CN112438998A

CN112438998A - Probiotics agent PAPH for preventing and treating helicobacter pylori infection and preparation method thereof

Info

Publication number: CN112438998A
Application number: CN202011359144.8A
Authority: CN
Inventors: 赵建; 彭静珊; 吴道艳; 于智豪; 李姝�; 严馨
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-11-27
Filing date: 2020-11-27
Publication date: 2021-03-05
Anticipated expiration: 2040-11-27
Also published as: CN112438998B

Abstract

The invention discloses a probiotic agent PAPH for preventing and treating helicobacter pylori infection and a preparation method thereof. The probiotic agent PAPH comprises Lactobacillus casei lyT-7 and a protective agent; the Lactobacillus casei lyT-7(Lactobacillus casei lyT-7) has a collection number of: CCTCC NO: m2010197, the preservation time is 2010, 8 months and 9 days, and the preservation addresses are as follows: china center for type culture Collection; the addition amount of the protective agent is 50-150 wt% of the dosage of the lactobacillus casei lyT-7. The microbial inoculum prepared by the application can effectively inhibit the adhesion of helicobacter pylori on gastric epithelial cells, inflammatory reaction and immune response caused by the helicobacter pylori and oxidation reaction induced by helicobacter pylori infection, can directly inhibit or even kill the helicobacter pylori, and simultaneously can improve the micro-ecological environment of gastrointestinal tracts and reduce the adverse reaction of antibiotics.

Description

Probiotics agent PAPH for preventing and treating helicobacter pylori infection and preparation method thereof

Technical Field

The invention belongs to the technical field of preparation of a probiotic agent PAPH (probiotic air preserved and Control of H.pylori, PAPH), and particularly relates to a probiotic agent PAPH for preventing and treating helicobacter pylori infection and a preparation method thereof.

Background

Helicobacter Pylori (HP) is a gram-negative bacterium that has a serpentine shape, and more than half of the world population is infected with Helicobacter pylori, of which 10-15% develop peptic ulcers and about 1-2% develop gastric cancers. Currently effective methods for treating H.pylori infection are triple therapy and quadruple therapy, where H.pylori is eliminated by the combined action of antibiotics. However, with the abuse of antibiotics and the increase of drug resistance of helicobacter pylori, the eradication rate of using antibiotics to treat helicobacter pylori infection has been reduced year by year, and the combined use of antibiotics causes various side effects such as gastrointestinal disorders. Probiotics (Probiotics) are active microorganisms which play a role by improving the balance of host intestinal microbial flora, are important physiological bacteria in human intestinal tract, can promote the ecological balance of intestinal microbial flora, improve the intestinal flora structure, promote the proliferation of beneficial bacteria in the intestinal tract, inhibit the growth of harmful bacteria and other important physiological effects. Some probiotics have been found to have inhibitory effects on H.pylori and to be able to achieve eradication rates of antibiotics and to reduce the occurrence of side effects, but few probiotics and their preparations are effective in preventing and treating H.pylori infection.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a probiotic agent PAPH for preventing and treating helicobacter pylori infection and a preparation method thereof, which can effectively inhibit and kill helicobacter pylori, inhibit inflammatory reaction and immune response caused by the helicobacter pylori and reduce the adhesion performance of the probiotic agent PAPH on gastric mucosal epithelial cells.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a probiotic agent PAPH (Probiotics aid at preservation and Control of H.pyrori, PAPH) for preventing and treating helicobacter pylori infection comprises Lactobacillus casei lyT-7 and protectant; the Lactobacillus casei lyT-7(Lactobacillus casei lyT-7) has a preservation number of: CCTCC NO: m2010197, the preservation time is 2010, 8 months and 9 days, and the preservation addresses are as follows: wuhan, Wuhan university, storage unit: china center for type culture Collection; the addition amount of the protective agent is 50-150 wt% of the dosage of the lactobacillus casei lyT-7.

Furthermore, the addition amount of the protective agent is 80-100% wt of the dosage of the lactobacillus casei lyT-7.

Further, the protective agent comprises the following components in percentage by weight:

10-15% of fructo-oligosaccharide, 10-15% of galacto-oligosaccharide, 10-20% of isomaltooligosaccharide, 1-5% of spirulina dry powder, 0.5-1% of glycerol and the balance of sterile water.

Further, the protective agent comprises the following components in percentage by weight: 15% of fructo-oligosaccharide, 15% of galacto-oligosaccharide, 10% of isomaltooligosaccharide, 2% of spirulina dry powder, 1% of glycerol and the balance of sterile water.

The preparation method of the probiotic agent comprises the following steps:

(1) inoculating lactobacillus casei lyT-7 on an MRS liquid culture medium in an inoculation amount of 4-10%, and then carrying out shake cultivation for 15-20 h at 35-37 ℃ and 150-200 rpm;

wherein, the Lactobacillus casei lyT-7(Lactobacillus casei lyT-7) preservation unit: china center for type culture Collection, the collection numbers are: CCTCC NO: m2010197. The method is characterized in that: round, milky-white, smooth colonies formed on MRS solid medium.

MRS liquid medium: weighing 12g of peptone, 6g of yeast extract, 6g of beef extract, 18g of glucose, 5g of sodium acetate, 2.15g of ammonium citrate, 0.58g of magnesium sulfate, 0.05g of manganese sulfate, 2g of dipotassium hydrogen phosphate and 801 mL of Tween respectively, adjusting the pH value to 6.2-6.6, and finally adding ultrapure water to 1000 mL. Heating, stirring to dissolve, packaging into test tube or bottle, and sterilizing with high pressure steam (121 deg.C, 103.4kPa, 20 min).

(2) Then washing with normal saline, centrifuging at 6000-8000 rpm for 10-15 min, and removing supernatant to obtain thalli;

(3) after the thalli and the protective agent are mixed evenly, the program is cooled to minus 80 ℃ for prefreezing, and then the probiotic agent is obtained by freeze-drying.

Further, the amount of Lactobacillus casei lyT-7 inoculated was 4%.

