CN114958650A - Lactobacillus paracasei for preventing and treating helicobacter pylori infection and composition and application thereof - Google Patents
Lactobacillus paracasei for preventing and treating helicobacter pylori infection and composition and application thereof Download PDFInfo
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- CN114958650A CN114958650A CN202210427202.9A CN202210427202A CN114958650A CN 114958650 A CN114958650 A CN 114958650A CN 202210427202 A CN202210427202 A CN 202210427202A CN 114958650 A CN114958650 A CN 114958650A
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- lactobacillus paracasei
- helicobacter pylori
- lactobacillus
- helicobacter
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Abstract
Lactobacillus paracasei for preventing and treating helicobacter pylori infection and a composition and application thereof, wherein the 16S rRNA gene sequence of the Lactobacillus paracasei S6 for preventing and treating the helicobacter pylori infection is shown as SEQ ID NO 1, and the preservation number is CCTCC NO: M20211627; the product has high bioactivity, wide carbon source utilization capacity, good acid production property, and tolerance to artificial gastric juice, intestinal juice, and bile salt. The Lactobacillus paracasei S6 can specifically adsorb helicobacter pylori to form copolymer, and can prevent or treat helicobacter pylori infection of human or animal by inhibiting growth of helicobacter pylori, adsorbing helicobacter pylori to form copolymer, and inhibiting urease activity. The lactobacillus paracasei S6 has good safety, has wide application range of the lactobacillus paracasei S6, and can be used for preparing functional food or medicine for preventing and treating helicobacter pylori infection and inhibiting the activity of uricase, or functional food or medicine for inhibiting pathogenic bacteria, or used as a leaven for preparing fermented food and health-care food.
Description
Technical Field
The invention relates to the technical field of microorganisms and food and medicine, in particular to lactobacillus paracasei for preventing and treating helicobacter pylori infection, a composition and application thereof.
Background
Helicobacter Pylori (HP) is a gram-negative bacterium that can colonize the gastrointestinal mucosa, and has characteristics of helicity, microaerophilic property, etc., wherein production of urease raises the pH in the stomach and in this way protects the bacterium against gastric acid. The nocell biomedical prize was obtained 2005 in 1982 by the successful isolation and culture from gastric mucosal specimens by Barry j. More and more studies have shown that HP is a major causative factor of chronic active gastritis, peptic ulcer, gastric MALT lymphoma and gastric cancer, and is also closely related to dyspepsia, unexplained anemia, poor response of immunotherapy, etc. (Shi, Yanyan, et al, "infection of Helicobacter pylori infection PD-1/PD-L1 block therapy genes attachment." Helicobacter 27.2(2022): e 12878.). HP was identified by WHO as a class I carcinogen in 1994, and HP chronic infection was classified as a clear human carcinogen by the U.S. department of health and public service in 12 months 2021. The HP has an infection rate of about 50% in the world, and has an infection rate of about 54.76% in China, and the eradication of HP not only can reduce the risk of gastric cancer, but also can effectively prevent diseases such as peptic ulcer and HP-related dyspepsia.
Currently, international Standard Triple Therapy (STT), i.e. a therapy using an antibiotic in combination with a proton pump inhibitor or a bismuth agent, is generally used clinically for patients infected with Hp. Antibiotics commonly used in triple therapy include metronidazole, tinidazole, clarithromycin, amoxicillin, and the like. The remaining alternative treatment regimens include "quadruple" therapy, sequential therapy and concomitant therapy. While there are many alternative treatment options, there are more and more cases of failure. As the resistance of Hp strains increases, adverse effects of therapeutic drugs and patient compliance are the main causes of treatment failure, researchers have to shift their efforts from looking for newer antibiotics to looking for other methods of prevention and treatment. Among the numerous alternatives, the properties possessed by probiotics and metazoans have become a focus of international research.
Among the probiotics for preventing HP infection which have been disclosed so far, most of them are isolated from animal sources and human bodies, less from plant sources, and most of them are lactobacillus strains reported, for example, CN103648511B discloses that lactobacillus reuteri DSM17646 inhibits helicobacter pylori by adsorption copolymerization; CN102174450B discloses that Lactobacillus plantarum CN2018 has a strong inhibition effect on helicobacter pylori adhering to human gastric epithelial cells, and has a reduction and prevention effect on mouse infection HP; from US5,716,615 pharmaceutical compositions are known which comprise inter alia lactobacillus. Such compositions are particularly useful for treating gastrointestinal disorders; from WO2004/087891 strains of the genus Lactobacillus are known which are suitable for the preparation of pharmaceutical or dietary compositions for the treatment of helicobacter pylori infections of the gastrointestinal tract. In addition, CN111849810B discloses that lactobacillus paracasei zjuid 03 can inhibit urease activity and prevent helicobacter pylori infection; CN109480231A discloses an anti-helicobacter pylori composition comprising 5-20 g of inactivated lactobacillus reuteri, 0.1-1 g of inactivated lactobacillus paracasei, 1.5-25 g of live animal bifidobacterium, 1.2-20 g of lactobacillus rhamnosus, 5-10 g of sea buckthorn fruit powder, 0.1-1 g of broccoli seed water extract, 20-30 g of fructo-oligosaccharide and 10-70 g of lactitol and application thereof.
However, the helicobacter pylori inhibiting strain reported in the prior art has a single action mechanism, a poor in-vivo action effect and an unobvious actual application effect, so that the development of a novel strain and a composition thereof for preparing a medicament or functional food for conveniently, conveniently and efficiently preventing, relieving and treating HP has important significance and great market value.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: overcomes the defects of the prior art, provides a lactobacillus paracasei S6 which is of plant source, can be separated and can effectively prevent and treat helicobacter pylori infection in human digestive tracts, particularly under the culture condition equivalent to stomach and intestine, further provides a food/medicine composition rich in lactobacillus paracasei S6 strain, and develops new application of the lactobacillus paracasei S6 and the composition containing the same in preventing and treating helicobacter pylori infection and the like.
One of the technical schemes adopted by the invention for solving the technical problems is as follows:
a Lactobacillus paracasei for preventing and treating helicobacter pylori infection is named as Lactobacillus paracasei (Lactcasei paracasei) S6, and is preserved in China Wuhan Chinese typical culture Collection in 12 months and 15 days of 2021, with the preservation number of CCTCC NO: M20211627.
Biological preservation description: lactobacillus paracasei (lactuca paracasei) S6, deposited in the chinese type culture collection center at the deposit address: eight-way 299 in Wuchang district, Wuhan city, Hubei province, the preservation organization is abbreviated as: CCTCC, preservation date: 12/15/2021 (registration received at 12/15/2021, survival was detected at 22/12, and preservation), and the biological preservation number is CCTCC NO: m20211627, strain number: lactobacillus paracasei S6.
The lactobacillus paracasei S6 is separated from a plant source, and specifically, the lactobacillus paracasei S6 is obtained by screening and separating from Sichuan Yibin farmhouse fermented sprouts.
