CN116103197B - Streptococcus thermophilus with helicobacter pylori inhibiting effect and application thereof - Google Patents
Streptococcus thermophilus with helicobacter pylori inhibiting effect and application thereof Download PDFInfo
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- CN116103197B CN116103197B CN202211638503.2A CN202211638503A CN116103197B CN 116103197 B CN116103197 B CN 116103197B CN 202211638503 A CN202211638503 A CN 202211638503A CN 116103197 B CN116103197 B CN 116103197B
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Abstract
The invention relates to the technical field of microorganisms, in particular to streptococcus thermophilus with an inhibiting effect on helicobacter pylori and application thereof. The streptococcus thermophilus with the inhibiting effect on helicobacter pylori is streptococcus thermophilus (Streptococcus thermophilus) FP13-03, and the strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms for 7 months and 15 days, and has a preservation address of Hospital No. 3 of North Chen West Lu 1 in the Korean region of Beijing city and a preservation number of CGMCC No.25311. The strain of the invention can eradicate helicobacter pylori in the body of a helicobacter pylori infected person, avoid various uncomfortable symptoms caused by medication on the body, and simultaneously reduce the recurrence rate and reinfection rate of the helicobacter pylori.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to streptococcus thermophilus with an inhibiting effect on helicobacter pylori and application thereof.
Background
Helicobacter pylori (Helicobacter pylori, hp) is a spiral, micro-anaerobic gram-negative bacillus capable of colonizing the gastric mucosa, which is a major causative factor for infectious halitosis, canker sores, chronic active gastritis, peptic ulcers, gastric mucosa-associated lymphomas and gastric cancer. Helicobacter pylori was first successfully isolated from gastric mucosal tissue of patients with chronic active gastritis in 1983, and is the only microorganism species currently known to survive in human stomach, and the international cancer center formally listed helicobacter pylori as a class i human carcinogen in 1994.
The pathogenic mechanism of the bacterium is mainly that the bacterium is spiral, one end of the bacterium is provided with flagella, the bacterium can penetrate through the acid environment and mucus layer of a gastric cavity to be colonised on the surface of gastric mucosa, meanwhile, the bacterium can metabolize urease, the urease can hydrolyze urea to generate ammonia and carbon dioxide, the acid environment around the bacterium is neutralized to protect the bacterium from being destroyed, and in addition, the bacterium can secrete pathogenic virulence factors such as vacuolate toxin A (VacA), cagA protein, heat shock protein, lipopolysaccharide, lewis antigen, iceA gene, lipase, protease, growth inhibition factor and the like.
How to inhibit the growth and proliferation of helicobacter pylori has become an important and hot spot for the study of the scholars. The prior art generally employs large doses of multiple antibiotics acting simultaneously in order to kill helicobacter pylori, for example:
1. triple therapy of levofloxacin: PPI (proton pump inhibitor) +levofloxacin+amoxicillin, seven days of treatment.
2. Sequential therapy: ppi+amoxicillin for the first five days; ppi+clarithromycin+metronidazole five days later.
3. Concomitant therapy: PPI, clarithromycin, amoxicillin and metronidazole for 5-10 days.
4. Standard triple therapy: PPI, clarithromycin, amoxicillin or metronidazole for 7-14 days.
5. Bismuth agent tetrad therapy: bismuth agent+PPI+two antibiotics (amoxicillin+clarithromycin or amoxicillin+levofloxacin or amoxicillin+furazolidone or tetracycline+metronidazole/furazolidone) for 7-14 days.
Although the cure rate of helicobacter pylori is greatly improved by the five methods, a plurality of problems still exist, such as rabeprazole and esomeprazole which are common proton pump inhibitors, and side effects such as anorexia, epigastric discomfort, nausea and the like are easy to occur after long-term application; bismuth is easy to cause constipation and other symptoms after long-term oral administration; the application of antibiotics can kill bacteria and cause problems of in vivo flora disorder, liver function damage and the like, and the use of antibiotics can also cause problems of enhanced drug resistance of helicobacter pylori, reduced eradication rate of helicobacter pylori and the like.
