Preparation, separation and purification method and application of lactobacillus casei IOB-P9 anti-helicobacter pylori active substance

Abstract

The invention belongs to the technical field of microorganisms, and discloses preparation, separation and purification methods and application of lactobacillus casei IOB-P9 anti-helicobacter pylori active substance separation.The preparation, separation and purification methods of the active substance separationcomprises the following steps that lactobacillus casei IOB-P9 is cultured, and active ingredients are prepared; performing macroporous adsorption chromatography separation on the active ingredients; separating active components by an anion exchange method; performing gel filtration chromatography separation on the active ingredients; and verifying the activity of the active component to obtain the active component of the lactobacillus casei IOB-P9. Active substances in the lactobacillus casei IOB-P9 obtained according to the method have certain sensitivity to protease, which indicates that the bacteriostatic active substances are protein or peptide substances. In addition, the bacteriostatic active substance has better thermal stability, is not easy to dissolve in organic reagents and has long preservation time, so that the bacteriostatic active substance is beneficial to storage and transportation and has higher production and application values.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular to a preparation and separation and purification method for separating and purifying active substances of Lactobacillus casei IOB-P9 against Helicobacter pylori and an application thereof. Background Art

[0002] Helicobacter pylori is a Gram-negative bacterium that grows in the gastric mucosa. It is the main pathogenic factor of chronic gastritis and peptic ulcer, and is also the main predisposing factor of gastric cancer. Helicobacter pylori infection is associated with the occurrence of gastric complications such as gastritis, gastric and duodenal ulcers. Among gastric cancers caused by carcinogenic infections, Helicobacter pylori-related gastric cancer accounts for the largest proportion. Studies have also shown that more than 77% of new gastric cancer cases and more than 89% of new non-cardiac gastric cancer cases are related to Helicobacter pylori infection. It can be seen that digestive system diseases caused by Helicobacter pylori have become a major global public health issue.

[0003] Over the past two decades, the recommended treatment for eradication of Helicobacter pylori has been standard triple therapy, which has an eradication rate of over 90% and is highly accepted among clinicians due to its safety. However, due to the increasing resistance of Helicobacter pylori to antibiotics, the efficacy of triple therapy has dropped to less than 70%, and it is no longer the best treatment for Helicobacter pylori. Some studies have begun to use probiotics associated with antibiotics as a treatment method. Probiotics are defined as "live beneficial microorganisms" that, when administered in adequate amounts, can benefit the host's health. They can reduce the colonization of Helicobacter pylori by secreting antimicrobial metabolites, modulate the host immune response, and reduce the expression of inflammatory factors, thereby alleviating gastric inflammation caused by Helicobacter pylori infection.

[0004] At present, there is still a lack of effective drugs for the treatment of this disease. Screening candidate compounds that have the ability to inhibit Helicobacter pylori is of great significance for the development of drugs for the treatment of gastrointestinal diseases such as gastritis. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a method for preparing and separating and purifying active substances of Lactobacillus casei IOB-P9 against Helicobacter pylori and its application.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A use of Lactobacillus casei IOB-P9 in the preparation of a drug for treating Helicobacter pylori, wherein the Lactobacillus casei IOB-P9 was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

[0008] A use of Lactobacillus casei IOB-P9 in the preparation of a drug for inhibiting and killing Helicobacter pylori. The Lactobacillus casei IOB-P9 was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

[0009] A method for separating, preparing and purifying active substances of Lactobacillus casei IOB-P9 against Helicobacter pylori comprises the following steps:

[0010] (1) Cultivation of Lactobacillus casei IOB-P9 and preparation of active ingredients;

[0011] (2) separation of active ingredients by macroporous adsorption chromatography;

[0012] (3) separation of active ingredients by anion exchange method;

[0013] (4) Gel filtration chromatography separation of active ingredients;

[0014] (5) verifying the activity of the active ingredient to obtain the active ingredient of Lactobacillus casei IOB-P9;

[0015] The Lactobacillus casei IOB-P9 was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

[0016] Furthermore, step (1) includes: bacterial activation and fermentation culture, the optimal seed age is 20-24h, the inoculation amount is 1%, and the fermentation time is 48h; wherein the seed culture medium is 40g / L defatted soybean powder, 10g / L glucose, and the solvent is water; the fermentation culture medium is 90g / L high-temperature soybean meal, 5g / L glucose, 1g / L bran powder, and the solvent is water.

[0017] In step (2), the operation steps of separating the active substance by macroporous adsorption chromatography are as follows:

[0018] a. XAD-16N macroporous adsorption resin was soaked in anhydrous ethanol for 24 hours to allow the resin to fully swell, then rinsed with ultrapure water, and soaked in 4% HCl and 4% NaOH solutions for 5 hours in turn; then, the resin was washed with ultrapure water until it reached a neutral state;

[0019] b. Rinse the column with ultrapure water;

[0020] c. The fermentation broth was centrifuged to obtain the supernatant, which was passed through the XAD-16N macroporous adsorption resin at a flow rate of 4 mL / min, and then the substances adsorbed on the macroporous resin were eluted with ethanol at a flow rate of 2 mL / min with a volume concentration of 10%, 20%, 40%, 60%, 80%, and 100% in 2 times the column volume, and the eluents of each gradient were collected;

[0021] In step (3), the operation steps of separating the active substance by anion exchange method are as follows:

[0022] d. crude separation of the eluent obtained from the XAD-16N macroporous adsorption resin;

[0023] e. Pre-treating the anion exchange filler, wet-packing the column and cleaning it;

[0024] f. Continue flushing with 20 mM Tris-HCl buffer for 3-5 column volumes to equilibrate the column;

[0025] g. After equilibration, load the sample, rinse the unbound proteins on the column with buffer, and then elute with different gradients of eluent: 20mM Tris-HCl+0.2M sodium chloride, 20mM Tris-HCl+0.5M sodium chloride, 20mM Tris-HCl+1.0M sodium chloride, and collect the eluent of each gradient.