Further, the process of programmed cooling comprises the following steps: preserving at 4 ℃ for 0.5 h; preserving at-20 ℃ for 0.5 h; preserving at-40 ℃ for 0.5 h; preserving at-80 ℃ for 1h, and then freezing and drying.

Furthermore, the number of viable bacteria in the lyophilized microbial inoculum is more than 1 multiplied by 10⁹CFU/g。

Further, the microbial inoculum is powder, tablet, pill or injection.

The invention has the beneficial effects that:

1. the microbial inoculum prepared by the application can effectively inhibit the adhesion of helicobacter pylori on gastric epithelial cells, inflammatory reaction and immune response caused by the helicobacter pylori and oxidation reaction induced by helicobacter pylori infection, can directly inhibit or even kill the helicobacter pylori, and meanwhile, the microbial inoculum can not damage the gastric epithelial cells and has good safety.

2. The microbial inoculum can improve the micro-ecological environment of the gastrointestinal tract and reduce the adverse reaction of antibiotics while directly inhibiting and even killing helicobacter pylori, thereby improving the treatment compliance of patients.

3. The protective agent used in the application is formed by compounding multiple components, wherein fructo-oligosaccharide, galacto-oligosaccharide and isomaltose hypgather have the function of helping probiotics to proliferate, the advantage of the probiotics in the gastrointestinal tract can be expanded, and meanwhile, the galacto-oligosaccharide has strong acid resistance and heat resistance and can provide good stability for the probiotic freeze-dried powder; the spirulina dry powder has the function of enhancing the human immunity, and promotes and improves the human immunity together with probiotics in the microbial inoculum; and glycerol is a lyoprotectant. Therefore, the protective agent formed by compounding multiple components can reduce or prevent the damage of the somatic cells of the probiotics, and the probiotics can keep the original physicochemical property and biological activity.

4. The viable bacteria number in the microbial inoculum prepared by the method is more than 1 multiplied by 10⁹CFU/g, the survival rate of the strain is high.

Drawings

FIG. 1 shows the acid resistance test of PAPCH;

FIG. 2 is a graph showing the inhibition assay of the growth of helicobacter pylori by PAPH; wherein, 100 mul, 200 mul and 300 mul are the amount of the used PAPH fermentation liquor; 1:200, 1:20 and 1:2 are the bacterial count ratio of the PAPCH to the helicobacter pylori;

FIG. 3 is the adhesion of PAPCH to GES-1 cells;

FIG. 4 is a graph showing the effect of PAPCH on H.pylori adhering GES-1 cells;

FIG. 5 shows the results of rapid urease detection;

FIG. 6 shows the effect of PAPH on the prevention of H.pylori infection on the content of pepsinogen I, II in mouse serum;

FIG. 7 is a graph showing the effect of PAPH in preventing helicobacter pylori infection on serum CRP levels in mice;

FIG. 8 shows the effect of PAPH on the prevention of H.pylori infection on the mouse serum MDA content;

FIG. 9 shows the effect of PAPH on the prevention of H.pylori infection in the serum GSH-PX content of mice;

FIG. 10 shows the effect of PAPCH on the prevention of H.pylori infection on the serum SOD level in mice;

FIG. 11 is a graph showing the effect of PAPH on the prevention of H.pylori infection on the amount of TNF-. alpha.expression in mouse gastric tissue;

FIG. 12 is a graph showing the effect of PAPH on the prevention of H.pylori infection on the expression level of MUC5AC in mouse gastric tissue;

FIG. 13 shows HE staining results of mouse gastric tissues;

fig. 14 is the results of detection of the h.pyri infection model;

FIG. 15 shows the results of the rapid urease assay;

figure 16 is a graph of the effect of PAPCH on the improvement of h.pyrori infection on pepsinogen I, II content in mouse serum;

figure 17 is the effect of PAPCH on ameliorating h.pyri infection on the level of C-reactive protein in mouse serum;

figure 18 is a graph of the effect of PAPCH on ameliorating h.pyri infection on malondialdehyde content in mouse serum;

figure 19 is a graph of the effect of PAPCH on ameliorating h.pyri infection on glutathione peroxidase content in mouse serum;

figure 20 is a graph of the effect of PAPCH on ameliorating h.

FIG. 21 is a graph of the effect of PAPCH on the improvement of H.pyrori infection on TNF- α mRNA expression in mouse gastric tissue;

figure 22 is a graph of the effect of PAPCH on ameliorating h.pyri infection on the expression of MUC5AC in mouse gastric tissue;

FIG. 23 shows HE staining results of mouse stomach tissue.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1 PAPCH performance detection

1. Acid resistance detection of PAPCH

The PAPH is acid-resistant as shown in FIG. 1, and the histogram in each set of data in FIG. 1 sequentially shows the detection results of pH6.5, pH3.5, pH3, pH2.5, and pH2 from left to right; 1h, 2h, 3h and 4h are the number of the viable PAPCH bacteria at the pH of 2, 2.5, 3, 3.5 and 6.5 respectively. Survival of PAPCH at pH3.5 and 3.0The amount of the active bacteria is almost not different from that of a blank group (pH6.5), the number of the active bacteria of the PAPCH is obviously reduced after 2 hours under the condition of pH2.5, and then the number of the active bacteria is stabilized at 10⁵On the order of magnitude; after 1h under the condition of pH2.0, the viable count of the PAPCH is obviously reduced, and after 2h, the viable count is stabilized at 10⁴On an order of magnitude.

Probiotics are defined as living microorganisms that provide benefits to the host when ingested in appropriate amounts by the body. The probiotics has good acid resistance, which is the basis of the colonization, survival and function of the probiotics in the stomach with high acidity, the survival rate of the PAPCH is not different from that of the blank group when the pH value is 3 and 3.5, and the survival rate of the PAPCH is half of that of the blank group when the pH value is 2.0, and the probiotics has certain survival capability, so the probiotics has the prospect of survival and function under the stomach condition.