In recent years, the rise of microecology and the application of microecology therapy provide a new treatment for helicobacter pyloriThe idea is as follows. There are now some probiotic preparations for the treatment of helicobacter pylori infection, which mostly belong to the pharmaceutical series, such as lejunkang (5×10) 6 CFU/tablet, compound Lactobacillus acidophilus tablet (0.25X10) 7 CFU/sheet), le Tuoer (1×10) 10 CFU/bag, inactivated), jin Shuangqi (0.5×10) 7 CFU/sheet), etc. Although these probiotic preparations have a certain effect in the treatment of helicobacter pylori, the number of viable bacteria in the existing dosage forms is mostly at a low level, and the therapeutic effect is limited. In addition, the probiotics used in these dosage forms have no discussion about resistance to antibiotics, and if the patients need to use the probiotics in combination with antibiotics, the effect of the probiotics may not be achieved when the probiotics are used alone, which is not beneficial to the treatment based on the condition differentiation of the patients.
Disclosure of Invention
Aiming at the problems that the existing medicines for treating helicobacter pylori infection are easy to cause side effects and the eradication rate of helicobacter pylori is reduced, the invention provides streptococcus thermophilus with an inhibiting effect on helicobacter pylori and application thereof, which can eradicate helicobacter pylori in a helicobacter pylori infected person, avoid various uncomfortable symptoms caused by medicine administration to the body and reduce the recurrence rate and reinfection rate of the helicobacter pylori.
In a first aspect, the present invention provides a streptococcus thermophilus (Streptococcus thermophilus) FP13-03 having an inhibitory effect on helicobacter pylori, which has been deposited at the general microbiological center of the chinese microbiological bacterial culture collection center for 7 months 15 days in 2022 with a deposit address of 1 st scholar No. 3 in the korean region of beijing and a deposit number of 25311 cgmcc.
In a second aspect, the present invention provides the use of the above Streptococcus thermophilus FP13-03 for the preparation of a product having an inhibitory effect on helicobacter pylori.
Further, the fermentation broth of streptococcus thermophilus FP13-03 is freeze-dried to obtain streptococcus thermophilus bacterial powder.
Further, the freeze-drying process is divided into two stages, wherein sterile maltodextrin is added into the fermentation broth after the strain is fermented and before centrifugation; after centrifugation, sequentially adding isomaltooligosaccharide, mannitol and skimmed milk powder into the bacterial mud emulsion.
Further, the final concentration of maltodextrin was 2g/L based on the volume of fermentation broth.
Further, the final concentration of isomaltooligosaccharide was 1.15g/L, the final concentration of mannitol was 1.15g/L, and the final concentration of skimmed milk powder was 3g/L.
Further, the viable count of FP13-03 in the Streptococcus thermophilus bacterial powder is 4.0X10 11 CFU/g。
Further, the viable count of Streptococcus thermophilus FP13-03 in the product was 5.0X10 9 CFU/g or 1.0X10 10 CFU/g。
The invention has the beneficial effects that:
the streptococcus thermophilus FP13-03 provided by the invention has the characteristics of high gastrointestinal survival rate, strong intestinal tract colonization capability and the like, can inhibit and kill helicobacter pylori, and can effectively reduce the recurrence rate and reinfection rate of the helicobacter pylori. When in use, the invention can avoid various adverse reactions of the body caused by using medicines, thoroughly eradicate helicobacter pylori in a patient, and simultaneously reduce the recurrence rate and reinfection rate of the helicobacter pylori. The invention also provides possibility for various application modes of the microbial inoculum through the resistance screening of antibiotics. In addition, the invention optimizes the usage and dosage of the microbial inoculum and avoids the influence on the body caused by improper use of the microbial inoculum.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
EXAMPLE 1 screening of Streptococcus thermophilus FP13-03
1. Isolation, purification and preservation of strains
(1) Isolation of strains
1mL of breast milk samples collected from Qingzhou gynostemma pentaphylla are respectively added into test tubes filled with 9mL of physiological saline, then 10 times of gradient dilution is carried out by using the physiological saline, proper dilution gradient is adopted to be coated on an MRS solid culture medium plate, after the culture is carried out for 48 hours at 37 ℃, 10 single colonies are selected for purification according to the form, the color and the microscopic examination form of the single colonies on the MRS solid culture medium plate.
(2) Purification of strains
Purifying the 10 single colonies by adopting a streaking method, and preserving the single colonies on the last streaking plate after twice streaking.
