JP4313767B2 - Polypeptide with Helicobacter pylori peeling action - Google Patents

Description

  The present invention relates to a polypeptide having a Helicobacter pylori peeling action. More specifically, the present invention relates to a polypeptide having a Helicobacter pylori peeling action, which is obtained by hydrolyzing buttermilk. The present invention also relates to a composition comprising the polypeptide as an active ingredient.

Helicobacter pylori (Helicobacter pylori; Hp) has since been found in stomach ulcer patient by Warren and Marshall (see Non-Patent Document 1), worldwide it is one of the most infected persons a large number of pathogenic bacteria Became clear (see Non-Patent Document 2). Currently, gastritis (see non-patent document 3), peptic ulcer (see non-patent document 1 and non-patent document 4), gastric cancer (see non-patent document 5, non-patent document 6 and non-patent document 7) and gastric lymphoma ( It is suggested to be involved in the onset of non-patent document 8).

Researches on therapeutic methods and medicines for eradicating Hp are actively conducted. Currently, treatment with antibiotics is the mainstream. In Japan, the Japan Helicobacter Society has prepared “Guidelines for Diagnosis and Treatment of H. pylori Infection”, and 1 proton pump inhibitor (lansoprazole) and two antibiotics (amoxicillin and clarithromycin) are the first choices for eradication treatment. Three-drug combination therapy administered weekly is recommended. However, side effects have been reported in 14.8-45.1% of patients, and it has been discussed as a problem that resistant bacteria are occurring at a frequency of 5-10%. On the other hand, sterilization methods that do not use antibiotics have also been studied, and representative examples include methods using probiotics (see Non-Patent Document 9). However, since the mechanism has not been elucidated, it has not yet been applied to medical treatment. Therefore, it is desired to develop a sterilization method (material) in consideration of the structure and properties of the gastric mucosa and the bacteriological characteristics of Hp in the mucosa.

  The gastric mucosa has a multi-layer structure in which the surface mucin layer and the gland mucin layer alternately overlap. Superficial mucin is always secreted from mucosal epithelial cells to protect the stomach from gastric acid, whereas glandular mucin is secreted from the fundus gland in the form of gastric acid and pepsin when ingested. The surface mucin is known to be a MUC5AC glycoprotein having a 1,300 amino acid polypeptide rich in serine, threonine, proline and cysteine as a basic skeleton (see Non-Patent Document 10). Hp grows and proliferates in the stomach, mainly in the surface mucin layer near the epithelial cells. When new mucin is secreted, it is pushed up and approaches the surface of the stomach lumen (upper layer of the mucosal layer). It is thought that the movement of moving to is repeated (see Non-Patent Document 11). Many studies have been conducted on the adhesion of Hp to gastric epithelial cells and gastric mucin covering the same, mainly sialic acid represented by 3′-sialyl lactose (NeuAcα2-3Galβ1-4Glc; 3′SL) (non- Patent Document 12 and Non-Patent Document 13), sulfated oligosaccharides of glycoproteins and glycolipids (see Non-Patent Document 14 and Non-Patent Document 15), and a large number of laminin and collagen as basement membrane constituents have been reported. (See Non-Patent Document 16).

  Utilizing such knowledge, in vitro Hp adhesion inhibitory action on epithelial cells (model) and gastric mucin is added to the above substances (see Non-Patent Document 17) and cranberry juice (Non-Patent Document 18 and Non-Patent Document 19). Reference) and milk-derived glycoproteins (see Non-Patent Document 20). These results were obtained by simultaneously adding Hp and an inhibitor and examining the agglutination reaction, or by mixing an inhibitor with cells or mucin immobilized on a plate at the same time as the cell suspension (non-) (See Patent Literature 20 and Non-Patent Literature 19). However, even if there is an Hp aggregation / adhesion inhibitory effect, it is unclear if the sterilization effect is actually obtained when administered to Hp carriers. There are almost no reports investigating Hp peeling action from gastric mucin, and in the report using cranberry juice (see Non-Patent Document 18), the Hp peeling action is observed in the polymer non-dialysis fraction in which Hp adhesion inhibitory action was observed. It is shown that there was no.