2. Papch inhibits the growth of helicobacter pylori

The inhibition of the PAPH on the helicobacter pylori is shown in figure 2, and as can be seen from figure 2, the fermentation liquor and the thallus of the PAPH have the inhibition effect on the growth of the helicobacter pylori, and the inhibition rate on the helicobacter pylori is increased along with the increase of the added fermentation liquor or the increasing proportion of the added bacteria. The inhibition rates were 28.9%, 56.9%, and 69.2% when the fermentation broth was added at 100. mu.L, 200. mu.L, and 69.2% when the ratio of the amount of PAPH to helicobacter pylori was 1:200, 1:20, and 1:2, respectively, and 15.8%, 59.1%, and 73.6% when the ratio of the amount of PAPH to helicobacter pylori was 1:200, 1:20, and 1: 2.

3. Ability to adhere to gastric epithelial cells

The adhesion of PAPCH to GES-1 cells is shown in FIG. 3, wherein A is the adhesion of probiotics to GES-1 cells, and a in A is a blank group; b is a PAPCH processing group; c is l.casei cic treatment group; d is the l.paracasei treatment group; b is the adhesion rate of the probiotics to GES-1 cells; wherein, the PAPCH is a PAPCH processing group; casei cic is the l. casei cic treatment group; l. paracasei is l.paracasei treatment group.

As shown in fig. 3(a), among the PAPCHs, l.casei CICC and l.paracasei, PAPCH was the strongest in adhesion to GES-1 cells, and l.casei CICC was the second, but most of the l.casei CICC cells were adhered to cell plates rather than cells, and l.paracasei was the weakest in adhesion, and these results also correspond to the adhesion rates of these three bacteria to GES-1 cells shown in fig. 3(B), with paphs of 10.8%, l.casei CICC of 6.5%, and l.paracasei of 3.8%.

4. Inhibiting the adhesion of helicobacter pylori to gastric epithelial cells

The effect of PAPCH on H.pylori-adhering GES-1 cells is shown in FIG. 4, where NC is a blank group; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

As shown in FIG. 4, the more helicobacter pylori adhered to the cells, the greater the urease activity, and the OD measured₅₅₀The larger the value at nm, it can be seen that the number of helicobacter pylori adhering to GES-1 cells was significantly reduced in the case of co-treatment with helicobacter pylori by adding PAPH, as compared with the helicobacter pylori-treated group alone, in which the helicobacter pylori-treated group alone had OD₅₅₀nm is 0.563, and H.pylori and PAPCH co-treated group is 0.255.

Example 2 establishment of model for PAPCH prevention of Hp infection

1. Preparation of bacterial suspension

(1) Preparation of PAPCH bacterial suspension

Centrifuging the subcultured PAPCH at 6000rpm for 10min in a centrifuge, removing the supernatant, washing with normal saline, and adjusting the concentration of the bacterial suspension to 2 × 10⁹CFU/mL, preparation before each gavage to ensure the viability of the bacteria.

(2) Preparation of helicobacter pylori suspension

Adding 1 mL/dish of normal saline into subcultured helicobacter pylori blood plate, scraping bacterial colony with coating rod, sucking into centrifuge tube with pipette, adjusting bacterial suspension concentration to 2 × 10⁹CFU/mL, preparation before each gavage to ensure the viability of the bacteria.

2. Mouse experiment

(1) First-stage intragastric administration: establishing a prevention model

After the mice are cultured for 7 days in an adaptive way, the mice are randomly divided into two groups, namely a blank group and a PAPCH intragastric group, and each group contains 16 mice.

The gavage method comprises the following steps:

blank group (prevention NC group): each mouse was gavaged with 0.5mL of physiological saline once every other day for 4 weeks.

PAPCH gavage group (prevention PAPCH group): each mouse was gavaged with 0.5mL of PAPCH bacterial suspension once every other day for 4 weeks.

(2) And (3) intragastric administration in the second stage: simulation of helicobacter pylori infection

After the first-stage intragastric administration is finished, randomly and averagely dividing blank groups (prevention NC groups) of the first stage into two groups, and intragastric administration (HP groups) of one group by using helicobacter pylori bacterial suspension; the other group was gavaged with physiological saline (NC group); the first-stage PAPCH intragastric administration group (the PAPCH prevention group) was randomly divided into two groups, one group was intragastric administered with helicobacter pylori suspension (pre-PAPCH/post-H. pylori group, pre-P/post-H group), and the other group was intragastric administered with physiological saline (PAPCH group).

The method for intragastric administration comprises the following steps:

NC group: each mouse was gavaged with 0.5mL of physiological saline once a day for 2 weeks.

HP group: each mouse was gavaged with 0.5mL of helicobacter pylori suspension once a day for 2 weeks.

PAPCH group: each mouse was gavaged with 0.5mL of physiological saline once a day for 2 weeks.

pre-P/post-H group: each mouse was gavaged with 0.5mL of helicobacter pylori suspension once a day for 2 weeks.

3. Mice sacrifice and sample collection

(1) Blood sampling from eyeball

After fasting for 12h, eyeball blood sampling is carried out, 100-fold blood sampling is carried out by an anticoagulation tube, 200 mu L of blood is stored at 4 ℃ for routine blood detection, the rest blood is put into a centrifuge tube, is placed at room temperature for standing for 4h and then is placed in a refrigerator at 4 ℃ for overnight, then is centrifuged at 3000rpm in a centrifuge (precooled to 4 ℃) for 20min after overnight, supernatant is carefully sucked, and serum is stored at-80 ℃ for ELISA detection.

(2) Mice sacrifice and tissue collection

The method comprises the following steps of taking blood from eyeballs of a mouse, immediately killing the blood by adopting a spondylodesis method, disinfecting the epidermis of the mouse by using 75% alcohol, placing the mouse on a dissection plate for dissection, taking out the stomach of the mouse, cutting the stomach of the mouse to be open along the greater curvature of the stomach, cleaning the stomach by using normal saline, cutting the stomach into 3 parts, performing rapid urease detection on one part, storing the other part in liquid nitrogen for extracting RNA, and storing the other part in 4% paraformaldehyde for HE staining and immunohistochemistry.