(3) Preservation of strains
Inoculating the single colony obtained above into MRS liquid culture medium, standing at 37deg.C for 24 hr, packaging into 5mL centrifuge tube, centrifuging at 5000rpm for 5min, discarding supernatant, adding 1mL25% glycerol into the centrifuge tube, shaking, placing into-18deg.C refrigerator for preservation, and numbering as FP13-01, FP13-02, FP13-03, FP13-04, FP13-05, FP13-06, FP13-07, FP13-08, FP13-09, and FP13-010.
2. Helicobacter pylori strain and preparation of culture solution thereof
The invention adopts helicobacter pylori clinical strain which is taken from gastric mucosa tissue of helicobacter pylori infection positive patient, and smear gram staining, oxidase, contact enzyme and urease of the helicobacter pylori clinical strain are identified to be positive.
The glycerin tube containing helicobacter pylori was removed from the refrigerator, and spread on a Columba blood agar medium plate containing 8% defibrinated sheep blood, and subjected to micro-oxygenation (5% O) at 37 ℃ 2 、10％CO 2 、85％N 2 ) After picking single colony and purifying twice on Columbia blood agar medium plate containing 8% defibrinated sheep blood, picking single colony and inoculating into helicobacter pylori liquid medium, culturing at 37deg.C and micro-oxygen (5% O) 2 、10％CO 2 、85％N 2 ) Is subjected to stationary culture under the condition of 2 to 3d, centrifuging at 8000rpm/min and 4deg.C for 15min, collecting precipitate, washing the precipitate with fresh helicobacter pylori liquid culture medium twice, and re-suspending helicobacter pylori thallus to reach viable bacteria concentration of 10 9 CFU/mL。
3. Inhibition of helicobacter pylori by the strain
(1) Preparation of probiotic fermentation broth
And inoculating the obtained ten strain of fungus glycerol tubes into an MRS liquid culture medium, and standing and culturing at 37 ℃ for 24 hours to obtain a fermentation broth for later use.
(2) Test of zone of inhibition
200. Mu.L of helicobacter pylori suspension was spread on a Columbia blood agar medium plate containing 8% defibrinated sheep blood, and then a sterilized oxford cup was placed on the plate, and 100. Mu.L of each of sterilized blank MRS liquid medium and ten bacteria fermentation broth was added to the oxford cup, respectively. Micro-oxygen (5% O) at 37deg.C 2 、10％CO 2 、85％N 2 ) After stationary culture for 2 to 3 days under the conditions of (2) and (1) the diameter of the zone of inhibition was measured by a vernier caliper, the results of which are shown in Table 1 below.
Table 1 inhibition of helicobacter pylori by each strain
It can be seen that the FP13-03 strain has the strongest inhibitory effect on helicobacter pylori and is significantly higher than other strains, so that the following experiment was performed with the FP13-03 strain.
4. Identification of strains
(1) Colony characterization
The colony form of the FP13-03 strain growing in the MRS solid culture medium is slightly yellowish at the bottom, the surface is mostly milky, the edges are irregular, the surface is smoother and moist, the middle is convex, and the colony is moderate.
(2) Morphology under microscope
The FP13-03 strain has positive gram staining, circular or elliptic shape, diameter of 0.7-0.9 microns, paired or chain arrangement, no spore or flagellum and no motility.
(3) Physiological and biochemical identification
Physiological and biochemical characterization of the FP13-03 strain is shown in Table 2 below.
TABLE 2 physiological and biochemical identification results of FP13-03 Strain
| Detection index | Biochemical reaction |
| Motility of | - |
| Hydrolysis of glucose | + |
| Galactose hydrolysis | + |
| Lactose hydrolysis | + |
| Sucrose hydrolysis | + |
| Starch hydrolysis | + |
| Catalase test | - |
| Gelatin liquefaction test | - |
| Indole test | - |
| V-P test | + |
Note that: in the table "+" represents positive reaction and "-" represents negative reaction.
(4) 16S rDNA identification
The glycerol of FP13-03 strain was sent to the Biotechnology (Shanghai) Co., ltd for sequencing, and the result was:
GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGCCGAAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCGAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGT。
the results were compared in the GenBank database by BLAST and the FP13-03 strain was identified as Streptococcus thermophilus (Streptococcus thermophilus).
