A strain of Saccharomyces boulardii and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a strain of Saccharomyces boulardii separated from fermented grains of Maotai-flavor liquor and its application. The strain is specifically Saccharomyces boulardii BD‑2, and its deposit number is CGMCC No.24930. Saccharomyces boulardii BD‑2 originates from the fermented grains in the fermentation process of the traditional fermented food Maotai-flavor liquor, and is a safe strain that can be used for food; it has strong acid resistance, bile salt resistance, high temperature resistance and gastrointestinal fluid resistance, as well as high self-aggregation, hydrophobicity, antioxidant activity and Helicobacter pylori inhibitory activity, has good in vivo survival ability and excellent prebiotic properties, and can be used in the fields of food, medicine, etc., such as winemaking, and the development of related functional foods and medicines.

Description

Technical field:

[0002] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Saccharomyces boulardii separated from fermented grains of Maotai-flavor liquor and an application thereof. Background technology:

[0004] As one of the three major flavors of Chinese liquor, Maotai-flavor liquor is widely loved by consumers. Its brewing process is the application of multiple microorganisms, which will directly affect the types and quantities of aroma substances. Therefore, microorganisms are an important factor in the formation of the quality and style of Maotai-flavor liquor, and brewer's yeast is a major functional bacteria in the liquor brewing process. Saccharomyces boulardii ) is a non-pathogenic yeast. Saccharomyces boulardii not only has the characteristics of ordinary yeast in providing nutrients, but also has probiotic effects such as increasing the activity of beneficial intestinal microorganisms, inhibiting the growth of pathogens and improving the immune function of intestinal mucosa. It is currently the only yeast used as a probiotic drug to treat intestinal diseases in clinical practice. In addition, Saccharomyces boulardii can also be used as microecological preparations, feed additives, etc., and has broad application prospects.

[0005] Saccharomyces boulardii was originally isolated from tropical fruits in India. Compared with the sources of yeast screening, the natural fermentation process of Maotai-flavor liquor is more likely to obtain high-quality Saccharomyces boulardii, because the environmental conditions have already screened the yeast population, from which better strains can be isolated than other sources. Moreover, the Saccharomyces boulardii screened from the mash is green and natural, and is a safe strain that can be used in food. This will provide a basis for the development of Chinese native Saccharomyces boulardii strains with probiotic potential.

[0006] In view of this, the present invention is proposed. Summary of the invention:

[0008] The purpose of the present invention is to enrich the existing bacterial strain resources, overcome the shortcomings of the existing technology, and provide a strain of Saccharomyces boulardii with excellent probiotic properties.

[0009] One of the technical solutions provided by the present invention is a strain of Saccharomyces boulardii with excellent probiotic properties, which is special in that it is selected from the fermented grains of Maotai-flavor liquor, specifically Saccharomyces boulardii ( Saccharomyces boulardii )BD-2, which was deposited on May 19, 2022 in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No.24930.

[0010] The BD-2 strain was screened, isolated, purified from fermented grains of Maotai-flavor liquor, and identified as Saccharomyces boulardii ( Saccharomyces boulardii ).

[0011] The boulardii yeast BD-2 has the following characteristics:

[0012] (1) The characteristics of the colony and the bacteria are as follows: after culturing on YPD medium for 24 h, the colonies are white, round, opaque, large and moist, with a smooth surface, a bulge in the middle, clear edges, easy to pick up, and emit a light wine aroma; under an optical microscope, the cell morphology is observed to be large and nearly spherical, and the reproduction method is single-terminal budding or multi-terminal budding;

[0013] (2) High temperature resistance: When cultured at 30-50 °C for 30 min, the survival rate was higher than 70%;

[0014] (3) Acid resistance: It can still grow normally after being cultured at pH 2.5 for 24 h, with a survival rate of 82.08%;

[0015] (4) Alkali resistance: It can still grow normally after being cultured at pH 9.0 for 24 h, with a survival rate of 84.37%;

[0016] (5) Bile salt tolerance: The survival rate of the strain still reached 97.77% after culturing in YPD medium containing 1.0% bile salt for 24 h;