[0026] Furthermore, in step (2), 2 times the column volume of ethanol with a volume concentration of 20% is used for elution;

[0027] In step (3), 20 mM Tris-HCl + 0.2 M sodium chloride was used for elution.

[0028] Furthermore, in step (4), the operation steps of separating the active substance by gel filtration are:

[0029] h. The active fraction obtained from the anion exchange was concentrated to 2 ml;

[0030] i. Treat the Sephadex G-75 gel and perform wet column packing, balancing more than 2 column volumes with ultrapure water;

[0031] j. After equilibration, load the sample, elute with buffer, i.e. ultrapure water, and collect the eluate.

[0032] Furthermore, the steps for preparing the fermentation broth in step (2) are as follows:

[0033] Strain activation and culture: taking out Lactobacillus casei IOB-P9 from a glycerol tube with an inoculating loop and streaking it on an MRS medium plate, and culturing it in a closed manner at 36±2°C for 20-22 hours; taking a single colony to a seed medium, and culturing it at 36±2°C for 24 hours to obtain a primary seed liquid, inoculating the primary seed liquid at a 1% rate to a secondary seed medium, and culturing it at 36±2°C for 24 hours, inoculating the secondary seed liquid at a 1% rate to a fermentation medium, and culturing it at 37°C for 48 hours, and collecting the fermentation liquid supernatant to obtain a fermentation liquid;

[0034] The seed culture medium is 40 g / L defatted soybean powder, 10 g / L glucose, and the solvent is water; the fermentation culture medium is 90 g / L high-temperature soybean meal, 5 g / L glucose, 1 g / L bran powder, and the solvent is water.

[0035] Further, step (5) includes preliminary activity detection of the active ingredient of Lactobacillus casei IOB-P9, using a filter paper disc method to measure the eluate of the macroporous adsorption chromatography, using an Oxford cup method to measure the eluate of the anion exchange method and the eluate of the gel filtration chromatography, using Sarcina luteus as an indicator bacterium;

[0036] Step (5) comprises HPLC analysis of the active ingredients of Lactobacillus casei IOB-P9: the active ingredients in the macroporous adsorption chromatography with a volume concentration of 20% ethanol eluent, the anion exchange chromatography with a volume concentration of 20mM Tris-HCl+0.2M sodium chloride eluent and the gel filtration chromatography are pre-treated and placed at 4°C for standby use, and the Agilent ZORBAXNH2 chromatographic column is equilibrated with mobile phase A, i.e., 100% acetonitrile+0.1% TFA, for 1 hour until the baseline is stable;

[0037] Step (5) comprises pretreatment of the active ingredient of Lactobacillus casei IOB-P9: freeze-drying the supernatant of the fermentation broth, weighing 6 portions at 0.2 g / portion, adding 1 ml each of water, methanol, ethanol, ethyl acetate, chloroform and acetone, mixing with ultrasound for 5 min, and centrifuging at 10000 r / min for 10 min;

[0038] Step (5) includes investigating the thermal stability of the active ingredient of Lactobacillus casei IOB-P9: treating with water bath at 25°C, 30°C, 37°C, 60°C, 80°C, 100°C, 115°C, and 121°C for 20 minutes respectively, cooling to room temperature, and using Sarcina lutea as an indicator bacteria;

[0039] Step (5) includes exploring the sensitivity of the active ingredient of Lactobacillus casei IOB-P9 to acid and alkali: at various pH values ​​from 3.0 to 9.0, the supernatant is mixed with each gradient pH solution, and then the pH value is adjusted to 4.0 with 1M NaOH or 1M HCl, and Sarcina luteus is used as an indicator bacteria;

[0040] Step (5) includes investigating the enzyme sensitivity of the active ingredient of Lactobacillus casei IOB-P9: treating the active ingredient with pepsin, trypsin, proteinase K, and α-chymotrypsin, using Sarcina luteus as an indicator bacterium;

[0041] Step (5) includes the inhibitory effect of the active ingredients of Lactobacillus casei IOB-P9 on the pathogenic bacteria Helicobacter pylori that causes intestinal diseases.

[0042] The anti-Helicobacter pylori active substance of Lactobacillus casei IOB-P9 prepared by the method as described above.

[0043] The use of the anti-Helicobacter pylori active substance of Lactobacillus casei IOB-P9 as described above in the preparation of drugs for inhibiting Helicobacter pylori.

[0044] The advantages and positive effects achieved by the present invention are:

[0045] 1, the preparation and separation method of lactobacillus casei 10B-P9 active ingredient of the present invention, explore and produce antibacterial substance optimum fermentation condition by experiment, to improve synthesis amount: the optimum seed age of seed is 20-24h, and inoculum size is 1%, and fermentation time is 48h.And use macroporous adsorption chromatography, anion exchange method and gel filtration chromatography to successfully realize the separation and purification of active substance, verified the antibacterial activity of active substance, therefore, above-mentioned lactobacillus casei active substance preparation and separation method, for the separation and extraction of this active substance and subsequent application in the field provide reference.