Example 3 Rapid urease reaction

The stomach tissue was placed in fast urease paper and the results were observed within 5 min. The result is shown in FIG. 5, where NC in FIG. 5 is a blank set; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

The stomach tissues of mice in the blank group and the PAPCH treatment group are placed in test paper, the color of the test paper has no obvious change, the helicobacter pylori treatment group and the pre-P/post-H group are placed in the test paper, the test paper rapidly turns red, and the color of the helicobacter pylori treatment group is purplish red compared with that of the pre-P/post-H group.

Example 4 blood routine

The blood of the mice is subjected to routine blood detection by using a full-automatic hematology analyzer, the numbers of leukocytes and neutrophils in the blood of the mice are shown in table 1, and the numbers of leukocytes and neutrophils in a pre-PAPCH/post-h.pylori treated group (pre-P/post-H group) and a helicobacter pylori treated group (HP group) are obviously higher than those in a blank group (NC group), but the numbers of the leukocytes and the neutrophils in the two groups are not significantly different, and the numbers of the leukocytes and the neutrophils in the PAPCH treated group (PAPCH group) are not significantly different from those in the blank group.

TABLE 1 number of leukocytes and neutrophils

Example 5 prevention of helicobacter pylori infection by PAPH on the Effect of the content of PG I, PG II, CRP, GSH-PX, SOD, and MDA in the serum of mice

1. ELISA detects the contents of PG I, PG II, CRP, GSH-PX, SOD and MDA in mouse serum, and the specific process is as follows:

(1) sample adding of the standard: adding 50 mu L of standard substance with different concentrations into the standard substance holes respectively;

(2) sample adding: respectively arranging a blank hole and a sample hole to be detected, adding 40 mu L of sample diluent into the sample hole to be detected on the enzyme-labeled coated plate, then adding 10 mu L of sample to be detected, and slightly shaking and uniformly mixing;

(4) adding an enzyme: adding 100 mu L of enzyme-labeled reagent into each hole except for blank holes;

(5) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 60 minutes at 37 ℃;

(6) preparing liquid: diluting 20 times of the concentrated washing liquid with 20 times of distilled water for later use;

(7) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;

(8) color development: adding 50 mu L of color-developing agent A into each hole, adding 50 mu L of color-developing agent B, shaking gently, mixing uniformly, and developing for 15 minutes at 37 ℃ in a dark place;

(9) and (4) terminating: adding 50 mu L of stop solution into each well to stop the reaction (at the moment, the blue color immediately turns to yellow);

(10) and (3) determination: after 15min, the wells were zeroed with blank wells and the absorbance of each well was measured at a wavelength of 450 nm.

2. The content of pepsinogen I (PG I) in mouse serum is shown in figure 6, and NC in figure 6 is a blank group; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

The content of PG I in the serum of mice in the blank group, the PAPH treatment group, the helicobacter pylori treatment group and the pre-P/post-H group is 187.26, 184.85, 148.22 and 157.32ng/mL respectively, and compared with the blank group, PG I in the helicobacter pylori treatment group and the pre-P/post-H group is remarkably reduced by 0.79 and 0.84 times of the blank group respectively, and PG I in the helicobacter pylori treatment group and the pre-P/post-H group is remarkably reduced, but the two groups have no remarkable difference. The content of pepsinogen II (PG II) in the mouse serum is shown in figure 6, the content of PG II in the mouse serum of the blank group, the PAPCH treatment group, the helicobacter pylori treatment group and the pre-P/post-H group is 137.23, 133.39, 110.55 and 120.63ng/mL respectively, and the content of PG II in the serum of the helicobacter pylori treatment group and the pre-P/post-H group is obviously reduced compared with the blank group and is 0.81 time and 0.88 time of the blank group respectively, but the content of PG II in the pre-P/post-H group is obviously higher than that of the helicobacter pylori treatment group.

3. The content of C-reactive protein (CRP) in mouse serum is shown in FIG. 7, and NC in FIG. 7 is blank group; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

The CRP content in the blood serum of mice in the blank group, the PAPCH treatment group, the helicobacter pylori treatment group and the pre-P/post-H group is 235.40, 243.33, 377.33 and 318.20ng/mL respectively, compared with the blank group, the CRP content in the blood serum of the helicobacter pylori treatment group and the pre-P/post-H group is obviously increased and is 1.43 times and 1.35 times of that of the blank group respectively, but the CRP content of the helicobacter pylori treatment group is obviously higher than that of the pre-P/post-H group and is 1.19 times of that of the pre-P/post-H group. The PAPCH treatment group was not significantly different from the blank group.

4. The content of Malondialdehyde (MDA) in mouse serum is shown in FIG. 8, and NC in FIG. 8 is blank group; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

The MDA content in the blood serum of mice in the blank group, the PAPCH treatment group, the helicobacter pylori treatment group and the pre-P/post-H group is respectively 16.09, 17.75, 27.60 and 20.78ng/mL, compared with the blank group, the MDA content in the blood serum of the helicobacter pylori treatment group and the pre-P/post-H group is obviously increased and is respectively 1.72 and 1.29 times of that of the blank group, but the MDA content of the helicobacter pylori treatment group is obviously higher than that of the pre-P/post-H group and is 1.33 times of that of the pre-P/post-H group. The PAPCH treatment group was not significantly different from the blank group.

5. The content of glutathione peroxidase (GSH-PX) in mouse serum is shown in FIG. 9, and NC in FIG. 9 is blank group; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

The content of GSH-PX in the blood serum of mice in a blank group, a PAPCH treatment group, a helicobacter pylori treatment group and a pre-P/post-H group is 122.02, 117.17, 90.03 and 107.14ng/mL respectively, and compared with the blank group, the content of GSH-PX in the blood serum of the helicobacter pylori treatment group and the pre-P/post-H group is remarkably reduced and is 0.73 times and 0.88 times of the blank group respectively, but the content of GSH-PX in the pre-P/post-H group is remarkably higher than that of the helicobacter pylori treatment group and is 1.19 times of that of the helicobacter pylori treatment group. The PAPCH treatment group was not significantly different from the blank group.