[0017] (6) Survival in simulated gastric fluid: After being treated in simulated gastric fluid at pH 3.0 for 3 h, the survival rate was 193.33%

[0018] (7) Survival in simulated intestinal fluid: After being treated in simulated intestinal fluid at pH 8.0 for 3 h, the survival rate was 79.15%;

[0019] (8) In vitro adhesion ability: After culturing at 30 °C for 3 h, the self-aggregation rate was still high, reaching 88.4%;

[0020] (9) Hydrophobicity of organic solvents: It has high hydrophobicity to n-hexane, dichloromethane, dodecane and chloroform, which are 52.01%, 54.13%, 51.4% and 54.25% respectively;

[0021] (10) Antioxidant activity: It has good antioxidant activity, with a DPPH free radical scavenging rate of 73.27%, a reducing capacity of 62.40%, and a hydroxyl free radical scavenging rate of 34.69%, all greater than 30%;

[0022] (11) Antibiotic sensitivity: The virus is resistant to 11 antibiotics. Testing showed that the virus was insensitive to 11 drug-susceptibility papers, including erythromycin, penicillin, chloramphenicol, cefotaxime, ceftriaxone, ciprofloxacin, tetracycline, kanamycin, streptomycin, gentamicin, and vancomycin.

[0023] The second technical solution provided by the present invention is the above-mentioned Saccharomyces boulardii ( Saccharomyces boulardii ) Applications of BD-2, especially in the fields of medicine, such as liquor or beer fermentation, development of drugs to inhibit Helicobacter pylori, and regulation of intestinal flora;

[0024] Furthermore, the anti-Helicobacter pylori therapeutic drug is Saccharomyces boulardii ( Saccharomyces boulardii ) BD-2 bacterial solution is used alone or in combination with standard quadruple therapy drugs;

[0025] Furthermore, the standard quadruple therapy drugs are: rabeprazole, amoxicillin, clarithromycin and bismuth potassium citrate;

[0026] Furthermore, the preparation of quadruple therapy drugs: 20 mg of rabeprazole, 1 g of amoxicillin, 0.5 g of clarithromycin, and 0.6 g of potassium citrate bismuth were prepared with water to make 100 mL of solution;

[0027] Furthermore, the above-mentioned quadruple therapy drugs are combined with Saccharomyces boulardii ( Saccharomyces boulardii ) The BD-2 bacterial solution is prepared into an anti-Helicobacter pylori therapeutic drug according to a volume ratio of 1:0.5-4;

[0028] Furthermore, the Saccharomyces boulardii ( Saccharomyces boulardii ) The concentration of BD-2 bacterial solution was 1.0×10 6 CFU / mL.

[0029] The advantages and positive effects achieved by the present invention are:

[0030] 1. The boulardii yeast provided by the present invention ( Saccharomyces boulardii BD-2 is derived from the mash during the fermentation of the traditional fermented food, Maotai-flavor liquor, and is a safe strain of bacteria.

[0031] 2. The boulardii yeast provided by the present invention ( Saccharomyces boulardii ) BD-2 has strong resistance to acid, bile salts, high temperature and gastrointestinal fluid. It also has high self-aggregation, hydrophobicity, antioxidant activity and Helicobacter pylori inhibitory activity. It has good survival ability in the body and excellent prebiotic properties. It can be used in the fields of medicine, such as brewing and developing related functional drugs. Description of the drawings:

[0033] Figure 1 Figure 2 is a colony morphology and cell morphology diagram of Saccharomyces boulardii BD-2 in the present invention;

[0034] Figure 2 It is a phylogenetic tree diagram of Saccharomyces boulardii BD-2 in the present invention. Specific implementation method:

[0036] For better understanding and implementation, the present invention will be described in detail below in conjunction with embodiments. Obviously, the implementation cases described are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0037] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0038] The technical solution of the present invention is further illustrated by the following examples.