[0046] 2. The active substances in Lactobacillus casei IOB-P9 obtained according to the method of the present invention have good inhibitory and killing effects on Helicobacter pylori, providing a new idea for improving the dilemma in the treatment of Helicobacter pylori.

[0047] 3, according to the active substance in the obtained Lactobacillus casei 10B-P9 of the inventive method, there is certain sensitivity to protease, indicating that the antibacterial active substance has protein or peptide substances. In addition, this antibacterial active substance has good thermal stability, is not easily soluble in organic reagents, has a long shelf life, is therefore conducive to storage and transportation, and has larger production application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : is a morphological characteristic diagram of Lactobacillus casei in the present invention; wherein: Figure a is a macroscopic morphological diagram; Figure b is a microscopic morphological diagram;

[0049] Figure 2 The inhibition zones of each gradient eluate of the macroporous adsorption resin effluent of the present invention;

[0050] Figure 3 The inhibition zone of each gradient eluent of the anion exchange column in the present invention;

[0051] Figure 4 This is the peak diagram of the gel filtration layer in the present invention;

[0052] Figure 5 The inhibition zones of each gradient eluent of the gel filtration chromatography column of the present invention;

[0053] Figure 6 The HPLC analysis chromatograms of the present invention; wherein: Figure a is a chromatogram of macroporous adsorption resin 20% ethanol; Figure b is a chromatogram of anion exchange resin 20mM sodium acetate + 0.2M sodium chloride; Figure c is a chromatogram of gel filtration chromatography elution components; Figure d is an antibacterial zone diagram of HPLC elution components, wherein y1 is the antibacterial activity of the elution component at about 8min, and y2 is the antibacterial activity of the elution component at about 11min;

[0054] Figure 7 The figure is a test diagram of the active ingredient extraction by different organic reagents in the present invention; wherein, the left column shows the antibacterial results of water, ethanol, and ethyl acetate extracts from top to bottom, and the right column shows the antibacterial effects of methanol, acetone, and chloroform extracts from top to bottom;

[0055] Figure 8 This is a graph showing the thermal stability test of the active ingredient in the present invention;

[0056] Fig. 9 This is a test diagram of the acid-base sensitivity of the active ingredient in the present invention;

[0057] Fig.10 This is an experimental diagram of the antibacterial effect of the active ingredients in the present invention on Helicobacter pylori.

[0058] Lactobacillus casei IOB-P9 (Lactobacillus casei) used in the present invention is isolated from healthy human feces and deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee on December 27, 2021, with a deposit number of CGMCC No. 24195. This strain is a well-known strain in the prior art and has been disclosed in patent publication CN114617265A, i.e., patent application number CN202210525330.7. DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0060] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0061] A use of Lactobacillus casei IOB-P9 in the preparation of a drug for treating Helicobacter pylori, wherein the Lactobacillus casei IOB-P9 was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

[0062] A use of Lactobacillus casei IOB-P9 in the preparation of a drug for inhibiting and killing Helicobacter pylori. The Lactobacillus casei IOB-P9 was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

[0063] A method for separating, preparing and purifying active substances of Lactobacillus casei IOB-P9 against Helicobacter pylori comprises the following steps:

[0064] (1) Cultivation of Lactobacillus casei IOB-P9 and preparation of active ingredients;

[0065] (2) separation of active ingredients by macroporous adsorption chromatography;

[0066] (3) separation of active ingredients by anion exchange method;

[0067] (4) Gel filtration chromatography separation of active ingredients;

[0068] (5) verifying the activity of the active ingredient to obtain the active ingredient of Lactobacillus casei IOB-P9;

[0069] The Lactobacillus casei IOB-P9 was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

[0070] Preferably, step (1) comprises: bacterial activation and fermentation culture, the optimal seed age is 20-24h, the inoculation amount is 1%, and the fermentation time is 48h; wherein the seed culture medium is 40g / L defatted soybean powder, 10g / L glucose, and the solvent is water; the fermentation culture medium is 90g / L high-temperature soybean meal, 5g / L glucose, 1g / L bran powder, and the solvent is water.

[0071] In step (2), the operation steps of separating the active substance by macroporous adsorption chromatography are as follows:

[0072] a. XAD-16N macroporous adsorption resin was soaked in anhydrous ethanol for 24 hours to allow the resin to fully swell, then rinsed with ultrapure water, and soaked in 4% HCl and 4% NaOH solutions for 5 hours in turn; then, the resin was washed with ultrapure water until it reached a neutral state;

[0073] b. Rinse the column with ultrapure water;

[0074] c. The fermentation broth was centrifuged to obtain the supernatant, which was passed through the XAD-16N macroporous adsorption resin at a flow rate of 4 mL / min, and then the substances adsorbed on the macroporous resin were eluted with ethanol at a flow rate of 2 mL / min with a volume concentration of 10%, 20%, 40%, 60%, 80%, and 100% in 2 times the column volume, and the eluents of each gradient were collected;

[0075] In step (3), the operation steps of separating the active substance by anion exchange method are as follows:

[0076] d. crude separation of the eluent obtained from the XAD-16N macroporous adsorption resin;

[0077] e. Pre-treating the anion exchange filler, wet-packing the column and cleaning it;

[0078] f. Continue flushing with 20 mM Tris-HCl buffer for 3-5 column volumes to equilibrate the column;

[0079] g. After equilibration, load the sample, rinse the unbound proteins on the column with buffer, and then elute with different gradients of eluent: 20mM Tris-HCl+0.2M sodium chloride, 20mM Tris-HCl+0.5M sodium chloride, 20mM Tris-HCl+1.0M sodium chloride, and collect the eluent of each gradient.