The biological properties of lactobacillus paracasei S6 of the present invention are as follows:
1) morphological characteristics: the bacterial colony on the MRS agar culture medium is circular, medium in size, milky white, upward convex, relatively tidy in the edge and easy to pick; positive after gram staining.
2) Biological identification: the 16S rRNA gene sequence of the lactobacillus paracasei S6 is shown as SEQ ID NO:1, the 16S rRNA sequence of the lactobacillus paracasei S6 is compared with NCBI BLAST, the similarity of the strain and lactobacillus paracasei (Lactcaseibacillus paracasei) in Genebank is more than 99 percent, and the strain is identified as lactobacillus paracasei (Lactcasei paracasei) S6.
The tolerance test of the lactobacillus paracasei S6 on gastric acid and bile salt shows that the survival rate of the strain reaches 47.29% when the pH of gastric acid is 2.5, the lactobacillus paracasei S6 is cultured; when the concentration of bile salt reaches 3.0g/L, the survival rate of the strain reaches 82.15 percent. The lactobacillus paracasei S6 has very good gastric acid and bile salt tolerance and is suitable for oral administration.
The other technical scheme adopted by the invention for solving the technical problem is as follows:
a composition comprising a viable strain of lactobacillus paracasei S6;
alternatively, a composition comprises a mixture of a viable strain of lactobacillus paracasei S6 with one or more of an inactivated strain of lactobacillus paracasei S6, a metabolite of the strain, or a post-natant of lactobacillus paracasei S6.
The lactobacillus paracasei S6 metagenesis of the invention refers to a general name of lactobacillus thallus and metabolic components after processing/inactivation, concentration and drying treatment of lactobacillus paracasei S6 fermentation liquor, and comprises thallus components and metabolic products.
The composition includes but is not limited to a biological agent, a functional food, a health product or a medicament.
The functional food is any one of ferment, pickle, solid beverage, pill, tablet or microcapsule crystal ball.
The medicine also contains a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carriers include, but are not limited to: one or more of a filler, a binder, a wetting agent, a disintegrant, or a lubricant.
The filler is one or more of fucoidin, trehalose, lactose, chitosan, starch or dextrin; the adhesive is one or more of liquid glucose, starch paste or syrup; the wetting agent is one or more of glycerol or ethanol; the disintegrant is one or more of crospovidone, sodium carboxymethyl starch or sodium croscarmellose; the lubricant is one or more of magnesium stearate silicon dioxide or sodium fumarate stearate.
In an exemplary embodiment, the composition further comprises one or more of lactobacillus rhamnosus strain, lactobacillus fermentum strain, lactobacillus acidophilus strain, lactobacillus reuteri, lactobacillus plantarum, etc. probiotic powder capable of inhibiting helicobacter pylori or reducing urease activity. Among them, lactobacillus plantarum is preferably lactobacillus plantarum LP 220.
The living bacteria, the dead bacteria and the supernatant metabolite of the lactobacillus paracasei S6 can reduce the urease activity, and the inhibition rates of the urease activity can reach 38.54%, 31.45% and 27.32% respectively.
The lactobacillus paracasei S6 can be specifically combined with helicobacter in both live bacteria and inactivated states, the 2-hour polymerization rate reaches the highest, and the helicobacter can be specifically adsorbed and eliminated by the lactobacillus paracasei S6. In addition, Lactobacillus paracasei S6 can dent and imperfectly make the cell wall and cell membrane of helicobacter pylori, and cytoplasmic contents leak out of the membrane. Meanwhile, the cell morphology of lactobacillus paracasei S6 is not affected by helicobacter pylori, and can inhibit helicobacter pylori by destroying its cell integrity.
The lactobacillus paracasei S6 can inhibit helicobacter pylori, remarkably reduce the number of helicobacter pylori colonized in the stomach of a mouse infected by the helicobacter pylori, reduce the relative expression of main pathogenic virulence factors CagA and VacA gene mRNA in stomach tissues, and simultaneously remarkably improve the pathological condition of the stomach tissues and reduce IL-1 beta and TNF-alpha inflammatory factors.
Based on the above characteristics of the lactobacillus paracasei S6 of the present invention, one of the applications of the lactobacillus paracasei and its metazoans for the control of helicobacter pylori infection of the present invention is:
the application of lactobacillus paracasei S6 in preparing functional food or medicine for preventing and treating helicobacter pylori infection and/or reducing urease activity; or the application of the composition formed by the lactobacillus paracasei S6 and the inactivated strain, the metabolite of the strain or the metagen of the lactobacillus paracasei S6 thereof in preparing functional food or medicine for preventing and treating helicobacter pylori infection and/or reducing urease activity.
In an exemplary embodiment, the amount of lactobacillus paracasei S6 is 1 × 10 8 ～5×10 10 The dosage of the inactivated strain of CFU/day and lactobacillus paracasei S6 is 3-8 multiplied by 10 10 The dosage of CFU/day and the postnatal biota of the lactobacillus paracasei S6 is 50-120 mg/day.
Secondly, bacteriostatic experiments show that: lactobacillus paracasei S6 is broadly bacteriostatic of pathogenic helicobacter. Specifically, the inhibitor has good inhibition effects on helicobacter pylori ATCC 26695, helicobacter pylori ATCC 25592, helicobacter pylori ATCC4356, Helminthobacter marini ATCC 49286 and helicobacter pylori ATCC 51449, wherein the inhibitor has the best inhibition effect on helicobacter pylori ATCC 26695.
Based on the above characteristics of lactobacillus paracasei S6, another application of the present invention for lactobacillus paracasei and its metazoans for the control of helicobacter pylori infection is:
application of Lactobacillus paracasei S6 and its metazoan in preparing functional food or medicine for inhibiting pathogenic helicobacter is provided.
In an exemplary embodiment, the pathogenic helicobacter species includes one or more of helicobacter pylori, helicobacter helmansoni, helicobacter hepaticus.
In an exemplary embodiment, the amount of lactobacillus paracasei S6 is 5 × 10 9 ～5×10 10 CFU/day, the dosage of the metazoan is 30-120 mg/day. The test shows that the lactobacillus paracasei S6 has strong inhibiting effect on helicobacter pylori, Helminthobacter and helicobacter hepaticum.
The lactobacillus paracasei S6 can produce acid by fermenting ribose, galactose, glucose, fructose, mannose, sorbose, sucrose, trehalose, mannitol and other substrates, and has wide carbon source utilization capacity; the acid production capacity is strong, the acid production is rapid in the initial stage of fermentation, the acid production amount is maximum, and the acid production amount tends to be stable along with the increase of the fermentation time. Based on the characteristics, the invention further applies the lactobacillus paracasei and the anagen thereof for preventing and treating the helicobacter pylori infection:
application of lactobacillus paracasei S6 and its metazoan as leaven in preparing fermented food, health food or dietary supplement.
The dosage of Lactobacillus paracasei S6 is 1 × 10 8 ～1×10 10 CFU/mL or 1X 10 8 ～1×10 10 CFU/g, the dosage of the postnatal is 1-30 mg/kg.