6. The content of superoxide dismutase (SOD) in mouse serum is shown in FIG. 10, wherein NC in FIG. 10 is blank group; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

The SOD content in the serum of mice in the blank group, the PAPCH treatment group, the helicobacter pylori treatment group and the pre-P/post-H group is respectively 14.46, 14.51, 12.30 and 12.66ng/mL, compared with the blank group, the SOD content in the serum of the helicobacter pylori treatment group is obviously reduced by 0.85 times, and compared with the blank group, the pre-P/post-H group and the PAPCH treatment group have no obvious difference.

Example 6 Effect of PAPCH on TNF-alpha and MUC5AC expression in mouse stomach tissue

1. Extraction of Total RNA from stomach tissue

(1) Precooling the mortar by using liquid nitrogen, submerging the stomach tissue in the mortar, and adding the liquid nitrogen in time until the stomach tissue is ground into powder;

(2) transferring the powder into a centrifugal tube without RNA enzyme, adding 1ml of TRIOL into each tube, repeatedly sucking the mixture for 5-6 times by using a pipette, uniformly mixing the mixture, and standing the mixture for 5min at room temperature;

(3) adding 200 μ L chloroform, vortex for 15s, standing at room temperature for 2min, and centrifuging in a centrifuge (precooling to 4 deg.C in advance) at 12000g for 10 min;

(4) carefully sucking 500 μ L of the centrifuged upper aqueous phase into a new RNase-free centrifuge tube, adding 500 μ L of isopropanol into each tube (precooling to-20 ℃ in advance), vortexing for 15s, standing at room temperature for 5min, and centrifuging for 5min at 12000g in a centrifuge (precooling to 4 ℃ in advance);

(5) directly pouring out the centrifuged supernatant, adding 75% ethanol (precooled to-20 ℃), vortexing for 15s, and centrifuging for 5min at 7500g in a centrifuge (precooled to 4 ℃);

(6) and pouring out the centrifuged supernatant, then carrying out air drying for 5-10min, and taking a proper amount of DEPC water for dissolving.

2. Reverse transcription of total RNA from gastric tissue

Genomic DNA was first removed using Prime script RT Reagent Kit with gDNA Eraser reverse transcription Kit, followed by reverse transcription.

3. Real time PCR reaction

The expression of TNF-alpha and MUC5AC transcription levels of GES-1 cells was determined using mouse GAPDH as an internal gene, and the primer sequences are shown in Table 2, and the results are shown in FIGS. 11 and 12.

Reactions for Real time PCR were as follows:

the reaction conditions were as follows:

TABLE 2 primer sequences

The effect of PAPCH and H.pylori on TNF- α mRNA expression in mouse stomach tissue is shown in FIG. 11, where NC is blank; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

Compared with the blank group, the helicobacter pylori treatment group and the pre-P/post-H group have the advantages that the expression level of TNF-alpha mRNA is obviously increased by 7.24 times and 2.91 times respectively. However, the expression level of TNF-alpha mRNA in the H.pylori-treated group was significantly higher than that in the pre-P/post-H group, which was 2.48 times higher than that in the pre-P/post-H group. The PAPCH treatment group was not significantly different from the blank group.

The effect of PAPCH and H.pylori on the expression of MUC5AC mRNA in mouse stomach tissue is shown in FIG. 12, where NC is a blank group; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

Compared with the blank group, the expression level of MUC5AC mRNA in the helicobacter pylori treated group and the pre-P/post-H group is remarkably increased by 3.03 times and 2.39 times respectively. However, the expression level of MUC5AC mRNA in the H.pylori-treated group was significantly higher than that in the pre-P/post-H group, which was 1.27 times higher than that in the pre-P/post-H group. P <0.05 indicates significant differences from the H.pylori-treated group, pre-P/post-H group and blank group, and P <0.05pre-P/post-H group and H.pylori-treated group.

Example 7 pathological analysis of HE staining of gastric tissue in mice

The results of HE staining of mouse stomach tissue are shown in FIG. 13, where NC in FIG. 13 is blank; the PAPCH is a PAPCH processing group; HP is h. pre-P/post-H is pre-PAPCH/post-H.

As shown in FIG. 13, red blood cells were observed in the gastric mucosa of the H.pylori-treated group, and necrosis and exfoliation of the surface layer of the mucosa were observed; red blood cells and a small number of cells were found to be exfoliated from the gastric mucosa in the pre-P/post-H group. The gastric mucosa of the normal group of mice and the PAPCH treatment group has no mucosa shedding and erosion phenomenon.

The following results were obtained by examining mice that had been protected from helicobacter pylori infection with PAPCH as described in examples 2 to 7:

1. the survival rate of the PAPCH is not different from that of a blank group when the pH value is 3 and 3.5, and the PAPCH has certain survival ability when the pH value is 2.5 and 2, so the PAPCH has the prospect of survival and function under stomach conditions.

2. The PAPCH and other fermentation liquid have better inhibiting effect on the growth of helicobacter pylori in vitro. The PAPCH adheres to gastric epithelial cells and reduces the adhesion of helicobacter pylori to GES-1 cells. The PAPCH shows good effect of antagonizing helicobacter pylori infection in vitro through regulating abnormal expression of TNF-alpha and MUC5AC after helicobacter pylori infection by an immune process with cells.

3. In vivo experiments show that the PAPCH has the effect of preventing and treating helicobacter pylori infection, probably because the PAPH occupies a certain site on the gastric mucosa, the colonization of the helicobacter pylori on the gastric mucosa is reduced, the immunity of the organism and the activity of antioxidant enzyme are improved, the immune state of the organism is adjusted, and the damage of the gastric mucosa and the reduction of the function of the gastric mucosa caused by the infection of the helicobacter pylori are reduced.

4. The PAPH probiotic has better viability and has important significance for developing a product of the probiotic for preventing and treating helicobacter pylori infection.