[0039] Example 1 Screening and Isolation of Saccharomyces boulardii BD-2

[0040] YPD medium composition: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L, agar 1.5 g / L, and the rest is water.

[0041] Preparation method: Dissolve the above ingredients in 1 L of distilled water, then dispense into 100 mL conical flasks and sterilize at 115°C for 20 min. In order to eliminate bacterial interference during the screening process, add 60 μg / mL of chloramphenicol.

[0042] Take 10 g of Lutaichun Maotai-flavor liquor mash in a 250 mL sterile triangular flask, add 90 mL of sterilized saline, seal the bottle mouth with a breathable sealing film, and activate at 30 °C for 30 min; take out the bacterial suspension and dilute it with sterile water to obtain a diluent;

[0043] The dilution was evenly spread on the YPD medium and cultured in a constant temperature incubator at 30°C for 1-2 days. A single colony with typical yeast characteristics was selected and the colony was streaked onto the YPD medium to further purify the yeast (purify 2-3 times depending on the separation effect) until a single strain was isolated for molecular identification.

[0044] The above process is a preliminary screening of yeast, which is to screen out single colonies with different appearance and aroma in the culture medium. Among them, the colony of strain BD-2 is white, round, opaque, large and moist, with a smooth surface, a bulge in the middle of the colony, clear edges, easy to pick up, and exudes a light wine aroma; its cell morphology is observed under an optical microscope, and the individual cells are large and nearly spherical, and the reproduction method is single-terminal budding or multi-terminal budding. Colony morphology and microscopic morphology are as follows Figure 1 .

[0045] Example 2 Identification of Saccharomyces boulardii BD-2

[0046] The pure strain BD-2 was selected and inoculated into YPD liquid medium and cultured at 30 °C for 24 h for activation. An appropriate amount of activated bacterial liquid was taken and centrifuged to collect the bacteria. The genomic DNA of the pure cultured target strain was extracted using the kit extraction method. The upstream primer NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3' and the downstream primer NL4: 5'-GGTCCGTGTTTCAAGACGG-3' were used to amplify the conserved region of the 26S rDNA gene and sent to a sequencing company for sequencing analysis.

[0047] The PCR reaction conditions were as follows: denaturation at 95 °C for 15 s; annealing at 55 °C for 15 s; extension at 72 °C for 15 s; and 30 cycles with extension at 72 °C for 10 min.

[0048] Sequence alignment was performed using the BLAST function of NCBI in the GenBank database, and the phylogenetic tree is shown in Figure 2 The analysis determined the classification of the strains as: Saccharomyces boulardii , so it was named Saccharomyces boulardii ( Saccharomyces boulardii )BD-2 and deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.24930.

[0049] Its 26S rDNA gene sequence is as follows (SEQ ID NO.1):

[0050] .

[0051] Example 3 Determination of tolerance and probiotic properties of Saccharomyces boulardii BD-2

[0052] The tolerance and probiotic properties of Saccharomyces boulardii BD-2 isolated from liquor mash were determined, and the acid resistance, bile salt resistance, high temperature resistance, gastrointestinal fluid resistance, self-aggregation, hydrophobicity, antioxidant activity, antibacterial and antibiotic sensitivity of Saccharomyces boulardii BD-2 were determined.

[0053] Preparation of Saccharomyces boulardii suspension: The strain was activated in YPD liquid medium at 30 ℃ for two generations, the supernatant was discarded after centrifugation to obtain the bacteria, and physiological saline was added to wash three times to remove the residual medium, and then physiological saline was added to make a bacterial suspension, and the bacterial concentration was adjusted to 1.0×10 6 CFU / mL(OD 595nm =1.0), and the bacterial suspension prepared by this method was used for subsequent performance tests.

[0054] Determination of tolerance of Saccharomyces boulardii BD-2 (1) Determination of high temperature tolerance

[0055] Take 100 μL of the suspension of Saccharomyces boulardii BD-2 and spread it on YPD solid culture medium. Incubate it at 35 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃ incubators for 30 min. At the same time, culture it at normal temperature (30 ℃) as a negative control. Observe whether there are colonies on the plate and count them to compare their growth conditions.