The lactobacillus paracasei for preventing and treating helicobacter pylori infection has the beneficial effects that:
the lactobacillus paracasei S6 has high bioactivity, wide carbon source utilization capacity, good acid production property, and tolerance to artificial gastric juice, intestinal juice, and bile salt.
The lactobacillus paracasei S6 can specifically adsorb helicobacter pylori to form a copolymer, and is discharged from the body through the lactobacillus paracasei S6, so that the planting quantity of the helicobacter pylori in the stomach is obviously reduced; the lactobacillus paracasei S6 and the supernatant can inhibit the activity of uricase, thereby weakening the protection mechanism of urease on helicobacter pylori; the lactobacillus paracasei S6 can make the cell wall and cell membrane of helicobacter pylori sunken and incomplete, the cytoplasmic content leaks out from the membrane, and the cell morphology of the lactobacillus paracasei S6 is not influenced by the helicobacter pylori, and can inhibit the growth of the helicobacter pylori by destroying the cellular integrity of the helicobacter pylori; the lactobacillus paracasei S6 can effectively reduce the expression of cytotoxin-related protein A and vacuolating toxin-related gene A in stomach tissues, obviously reduce the expression of inflammatory factors IL-1 beta and TNF-alpha, and improve the stomach injury and inflammatory reaction; therefore, Lactobacillus paracasei S6 can prevent and treat helicobacter pylori infection of human or animals by various ways such as inhibiting the growth of helicobacter pylori, adsorbing helicobacter pylori to form copolymer, and inhibiting urease activity.
The lactobacillus paracasei S6 has better inhibiting effect on common pathogenic bacteria of helicobacter pylori, Helminthobacter marini and helicobacter hepaticum.
The lactobacillus paracasei S6 and metazoan group can obviously improve the pathological condition of gastric tissue, and improve inflammatory reaction by reducing IL-1 beta and TNF-alpha inflammatory factors.
The lactobacillus paracasei S6 has good safety, has wide application range of the lactobacillus paracasei S6, and can be used for preparing functional food or medicine for preventing and treating helicobacter pylori infection and inhibiting the activity of uricase, or functional food or medicine for inhibiting pathogenic bacteria, or used as a leaven for preparing fermented food and health-care food.
Drawings
FIG. 1 is a colony morphology of Lactobacillus paracasei S6 on MRS agar medium;
FIG. 2 is a light microscopic image (1000X) of Lactobacillus paracasei S6;
FIG. 3 is a graph showing the analysis of the bacteriostatic effect of Lactobacillus paracasei S6 on pathogenic bacteria such as helicobacter pylori;
FIG. 4 is a graph of acid production by Lactobacillus paracasei S6;
FIG. 5 is a graph showing the analysis of the gastric juice-resistant ability of Lactobacillus paracasei S6;
FIG. 6 is a graph showing the analysis of the bile salt tolerance of Lactobacillus paracasei S6;
FIG. 7 is a scanning electron microscope showing co-culture of Lactobacillus paracasei S6 with helicobacter pylori;
wherein (A) is helicobacter pylori under microscopic scanning microscope; (B) is microscopic lactobacillus paracasei S6 under a scanning electron microscope; (C) and (D) H.pylori and L.paracasei S6 under scanning electron microscopy at 2 and 5 micron fields, respectively.
FIG. 8 is a graph showing the effect of Lactobacillus paracasei S6 on the colonization of H.pylori in mice;
wherein: (A) the number of helicobacter pylori viable bacteria planted in the stomach (n is 10); (B) helicobacter pylori 16S rRNA expression levels in mouse stomach tissue (n ═ 3); (C) the ratio of helicobacter pylori antigen to total protein content in gastric tissue (n-10); (D) helicobacter pylori antigen content (n ═ 10) in mouse feces.
FIG. 9 is a graph showing the analysis of the effect of Lactobacillus paracasei on the pathological condition of the gastric tissue of the mouse stomach tissue;
wherein: (A) h & E pathological staining of gastric tissue (n ═ 5); (B) colon H & E stained histopathology score (n ═ 5)
FIG. 10 is a graph showing the analysis of the mRNA expression levels of VacA and CagA in mouse stomach tissue by Lactobacillus paracasei;
wherein: (A) VacA; (B) CagA
FIG. 11 is a graph showing the expression level of inflammatory factor mRNA in mouse stomach tissue analyzed by Lactobacillus paracasei S6;
Detailed Description
The invention is further explained with reference to the drawings and the embodiments.
The culture medium formulation involved in the examples of the present invention:
MRS medium (lactobacillus paracasei S6): 10.0g/L of peptone, 5.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 801.0 g/L of Tween and K 2 HPO 4 ·7H 2 O2.0 g/L, anhydrous sodium acetate 5g/L, citric acid diammine 2.0g/L, MgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O0.038 g/L (agar powder 15g/L as solid medium).
BHI medium (helicobacter): tryptone 10.0g/L, bovine heart extract powder 17.5g/L, sodium chloride 5g/L, disodium hydrogen phosphate (12H) 2 O)2.5g/L and glucose 2g/L, and adjusting the pH value to 7.2 +/-0.2, (20 g/L of agar powder is added to be a solid culture medium).
Example 1
The invention relates to a lactobacillus paracasei S6 (Lactcaseibacillus paracasei S6) for preventing and treating helicobacter pylori infection, which has a preservation number: CCTCC NO: M20211627.
1) Separation and screening of Lactobacillus paracasei S6
Collecting microorganism screening samples from Sichuan Yibin farmhouse fermented sprouts, shearing the collected samples, weighing 1g of the samples, putting the samples into 9mL of sterile physiological saline, fully shaking and uniformly mixing the samples, diluting the samples by 10 times, coating the diluted samples in an MRS solid culture medium, and culturing the samples for 48 hours at 37 ℃. Observing with naked eyes, picking single colonies with different shapes and sizes in a culture medium, and repeatedly streaking, purifying and culturing; then primarily determining the strain as lactobacillus by gram staining and calcium dissolving method, and storing the purified strain in a refrigerator at-80 deg.C with 45% glycerol for use.
a) Morphological observation
The colony morphology of the purified lactobacillus paracasei S6 was observed after streaking on MRS agar medium and culturing for 48 hours at 37 ℃ in an inverted manner, and the results are shown in fig. 1: the strain grows well on an MRS agar culture medium, and a bacterial colony is circular, medium in size, milky white, convex upwards, relatively neat in edge and easy to pick; gram staining was positive and microscopically rod-shaped (as shown in FIG. 2).
b) Molecular biological identification of strains
The purified strain is sent to China center for type culture collection to carry out 16S rRNA identification, the measured 16S rRNA sequence is compared with NCBI BLAST, the similarity with lactobacillus paracasei in Genebank is more than 99 percent, and the strain can be preliminarily identified as lactobacillus paracasei (Lactcaseibacterium paracasei). The 16S rRNA identification sequence of the strain is shown as SEQ ID NO:1 and is named as lactobacillus paracasei S6 (Lactcaseibacillus paracasei S6).
