented milk is as follows:

(65) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(66) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(67) The components of the protectant include: 130 g/L skimmed milk powder.

(68) The Lactobacillus rhamnosus CCFM1119 bacterial powder was mixed with a commercial dry powder starter Lactobacillus bulgaricus and a commercial dry powder starter Streptococcus thermophilus at a mass ratio of 1:1:1 to obtain a starter. Sugar was added to fresh milk to a concentration of 50 g/L to obtain a mixed solution. The mixed solution was homogenized at 65 C. and 20 MPa, and then heated and sterilized at 95 C. for 5 min to obtain a fermentation raw material. After the fermentation raw material was cooled to 35 C., the starer was inoculated in the fermentation raw material at an inoculation amount of 0.03% (v/v), and fermentation was performed at 35 C. for 16 h to obtain the fermented milk. After the fermented milk was stood at 42 C. for 4 h for curdling, the fermented milk was refrigerated at 4 C. for 24 h for aging to obtain the fermented milk product.

Example 10: Application of Lactobacillus rhamnosus

(69) Lactobacillus rhamnosus CCFM1119 can be used to prepare soybean milk. The specific preparation process of the soybean milk is as follows:

(70) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(71) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(72) The components of the protectant include: 130 g/L skimmed milk powder.

(73) Soybeans were soaked at a temperature of 80 C. for 2 h and then soybean hulls were removed to obtain dehulled soybeans. The dehulled soybeans were drained to remove soaking water and boiling water was added for performing pulping to obtain soybean milk. The soybean milk was kept at a temperature higher than 80 C. for 12 min to obtain cooked soybean milk. The cooked soybean milk was filtered with a 150-mesh screen and centrifugally separated to obtain crude soybean milk. The crude soybean milk was heated to a temperature of 140-150 C. and then quickly introduced into a vacuum cooling chamber and vacuumized, so that off-flavor substances in the crude soybean milk were quickly discharged with water vapor, and the cooked soybean milk was obtained. After the cooked soybean milk was cooled to about 37 C., the Lactobacillus rhamnosus CCFM1119 bacterial powder was added to the cooked soybean milk to a concentration of not less than 110.sup.6 CFU/mL to obtain the soybean milk (the soybean milk needs to be stored under refrigeration at 4 C.).

Example 11: Application of Lactobacillus rhamnosus

(74) Lactobacillus rhamnosus CCFM1119 can be used to prepare a fruit and vegetable beverage. The specific preparation process of the fruit and vegetable beverage is as follows:

(75) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(76) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(77) The components of the protectant include: 130 g/L skimmed milk powder.

(78) Fresh fruits and vegetables were washed and squeezed to obtain fruit and vegetable juice. The fruit and vegetable juice was heated and sterilized at a high temperature of 140 C. for 2 seconds to obtain the sterilized fruit and vegetable juice. After the sterilized fruit and vegetable juice was cooled to about 37 C., the Lactobacillus rhamnosus CCFM1119 bacterial powder was added to the sterilized fruit and vegetable juice to a concentration of not less than 110.sup.6 CFU/mL to obtain the fruit and vegetable beverage (the fruit and vegetable beverage needs to be stored under refrigeration at 4 C.).

Example 12: Application of Lactobacillus rhamnosus

(79) Lactobacillus rhamnosus CCFM1119 can be used to prepare a milk beverage. The specific preparation process of the milk beverage is as follows:

(80) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(81) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(82) The components of the protectant include: 130 g/L skimmed milk powder.

(83) The skimmed milk was heated and sterilized at 95 C. for 20 min and then cooled to 4 C. to obtain the raw material. The Lactobacillus rhamnosus CCFM1119 bacterial powder was added to the raw material to a concentration of not less than 110.sup.6 CFU/mL to obtain the milk beverage (the milk beverage needs to be stored under refrigeration at 4 C.).

Example 13: Application of Lactobacillus rhamnosus

(84) Lactobacillus rhamnosus CCFM1119 can be used to prepare chocolate. The specific preparation process of the chocolate is as follows:

(85) Lactobacillus rhamnosus CCFM1119 was streaked on an MRS solid medium and cultured at 37 C. for 48 h to obtain a single colony. The single colony was picked and inoculated in MRS liquid medium, and cultured at 37 C. for 18 h for activation, and activated for two consecutive generations to obtain an activation solution. The activation solution is inoculated in medium at an inoculation amount of 2% (v/v), and the activation solution was cultured at 37 C. for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8,000 g for 10 min to obtain bacterial sludge. The bacterial sludge was washed with physiological saline 3 times and then resuspended with a protectant to a concentration of 110.sup.10 CFU/mL to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37 C. for 60 min and then the culture was freeze-dried to obtain Lactobacillus rhamnosus CCFM1119 bacterial powder.

(86) The preparation method of the medium is as follows: 10% of enzymatically hydrolyzed skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved in water accounting for 87.7% of the total weight of the medium. Then the pH value of the solution was adjusted to 6.8 to obtain the medium.

(87) The components of the protectant include: 130 g/L skimmed milk powder.

(88) Cocoa mass and white granulated sugar were mixed in a mass ratio of 1:1 to 1:3, and then heated and stirred evenly to obtain a chocolate melt. First, emulsifiers (liquid lecithin, soybean phospholipid, and sorbitan monolaurate) and the Lactobacillus rhamnosus CCFM1119 bacterial powder were mixed uniformly in a mass ratio of emulsifiers:bacterial powder=(80-90):(10-20). Then fine grinding, acid removal, water removal, crystallization, and temperature adjustment were performed. Finally, a suitable model is selected for pouring and forming to obtain the chocolate (the chocolate needs to be stored under refrigeration at 4 C.).

(89) Although the disclosure has been disclosed as above in preferred examples, it is not intended to limit the disclosure. Those skilled in the art can make various alterations and modifications without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure should be defined by the claims.