Then, the mice infected with helicobacter pylori are improved by using PAPH, and the specific process is shown in examples 8-13.

Example 8 establishment of PAPCH improved HP infected mouse model

The procedure for establishing a mouse model with PAPCH-modified HP infection was the same as in example 2, except that this example replaced the PAPCH lavage group (prevention PAPCH group) in its first stage lavage with the h.pyrori lavage group (modified HP group) in the difference from example 2. In the second stage of gavage, only the h.pylori gavage group (HP improvement group) was randomly divided into two groups, one group was gavage with a PAPCH bacterial suspension (pre-h.pylori/post-PAPCH group, pre-H/post-P group), and the other group was gavage with physiological saline (HP group).

First-stage intragastric perfusion method for improving model

Blank group (improved NC group): each mouse was gavaged with 0.5mL of saline once every other day for 4 weeks in the h.pyri gavage group (improve HP group): each mouse was gavaged with 0.5ml h. pyri bacterial suspension once every other day for 4 weeks.

Second-stage intragastric lavage method for improving model

HP group: each mouse was gavaged with 0.5mL of physiological saline once a day for 2 weeks.

pre-H/post-P group: each mouse was gavaged with 0.5mL of PAPCH bacterial suspension once a day for 2 weeks.

Performing a test of an h.pyrori infection model on the model established after the first stage of gavage, comprising rapid urease detection and specific primer detection, wherein:

1. rapid urease detection and specific primer detection

One week after the first-stage gavage is finished, after fasting for 12 hours, the mice of the H.pyleri gavage group (improved HP group) are randomly killed for 6, one part of stomach tissues are taken for rapid urease detection, one part of stomach tissues are used for extracting DNA for specific primer detection, whether the stomach tissues of the mice are infected by H.pyleri is detected, the result is shown in figure 14, and A in the figure is the rapid urease detection result; b is the detection result of the specific primer.

As shown in fig. 14(a), stomach tissue of the blank group h. DNA of h.pyloi was extracted, PCR was performed using primers specific for h.pyloi VacA gene, and 1% agarose gel electrophoresis was performed after the reaction to detect that the product coincided with the expected at 678bp (see fig. 14 (B)).

Example 9 Rapid urease reaction

The mice after death were treated in the same manner as described in example 2, and their stomach tissues were placed in a quick urease test paper, and the results were observed within 5min, as shown in FIG. 15; in the figure, A: blank group; b: the pyrori treatment group; c: pyri/post-PAPCH treatment group.

As shown in fig. 15, the stomach tissue of the blank group of mice was placed in the test paper with no significant change in the color of the test paper, and the h.pyri-treated group and the pre-h.pyri/post-PAPCH (pre-H/post-P group) were placed in the test paper with the test paper rapidly turning red, and the h.pyri-treated group was more purplish red in color than the pre-H/post-P group.

Example 10 blood routine

And carrying out conventional blood detection on the blood of the mouse by using a full-automatic blood cell analyzer. The blood of the mice is subjected to blood routine detection, the numbers of the white blood cells and the neutrophils in the blood of the mice are shown in table 3, and the numbers of the white blood cells and the neutrophils in the pre-H.pyrori/post-PAPCH (pre-H/post-P group) and the H.pyrori treatment group (HP group) are obviously higher than those in the blank group (NC group), but the numbers of the white blood cells and the neutrophils in the H.pyrori treatment group are not obviously different between the two groups. P<0.05 indicates that the numbers of leukocytes in the h.pyri-treated group and pre-H/post-P group were significantly different from those in the blank group,^#P<0.05 indicated that there was a significant difference in the numbers of neutrophils between the H.pyri-treated group and the pre-H/post-P group compared to the blank group.

TABLE 3 leukocyte and neutrophil counts

EXAMPLE 11 ELISA assay of the levels of PG I, PG II, CRP, GSH-PX, SOD, and MDA in mouse sera as in example 5

1. Effect of PaPCH on improving H.pyrori infection on PG I and PG II in mouse serum

The content of pepsinogen I (PG I) in mouse serum is shown in FIG. 16, wherein NC is blank group; HP is h. pre-H/post-P is pre-H.

In fig. 16, the content of pgi in the serum of mice in the blank group, the h.pyri-treated group, and the pre-h.pyri/post-PAPCH group (pre-H/post-P group) was 187.26, 125.03, and 137.12ng/mL, and the pgi in the serum of the h.pyri-treated group and the pre-H/post-P group was significantly reduced compared to the blank group, and was 0.67 and 0.73 times of the blank group, respectively. P<0.05 indicates that the H.pyri-treated group, pre-H/post-P group, was significantly different from the blank group. The content of PG II in the serum of mice in the blank group, the H.pyri treatment group and the pre-H.pyri/post-PAPCH group (the pre-H/post-P group) is 137.23, 84.51 and 106.36ng/mL, compared with the blank group, the content of PG II in the serum of the H.pyri treatment group and the pre-H/post-P group is obviously reduced and is respectively 0.61 and 0.77 times of that of the blank group, and the content of PG II in the pre-H/post-P group is obviously higher than that of the H.pyri treatment group and is 1.28 times of that of the H.pyri treatment group. P<0.05 indicates that the h.pyri-treated group and the pre-H/post-P group were significantly different from the blank group,^#P<0.05 indicated a significant difference between the pre-H/post-P group and the H.pyri-treated group.

2. Effect of PAPCH on ameliorating h

The content of C-reactive protein (CRP) in mouse serum is shown in FIG. 17, wherein NC is blank group; HP is h. pre-H/post-P is pre-H.

In FIG. 17, the CRP content in the serum of mice in the blank group, H.pyri-treated group, pre-H.pyri/post-PAPCH group (pre-H/post-P group) was 235.40, 488.75, 302.00ng/mL, H.pyri-treated group, pre-H/post-P group and empty groupCompared with the white group, the content of CRP in serum is obviously increased by 2.07 times and 1.2 times of that in the blank group, and the content of CRP in the H.pyri treatment group is obviously higher than that in the pre-H/post-P group and is 1.62 times of that in the pre-H/post-P group. P<0.05 indicates that the H.pyri-treated group and the pre-H/post-P group were significantly different from the blank group,^#P<0.05 indicated a significant difference between the pre-H/post-P group and the H.pyri-treated group.