  Furthermore, regarding the infection prevention effect against H. pylori, it has been disclosed that mucin derived from milk has an inhibitory effect on colonization (see Patent Document 1). However, the described method is to separate milk-derived mucin on a Sepharose column after removing milk fat and casein, as well as lipoproteins. In addition, it is described that whey that is produced in large quantities as a by-product in the production process of cheese or the like can be used as a raw material for milk-derived mucin, which is advantageous in terms of price and practicality. Has a very low content in milk (0.01% yield in Patent Document 1), and is expected to be an expensive material, which is problematic in profitability and feasibility.

Buttermilk (BM) is a by-product of charring during butter production. Most of milk fat globule membrane (MFGM) contained in BM is very small in normal skim milk (see Non-Patent Document 21), and MFGM contains a large amount of sialic acid, serine, It is composed of a glycopeptide containing a large amount of threonine (see Non-Patent Document 22). Until now, it has been reported that a glycopolypeptide obtained by digesting BM with bromelain has an anti-infection action against bovine rotavirus having a cell surface sialic acid residue as a receptor (see Non-patent Document 23). .
Japanese Patent No. 3255161 Warren JR and Marshall B. 1983, Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 321: 1273-1274. Taylor DN, Blaser MJ. 1991, The epidemiology of Helicobacter pylori infection. Epidemiol. Rev. 13: 42-59. Lee A, Fox J, Hazell S. 1993, Pathogenicity of Helicobacter pylori: a perspective. Infect. Immun. 61: 1601-1610. Buck GE, Gourely EG, Lee WK, Subramanyam K, Latimer LM, Dinuzzo AR. 1986, Relation of Campylobacter pyloridis to gastritis and peptic ulcer. J. Infect. Dis. 153: 664-669. Goodwin CS. 1993, Gastric cancer and Helicobacter pylori: the whispering killer? Lancet 342: 507-508. Isaacson PG. 1994, Gastric lymphoma and Helicobacter pylori. N. Engl. J. Med. 330: 1310-1311. The Eurogust Study Group. 1993, An international association between Helicobacter pylori infection and gastric cancer. Lancet 341: 1356-1362. Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Warnke RA, Jelum E, Orentreich N, Vogelman JH, Friedman GD. 1994, Helicobacter pylori infection and gastric lymphoma. N. Eng. J. Med. 330: 1267-1271. Sakamoto I, Igarashi M, Kimura K, Takagi A, Miwa T, Koga Y. 2001, Suppressive effect of Lactobacillus gasseri OLL 2716 (LG21) on Helicobacter pylori infection in humans.J. Antimicrobial Chemotherapy 47: 709-710. Van Klinken Bj, Dekker J, Buller HA, de Bolos C, Einerhand AW. 1997, Biosynthesis of mucins (MUC2-6) along the longitudinal axis of the human gastrointestinal tract. Am. J. Physiol. Gastrointest. Liver Physiol. G296-G302. Masako Nakazawa. 2001. Why can Helicobacter pylori bite into the stomach? Protein Nucleic acid Enzyme 46: 2025-2033. Evans DG, Evans DJ, Molds JJ, Graham DY. 1988, N-acetylneuraminyllactose-binding fibrillar hemagglutinin of Campylobacter pylori: a putative colonization factor antigen. Infect. Immun. 56: 2896-2906. Evans DG, Evans DJ, Graham DY. 1989, Receptor-mediated adherence of Campylobacter pylori to mouse Y-1 adrenal cell monolayers. Infect. Immun. 57: 2272-2278. Tsouvelekis LS, Mentis AG, Makris AM, Spiliadis C, Blackwell C, Weir DM. 1991, In vitro binding of Helicobacter pylori to human gastric mucin. Infect. Immun. 59: 4252-4254. Saitoh T, Natomi H, Zhao W, Okuzumi K, Sugano K, Iwamori M, Nagai Y. 1991, Identification of glycolipid receptors for Helicobacter pylori by TLC-immunostaining.FEBS Lett. 282: 385-387. Trust TJ, Doig P, Emody L, Dienle Z, Wadstrom T, O'Toole P. 1991, High-affinity binding of the basement membrane proteins collagen type IV and laminin to the gastric pathogen Helicobacter pylori. Infect. Immun. 59: 4398 -4404. Simon PM, Goode PL, Mobasseri A, Zopf D. 1997, Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides. Infect. Immun. 65: 750-757. Burger O, Ofek T, Tabak M, Weiss EI, Sharon N, Neeman. 2000, A high molecular mass constituent of cranberry juice inhibits Helicobacter pylori adhesion to human gastric mucus. FEMS Immunol. Med. Microbiol. 29: 295-301. Burger O, Weiss E, Sharon N, Tabak M, Neeman I, Ofek I. 2002, Inhibition of Helicobacter pylori adhesion to human gastric mucus by a high-molecular-weight constituent of cranberry juice. Critical Rev. Food Sci. Nutr. 42 : 279-284. Hirmo S, Kelm S, Iwersen M, Hotta K, Goso Y, Ishihara K, Suguri T, Morita M, Wadstrom T, Schauer R. 1998, Inhibition of Helicobacter pylori sialic acid-specific haemagglutination by human gastrointestinal mucins and milk glycoproteins. Immunol. Med. Microbiol. 20: 275-281. Adachi, Satoshi Ito 1987, Milk and its processing, p. 52-70, Kenshisha, Tokyo. McPherson AV, Kitchen BJ. 1983, Reviews of the progress of dairy science: The bovine milk fat globule membranepits formation, composition, structure and behavior in milk and dairy products.J. Dairy Res. 50: 107-133. Mitsuharu Matsumoto, Kazuki Oishi, and Akiyoshi Hosono. 2002. Anti-bovine rotavirus peptide derived from buttermilk. Daily newsletter 73: 49-56. Marshall BJ, Warren JR. 1984, Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet i: 1311-1314.