2) Growth capacity and physiological and biochemical characteristics of lactobacillus paracasei S6
First, the ability to utilize fermentation substrate
Puncturing and inoculating a fresh slant culture of the lactobacillus to be tested in a culture medium, or dripping a fresh culture bacterium solution into a melted soft agar column (at the temperature of 47 +/-1 ℃), mixing uniformly, covering a layer of 2% agar with the thickness of about 7mm above, placing at the temperature of 37 ℃ for culturing for 48h, and judging the strain to produce acid by utilizing a carbon source by using the culture medium from purple yellowing.
As shown in Table 1, Lactobacillus paracasei S6 strain can produce acid by fermentation using 15 carbon source substrates such as ribose, galactose, glucose, fructose, mannose, sorbose, sucrose, trehalose, and mannitol.
TABLE 1 physiological and biochemical characteristics of Strain S6-production of acid Using carbon Source
Note: +: positive reaction; negative reaction; w-weakly positive reaction
② test of bacteriostasis ability of lactobacillus paracasei S6 to pathogenic helicobacter
And (3) carrying out bacteriostatic experiments on helicobacter: pouring 10mL of water agar culture medium in a sterile plate, placing an Oxford cup after cooling and solidifying, respectively adding indicator bacteria suspension (helicobacter pylori ATCC 26695, helicobacter pylori ATCC 25592, helicobacter pylori ATCC4356, Helminthobacter marini ATCC 49286 and helicobacter hepaticum ATCC 51449) into agar culture medium correspondingly grown with indicator bacteria cooled to 50 ℃ to make the concentration of the indicator bacteria 10 6 And mixing the CFU/mL, pouring the mixture on water agar at the bottom layer, taking out the Oxford cup by using forceps after the mixture is solidified to form holes, adding 200 mu L of a sample to be detected into each hole, diffusing for 30min, and culturing for 15-24h at 37 ℃. Observing whether a bacteriostatic circle appears around the culture hole, measuring the diameter of the bacteriostatic circle by using a vernier caliper, recording the diameter of the bacteriostatic circle, and finally evaluating bacteriostatic activity according to the existence and the size of the bacteriostatic circle.
The results are shown in figure 3, and show that the lactobacillus paracasei S6 has better inhibition effects on helicobacter pylori ATCC 26695, helicobacter pylori ATCC 25592, helicobacter pylori ATCC4356, Helminthobacter marini ATCC 49286 and helicobacter hepaticum ATCC 51449, wherein the inhibition effect on the helicobacter pylori ATCC 26695 is optimal, and the lactobacillus paracasei S6 has extensive bacteriostasis on pathogenic helicobacter.
③ acid production ability test of Lactobacillus paracasei S6
The lactobacillus paracasei S6 strain is inoculated into MRS liquid culture medium according to the inoculation amount of 5 percent, activated and cultured for 24 hours at 37 ℃, and activated twice continuously. Inoculating the activated S6 bacterial liquid into MRS liquid culture medium according to the inoculation amount of 5%, uniformly mixing, and subpackaging into sterile test tubes (18mm multiplied by 180mm test tubes) according to 8ml per tube. Placing the subpackaged S6 bacterial liquid in a constant-temperature incubator at 37 ℃ for standing culture, taking 3 test tubes to measure the total acid of the bacterial liquid, and calculating the average acid-producing value; and (3) measuring the total acid of the bacterial liquid by taking 3 test tubes at regular intervals, and drawing an acid production curve by taking the time as an abscissa and the acid production amount as an ordinate, wherein the acid production curve is shown in an attached figure 4.
As can be seen from FIG. 4, the acid production of the strain at the initial stage of fermentation is rapid, and reaches 1.08g/100g in 8 hours and reaches the maximum value of 1.98g/100g in 22 hours; with the continuous increase of time, the acid production amount of the lactobacillus paracasei tends to be stable, which shows that the lactobacillus paracasei S6 has fast acid production and strong acid production capability and can form an inhibiting effect on helicobacter pylori through produced organic acid and other substances.
3) Lactobacillus paracasei S6 test for human gastrointestinal tolerance
Tolerance of lactobacillus paracasei S6 to gastric juice and intestinal juice
Preparing simulated gastric juice: NaCl 2.0g/L, adjusting pH to 2.0, 3.0 and 4.0 with HCl, respectively, and autoclaving with pepsin 3.2g/L, wherein pepsin is added at present during the experiment; preparing simulated intestinal juice: 6.8g/L potassium dihydrogen phosphate, adjusting the pH value to 7.5 by NaOH, and autoclaving, wherein the concentration of trypsin is 10.0g/L, and the trypsin is added at the moment of experiment; inoculating the S6 strain stored in the glycerin tube into an MRS culture medium at the temperature of 37 ℃ for activation for 24h in an inoculation amount of 10%; adding the same amount of S6 bacterial liquid into 50mL simulated gastric juice of the system, recording initial viable bacteria, and determining the number of viable bacteria after culturing at constant temperature of 37 ℃ for 3 h. Counting the detected live lactobacillus, and calculating the survival rate, wherein the survival rate of the strain is equal to that of the test group/the control group multiplied by 100%.
When food enters the stomach, gastric acid begins to be secreted. The pH value of the gastric acid which is normally secreted by the stomach of a human body is about 1.0 to 2.5. The pH of the stomach is between about 7.0 and 7.2 when empty, and drops rapidly to 2.0-3.0 when food enters the stomach. After meal, the gastric juice is diluted and the pH rises to about 3.5.
Referring to fig. 5, experimental results show that: the survival rate of the lactobacillus paracasei S6 is 47.29% when the pH is 2.50; the survival rate of lactobacillus paracasei S6 was 95.42% at ph3.00, indicating that the lactobacillus paracasei S6 strain taken after eating was able to tolerate gastric juices.
The pH in the intestine was about 7.50, and the survival rate of S6 was 103.2% at pH7.50, indicating that the strain Lactobacillus paracasei S6 was able to tolerate intestinal juice.
② tolerance of lactobacillus paracasei S6 to bile salt
The lactobacillus paracasei S6 strain is inoculated into MRS liquid culture medium according to the inoculation amount of 5 percent, activated and cultured for 24 hours at 37 ℃, and activated twice continuously. Inoculating the activated S6 bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 5%, and performing static culture for 15h at 37 ℃ in a constant-temperature incubator. And centrifuging the cultured bacterial liquid at 5000rpm for 10min to collect thalli, and shaking the thalli uniformly by using sterile physiological saline.
Adding the uniformly shaken bacterial liquid into MRS culture media with cholate concentrations of 1.0g/L, 2.0g/L and 3.0g/L according to the addition amount of 10 percent, and taking the cholate concentration of 0.0g/L as a control group. Then incubated in a 37 ℃ incubator for 3 h. Taking out the incubated bacterial liquid, immediately diluting according to 10 times, adding sterile normal saline, beating and uniformly mixing, and detecting the number of lactic acid bacteria; counting the detected live lactobacillus, and calculating the survival rate according to the following formula:
the survival rate (%) of the strain was 100% for the test group/control group.