[0056] Table 1 Relative survival rate of Saccharomyces boulardii at different temperatures

[0057]

[0058] The growth of strain BD-2 is shown in Table 1. When the growth temperature is between 30°C and 50°C, the survival rate of the strain can reach more than 70%. At the human body temperature of 37°C, the survival rate is as high as 86.67%, and at 50°C, the survival rate reaches 70%. When the treatment temperature is higher than 50°C, the number of live bacteria of BD-2 drops sharply, and the survival rate drops to 16.67%. This shows that strain BD-2 has strong heat resistance.

[0059] (2) Determination of acid resistance

[0060] The suspension of Saccharomyces boulardii was inoculated into YPD liquid medium with pH values ​​of 2.5, 3.0, and 3.5 at a 2% inoculation rate, and cultured in a shaking incubator at 30 °C and 180 r / min for 24 h. At the same time, YPD liquid medium with a normal pH value (pH 5.3) was used as a negative control, and the OD was measured using a UV spectrophotometer. 595nm , and compare their growth conditions.

[0061] Table 2 Growth of Saccharomyces boulardii at different pH values

[0062]

[0063] As shown in Table 2, strain BD-2 can grow normally at pH 3.5 and pH 3.0. Although the OD value is slightly reduced at pH 2.5, it can still grow, and the survival rate reaches 82.08%, indicating that strain BD-2 has good acid resistance. The pH of human gastric juice changes according to the environment and usually remains at around 3.0, so strain BD-2 can survive in human gastric juice and play a probiotic role.

[0064] (3) Determination of alkali resistance

[0065] The suspension of Saccharomyces boulardii was inoculated at a 2% inoculum into YPD liquid medium with pH values ​​of 7.5, 8.0, 8.5, and 9.0, respectively, and cultured in a shaking incubator at 30 °C and 180 r / min for 24 h. At the same time, YPD liquid medium with a normal pH value (pH 5.3) was used as a negative control, and the OD was measured using a UV spectrophotometer. 595nm , and compare their growth conditions.

[0066] Table 3 Growth of Saccharomyces boulardii at different pH values

[0067]

[0068] As shown in Table 3, strain BD-2 can grow normally at pH 7.5 and pH 8.0, but the OD value begins to decrease at pH 8.5, but it can still grow, and the survival rate reaches 87.70%, indicating that strain BD-2 has good alkali resistance. The pH of human intestinal fluid is usually maintained at around 8.0, so strain BD-2 can survive in human intestinal fluid and play a probiotic role.

[0069] (4) Determination of bile salt tolerance

[0070] 100 μL of the Saccharomyces boulardii suspension was spread on YPD solid culture medium with bile salt content of 0.3%, 0.5%, 1.0%, 1.2%, 1.5%, 1.8% and 2.0%, respectively, and cultured in a 30 ℃ incubator for 24 h. At the same time, conventional YPD culture medium without bile salt was used as a negative control to observe whether colonies were generated on the plate and count them, and compare their growth conditions.

[0071] Table 4 Survival rate of Saccharomyces boulardii at different bile salt contents

[0072]

[0073] As an intestinal probiotic, it needs to have a certain bile salt tolerance to ensure that it can pass through the digestive tract smoothly, survive and colonize in the intestine, and play a beneficial role. The bile salt tolerance test results of strain BD-2 are shown in Table 4. When the bile salt content is between 0 and 1.0%, the number of live bacteria of strain BD-2 basically does not change, and the survival rate is high; when the bile salt content exceeds 1.0%, the survival rate decreases slightly, but still exceeds 60%. Therefore, strain BD-2 has a high bile salt tolerance. The normal bile salt content in the human body is only 0.3%-0.5%, and strain BD-2 can survive well in the human body and play a probiotic role.

[0074] Determination of the probiotic properties of Saccharomyces boulardii BD-2 (1) Determination of resistance to gastrointestinal fluid

[0075] Simulated artificial gastric juice: 8 g sodium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, 3.5 g pepsin, 1000 mL distilled water, adjust the pH to 3.0 with 1 mol / L hydrochloric acid, and sterilize at 115 ℃ for 20 min.