[0080] Preferably, in step (2), 2 times the column volume of ethanol with a volume concentration of 20% is used for elution;

[0081] In step (3), 20 mM Tris-HCl + 0.2 M sodium chloride was used for elution.

[0082] Preferably, in step (4), the operation steps of separating the active substance by gel filtration are:

[0083] h. The active fraction obtained from the anion exchange was concentrated to 2 ml;

[0084] i. Treat the Sephadex G-75 gel and perform wet column packing, balancing more than 2 column volumes with ultrapure water;

[0085] j. After equilibration, load the sample, elute with buffer, i.e. ultrapure water, and collect the eluate.

[0086] Preferably, the steps for preparing the fermentation broth in step (2) are as follows:

[0087] Strain activation and culture: taking out Lactobacillus casei IOB-P9 from a glycerol tube with an inoculating loop and streaking it on an MRS medium plate, and culturing it in a closed manner at 36±2°C for 20-22 hours; taking a single colony to a seed medium, and culturing it at 36±2°C for 24 hours to obtain a primary seed liquid, inoculating the primary seed liquid at a 1% rate to a secondary seed medium, and culturing it at 36±2°C for 24 hours, inoculating the secondary seed liquid at a 1% rate to a fermentation medium, and culturing it at 37°C for 48 hours, and collecting the fermentation liquid supernatant to obtain a fermentation liquid;

[0088] The seed culture medium is 40 g / L defatted soybean powder, 10 g / L glucose, and the solvent is water; the fermentation culture medium is 90 g / L high-temperature soybean meal, 5 g / L glucose, 1 g / L bran powder, and the solvent is water.

[0089] Preferably, step (5) comprises a preliminary activity test of the active ingredient of Lactobacillus casei IOB-P9, using a filter paper method to measure the eluate of the macroporous adsorption chromatography, using an Oxford cup method to measure the eluate of the anion exchange method and the eluate of the gel filtration chromatography, using Sarcina luteus as an indicator bacterium;

[0090] Step (5) comprises HPLC analysis of the active ingredients of Lactobacillus casei IOB-P9: the active ingredients in the macroporous adsorption chromatography with a volume concentration of 20% ethanol eluent, the anion exchange chromatography with a volume concentration of 20mM Tris-HCl+0.2M sodium chloride eluent and the gel filtration chromatography are pre-treated and placed at 4°C for standby use, and the Agilent ZORBAXNH2 chromatographic column is equilibrated with mobile phase A, i.e., 100% acetonitrile+0.1% TFA, for 1 hour until the baseline is stable;

[0091] Step (5) comprises pretreatment of the active ingredient of Lactobacillus casei IOB-P9: freeze-drying the supernatant of the fermentation broth, weighing 6 portions at 0.2 g / portion, adding 1 ml each of water, methanol, ethanol, ethyl acetate, chloroform and acetone, mixing with ultrasound for 5 min, and centrifuging at 10000 r / min for 10 min;

[0092] Step (5) includes investigating the thermal stability of the active ingredient of Lactobacillus casei IOB-P9: treating with water bath at 25°C, 30°C, 37°C, 60°C, 80°C, 100°C, 115°C, and 121°C for 20 minutes respectively, cooling to room temperature, and using Sarcina lutea as an indicator bacteria;

[0093] Step (5) includes exploring the sensitivity of the active ingredient of Lactobacillus casei IOB-P9 to acid and alkali: at various pH values ​​from 3.0 to 9.0, the supernatant is mixed with each gradient pH solution, and then the pH value is adjusted to 4.0 with 1M NaOH or 1M HCl, and Sarcina luteus is used as an indicator bacteria;

[0094] Step (5) includes investigating the enzyme sensitivity of the active ingredient of Lactobacillus casei IOB-P9: treating the active ingredient with pepsin, trypsin, proteinase K, and α-chymotrypsin, using Sarcina luteus as an indicator bacterium;

[0095] Step (5) includes the inhibitory effect of the active ingredients of Lactobacillus casei IOB-P9 on the pathogenic bacteria Helicobacter pylori that causes intestinal diseases.

[0096] The anti-Helicobacter pylori active substance of Lactobacillus casei IOB-P9 prepared by the method as described above.

[0097] The use of the anti-Helicobacter pylori active substance of Lactobacillus casei IOB-P9 as described above in the preparation of drugs for inhibiting Helicobacter pylori.

[0098] Specifically, the relevant preparation and detection are as follows:

[0099] Example 1: Cultivation of Lactobacillus casei IOB-P9 and preparation of fermentation samples

[0100] (1) Activation and culture of strains: Use an inoculation loop to take out Lactobacillus casei from the glycerol tube and streak it on an MRS medium plate, and culture it in a closed environment at 36±2°C for 20-22 hours. Take a single colony and add it to a seed medium, culture it at 36±2°C for 24 hours to obtain a first-level seed solution, inoculate the first-level seed solution at a 1% rate to a second-level seed medium, culture it at 36±2°C for 24 hours, inoculate the second-level seed solution at a 1% rate to a fermentation medium at 37°C for 48 hours, and collect the fermentation supernatant.

[0101] The seed culture medium is 40g / L defatted soybean powder, 10g / L glucose, and the solvent is water; the fermentation culture medium is 90g / L high-temperature soybean meal, 5g / L glucose, 1g / L bran powder, and the solvent is water.