S6 bile salt tolerance data are shown in figure 6: when the concentration of the bile salts is 1.0g/L and 2.0g/L, the survival rate of the strain is 101.23 percent and 99.17 percent respectively, but when the concentration of the bile salts reaches 3.0g/L, the survival rate of the strain still reaches 82.15 percent. Since the intestinal bile salt concentration is generally not more than 3.0g/L, the Lactobacillus paracasei S6 strain is able to tolerate intestinal bile salts.
4) Determination of the inhibitory Rate of urease activity produced by helicobacter pylori by Lactobacillus paracasei S6
Carrying out room-temperature centrifugation on live bacteria of lactobacillus paracasei S6 and inactivated lactobacillus paracasei S6 at 5000r/min for 5min, washing twice by PBS buffer solution, and suspending by BHI culture medium for later use; respectively adding the helicobacter pylori resuspension and the same amount of mixed bacterial liquid of the viable bacterial thallus of the lactobacillus paracasei S6, the inactivated lactobacillus paracasei S6 and the supernatant of the lactobacillus paracasei S6 into a 96-pore plate, and incubating for 3 hours at 37 ℃ under the condition of micro-oxygen; and adding urea-phenol red solution into each group, and measuring the absorbance of the urea-phenol red solution at 550nm by using an enzyme-labeling instrument. The control was BHI instead of sample solution.
Urease is capable of breaking down urea to raise the pH of the solution, discolouring the indicator solution, the activity of which can be reflected by measuring the value of the solution at OD 550 nm. As can be seen from Table 2, the OD of H.pylori after the S6 live cell inactivation S6 and S6 supernatant treatment 550 Value respectively2.011 +/-0.04, 2.243 +/-0.03 and 2.378 +/-0.04 which are all significantly lower than the control group value 3.272 +/-0.02 (P)<0.01), the inhibition rates respectively reach 38.54%, 31.45% and 27.32%, which shows that the living bacteria, the dead bacteria and the supernatant metabolites of the lactobacillus paracasei S6 can effectively inhibit the urease activity of the helicobacter pylori. The urease of pyloric helicobacter can decompose urea in environment to generate NH 3 Thus, the living bacteria, dead bacteria and supernatant metabolites of Lactobacillus paracasei S6 inhibit the growth of helicobacter pylori by inhibiting the activity of uricase.
TABLE 2 inhibition of uricase Activity by Lactobacillus paracasei S6
5) Co-culture test of Lactobacillus paracasei S6 with helicobacter pylori
Determination of the Co-culture growth of Lactobacillus paracasei S6 with helicobacter pylori: two generations of fresh H.pylori (1X 10) 7 CFU/mL) was resuspended in BHI, and 10% live cells of lactic acid bacteria (1X 10) 7 CFU/mL)/supernatant for 6 hours. And (3) taking the lactobacillus paracasei S6 cultured independently and helicobacter pylori as a control, centrifuging the mixed solution after the culture is finished to obtain a culture, placing the culture in a 2.5% glutaraldehyde solution for fixing overnight at 4 ℃, collecting the fixed aggregate, performing gradient dehydration on the collected aggregate by using 70-100% ethanol, and performing freeze drying for 48 hours to obtain a co-culture sample to be photographed. After the palladium sputtering gold spraying treatment, the film was photographed by a cold field emission scanning electron microscope to obtain fig. 7.
The morphological change of H.pylori cells after 6 hours of exposure to the strain Lactobacillus paracasei S6 at 37 ℃ was continuously observed by scanning electron microscopy, as shown in FIG. 7A, the untreated H.pylori cells had intact morphology with slightly curved rods or short arcs; the cells of Lactobacillus paracasei S6 have an intact rod-like morphology (as shown in FIG. 7B), and comparing FIGS. 7A and 7B, it can be seen that the cell wall of Lactobacillus paracasei S6 (gram-positive bacteria) is much thicker than the cell wall of helicobacter pylori (gram-negative bacteria); referring to FIGS. 7C and 7D, after treatment with Lactobacillus paracasei S6, most of the cell structures such as cell walls and cell membranes of helicobacter pylori are depressed and incomplete or disappear, and cytoplasmic contents leak out of the membranes, and sometimes collapse into residues, whereas the cell morphology of Lactobacillus paracasei S6 is not affected by helicobacter pylori, so that Lactobacillus paracasei S6 can well inhibit the growth of helicobacter pylori.
6) Study on adsorption copolymerization of Lactobacillus paracasei S6 and helicobacter pylori
Culturing helicobacter: streaking helicobacter on a BHI solid culture medium, culturing for 72h in an incubator at 37 ℃, and picking a single colony; inoculating the single colony in a BHI liquid culture medium, and culturing in an incubator at 37 ℃ for 48h to obtain a seed solution; inoculating the seed liquid into a BHI liquid culture medium with the inoculation amount of 3% (v/v), and culturing for 72h in a three-air culture box at 37 ℃ to obtain a helicobacter liquid; centrifuging helicobacter bacteria liquid 10000g for 5min to obtain helicobacter thallus, and adjusting bacteria solution concentration to OD with artificial gastric juice (pH 4) 600 When the concentration was 0.5, a helicobacter suspension was obtained.
The bacterial suspensions were prepared from 5 strains of pathogenic helicobacter pylori ATCC 26695, helicobacter pylori ATCC 25592, helicobacter pylori ATCC4356, Helminthobacter marini ATCC 49286, helicobacter pylori ATCC 51449, respectively, according to the above-described method.
Streaking lactobacillus paracasei S6 to be tested on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 24h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated solution into an MRS liquid culture medium according to the inoculation amount of 3% (v/v), and culturing for 24h at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial solution at 8000g for 5min to obtain Lactobacillus thallus, and adjusting the concentration of the bacterial solution to OD with PBS (pH 7) 600 ＝0.5。
③ taking 2mL of the suspension of the lactobacillus paracasei S6 and the 5 strains of the helicobacter sp with the adjusted bacterium concentration, and respectively measuring the initial OD 600 Equal volume mixing lactobacillus paracasei S6 with helicobacter ratio and shaking well for 15S to mix well. Standing and incubating for 4h at the room temperature of 37 DEG C. The OD (OD) values of the reaction mixture of Lactobacillus paracasei S6 and helicobacter were measured every 1 hour 600 Blend tmin), the copolymerization ratio was calculated according to the following formula.