The streptococcus thermophilus (Streptococcus thermophilus) FP13-03 is sent to China general microbiological culture Collection center for preservation, wherein the preservation address is North Chen Silu No. 1, 3 in the Korean area of Beijing city, the preservation number is CGMCC No.25311, and the preservation date is 2022, 7, 15 days.
EXAMPLE 2 stomach acid and bile salt tolerance test of Streptococcus thermophilus FP13-03
1. Gastric acid tolerance test of strains
(1) Preparing artificial gastric juice: 10% hydrochloric acid is added into MRS liquid culture medium to make its pH value be 1.5, 2.0, 2.5 and 3.0 respectively, then pepsin is added into every group to make its final concentration be 10g/L, after it is fully dissolved, the above-mentioned materials are filtered and sterilized by using microporous filter membrane with 0.22 micrometers, and used.
(2) Preparation of FP13-03 Strain fermentation broth: and inoculating the glycerol tube of the FP13-03 strain into an MRS liquid culture medium, and standing and culturing at 37 ℃ for 24 hours for later use.
(3) According to the fermentation broth of FP13-03 strain and artificial gastric juice with different pH values, the ratio is 1:9 (V/V) and then placing the mixture in an incubator at 37 ℃ for static culture, and then sampling the mixture at 0h, 1h, 2h and 3h respectively to detect the viable count of FP13-03 bacteria in the mixture. The results are shown in Table 3 below.
TABLE 3 viable count of FP13-03 Strain in Artificial gastric juice at various time points (unit: CFU/mL)
| pH value of gastric acid | 0h | 1h | 2h | 3h |
| pH3.0 | 2.0×10 8 | 1.95×10 8 | 1.92×10 8 | 1.86×10 8 |
| pH2.5 | 2.0×10 8 | 1.92×10 8 | 1.87×10 8 | 1.76×10 8 |
| pH2.0 | 2.0×10 8 | 1.89×10 8 | 1.80×10 8 | 1.73×10 8 |
| pH1.5 | 2.0×10 8 | 1.87×10 8 | 1.78×10 8 | 1.70×10 8 |
As can be seen from Table 3 above, after 3 hours in artificial gastric juice, the survival rates of the FP13-03 strain in pH1.5, pH2.0, pH2.5 and pH3.0 were: 85%, 86.5%, 88%, 93%. Thus, it was found that the FP13-03 strain had good gastric acid resistance.
2. Bile salt tolerance test of strains
(1) Preparing a bile salt solution: adding oxgall powder into MRS liquid culture medium to make its mass fractions be 0.0%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, then adding trypsin into every group to make final concentration be 1.0g/L, after fully dissolving, filtering and sterilizing by using microporous filter membrane with 0.22 micrometers for later use.
(2) Preparation of FP13-03 Strain fermentation broth: and inoculating the glycerol tube of the FP13-03 strain into an MRS liquid culture medium, and standing and culturing at 37 ℃ for 24 hours for later use.
(3) Fermentation broth of FP13-03 strain and bile salt solution with different mass fractions are 1:9 (V/V) and then placing the mixture in an incubator at 37 ℃ for static culture, and then sampling the mixture at 0h, 2h, 4h and 6h respectively to detect the viable count of FP13-03 bacteria in the mixture. The results are shown in Table 4 below.
TABLE 4 viable count of FP13-03 Strain in bile salt solution at various time points (unit: CFU/mL)
| Concentration of bile salts | 0h | 2h | 4h | 6h |
| 0.0％ | 2.0×10 8 | 2.01×10 8 | 2.04×10 8 | 2.10×10 8 |
| 0.1％ | 2.0×10 8 | 2.0×10 8 | 2.01×10 8 | 2.02×10 8 |
| 0.2％ | 2.0×10 8 | 2.0×10 8 | 1.99×10 8 | 1.97×10 8 |
| 0.3％ | 2.0×10 8 | 1.99×10 8 | 1.98×10 8 | 1.97×10 8 |
| 0.4％ | 2.0×10 8 | 1.99×10 8 | 1.97×10 8 | 1.96×10 8 |
| 0.5％ | 2.0×10 8 | 1.98×10 8 | 1.96×10 8 | 1.95×10 8 |
As shown in the table, after the FP13-03 strain is treated for 6 hours at the highest bile salt concentration (the mass fraction is 0.5%), the survival rate of the strain is still up to 97.5%, so that the strain has higher tolerance to bile salts, has high survival rate in intestinal tracts and can play a longer-lasting role.