[0102] Experimental results:

[0103] Lactobacillus casei strains activated Figure 1 As shown, the colony is round, with neat edges, smooth surface, opaque and milky white (see Figure 1 Figure a), the colony diameter is 1.0-2.0mm, with typical growth characteristics of lactic acid bacteria; the bacteria are purple after staining, and are Gram-positive rods. The bacteria are short rod-shaped, blunt at both ends, and have no spores (see Figure 1 (Fig. b).

[0104] Lactobacillus casei IOB-P9 (Lactobacillus casei) used in the present invention is isolated from healthy human feces and deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee on December 27, 2021, with a deposit number of CGMCC No. 24195. This strain is a well-known strain in the prior art and has been disclosed in patent publication CN114617265A, i.e., patent application number CN202210525330.7.

[0105] Example 2: Analysis and separation of active ingredients of Lactobacillus casei IOB-P9 soybean fermentation product

[0106] Experimental methods:

[0107] (1) Preparation of indicator bacteria suspension: Take 0.1 ml of fresh culture of Sarcina lutea and inoculate it into LB liquid culture medium in a 50 / 250 ml Erlenmeyer flask, culture it at 36±2°C for 12 h, centrifuge it, discard the supernatant, add sufficient amount of sterile 0.9% saline to resuspend the precipitate, centrifuge it, repeat washing three times, discard the supernatant, and finally resuspend it in 25 ml of 0.9% sterile saline and store it at 4°C for future use;

[0108] (2) Preparation of double-layer identification plates: Weigh 5 g of agar powder into a 500 ml conical flask and add 250 ml of deionized water; weigh 3.05 g of the finished identification culture base into a 250 ml conical flask and add 120 ml of deionized water. After sterilization, pour an appropriate amount of heated and melted plain agar into each glass plate. After cooling and solidifying, heat and melt the identification culture medium, cool to 45-50°C, add 1 ml of bacterial suspension, shake well, add 10 ml of bacterial identification culture medium to the solidified plain agar plate, shake well, and after cooling and solidifying, store at 4°C for later use.

[0109] (3) Component analysis by macroporous adsorption chromatography: The macroporous resin was pretreated according to the method described by the predecessors with slight modifications: the macroporous resin was soaked in anhydrous ethanol for 24 hours to fully swell the resin, then rinsed with ultrapure water, and soaked in 4% HCl and 4% NaOH solutions for 5 hours. Subsequently, the resin was washed with ultrapure water until it reached a neutral state. The column was filled with wet method, and the macroporous resin was filled to 2 / 3 of the column volume to ensure that the resin was completely immersed in the liquid. The fermentation liquid supernatant prepared in Example 1 was passed through the macroporous resin column at a flow rate of 3-5 mL / min, and there should be no bubbles in the resin layer. After all the fermentation supernatant was drained, the unadsorbed stock solution was collected and all the effluent was collected. The substances adsorbed on the macroporous resin were eluted with 10%, 20%, 40%, 60%, 80% and 100% ethanol solutions of 2 times the column volume at a flow rate of 2 mL / min, and the eluents of each gradient were collected.

[0110] The activity was determined by the filter paper method. 30 μL of each gradient elution solution was spotted on each filter paper. After air drying, it was clamped to a double-layer identification plate with tweezers. Sarcina luteus was used as an indicator bacteria, and the activity was measured in three parallel groups.

[0111] (4) Component analysis by anion exchange method: The active part obtained from the crude separation of XAD-16N macroporous adsorption resin is subjected to anion exchange. The anion exchange filler is rinsed with ultrapure water and then wet packed into the column. The storage solution in the chromatography column is rinsed with ultrapure water, and then the buffer (20mM Tris-HCl) is used to balance the chromatography column for about 3-5 column volumes. After equilibration, the sample is loaded. After loading, the unbound impurities are washed away with the buffer for about 3-5 column volumes. Then, different gradient eluents are used for elution: 20mM Tris-HCl+0.2M sodium chloride, 20mM Tris-HCl+0.5M sodium chloride, 20mMTris-HCl+1.0M sodium chloride, and the eluents of each gradient are collected and detected.

[0112] The activity was determined by the Oxford cup method. 100 μL of each gradient elution was added to a double-layer identification plate. Sarcina luteus was used as an indicator bacterium, and the activity was measured in three parallel groups.

[0113] (5) Gel filtration chromatography for component analysis: The active ingredients eluted from the anion exchange chromatography were concentrated to 2 mL by rotary evaporation for gel filtration chromatography. The Sephadex G-75 gel was treated and wet packed into the column, which was rinsed with ultrapure water. The active ingredients were injected into the column and eluted with ultrapure water. The active substances were collected and detected.

[0114] The absorbance of the eluate in each collection tube was measured by a spectrophotometer at a wavelength of 254 nm, and a curve of absorbance versus tube number was drawn based on the data. The eluates with higher absorbance were combined according to the curve and concentrated by rotary evaporation, and the antibacterial activity was detected by the Oxford cup agar diffusion method using Micrococcus luteus as the indicator bacteria.