3. Effect of PAPCH on ameliorating h

The content of Malondialdehyde (MDA) in mouse serum is shown in FIG. 18, wherein NC is blank group; HP is h. pre-H/post-P is pre-H.

In fig. 18, the serum MDA content of mice in the blank group, the h.pyri-treated group, and the pre-h.pyri/post-PAPCH group (pre-H/post-P group) was 16.09, 40.80, and 25.97ng/mL, and the serum MDA content of the h.pyri-treated group and the pre-H/post-P group was significantly increased compared to the blank group, and was 2.55 and 1.62 times higher than that of the blank group, respectively, and the MDA content of the h.pyri-treated group was significantly higher than that of the pre-H/post-P group, and was 1.57 times higher than that of the pre-H/post-P group. P<0.05 indicates that the h.pyri-treated group and the pre-H/post-P group were significantly different from the blank group,^#P<0.05 indicated a significant difference between the pre-H/post-P group and the H.pyri-treated group.

4. Effect of PAPCH on GSH-PX in mouse serum to ameliorate h

The content of glutathione peroxidase (GSH-PX) in mouse serum is shown in FIG. 19, wherein NC is blank group; HP is h. pre-H/post-P is pre-H.

In fig. 19, the content of GSH-PX in the sera of mice in the blank group, h.pyri-treated group, pre-h.pyri/post-PAPCH group (pre-H/post-P group) was 120.02, 63.35, 106.71ng/mL, and both the content of GSH-PX in the sera of h.pyri-treated group and pre-H/post-P group were significantly reduced compared to the blank group, respectively 0.52 and 0.87 times of the blank group, and the content of GSH-PX in the pre-H/post-P group was significantly higher than that of h.pyri-treated group, and was 1.68 times of that of h.pyri-treated group. P<0.05 means that the H.pyri-treated group and the pre-H/post-P group were significantly compared with the blank groupThe difference in the degree of tackiness is large,^#P<0.05 indicated a significant difference between the pre-H/post-P group and the H.pyri-treated group.

5. Effect of PAPCH on ameliorating h

The content of superoxide dismutase (SOD) in mouse serum is shown in figure 20, wherein NC is blank group; HP is h. pre-H/post-P is pre-H.

In fig. 20, the serum SOD content of mice in the blank group, h.pyri-treated group, pre-h.pyri/post-PAPCH group (pre-H/post-P group) was 14.46, 9.79, 13.24ng/mL, the serum SOD content of h.pyri-treated group was significantly reduced to 0.68 times that of the blank group compared to the blank group, and the pre-H/post-P group was not significantly different from the blank group. And the SOD content of pre-H/post-P group is obviously higher than that of H.pyri treated group and is 1.35 times of that of H.pyri treated group. P<0.05 indicates that the h.pyri treated group was significantly different from the blank group.^#P<0.05 indicated that the pre-H/post-P group was significantly different from the H.pyri-treated group.

Example 12 the Effect of PAPCH on ameliorating the effects of H.pyrori infection on TNF-alpha and MUC5AC expression in mouse gastric tissue

1. Extraction of total RNA from GES-1 stomach tissue

Same procedure as in step 1 of example 6.

2. Reverse transcription of total RNA from gastric tissue

Same procedure as in step 2 of example 6.

3. Real time PCR reaction

The same procedure as in step 3 of example 6.

4. Effect of PAPCH on TNF-alpha expression in mouse stomach tissue

The effect of PAPCH and h.pyri on TNF- α mRNA expression in mouse gastric tissue is shown in fig. 21, where NC is blank; HP is h. pre-H/post-P is pre-H.

In fig. 21, TNF- α mRNA expression was significantly increased 2.71 and 2.27 times higher in both the h.pyri-treated group and the pre-h.pyri/post-PAPCH (pre-H/post-P) blank group than in the blank group. But p isThe expression level of TNF-alpha mRNA in the re-H/post-P group was significantly lower than that in the H.pyri-treated group, which was 0.84 times that in the H.pyri-treated group. P<0.05 indicates that the h.pyri treated group was significantly different from the pre-H/post-P group compared to the blank group,^#P<0.05 indicated that the pre-H/post-P group was significantly different from the H.pyri-treated group.

5. Effect of PAPCH on ameliorating h. pyri infection on the expression of MUC5AC in gastric tissue of mice

The effect of PAPCH and h. pyri on the expression of MUC5AC mRNA in mouse gastric tissue is shown in fig. 22, where NC is blank; HP is h. pre-H/post-P is pre-H.

In fig. 22, compared to the blank group, both the h.pyri-treated group and the pre-h.pyri/post-PAPCH group (pre-H/post-P group) significantly increased the expression level of MUC5AC mRNA 2.37-fold and 1.48-fold, respectively, compared to the blank group. However, the expression level of MUC5AC mRNA in the pre-H/post-P group was significantly lower than that in the H.pyri-treated group, which was 0.63 times that in the H.pyri-treated group. P<0.05 indicates a significant difference from the h.pyri-treated group and the pre-H/post-P group compared to the blank group,^#P<0.05 indicated that the pre-H/post-P group was significantly different from the H.pyri-treated group.

Example 13 HE staining of stomach tissue

The procedure was the same as in example 7. The results of HE staining of mouse stomach tissue are shown in FIG. 23, in which A is a blank group; b is h. C is pre-H.pyri/post-PAPCH treatment group

In fig. 23, red blood cells were observed in the gastric mucosa of the h.pyri treated group, and there was necrosis and exfoliation of the mucosal surface; red blood cells were visible on the gastric mucosa of the pre-h, pylori/post-PAPCH group, with a small number of cells exfoliated. The gastric mucosa of the blank mice has no mucosa shedding and erosion phenomenon.