Table 3: copolymerization rate of lactobacillus paracasei S6 after reaction with mixed bacteria liquid of helicobacter
From the experimental results table 3, it can be seen that: the lactobacillus paracasei S6 can specifically form a copolymer with helicobacter (helicobacter pylori ATCC 26695, helicobacter pylori ATCC 25592, helicobacter pylori ATCC4356, Helminthobacter marini ATCC 49286 and helicobacter pylori ATCC 51449), and after mixed co-culture is carried out for 2 hours, the copolymerization rate of the lactobacillus paracasei S6 on the helicobacter reaches the highest value, and is stable and maintained to be more than 45 percent along with the prolonging of time, wherein the copolymerization effect of the lactobacillus paracasei S6 and the helicobacter pylori ATCC 26695 is the best, and the copolymerization rate of 2 hours reaches 57.71 percent.
7) Study on adsorption copolymerization of inactivated strain of lactobacillus paracasei S6 and helicobacter pylori
Culturing helicobacter: according to 6) method (1) in the research on the adsorption copolymerization of lactobacillus paracasei S6 and helicobacter pylori, 5 strains of helicobacter were prepared into helicobacter bacterial suspensions according to the above method.
Secondly, culturing inactivated lactobacillus paracasei S6: streaking lactobacillus paracasei S6 to be tested on an MRS solid culture medium, and culturing for 48h at 37 ℃ to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 24h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated solution into an MRS liquid culture medium according to the inoculation amount of 3% (v/v), culturing for 24h at 37 ℃ to obtain a bacterial solution, and sterilizing the bacterial solution for 5min at 125 ℃; centrifuging the inactivated bacterial liquid at 8000g for 5min to obtain Lactobacillus paracasei S6The concentration of the inactivated strain of (4) was OD adjusted with PBS (pH 7) 600 ＝0.5。
③ according to 6) the method for the study on the adsorption copolymerization of Lactobacillus paracasei S6 and helicobacter pylori, and the copolymerization rate of the inactivated strain of Lactobacillus paracasei S6 after the reaction with the mixed bacterial liquid of pathogenic helicobacter, the experimental results are shown in Table 3.
Table 3: copolymerization ratio of inactivated strain of Lactobacillus paracasei S6 and mixed bacterium solution of helicobacter after reaction
The experimental results show that: the inactivated strain of lactobacillus paracasei S6 can specifically form a copolymer with helicobacter (helicobacter pylori ATCC 26695, helicobacter pylori ATCC 25592, helicobacter pylori ATCC4356, Helminthobacter marini ATCC 49286 and helicobacter hepatica ATCC 51449), the copolymerization rate of the inactivated strain of lactobacillus paracasei S6 and the helicobacter pylori reaches the highest 2h after mixing, the copolymerization rate is lower than that of the inactivated strain of lactobacillus paracasei S6, the copolymerization rate is still high, the copolymerization rate is stable along with the prolonging of time and is maintained at about 45%, the copolymerization rate of the inactivated strain of lactobacillus paracasei S6 to the helicobacter pylori ATCC 25592 is the best, and the copolymerization rate of the inactivated strain of lactobacillus paracasei S6 reaches 51.56% in 2 h.
In summary, the Lactobacillus paracasei S6 can specifically combine with helicobacter to form copolymer in live and inactivated states, and the Lactobacillus paracasei S6 can be partially excluded from the body by colonization in vivo, so that the helicobacter can be specifically adsorbed and excluded by the Lactobacillus paracasei S6.
Example 2
A composition comprising the Lactobacillus paracasei S6 strain of example 1 with a viable count of 9X 10 of Lactobacillus paracasei S6 10 CFU/g。
The composition is applied to the preparation of functional food or medicine for preventing and treating helicobacter pylori infection and/or reducing urease activity, and the dosage of the lactobacillus paracasei S6 is 3.0 multiplied by 10 10 CFU/day。
Study on inhibition of helicobacter pylori infection by Lactobacillus paracasei S6
And (3) testing a sample: lactobacillus paracasei S6 powder (9X 10) of Sichuan Gaofu Ji Biotech Co 10 CFU/g)；
Grouping tests: 30 6-8 week old C57BL/6J female mice purchased from Beijing Wakuukang were randomly divided into three groups of 10 mice each. The grouping situation is as follows:
and (2) CON group: normal drinking water, and perfusing the same amount of normal saline;
HP group: perlongday gavage of helicobacter pylori (10) 9 CFU/per mouse/day), and irrigating the stomach for 5 times in total, wherein the normal saline with the same amount is irrigated to the stomach for 14 consecutive days;
HP-L.PA group: perlongday gavage of helicobacter pylori (10) 9 CFU/per mouse/day), 5 times total intragastric administration, and 14 consecutive days of intragastric Lactobacillus paracasei S6 (6X 10) 8 CFU/per mouse/day)。
The results of the test for inhibition of H.pylori infection by Lactobacillus paracasei S6 of this example are expressed as mean values. + -. standard error; inter-group significance is indicated in the figures as: HP <0.05, HP <0.01 in the HP group compared to the CON group, HP <0.05, # p <0.01 in the HP-l.pa group compared to the HP group.
1) Effect of Lactobacillus paracasei S6 on in vivo colonization by helicobacter pylori in mice infected with helicobacter pylori
Homogenates of stomach tissue, after dilution by gradient, were plated onto Glax Selective Supplement A (GSSA) plates (7% sheep blood, 2.5. mu.g/mL amphotericin, 200. mu.g/mL bacitracin, 6. mu.g/mL vancomycin, 2. mu.g/mL nalidixic acid and 0.5. mu.g/mL polymyxin) and cultured for 48h to calculate the amount of H.pyrori. The content of helicobacter pylori 16S rRNA, antigen in the stomach and antigen in the feces was determined using a commercial kit.
The experimental results are shown in fig. 8A-D, and compared with CON group, the number of colonization by helicobacter pylori and the expression level of helicobacter pylori gene in stomach tissue of HP mice were significantly increased (p <0.01), and the relative content of helicobacter pylori antigen and the content of antigen in feces in stomach tissue were significantly increased (p <0.05), and the above results indicate that: h.pylori bacterial liquid was perfused into C57BL/6 mice to successfully infect H.pylori. Secondly, compared with HP mice, the colonization quantity of helicobacter pylori in stomach tissues of HP-L.PA mice (fig. 8A, p <0.01), the gene expression level of helicobacter pylori (fig. 8B, p <0.01), the relative content of helicobacter pylori antigen in stomach tissues and the content of helicobacter pylori antigen in feces (fig. 8C, D, p <0.01) are obviously reduced, and the fact that the mice are irrigated with lactobacillus paracasei S6 can effectively inhibit the helicobacter pylori from colonizing the stomach tissues of the mice such as stomach mucous membranes, stomach epithelial cells and the like.
2) Effect of Lactobacillus paracasei S6 on gastric histopathological conditions of mice infected with helicobacter pylori
Dissected mouse stomach tissue was fixed with 4% paraformaldehyde solution at room temperature for more than one day, and the fixed samples were prepared into physiological sections and stained with H & E. After the section is made, the gastric tissue damage of different groups of mice is observed under a microscope.