EXAMPLE 3 antibiotic resistance test of Streptococcus thermophilus FP13-03
The final concentrations of levofloxacin, tetracycline, clarithromycin and amoxicillin were respectively 0mg/L, 0.05mg/L, 0.10mg/L and 0.15mg/L in MRS liquid medium, and after complete dissolution, the solution was filtered and sterilized with a microporous filter membrane of 0.22 μm, and then, one of the glycerol tubes of FP13-03 strain was inoculated, and after standing culture at 37℃for 12 hours, samples were taken and the number of bacteria was measured, and the results of the test are shown in Table 5 below.
TABLE 5 live bacterial count (unit: CFU/mL) of FP13-03 Strain in antibiotics at various concentrations
| Antibiotic concentration | Levofloxacin | Tetracycline | Clarithromycin | Amoxicillin |
| 0mg/L | 5.0×10 8 | 5.0×10 8 | 5.0×10 8 | 5.0×10 8 |
| 0.05mg/L | 4.0×10 8 | 4.3×10 8 | 3.9×10 8 | 4.2×10 8 |
| 0.10mg/L | 5.0×10 6 | 6×10 6 | 4.6×10 6 | 5.7×10 6 |
| 0.15mg/L | - | - | - | - |
As shown in Table 5, the FP13-03 strain has a certain resistance to antibiotics used in the treatment of helicobacter pylori, but the growth of the helicobacter pylori can be inhibited when the concentration of the antibiotics is too high, which not only provides possibility for personalized medication of different patients, but also ensures the safety of the helicobacter pylori during application.
Meanwhile, the antagonism of the FP13-03 strain on other probiotics in the intestinal tract (bifidobacterium lactis, bifidobacterium longum, lactobacillus plantarum, lactobacillus reuteri and other strains) is explored through an antagonism test, and test results show that the strain has no antagonism on the other probiotics in the intestinal tract, so that the strain can not inhibit the growth of the other probiotics in the intestinal tract, and the combined application of various probiotics is possible.
EXAMPLE 4 safety toxicology evaluation of Streptococcus thermophilus FP13-03
Safety test researches on streptococcus thermophilus FP13-03 are carried out by adopting a male and female mouse acute toxicity test, a mouse bone marrow cell micronucleus test, a mouse sperm malformation test, an Ames test, a rat 30-day feeding test and the like. The acute oral toxicity test shows that the acute oral LD50 of streptococcus thermophilus FP13-03 on male and female mice is larger than 22000mg/kg, and the toxicity is classified by the acute toxicity half-lethal dose, which belongs to a nontoxic class substance. The results of the micronucleus test of the bone marrow cells and the sperm malformation test of the mice and the Ames test are all negative. The test results of 30 days of feeding show that the rats have good growth conditions, and the results of the hematological examination, the biochemical examination, the main viscera and the histological examination have no statistical significance compared with the control group. As can be seen from this, the streptococcus thermophilus FP13-03 has no genetic toxicity, and the use of the streptococcus thermophilus FP13-03 is safe and reliable.
EXAMPLE 5 preparation of products containing Streptococcus thermophilus FP13-03
Inoculating strain activated twice in MRS solid culture medium, culturing at 37deg.C for 24 hr, inoculating in fermentation culture medium, and culturing at 36deg.C for 24 hr to obtain Streptococcus thermophilus FP13-03 with viable count of 1.2X10 10 CFU/mL fermentation broth; adding sterile maltodextrin serving as a first freeze-drying protective agent into the fermentation liquor to enable the final concentration of the maltodextrin to be 2g/L, stirring the mixture uniformly, and centrifuging the mixture to obtain a bacterial mud emulsion; sequentially adding isomaltooligosaccharide and mannitol into the bacterial sludge emulsion to obtain the isomaltooligosaccharide with the final concentration of 1.15g/L (calculated according to the volume of fermentation broth), adding skimmed milk powder with the final concentration of 3g/L (calculated according to the volume of fermentation broth) after the isomaltooligosaccharide and mannitol are fully dissolved and uniformly stirred, and then adopting a freeze-drying method (the prefreezing temperature is maintained at-42 ℃ for 2h, the sublimation drying temperature is maintained at-16 ℃ for 37h, and the analytic drying temperature is maintained at 20 ℃ for 5 h) to obtain the bacterial powder raw powder. Meanwhile, the freeze-drying condition is controlled unchanged, and the fermentation broth is directly centrifuged and then freeze-dried to serve as a control.