Examples 8-13 are procedures for treating H.pylori-infected mice with PAPH. Mice one month after gavage with h.pyri were divided into two groups, one treated with PAPCH for 14d and one untreated. Mice in the h.pyri treated group and pre-h.pyri/post-PAPCH group (pre-H/post-P group) were routinely found to have significantly elevated leukocytes and neutrophils compared to the blank group by testing the blood of the mice, suggesting bacterial infection in vivo. H. pylori colonization in gastric tissues of h.pylori treated and pre-H/post-P mice was also shown by rapid urease experiments.

The content of PG I and PG II in mouse serum is detected by an ELISA method, and the PG I in the serum of the H.pyriori treatment group and the PG I in the pre-H/post-P group are reduced compared with the blank group and are respectively 0.67 and 0.73 times of the blank group. The content of PG II in the serum is reduced, which is respectively 0.61 and 0.77 times of that in the blank group. C-reactive protein (CRP) is a very sensitive reactive protein in the inflammatory phase and is clinically closely related to infection, tissue necrosis, tumor and the like. The CRP content in the serum of the H.pyriri-treated group and the pre-H/post-P group is increased compared with the blank group by detecting the CRP content in the serum of the mice by an ELISA method, but the CRP content of the H.pyriri-treated group is obviously higher than that of the pre-H/post-P group and is 1.62 times higher than that of the pre-H/post-P group. The content of SOD, MDA and GSH-PX in the blood serum of the mouse is detected by an ELISA method, so that the content of SOD in the blood serum of the H.pyleri treatment group is obviously reduced to 0.68 time of that of the blank group compared with the blank group, the content of SOD in the pre-H/post-P group is not obviously different from that of the blank group, and the content of SOD in the pre-H/post-P group is obviously higher than that of the H.pyleri treatment group and is 1.35 times of that of the H.pyleri treatment group; the content of MDA in serum of the H.pyri treated group and the pre-H/post-P group is obviously increased compared with that of the blank group, but the content of MDA of the H.pyri treated group is obviously higher than that of the pre-H/post-P group and is 1.57 times that of the pre-H/post-P group; the content of GSH-PX in serum was significantly reduced in both the h.pyri-treated group and the pre-H/post-P group compared to the blank group, but the content of GSH-PX in the pre-H/post-P group was significantly higher than that in the h.pyri-treated group, which was 1.68 times that in the h.pyri-treated group.

The expression levels of TNF-alpha mRNA and MUC5AC mRNA in mouse gastric tissues are detected by a qRT-PCR method, so that the expression levels of the TNF-alpha mRNA in the H, pylori-treated group and the pre-H/post-P group are remarkably increased compared with that in the blank group, but the expression level of the TNF-alpha mRNA in the pre-H/post-P group is remarkably lower than that in the H, pylori-treated group and is 0.84 times that in the H, pylori-treated group; compared with the blank group, the expression level of MUC5AC mRNA in the H.pyri treated group and the pre-H/post-P group is obviously increased, but the expression level of MUC5AC mRNA in the pre-H/post-P group is obviously lower than that in the H.pyri treated group and is 0.63 times that in the H.pyri treated group.

The gastric mucosa of the H.pyri treated group can see red blood cells after the stomach tissue is subjected to HE staining, and the surface layer of the mucosa has cell necrosis and desquamation; red blood cells are visible in the gastric mucosa of pre-H/post-P, and a small number of cells are shed. The results show that the pathological state of the stomach tissue in the pre-H/post-P treated group is better than that in the non-PAPCH improved group.

The detection results of the examples 1 to 13 show that the PAPCH has a certain improvement effect on the infection of the H.pyriri, probably because the PAPCH enters the stomach and secretes antibacterial substances to inhibit the growth and activity of the H.pyriri and inhibit the generation of local proinflammatory cytokines. Through local or systemic immunoregulation and improvement of activities of antioxidant enzymes, the immune state of an organism is regulated, the clearing capacity of an immune system on H.

Claims

1. A probiotic agent PAPH for preventing and treating helicobacter pylori infection is characterized by comprising lactobacillus casei and a protective agent; the addition amount of the protective agent is 50-150 wt% of the dosage of the lactobacillus casei.

2. The probiotic agent PAPH for preventing and treating helicobacter pylori infection according to claim 1, wherein the Lactobacillus casei is Lactobacillus casei lyT-7 with the deposit number: CCTCC NO: m2010197.

3. The probiotic agent PAPCH for preventing and treating helicobacter pylori infection according to claim 1, wherein the addition amount of the protective agent is 80-100% wt of the amount of lactobacillus casei.

4. The probiotic agent PAPH for preventing and treating helicobacter pylori infection according to claim 1 or 3, wherein the protective agent comprises the following components in percentage by weight:

5. The probiotic agent PAPH for preventing and treating helicobacter pylori infection according to claim 4, wherein the protective agent comprises the following components in percentage by weight: 15% of fructo-oligosaccharide, 15% of galacto-oligosaccharide, 10% of isomaltooligosaccharide, 2% of spirulina dry powder, 1% of glycerol and the balance of sterile water.

6. The method for preparing the probiotic agent PAPH according to any one of claims 1 to 5, comprising the following steps:

(1) inoculating lactobacillus casei on an MRS liquid culture medium in an inoculation amount of 4-10%, and then carrying out shake cultivation for 15-20 h at 35-37 ℃ and 150-200 rpm;

7. The method according to claim 6, wherein the lactobacillus casei is inoculated in an amount of 4%.

8. The method according to claim 6, wherein the MRS liquid medium comprises peptone, yeast extract, beef extract, glucose, sodium acetate, ammonium citrate, magnesium sulfate, manganese sulfate, dipotassium phosphate, Tween, and ultrapure water.

9. The preparation method according to claim 6, wherein the number of viable bacteria in the lyophilized microbial inoculum is more than 1 x 10⁹CFU/g。

10. The probiotic agent PAPH for preventing and treating helicobacter pylori infection according to claims 1-5, wherein the agent is powder, tablet, pill or injection.