[0076] Simulated artificial intestinal fluid: 8 g sodium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, 1 g trypsin, 3 g bile salt, 1000 mL distilled water, adjust the pH to 8.0 with 1 mol / L sodium hydroxide, and sterilize at 115 ℃ for 20 min.

[0077] ① Take 1 mL of Saccharomyces boulardii suspension and add it to 9 mL of simulated artificial gastric fluid. Place it in a 30 ℃ incubator. Take samples for gradient dilution after 0 h and 3 h, spread them on YPD solid plates, and calculate the survival rate.

[0078] ② Take 1 mL of Saccharomyces boulardii suspension and add it to 9 mL of simulated artificial intestinal fluid. Place it in a 30 ℃ incubator. Take samples after 0 h and 3 h, perform gradient dilution, spread them on YPD solid plates, and calculate the survival rate.

[0079] Table 5 Survival rate of Saccharomyces boulardii in simulated artificial gastrointestinal fluid

[0080]

[0081] The growth of the strains after treatment with simulated artificial intestine and gastric juice is shown in Table 5. After being treated in simulated gastric juice at pH 3.0 for 3 h, the survival rate of strain BD-2 in gastric juice was 193.33%, showing excellent tolerance; after being treated in simulated intestinal juice at pH 8.0 for 3 h, the survival rate of strain BD-2 in intestinal juice was 79.15%, also showing good tolerance.

[0082] (2) Determination of self-cohesion

[0083] The boulardii yeast suspension was cultured in a 30 °C incubator for 0 h, and the OD of the bacterial suspension was measured using a spectrophotometer. 595nm Then, after culturing in a 30°C incubator for 3 h, carefully aspirate the supernatant of the solution and measure the OD value again. 595nm Value. The self-cohesion is calculated as follows:

[0084] Self-aggregation rate % = [1-(A j / A i )×100]

[0085] Among them, A i is the OD at 0 h of culture 595 nm Value, A j OD after 3 h of culture595 nm value.

[0086] The evaluation of the self-aggregation ability of strains has been used as an in vitro method to screen strains with probiotic potential. The formation of self-aggregates can shield the central cells from harmful external environments. 595nm The self-aggregation rate of strain BD-2 was found to be higher, reaching 88.4%.

[0087] (3) Determination of hydrophobicity

[0088] Add 3 mL of Saccharomyces boulardii suspension to 1 mL of solvent (n-hexane, dichloromethane, ethyl acetate, dodecane, chloroform), react at 30 °C for 10 min, shake and mix, balance the temperature, and then continue to culture at 30 °C for 3 h. Take 1 mL of the aqueous phase at 0 h and 3 h to measure the OD 595nm Value. The hydrophobicity is calculated as follows:

[0089] Hydrophobicity%=[1-(A j / A i )×100]

[0090] Among them, A i is the OD at 0 h of culture 595 nm Value, A j The OD after 3 h of culture 595 nm value.

[0091] Hydrophobicity is widely used to evaluate the adhesion of probiotics, and the specific adhesion of probiotics to intestinal epithelial cells helps them colonize in the intestine, inhibit the colonization of pathogens in the intestine, and improve the body's immunity. The test results showed that strain BD-2 had high hydrophobicity to n-hexane, dichloromethane, dodecane and chloroform, which were 52.01%, 54.13%, 51.4% and 54.25% respectively, and its hydrophobicity to ethyl acetate was 19.52%, which was at a relatively low level.