  An object of the present invention is to provide an inexpensive polypeptide having not only the effect of suppressing the adhesion of Helicobacter pylori (Hp) to the gastric mucosa but also the effect of removing Hp from the gastric mucosa of Hp carriers.

  The present inventors examined the effect of buttermilk (BM) on Hp. As a result, it was found that a BM degradation product having improved adhesion to Hp can be obtained by treating BM with protease. It was also found that this BM degradation product actually has a remarkable release activity from the gastric mucosa against Hp clinical isolates. The present invention has been completed based on these findings.

That is, the present invention provides a polypeptide having a molecular weight of 5 to 900 kDa obtained by subjecting a solution of BM or a dried product thereof to protease treatment.
The present invention also provides a composition having not only a Helicobacter pylori adhesion inhibitory action but also a peeling action, comprising the above polypeptide as an active ingredient.

The present invention also provides a composition comprising a polypeptide having a molecular weight of 5 to 900 kDa obtained by treating a solution of BM or a dried product thereof with a protease.
Furthermore, the present invention provides a food for preventing or improving gastrointestinal ulcer comprising the above polypeptide as an active ingredient.

The present invention also provides a preventive or therapeutic agent for gastrointestinal ulcer comprising the above polypeptide as an active ingredient.
Furthermore, the present invention provides a food for preventing or ameliorating Helicobacter pylori infection comprising the polypeptide as an active ingredient.

  The present invention also provides a preventive or therapeutic agent for Helicobacter pylori infection comprising the above polypeptide as an active ingredient.

  The polypeptide of the present invention has a remarkably high activity to peel off Hp infected to the gastric mucosal layer. As shown in Example 3, this activity is clearly higher than milk mucin having Hp colonization inhibitory activity.

The polypeptide of the present invention can be prepared from an inexpensive raw material in a high yield of 50% or more, and solves the above-mentioned profitability problem.
Therefore, by using the polypeptide of the present invention, it becomes possible to develop foods and pharmaceuticals having preventive and / or therapeutic effects against Hp infection. Furthermore, these foods and pharmaceuticals are effective for the prevention and / or treatment of diseases such as digestive ulcers associated with Hp infection.