As can be seen from the coating count, no addition ofThe number of bacteria of the freeze-dried protective agent is 1.0X10 after freeze-drying 11 CFU/g, and the number of freeze-dried bacteria after adding the freeze-dried protective agent is 4.0X10 11 Compared with the CFU/g without the protective agent, the survival rate of thalli in the freeze-drying process can be improved to 4 times by adding the freeze-drying protective agent.
A proper amount of bacterial powder is taken for carrying out helicobacter pylori inhibition effect test and gastric acid and bile salt tolerance test, and test results show that after the freeze-drying protective agent is added, the inhibition effect of the streptococcus thermophilus FP13-03 strain on helicobacter pylori is not influenced, and even after the freeze-drying protective agent is added, the gastric acid and bile salt tolerance of the streptococcus thermophilus FP130-03 strain is slightly enhanced.
The product added with the freeze-drying protective agent in the freeze-drying process is preserved in a refrigeration house for 12 months, the degradation rate of the effective viable count is 5.7%, and the degradation rate of the effective viable count is 11.3% after the product without the freeze-drying protective agent is preserved in the refrigeration house for 12 months.
Therefore, the addition of the cryoprotectant in the freeze-drying process can not only protect the thalli from being damaged in the freeze-drying process, but also protect the microbial inoculum in the microbial inoculum storage process.
Example 6 clinical efficacy verification of Streptococcus thermophilus FP13-03
Clinical random selection to hospital visit, gastroscopy, histological examination 14 C Urea expiration test 120 patients diagnosed as positive for helicobacter pylori were the subject of the study, and the test was developed with the patient fully informed of the purpose of the test and fully voluntary. Meanwhile, after comprehensively considering the age, sex, course of disease, illness state, primary diseases and other factors of patients, 120 study objects are equally divided into 4 groups.
The first group adopts bismuth agent tetrad therapy, and the specific implementation modes are as follows: 2 times per day of amoxicillin, 1g each time; clarithromycin is 2 times per day, 0.25g each time; the colloidal bismuth pectin is 150mg for each time, and the bismuth pectin is 4 times per day; omeprazole is administered 2 times daily, 20mg each time. 7d as a course of treatment, the treatment of H.pylori was observed.
The second group is orally administered 5.0X10 before meal 9 CFU/g of Streptococcus thermophilus FP13-03 bacteria group havingThe body embodiment is as follows: firstly, the streptococcus thermophilus FP13-03 bacterial agent is diluted to 5.0X10 by maltodextrin 9 CFU/g, which was then divided into 2 g/pack packets. The patient took 1 bag 1 hour before meal, 1 bag each in the morning and evening. 7d as a course of treatment, the treatment of H.pylori was observed.
The third group is orally administered 5.0X10 after meal 9 The specific implementation mode of the CFU/g streptococcus thermophilus FP13-03 microbial inoculum group is as follows: firstly, the streptococcus thermophilus FP13-03 bacterial agent is diluted to 5.0X10 by maltodextrin 9 CFU/g, which was then divided into 2 g/pack packets. The patient took 1 bag 1h after meal, 1 bag each in the morning and evening. 7d as a course of treatment, the treatment of H.pylori was observed.
The fourth group is orally administered 1.0X10 after meal 10 The specific implementation mode of the CFU/g streptococcus thermophilus FP13-03 microbial inoculum group is as follows: firstly, the streptococcus thermophilus FP13-03 bacterial agent is diluted to 1.0X10 by maltodextrin 10 CFU/g, which was then divided into 2 g/pack packets. The patient took 1 bag 1h after meal, 1 bag each in the morning and evening. 7d as a course of treatment, the treatment of H.pylori was observed.
The cure standard in the invention is: clinical symptoms and signs all disappeared, gastroscopy, histological examination 14 The urea breath test was all Hp negative and had no symptoms of relapse and reinfection within 3 months after negative transfer.
The cumulative cure rates for H.pylori for the different groups during the test are shown in Table 6 below.