[0092] (4) Determination of antioxidant activity

[0093] ① Determination of reducing power

[0094] The reducing ability was determined by the potassium ferrocyanide method. Take 0.5 mL of the boulardii yeast suspension, 0.2 mol / L PBS, and 1 g / 100 mL potassium ferrocyanide solution, mix them evenly in an EP tube, react in a 50 ℃ water bath for 20 min, and quench in ice. Then add 0.5 mL of 10 g / 100 mL trichloroacetic acid solution, centrifuge at 4000 r / min for 5 min, take 1 mL of the supernatant, mix it with 1 mL of ultrapure water and 1 mL of 0.1 g / 100 mL ferric chloride solution, and let it stand for 10 min. Use PBS buffer instead of the bacterial suspension as a blank control, and measure the OD by UV spectrophotometry. 700nm Numerical,reduction capability is calculated as follows:

[0095] Reduction ability (%) = (A1-A0) / A0×100

[0096] Among them, A0 is the absorbance value of the blank group; A1 is the absorbance value of the sample.

[0097] ② Ability to scavenge hydroxyl free radicals

[0098] Take 1 mL of 5 mmol / L ferrous sulfate solution, 5 mmol / L salicylic acid ethanol solution, and 3 mmol / L hydrogen peroxide solution into a centrifuge tube, add 2 mL of Saccharomyces boulardii suspension, add 5 mL of distilled water, react in a 30 ℃ water bath for 15 min, centrifuge at 6000 r / min for 10 min, take the supernatant and measure OD with a UV spectrophotometer 510 nm The absorbance value was measured and distilled water was used as a blank control. The clearance rate was calculated as follows:

[0099] Clearance rate (%) = (A0-A1) / A0×100

[0100] Among them, A0 is the OD value of the control group; A1 is the OD value of the sample.

[0101] ③ DPPH free radical scavenging ability

[0102] Take 2 mL of Saccharomyces boulardii suspension and add it to 1 mL of 0.2 mmol / L DPPH solution and mix well. Incubate the mixture at 30°C in the dark for 30 min and centrifuge at 6000 r / min for 10 min. Take the supernatant and use an equal volume of anhydrous ethanol instead of DPPH as a blank. Use distilled water as a control and measure the OD 517 nm The clearance rate is calculated as follows:

[0103] Clearance rate (%) = [1-(A0-A1) / A2] × 100

[0104] Among them, A0 is the OD value of the blank group, A1 is the OD value of the sample group, and A2 is the OD value of the control group.

[0105] Calculations show that strain BD-2 has the highest DPPH free radical scavenging activity of 73.27%, a reducing capacity of 62.40%, and a hydroxyl free radical scavenging activity of 34.69%, all of which are greater than 30%, indicating that the boulardii yeast of the present invention has good antioxidant activity.

[0106] (5) Antibiotic sensitivity test

[0107] Take 100 μL of Saccharomyces boulardii suspension and spread it evenly on YPD solid medium. Stick antibiotic paper on the surface, incubate at 30 ℃ for 24 h, measure the diameter of the inhibition zone with a vernier caliper, and evaluate and compare the results with the CLSI (Clinical Laboratory Standards Institute) 2012 standard.

[0108] Table 6 Antibiotic sensitivity of Saccharomyces boulardii

[0109]

[0110] Note: “R” indicates resistance.

[0111] The results of antibiotic sensitivity experiments showed that Saccharomyces boulardii BD-2 was insensitive to 11 drug-susceptibility papers, including erythromycin, penicillin, chloramphenicol, cefotaxime, ceftriaxone, ciprofloxacin, tetracycline, kanamycin, streptomycin, gentamicin and vancomycin, and had strong resistance to these 11 antibiotics.

[0112] Example 4 Inhibitory Effect of Saccharomyces boulardii BD-2 on Helicobacter pylori

[0113] The Helicobacter pylori inhibitory activity of Saccharomyces boulardii BD-2 was determined to determine the antibacterial ability of Saccharomyces boulardii BD-2 and its inhibitory ability against Helicobacter pylori in combination with quadruple therapy drugs used in medicine to treat Helicobacter pylori.

[0114] Preparation of Saccharomyces boulardii suspension: The strain was activated in YPD liquid medium at 30 ℃ for two generations, the supernatant was discarded after centrifugation to obtain the bacteria, and physiological saline was added to wash three times to remove the residual medium, and then physiological saline was added to make a bacterial suspension, and the bacterial concentration was adjusted to 1.0×10 6 CFU / mL(OD 595nm =1.0).