  The polypeptide of the present invention can be obtained from BM or a dried product obtained by removing low molecular components such as lactose and salts by diafiltration by ultrafiltration. By subjecting a protein component containing milk fat globule membrane (MFGM) contained in BM to hydrolysis treatment with a protease, the polypeptide of the present invention which is a glycopeptide constituting them can be prepared. Hydrolysis can be carried out not only by protease treatment but also by chemical treatment such as various acids and alkalis. In consideration of inclusion in food, protease treatment is preferred. Examples of proteases used for hydrolysis include trypsin, papain, pancreatin, pepsin, etc., but it is also possible to use lactic acid bacteria and other microorganisms having protease activity, and extracts thereof, and those having protease activity As long as it is, it is not limited to these.

  The polypeptide of the present invention has a molecular weight of 5 to 900 kDa. For example, if it is a low molecular component, it is 5 to 45 kDa, preferably 10 to 40 kDa, and more preferably 10 to 30 kDa. For example, in the case of a polymer component, it is 400 to 900 kDa, preferably 500 to 800 kDa, and more preferably 550 to 750 kDa.

  The polypeptide of the present invention can be prepared, for example, as follows. Suspend BM or BM powder in deionized water and heat sterilize at 85-90 ° C. After cooling the suspension, sodium hydroxide or hydrochloric acid is added to adjust the pH to the optimum value for the protease used. Thereafter, protease powder or suspension is added, and the reaction is carried out with gentle stirring at the optimum temperature. After the reaction, the pH is adjusted to 6.8 to 7.0 with sodium hydroxide or hydrochloric acid, and the protease is inactivated by heating at 90 ° C. for 10 minutes. After cooling, it is freeze-dried to obtain a BM hydrolyzate. Specific reaction conditions such as the amount of protease used, the reaction temperature, and the reaction time can be easily set by those skilled in the art. The polypeptide of the present invention contained in this BM hydrolyzate may be used after purification, or may be used as it is without purification.

  The polypeptide of the present invention prepared as described above has a high ability to bind to the Hp surface layer component and has an action of peeling Hp from the gastric mucosa. Moreover, Hp peeling activity does not exist in BM and BM powder which are starting materials, but the activity is manifested only by decomposition with protease.

Therefore, this invention provides the composition which has Hp peeling action which uses the polypeptide of this invention as an active ingredient.
The composition of the present invention can be prepared from the BM hydrolyzate by, for example, the above method.

Furthermore, a liquid from which a precipitate has been removed by centrifugation or the like, or a dry powder thereof, or a precipitate thereof can also be used.
The polypeptide of the present invention can be used as a food for preventing / ameliorating Hp infection or digestive ulcer by containing it in food as an active ingredient.

  The food for preventing / ameliorating gastrointestinal ulcer of the present invention may be a food containing substantially only the polypeptide of the present invention, or may be in the form of a supplement combined with other dietary supplements. Moreover, what added the said polypeptide directly to the existing foodstuff, a drink, etc. may be used. For example, peptides such as gums, candies, jellies, gummies, cookies, biscuits, chocolate and other sweets, juices and other dairy products, cheese, butter, yogurt and other dairy products, ice cream, ham and other agricultural processed products, chikuwa, Can be added directly to processed fishery products such as hampen, noodles such as buckwheat and udon, processed flour products such as bread and cake, canned or miso, soy sauce, salt, pepper, sugar, and artificial foods . Polypeptides may also be mixed during the processing of these foods. When processing a polypeptide into food, it can be performed based on a normal food processing method. In addition, when adding or mixing a polypeptide to a food, the polypeptide may be used in a solid form such as a powder, granule or fine particle, or in a liquid form. The food for preventing / ameliorating gastrointestinal ulcer according to the present invention can also be applied as a food for specified health use, functional food, and health food for preventing or improving gastrointestinal ulcer. “Functional food” means food containing a substance with a bioregulatory function, and the functionality can be displayed by obtaining individual permission for each food as food for specified health use from the Ministry of Health, Labor and Welfare. .