[0115] Experimental results:

[0116] (1) In this experiment, the fermentation broth supernatant was passed through a macroporous resin adsorption column, equilibrated with ultrapure water, and then eluted with different gradients of ethanol. The supernatant and each gradient eluate were collected, and the antibacterial activity of each gradient eluate was detected by the filter paper method. The results are as follows: Figure 2 As shown, only the 20% ethanol eluate had good antibacterial activity against the indicator bacteria, and no antibacterial activity was detected in the 10%, 40%, 60%, and 80% ethanol eluates. Figure 2Among them, A1 corresponds to the antibacterial result of the supernatant passing through the macroporous adsorption chromatography column effluent on the indicator bacteria, and there is no obvious inhibition zone, indicating that the active substances in the supernatant have been adsorbed by the macroporous adsorption resin. A2 and A3 correspond to the antibacterial results of the 10% ethanol eluate on the indicator bacteria. B1, B2, and B3 are 20% ethanol eluates with obvious inhibition zones, and the diameter of the inhibition zone is 13.56 mm, which have good antibacterial activity against Sarcina luteus. C1, C2, and C3 are 40% ethanol eluates, 60% ethanol eluates, and 80% ethanol eluates, respectively, and all have no antibacterial activity.

[0117] (2) In this experiment, 20 ml of the 20% ethanol eluate sample of XAD-16N macroporous resin was loaded and eluted with different concentrations of salt buffer. The eluate was collected and the antibacterial activity against the indicator bacteria was detected by the Oxford cup method. The results are as follows Figure 3 As shown: t1 is the buffer eluent, and there is no obvious inhibition zone, indicating that the antibacterial active substance is bound to the ion exchange resin; t2, t3, and t4 are the eluents of 20mM Tris-HCl+0.2M sodium chloride, and there are obvious transparent zones, indicating that the active ingredients are eluted.

[0118] (3) The active ingredients eluted from the anion exchange chromatography were concentrated and purified using a Sephadex G-75 gel column. One tube was collected for every 3 ml of eluate, and a total of 30 tubes were collected. The absorbance of each tube of eluate was measured at a detection wavelength of 254 nm and a curve was drawn. The results are shown in FIG. Figure 4 As shown, the absorbance of tubes 15-19 was significantly higher. The eluates from these five tubes were combined, concentrated by rotary evaporation, and the antibacterial activity was tested by the Oxford cup agar diffusion method. The results are as follows Figure 5 , and was found to have significant antibacterial activity.

[0119] Example 3: HPLC analysis of active ingredients

[0120] (1) HPLC detection conditions: The active ingredients in the macroporous adsorption chromatography 20% ethanol eluent, the anion exchange chromatography 20mM Tris-HCl + 0.2M sodium chloride eluent and the gel filtration chromatography were concentrated by rotary evaporation and placed at 4°C for use. The Agilent ZORBAX NH2 column was equilibrated with mobile phase A (100% acetonitrile + 0.1% TFA) for 1 hour until the baseline was stable. The method was set up, after injection, 85% mobile phase A: 15% mobile phase B (100% ultrapure water + 0.05% TFA) was used for isocratic elution for 20 minutes, and 30 μL of the pretreated sample was injected, and the UV detection wavelength was 254 nm.

[0121] (2) HPLC analysis results: The active ingredients in the macroporous adsorption chromatography 20% ethanol eluate, the anion exchange chromatography 20mMTris-HCl+0.2M sodium chloride eluate and the gel filtration chromatography were analyzed using an Agilent ZORBAX NH2 column. The chromatograms obtained are as follows: Figure 6 As shown, the chromatogram of the macroporous adsorption resin 20% ethanol eluate (see Figure 6 a) Multiple absorption peaks appear; chromatogram of anion exchange resin eluate (see Figure 6 b) The absorption peaks in the chromatographic peaks are significantly reduced, and the retention time of the chromatographic peaks is concentrated at 8-9min and 11-12min; the chromatogram of the active ingredient in gel filtration chromatography (see Figure 6 The absorption peak of c) is more single, with obvious single peaks at 7.95min and 11.341min. The collected liquid of these two peaks was freeze-dried and concentrated, adjusted to the same pH as the fermentation liquid, and tested for antibacterial activity. The results showed that both had antibacterial activity (see Figure 6 d).

[0122] Example 4: Analysis of the physicochemical properties of active ingredients in fermentation products

[0123] (1) Activity analysis of Lactobacillus casei IOB-P9 soybean fermentation product: After freeze-drying the fermentation broth supernatant obtained in Example 1, 6 portions were weighed at 0.2 g / portion, and 1 ml of water, methanol, ethanol, ethyl acetate, chloroform, and acetone were added respectively, mixed with ultrasound for 5 min, and centrifuged at 10000 r / min for 10 minutes. 30 uL of the supernatant was taken and added to a double-layer circular filter paper with a diameter of 5 mm. After drying in a fume hood, these filter papers were respectively attached to a double-layer identification plate containing indicator bacteria, and cultured in an incubator at 36±2° C. for 12 h to observe the inhibitory activity of different extracts on the indicator bacteria.

[0124] (2) To investigate the thermal stability of the active ingredient: the samples were treated in a water bath at 25°C, 30°C, 37°C, 60°C, 80°C, 100°C, 115°C, and 121°C for 20 min, respectively, and then cooled to room temperature. Sarcina luteus was used as an indicator bacteria, and the activity was measured in three parallel groups.

[0125] (3) The sensitivity of the active ingredients to acid and alkali was investigated: the initial pH of the fermentation broth supernatant was around 4. The fermentation broth was adjusted to various pH values ​​from 3.0 to 9.0 using 1 M NaOH or 1 M HCl. Sarcina luteus was used as an indicator bacterium, and the activity was measured in three parallel groups.

[0126] (4) To investigate the enzymatic sensitivity of the active ingredients: the active ingredients were treated with pepsin, trypsin, proteinase K, and α-chymotrypsin, and the activity was measured in three parallel groups using Sarcina luteus as an indicator bacterium.