H & E staining of mouse gastric tissue as shown in fig. 9A, CON group mice had normal gastric epithelium and no significant inflammation, while HP group showed gland atrophy and hyperplasia and significant mucosal thickening compared to CON group (p <0.05 as shown in fig. 9B). After lactobacillus paracasei S6 is dried, the pathological condition of the stomach tissue of mice in the HP-L.PA group is improved to a certain extent, and compared with the HP group, the mucous membrane thickness is reduced; as shown in fig. 9B, the gastric epithelial tissue structure of CON group mice was normal with no significant inflammatory response; the HP group had atrophic and hyperplastic stomach tissue glands, infiltrated with inflammatory cells, and the mucosa was significantly thickened compared to the CON group (fig. 9B, p < 0.05). After lactobacillus paracasei S6 is dried, the pathological condition of the stomach tissue of mice in the HP-L.PL group is obviously improved.
3) Effect of Lactobacillus paracasei S6 on expression of virulence factor in mouse stomach tissue infected with helicobacter pylori
After H.pyrori successfully infects mice, the mice were gavaged with Lactobacillus paracasei S6 for 14 days, and then the expression of H.pyrori major virulence factor, Cytotoxin-associated protein A (CagA) and vacuolar toxin-associated gene A (VacA), in the stomach tissue was examined by qRT-PCR.
As shown in FIG. 10, the relative expression levels of the mRNA of the genes of CagA and VacA in stomach tissue of HP mice were significantly increased (p <0.01) compared with those of normal mice, while the expression levels of the mRNA of the genes of VacA and CagA in stomach tissue of HP-L.PA mice were significantly decreased (p <0.01) compared with those of mice with dry prognosis of Lactobacillus paracasei S6.
4) Effect of Lactobacillus paracasei S6 on the expression of inflammatory factors in stomach tissue of mice infected with helicobacter pylori
As shown in FIG. 11, the levels of inflammatory factors such as IL-1 β, TNF- α, TLR4 were significantly increased in the stomach tissue of HP mice compared to normal mice (p < 0.05). Compared with the HP group, the IL-1 beta and TNF-alpha expression level of mice in the HP-L.PA group is remarkably reduced after the lactobacillus paracasei S6 is dried (p is less than 0.01).
In conclusion, the lactobacillus paracasei S6 can inhibit helicobacter pylori, remarkably reduce the number of helicobacter pylori colonized on gastric tissues such as gastric mucosa, gastric epithelial cells and the like of H.pyrori infected mice, reduce the expression of main pathogenic virulence factors in the gastric tissues and improve the inflammatory response of the gastric tissues; lactobacillus paracasei S6 inhibits helicobacter pylori infection through various routes, i.e., Lactobacillus paracasei S6 has preventive and/or therapeutic effects on helicobacter pylori infection in a subject infected with helicobacter pylori.
Example 3
A composition of this example comprising a live strain of lactobacillus paracasei S6 and an inactivated strain of lactobacillus paracasei S6, wherein the live strain of lactobacillus paracasei S6 is as described in example 1; the inactivated strain of lactobacillus paracasei S6 was as described in 6) of example 1; the weight ratio of the live strain of the lactobacillus paracasei S6 to the inactivated strain of the lactobacillus paracasei S6 is 1: 3.
As is clear from 6) and 7) in example 1, both of the live strain of Lactobacillus paracasei S6 and the inactivated strain of Lactobacillus paracasei S6 can specifically adsorb and bind to helicobacter pylori to form a copolymer, and then the pathogenic helicobacter is excreted out of the body, so that the composition of this example can be used for the prevention/treatment of helicobacter pylori infection, and is safe.
The use of a composition according to this example for the preparation of a functional food or medicament for the infection by helicobacter pylori and/or for reducing urease activity, wherein the amount of lactobacillus paracasei S6 is 2.0 x 10 10 CFU/day, inactivated paracaseaThe amount of Lactobacillus sp 6 is 6.0 × 10 10 CFU/day。
Example 4
The composition of this example comprises live Lactobacillus paracasei S6 strain and postbiotic strain of Lactobacillus paracasei S6, wherein the live Lactobacillus paracasei S6 strain has a viable cell content of 3.0 × 10 or more as described in example 1 10 CFU/g; preparation of postnatal lactobacillus paracasei S6: the lactobacillus paracasei S6 is prepared by inactivation, concentration and spray drying after fermentation culture for 24h (the cell number is 5.0 multiplied by 10) 10 CFU/g, batch number: 20220124); preferably, the content ratio of the viable strain of lactobacillus paracasei S6 and the metazoan S6 in terms of the number of cells contained in the strain is 1: 1.
the composition of this example is used for the preparation of functional food or medicament for the inhibition of pathogenic helicobacter pylori ATCC 25592, in an amount of 3.0 x 10 Lactobacillus paracasei S6 10 CFU/day, inactivated Lactobacillus paracasei S6 in an amount of 5 x 10 10 CFU/day。
Example 5
The composition comprises a live strain of lactobacillus paracasei S6, an inactivated strain of lactobacillus paracasei S6 and a postbiotic strain of lactobacillus paracasei S6 in a mass ratio of 1:2: 1.
As is clear from the test of the bacteriostatic ability of the Lactobacillus paracasei S6 of example 1 against pathogenic helicobacter, the viable strain of Lactobacillus paracasei S6 has a good inhibitory effect against pathogenic helicobacter such as the common helicobacter pylori ATCC 26695, the common helicobacter pylori ATCC 25592, the common helicobacter pylori ATCC4356, the common Helminthobacter marini ATCC 49286, and the common helicobacter pylori ATCC 51449.
The composition of this example has bacteriostatic effects and is used for preparing functional foods or medicines for inhibiting pathogenic helicobacter including helicobacter pylori, Helminthobacter hepaticus and helicobacter hepaticus. When in use, the dosage of the viable lactobacillus paracasei S6 strain is 1 multiplied by 10 10 The inactivated strain of CFU/day, Lactobacillus paracasei S6 was 2X 10 10 CFU/day, the amount of metazoan is 50 mg/day.
Example 6
A composition of this example comprises a viable strain of Lactobacillus paracasei S6 and a metabolite of the strain of Lactobacillus paracasei S6.
According to the embodiment 1, the live lactobacillus paracasei S6 strain in the composition has better acid production and bacteriostasis capacity, and the composition of the embodiment is used as a leavening agent in the preparation of fermented food and health-care food.
The fermented food is pickled vegetables, and the application method of the composition serving as the leavening agent in preparing the pickled vegetables specifically comprises the following steps:
cleaning fresh vegetables, adding into 4-5 times of drinking water, adding edible glucose 1% of total volume and edible sodium chloride 0.4-0.6% of total volume, inoculating Lactobacillus paracasei S6 prepared in the embodiment 1 of the invention, and making its concentration reach 10 7 Fermenting at room temperature for 5-15 hr at CFU/mL or above to obtain fermented sauerkraut containing composition containing Lactobacillus paracasei S6 and Lactobacillus paracasei S6 strain metabolite. The fermented sauerkraut has crisp taste and unique flavor, contains Lactobacillus paracasei S6 thallus and metabolite, and has good safety and probiotic function.