[0115] Preparation of quadruple therapy drugs: 20 mg of rabeprazole, 1 g of amoxicillin, 0.5 g of clarithromycin, and 0.6 g of potassium citrate bismuth were prepared with water to make 100 mL of solution.

[0116] The antibacterial activity of Saccharomyces boulardii strains was determined by double-layer agar diffusion method. First, 10 mL of 2% (w / v) water agar medium was added to the sterilized plate. After the water agar solidified, it was placed in a sterilized Oxford cup. Helicobacter pylori (1×10 8 CFU / mL, add 1mL), mix well and pour into the plate. After the upper culture medium solidifies, take out the Oxford cup and add each sample into different wells: 180 μL BD-2 bacterial suspension, 180 μL quadruple drug, 120 μL quadruple drug + 60 μL BD-2 bacterial suspension (1:0.5), 90 μL quadruple drug + 90 μL BD-2 bacterial suspension (1:1), 60 μL quadruple drug + 120 μL BD-2 bacterial suspension (1:2), 45 μL quadruple drug + 135 μL BD-2 bacterial suspension (1:3) and 36 μL quadruple drug + 144 μL BD-2 bacterial suspension (1:4). The plates were cultured at 37 ℃ under microaerobic conditions for 3 days, and the diameter of the transparent inhibition zone was measured.

[0117] Table 7 Inhibitory activity of Saccharomyces boulardii against Helicobacter pylori

[0118]

[0119] The experimental results show that strain BD-2 has a certain inhibitory activity against Helicobacter pylori. Although the diameter of the inhibition zone is not as large as that of the positive control quadruple drug, when the ratio of quadruple drug: BD-2 is 1:0.5 and 1:1, it can basically achieve the same effect as the positive control. It is preliminarily inferred that Saccharomyces boulardii can replace part of the quadruple drug to treat Helicobacter pylori. Moreover, according to the antibiotic sensitivity results, Saccharomyces boulardii is resistant to antibiotics and can be used in combination with antibiotics to reduce the imbalance of intestinal flora caused by antibiotic treatment. Therefore, Saccharomyces boulardii can be tried in combination with quadruple therapy to treat Helicobacter pylori, in order to reduce the incidence of adverse reactions caused by drugs and improve the safety of Helicobacter pylori treatment.

[0120] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, any modification, equivalent replacement, improvement, etc. made without departing from the technical concept of the present invention should be included in the protection scope of the present invention.

Claims

1. A boulardii yeast, characterized in that Specifically, Saccharomyces boulardii ( Saccharomyces boulardii )BD-2, the accession number is CGMCC No.24930.

2. The boulardii yeast according to claim 1 ( Saccharomyces boulardii ) Application of BD-2, characterized in that, It is used in the preparation of drugs for inhibiting Helicobacter pylori.

3. The use according to claim 2, characterized in that The drug for inhibiting Helicobacter pylori is Saccharomyces boulardii ( Saccharomyces boulardii )BD-2 bacterial solution.

4. The use according to claim 2, characterized in that The drug for inhibiting Helicobacter pylori is Saccharomyces boulardii ( Saccharomyces boulardii ) BD-2 bacterial solution combined with standard quadruple therapy drugs; The standard quadruple therapy drugs are: rabeprazole, amoxicillin, clarithromycin and bismuth potassium citrate.

5. The use according to claim 4, characterized in that The quadruple therapy drug and the Saccharomyces boulardii BD-2 bacterial solution are prepared into an anti-Helicobacter pylori therapeutic drug in a volume ratio of 1:0.5-4; Preparation of the quadruple therapy drug: 20 mg of rabeprazole, 1 g of amoxicillin, 0.5 g of clarithromycin, and 0.6 g of potassium citrate bismuth are prepared into 100 mL of liquid with water; The Saccharomyces boulardii ( Saccharomyces boulardii ) The concentration of BD-2 bacterial solution was 1.0×10 6 CFU / mL.

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