[0127] Experimental results:

[0128] (1) Figure 7 As shown, there is no obvious transparent circle in the ethanol, ethyl acetate, acetone and chloroform extracts, and only the water and methanol extracts have obvious antibacterial circles, indicating that there are antibacterial active substances in the water and methanol extracts. The polarity of the active substances is relatively large and it is not easily soluble in organic reagents.

[0129] (2) Figure 8 As shown in the figure, in the thermal stability test, after being treated in a water bath at 25℃, 30℃, 37℃, 60℃, 80℃, 100℃, 115℃, and 121℃ for 20min, it was found that the temperature had little effect on the activity of the antibacterial substance. Compared with room temperature, the antibacterial activity after treatment can still be maintained at 90.06%, without significant decrease. This shows that the antibacterial substance has good thermal stability and still has antibacterial activity under high temperature conditions. It may be because the antibacterial substance does not have a complex spatial structure, so it can still maintain activity when the temperature changes.

[0130] (3) Fig. 9 As shown in the figure, the antibacterial substances are stable under acidic conditions, but easily lose their activity under neutral or alkaline conditions. The initial pH of the fermentation broth supernatant is around 4. When the pH is adjusted to 3.0, the diameter of the antibacterial zone of the antimicrobial peptide does not change significantly. When the pH drops to 5.0, the activity of the antibacterial substances has been completely lost, indicating that the antibacterial substances have been denatured and inactivated under higher pH conditions.

[0131] (4) As shown in Table 1, after the active ingredients were treated with pepsin, trypsin, proteinase K and α-chymotrypsin, it was found that the antibacterial substances showed different sensitivities to different proteases, and the diameters of the inhibition zones were reduced to varying degrees, indicating that the antibacterial active substances in the cell-free supernatant of Lactobacillus casei include protein or peptide substances.

[0132] Table 1 Sensitivity test of Lactobacillus casei soybean fermentation active products to different proteases

[0133]

[0134] Example 5: Antibacterial effect of active ingredients in fermentation products on Helicobacter pylori

[0135] Using the culture medium dilution method, 1 mL of liquid culture medium (special broth culture medium) was first added to each well of the 24-well plate, and then 1 mL of the active ingredient prepared in Example 2 was added to the first well. After blowing and mixing, 1 mL of the mixed solution in the first well was drawn and added to the second well. The same method was then used to repeat the multiple dilutions in sequence, and 1 mL was drawn from the last well and discarded. Samples were diluted at different multiples, and three parallel wells were set for each concentration. Broth culture medium plus ampicillin standard solution (3.5×10- 5 mg / ml) as the control group. Add 10 μL of the prepared 0.5 McFarland concentration of Helicobacter pylori to each well (except the blank control well), place the 24-well plate in a constant temperature incubator at 37°C for 16-20 hours, measure the OD600 results, and analyze.

[0136] Experimental results:

[0137] The results are as follows Fig.10 As shown, when the dilution factor is four times or less, each well has a clear appearance, no sterile growth, and the inhibition rate against Helicobacter pylori is more than 94.45%. Therefore, the concentration when diluted four times is taken as the minimum inhibitory concentration (MIC), and the minimum inhibitory concentration is 1.75 mg / ml.

[0138] In summary, the Lactobacillus casei IOB-P9 soybean fermentation product has a good inhibitory and killing effect on the pathogenic bacteria Helicobacter pylori that causes intestinal diseases. In addition, after the active ingredient is treated at high temperature, its activity is almost unaffected, it has good thermal stability, is easy to store, and has a certain sensitivity to proteases. After taking it, it is not easy to form residues when flowing through the stomach and intestines, so it is not easy to produce side effects on the human body, nor will it cause adverse effects on the environment, and has good application potential.

[0139] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. An application of Lactobacillus casei IOB-P9 in the preparation of a medicament for treating Helicobacter pylori, wherein the Lactobacillus casei IOB-P9 was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

2. An application of Lactobacillus casei IOB-P9 in the preparation of a drug for inhibiting and killing Helicobacter pylori, wherein the Lactobacillus casei IOB-P9 was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

3. A method for preparing and separating and purifying the anti-Helicobacter pylori active substance of Lactobacillus casei IOB-P9, characterized in that: The steps include: (1) Cultivation of Lactobacillus casei IOB-P9 and preparation of active ingredients; (2) separation of active ingredients by macroporous adsorption chromatography; (3) separation of active ingredients by anion exchange method; (4) Gel filtration chromatography separation of active ingredients; (5) verifying the activity of the active ingredient to obtain the active ingredient of Lactobacillus casei IOB-P9; The Lactobacillus casei IOB-P9 was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on December 27, 2021, and its deposit number is CGMCC No.24195.