Example 7
A composition of this example comprises, in parts by weight, powder of Lactobacillus paracasei S6 (2.0X 10) 10 CFU/g)10 parts, Lactobacillus paracasei S6 inactivated powder (3.0X 10) 10 CFU/g)21 parts by weight, Lactobacillus paracasei S6 metazoa (cell number 4.0X 10) 10 CFU/g)1 part, lactobacillus plantarum LP220 powder (2.0X 10) 10 CFU/g)4 parts and auxiliary materials, wherein the auxiliary materials comprise 1 part of magnesium stearate, 23 parts of lactose, 2 parts of fucoidin, 12 parts of resistant starch, 5 parts of microcrystalline cellulose, 10 parts of maltodextrin, 9 parts of glucose, 1 parts of vitamin C and 1 part of folic acid.
Weighing 23 parts by weight of lactose, 2 parts by weight of fucoidin, 12 parts by weight of resistant starch, 5 parts by weight of microcrystalline cellulose, 10 parts by weight of maltodextrin, 9 parts by weight of glucose, 1 parts by weight of vitamin C and 1 part by weight of folic acid, uniformly mixing, adopting 30% alcohol wet method and 20-mesh screen mesh to granulate into wet granules, drying for 3.5 hours at 55 ℃, adding the 20-mesh screen mesh after granulating, and then adding the wet granulesAdding Lactobacillus paracasei S6 bacterial powder (2.0 × 10) 10 CFU/g)10 parts, Lactobacillus paracasei S6 inactivated powder (3.0X 10) 10 CFU/g)21 parts by weight, Lactobacillus paracasei S6 metazoa (cell number 4.0X 10) 10 CFU/g)1 part, lactobacillus plantarum LP220 powder (2.0 x 1010CFU/g)4 parts and magnesium stearate 1 part, and the components are uniformly mixed and then tabletted by a rotary tablet press to obtain the tablets of the lactobacillus paracasei S6 dietary supplement for preventing and treating helicobacter pylori infection.
The composition of the embodiment is used for preparing the dietary supplement (tablet) for preventing and treating helicobacter pylori infection, can effectively prevent and treat the helicobacter pylori infection and improve the stomach inflammation.
Example 8
The composition of this example comprises, in parts by weight, Lactobacillus paracasei S6 (3.0X 10) 10 10 parts of CFU/g and 13 parts of inactivated powder of lactobacillus paracasei S6 (not less than 3.0 multiplied by 10) 10 CFU/g), Lactobacillus plantarum LP220 (1.0X 10) 11 CFU/g)10 parts by weight, maltodextrin 12 parts by weight, sorbitol 11 parts by weight, galacto-oligosaccharide 8 parts by weight, corn peptide 10 parts by weight, anserine 1 part by weight, soybean peptide 5 parts by weight, xylo-oligosaccharide 4 parts by weight, broccoli seed water extract 4 parts by weight, selenium-enriched yeast 3 parts by weight, sucralose 2 parts by weight, malic acid 2 parts by weight, glutathione 2 parts by weight, vitamin E1 parts by weight, vitamin C1 parts by weight and folic acid 1 parts by weight.
The raw materials of the composition are uniformly mixed according to the proportion after passing through a 40-mesh screen, and are bagged by a screw back sealing packaging machine to prepare 2g of solid beverage for preventing and treating helicobacter pylori infection per bag.
Example 9
Application of lactobacillus paracasei S6 in preparing functional food or medicine for inhibiting pathogenic helicobacter, wherein the dosage of lactobacillus paracasei S6 is 5.0 × 10 10 CFU/day。
Wherein, lactobacillus paracasei S6 was obtained by culturing as described in example 1.
Example 10
Preparation of lactobacillus paracasei S6 and its metazoan for preventing and treating helicobacter pylori infection and/or inhibiting urease activityUse of lactobacillus paracasei S6 in functional food or medicine in an amount of 4 x 10 10 The amount of post-natal biomass of CFU/day, Lactobacillus paracasei S6 was 70 mg/day.
In this case, Lactobacillus paracasei S6 was obtained by culturing as described in example 1, and the postnatal preparation of Lactobacillus paracasei S6 was the same as in example 4.
Claims (10)
1. The lactobacillus paracasei for preventing and treating helicobacter pylori infection is named as lactobacillus paracasei (Lactcaseibacillus paracasei) S6 and is preserved in China type culture Collection of Wuhan, China at 12-15 months in 2021 with the preservation number of CCTCC NO: M20211627.
2. Lactobacillus paracasei for controlling helicobacter pylori infection according to claim 1, wherein the 16S rRNA gene sequence of Lactobacillus paracasei S6 is shown in SEQ ID NO 1.
3. A composition comprising a live lactobacillus paracasei S6 strain as claimed in claim 1 or 2, or a mixture of one or more of a live lactobacillus paracasei S6 strain as claimed in claim 1 or 2, an inactivated strain of lactobacillus paracasei S6 as claimed in claim 1 or 2, a metabolite of the strain, or a post-natant of lactobacillus paracasei S6.
4. The composition of claim 3, wherein the composition further comprises one or more of a probiotic powder selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus plantarum, and the like, which inhibits helicobacter pylori or reduces urease activity.
5. The composition of claim 3 or 4, wherein the composition comprises but is not limited to a biological agent, a functional food, a health product or a drug.
6. Use of lactobacillus paracasei for the control of helicobacter pylori infection according to claim 1 or 2 and/or a composition according to any one of claims 3 to 5 for the preparation of a functional food or medicament for the control of helicobacter pylori infection and/or for inhibiting urease activity.
7. The use of claim 6, wherein said Lactobacillus paracasei adsorbs and binds with pathogenic helicobacter to form a copolymer, which simultaneously inhibits urease activity, reduces the relative expression of the major pathogenic virulence factors CagA and VacA gene mRNA in gastric tissue, significantly improves gastric histopathology and reduces IL-1 β and TNF- α inflammatory factors, and the dose of Lactobacillus paracasei S6 is 1 x 10 8 ～5×10 10 The dosage of the inactivated strain of CFU/day and lactobacillus paracasei S6 is 3-8 multiplied by 10 10 The dosage of CFU/day and the postnatal biota of the lactobacillus paracasei S6 is 50-120 mg/day.
8. Use of lactobacillus paracasei for the control of helicobacter pylori infection according to claim 1 or 2 and/or of a composition according to any one of claims 3 to 5 for the preparation of a functional food or medicament for the inhibition of pathogenic helicobacter pylori.
9. The use according to claim 8, wherein the pathogenic helicobacter species comprises one or more of helicobacter pylori, helicobacter helmansoni, helicobacter hepaticus.
10. Use of lactobacillus paracasei S6 for the control of helicobacter pylori infection according to claim 1 or 2 and/or a composition according to any one of claims 3 to 5 as a starter in the preparation of fermented foods, health foods or dietary supplements.
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