TABLE 6 cumulative cure rate of helicobacter pylori (unit:%)
| Group of | First course of treatment | Second course of treatment | The third course of treatment | Fourth course of treatment |
| First group of | 20.0 | 40.0 | 66.7 | 90.0 |
| Second group of | 23.3 | 46.7 | 76.7 | 96.7 |
| Third group of | 26.7 | 63.3 | 90.0 | 100.0 |
| Fourth group | 33.3 | 70.0 | 93.3 | 100.0 |
As can be seen from the analysis of Table 6, the Streptococcus thermophilus FP13-03 preparation is superior to the bismuth quadruple therapy in terms of eliminating helicobacter pylori in patients, and is mainly characterized by rapid action and high eradication rate of Streptococcus thermophilus FP13-03 preparation. In terms of the application mode of the product, the effect of taking the product after meals is better than that of taking the product before meals; from the viewpoint of the bacterial count contained in the product, increasing the bacterial count of the bacterial agent has little influence on the treatment effect of helicobacter pylori. Thus, it may contain 5.0X10 9 The CFU/g product of Streptococcus thermophilus FP13-03 is used as a therapeutic agent and taken orally 2g 1h after meals 2 times a day as a standard use of the product for the treatment of helicobacter pylori.
The occurrence of adverse reactions in the different groups during the test are shown in table 7 below.
TABLE 7 Total incidence of adverse reactions (unit:%)
| Group of | Abdominal pain and diarrhea | Nausea and vomiting | Headache and dizziness | Insomnia and hypodynamia |
| First group of | 33.3 | 36.7 | 30.0 | 43.3 |
| Second group of | 10.0 | 13.3 | 10.0 | 10.0 |
| Third group of | 6.7 | 3.3 | 3.3 | 3.3 |
| Fourth group | 3.3 | 3.3 | 3.3 | 3.3 |
As shown in the table above, taking the product containing streptococcus thermophilus FP13-03 can obviously reduce the incidence of adverse reactions in the treatment process; and the incidence of adverse reactions is not as good as that of taking before meals, and the incidence of adverse reactions is not obviously changed along with the increase of the number of viable bacteria in the product. In addition, after the treatment course is finished, all the tested subjects do not have allergy and other similar symptoms, which indicates that the microbial inoculum provided by the invention is safe and has no toxic or side effect.
In conclusion, the product containing streptococcus thermophilus FP13-03 has good curative effect in treating helicobacter pylori infection and can furthest reduce adverse reaction, so that the microbial inoculum has wide application prospect in the aspect of treating helicobacter pylori infection.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (8)
1. Streptococcus thermophilus (Streptococcus thermophilus) FP13-03 with helicobacter pylori inhibiting effect is characterized in that the streptococcus thermophilus is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 7 months 15 of 2022, and the preservation address is 1 # 3 of North Chen West Lu in the Korean region of Beijing city, and the preservation number is CGMCC No.25311.
2. Use of streptococcus thermophilus FP13-03 according to claim 1 for the preparation of a product having an inhibitory effect on helicobacter pylori.
3. Use according to claim 2, characterized in that the fermentation broth of streptococcus thermophilus FP13-03 is lyophilized to obtain streptococcus thermophilus bacterial powder.
4. Use according to claim 3, wherein the freeze-drying process comprises the addition of a lyoprotectant in two stages, wherein sterile maltodextrin is added to the fermentation broth after fermentation of the strain and before centrifugation; after centrifugation, sequentially adding isomaltooligosaccharide, mannitol and skimmed milk powder into the bacterial mud emulsion.
5. The use according to claim 4, wherein the final concentration of maltodextrin is 2g/L based on the volume of the fermentation broth.
6. The use according to claim 4, wherein the final concentration of isomaltooligosaccharide is 1.15g/L, the final concentration of mannitol is 1.15g/L and the final concentration of skimmed milk powder is 3g/L by volume of the fermentation broth.
7. The use according to claim 3, wherein the live bacteria count of FP13-03 in the Streptococcus thermophilus bacterial powder is 4.0X10 11 CFU/g。
8. The use according to claim 2, wherein the viable count of streptococcus thermophilus FP13-03 in the product is 5.0 x 10 9 CFU/g or 1.0X10 10 CFU/g。
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| CN113736683A (en) * | 2020-08-03 | 2021-12-03 | 郑州大学 | Streptococcus thermophilus for inhibiting helicobacter pylori and application thereof |
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