4. The method according to claim 3, characterized in that: Step (1) comprises: bacterial strain activation and fermentation culture, wherein the optimal seed age is 20-24 hours, the inoculation amount is 1%, and the fermentation time is 48 hours; wherein the seed culture medium is 40 g / L defatted soybean powder, 10 g / L glucose, and the solvent is water; and the fermentation culture medium is 90 g / L high-temperature soybean meal, 5 g / L glucose, 1 g / L bran powder, and the solvent is water. In step (2), the operation steps of separating the active substance by macroporous adsorption chromatography are as follows: a. XAD-16N macroporous adsorption resin was soaked in anhydrous ethanol for 24 hours to allow the resin to fully swell, then rinsed with ultrapure water, and soaked in 4% HCl and 4% NaOH solutions for 5 hours in turn; then, the resin was washed with ultrapure water until it reached a neutral state; b. Rinse the column with ultrapure water; c. The fermentation broth was centrifuged to obtain the supernatant, which was passed through the XAD-16N macroporous adsorption resin at a flow rate of 4 mL / min, and then the substances adsorbed on the macroporous resin were eluted with ethanol at a flow rate of 2 mL / min with a volume concentration of 10%, 20%, 40%, 60%, 80%, and 100% in 2 times the column volume, and the eluents of each gradient were collected; In step (3), the operation steps of separating the active substance by anion exchange method are as follows: d. crude separation of the eluent obtained from the XAD-16N macroporous adsorption resin; e. Pre-treating the anion exchange filler, wet-packing the column and cleaning it; f. Continue flushing with 20 mM Tris-HCl buffer for 3-5 column volumes to equilibrate the column; g. After equilibration, load the sample, rinse the unbound proteins on the column with buffer, and then elute with different gradients of eluent: 20mM Tris-HCl+0.2M sodium chloride, 20mM Tris-HCl+0.5M sodium chloride, 20mM Tris-HCl+1.0M sodium chloride, and collect the eluent of each gradient.

5. The method according to claim 4, characterized in that: In step (2), 2 times the column volume of ethanol with a volume concentration of 20% is used for elution; In step (3), 20 mM Tris-HCl + 0.2 M sodium chloride was used for elution.

6. The method according to claim 4, characterized in that: In step (4), the operation steps of separating the active substance by gel filtration are as follows: h. The active fraction obtained from the anion exchange was concentrated to 2 ml; i. Treat the Sephadex G-75 gel and perform wet column packing, balancing more than 2 column volumes with ultrapure water; j. After equilibration, load the sample, elute with buffer, i.e. ultrapure water, and collect the eluate.

7. The method according to claim 4, characterized in that: The steps for preparing the fermentation broth in step (2) are as follows: Strain activation and culture: taking out Lactobacillus casei IOB-P9 from a glycerol tube with an inoculating loop and streaking it on an MRS medium plate, and culturing it in a closed manner at 36±2°C for 20-22 hours; taking a single colony to a seed medium, and culturing it at 36±2°C for 24 hours to obtain a primary seed liquid, inoculating the primary seed liquid at a 1% rate to a secondary seed medium, and culturing it at 36±2°C for 24 hours, inoculating the secondary seed liquid at a 1% rate to a fermentation medium, and culturing it at 37°C for 48 hours, and collecting the fermentation liquid supernatant to obtain a fermentation liquid; The seed culture medium is 40 g / L defatted soybean powder, 10 g / L glucose, and the solvent is water; the fermentation culture medium is 90 g / L high-temperature soybean meal, 5 g / L glucose, 1 g / L bran powder, and the solvent is water.

8. The method according to any one of claims 4 to 7, characterized in that: Step (5) comprises a preliminary activity detection of the active ingredient of Lactobacillus casei IOB-P9, using a filter paper disc method to determine the eluate of the macroporous adsorption chromatography method, using an Oxford cup method to determine the eluate of the anion exchange method and the eluate of the gel filtration chromatography method, and using Sarcina luteus as an indicator bacterium; Step (5) comprises HPLC analysis of the active ingredients of Lactobacillus casei IOB-P9: the active ingredients in the macroporous adsorption chromatography with a volume concentration of 20% ethanol eluent, the anion exchange chromatography with a volume concentration of 20mM Tris-HCl+0.2M sodium chloride eluent and the gel filtration chromatography are pre-treated and placed at 4°C for standby use, and the Agilent ZORBAX NH2 chromatographic column is equilibrated with mobile phase A, i.e., 100% acetonitrile+0.1% TFA, for 1 hour until the baseline is stable; Step (5) comprises pretreatment of the active ingredient of Lactobacillus casei IOB-P9: freeze-drying the supernatant of the fermentation broth, weighing 6 portions at 0.2 g / portion, adding 1 ml each of water, methanol, ethanol, ethyl acetate, chloroform and acetone, mixing with ultrasound for 5 min, and centrifuging at 10000 r / min for 10 min; Step (5) includes investigating the thermal stability of the active ingredient of Lactobacillus casei IOB-P9: treating with water bath at 25°C, 30°C, 37°C, 60°C, 80°C, 100°C, 115°C, and 121°C for 20 minutes respectively, cooling to room temperature, and using Sarcina lutea as an indicator bacteria; Step (5) includes exploring the sensitivity of the active ingredient of Lactobacillus casei IOB-P9 to acid and alkali: at various pH values ​​from 3.0 to 9.0, the supernatant is mixed with each gradient pH solution, and then the pH value is adjusted to 4.0 with 1M NaOH or 1M HCl, and Sarcina luteus is used as an indicator bacteria; Step (5) includes investigating the enzyme sensitivity of the active ingredient of Lactobacillus casei IOB-P9: treating the active ingredient with pepsin, trypsin, proteinase K, and α-chymotrypsin, using Sarcina luteus as an indicator bacterium; Step (5) includes the inhibitory effect of the active ingredients of Lactobacillus casei IOB-P9 on the pathogenic bacteria Helicobacter pylori that causes intestinal diseases.

9. the anti-Helicobacter pylori active substance of Lactobacillus casei 10B-P9 prepared by any one of claims 4 to 8.

10. Use of the anti-Helicobacter pylori active substance of Lactobacillus casei IOB-P9 as claimed in claim 9 in preparing a medicine for inhibiting Helicobacter pylori.

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