  In the food of the present invention, it is generally preferable to mix the polypeptide in a range of about 0.1 to 50 g per 100 g of food. The amount of intake varies depending on the sex, weight, constitution, age, other ingested foods, etc. of the ingested subject, but in general, in the case of human adult subjects, the ingested amount is within a range of 3 to 15 g per day. It is preferable to make it. The number of times of intake is appropriate several times a week to once a day.

The polypeptide of the present invention can also be used as a pharmaceutical composition such as a preventive or therapeutic agent for Hp infection or digestive ulcer.
When the preventive or therapeutic agent for digestive ulcer of the present invention is administered to a living body, it is administered orally.

  The composition of the present invention may be formulated into an appropriate dosage form and used. For example, it can be used in preparations such as tablets, powders, granules, fine granules, pills, capsules, troches, chewables, solutions, emulsions, suspensions and syrups. Formulation into these dosage forms can be performed using appropriate pharmaceutically acceptable carriers, excipients, additives and the like.

  In order to prepare solid preparations such as tablets, pills, powders, granules, fine granules, troches, chewables, carriers such as sodium bicarbonate, calcium carbonate, starch, sucrose, mannitol, carboxymethylcellulose, etc. In addition, an additive such as calcium stearate, magnesium stearate, glycerin and the like can be added and used in a conventional manner.

  When the composition of the present invention is used as a medicine, the dosage varies depending on the sex, body weight, constitution, age, symptom, dosage form, etc. of the patient. It can be suitably selected within the range of 0.1 to 50 g, preferably 3 to 15 g. The number of times of administration varies depending on the patient's symptoms or dosage form, etc., but several times a week to once a day is appropriate.

Hp adhesion test
Materials and Methods (1) Strains Used Helicobacter pylori ATCC 43504 ^T and ATCC 43579 used in this test were purchased from the American Type Culture Collection.

(2) Preparation of BM hydrolyzate BM powder was suspended in deionized water so that it might become 60 g / L, and it sterilized at 85-90 degreeC for 15 minutes. After cooling, the suspension was adjusted to an optimum pH with sodium hydroxide or hydrochloric acid (1-6N, concentrated hydrochloric acid in the case of pepsin), and 0.3 g of various protease powders (manufactured by Nippon Bicon Co., Ltd.) were added. The reaction was allowed to proceed with gentle stirring for 24 hours at the optimum temperature. The pH was readjusted 12 hours after the start of the reaction, and 0.3 g of protease powder was added again. After the reaction, the pH was adjusted to 6.8 to 7.0 with sodium hydroxide or hydrochloric acid, and the enzyme was inactivated by heating at 90 ° C. for 10 minutes. After cooling, it was freeze-dried to obtain a BM hydrolyzate. Table 1 shows the enzyme used, its optimum temperature and optimum pH.

(3) Test method BM hydrolyzate by various proteases, untreated BM, porcine and mouse stomach crude mucin were suspended / dissolved in 0.1 M carbonate buffer (pH 9.6) so as to be 10 mg / mL, respectively. 75 μL / well was added to a 96-well microplate (Nunc A / S) and coated at 37 ° C. for 2 hours. Each well was washed 3 times with 250 μL of sterile PBS (pH 7.2), Hp suspension was added at 50 μL / well, and gently shaken under microaerobic conditions (30 rpm) at 37 ° C. for 2 hours. Cultured. Non-adherent cells were removed by washing 5 times with sterile PBS. Thereafter, 100 μL of PBS was added and vigorous pipetting was performed to peel the adherent cells, and 50 μL was smeared on a Tryptic soy agar medium supplemented with 5% sheep defibrillated blood. After microaerobic culture at 37 ° C. for 96 hours, the number of appearing colonies was counted, and the number of adherent bacteria per well was calculated.

  Porcine gastric mucin was purchased from Sigma, and mouse gastric mucin was prepared from BALB / c mice (6 weeks old, rabbit). That is, after cervical dislocation, the stomach was removed and opened. After removing the contents and washing with PBS, the inner surface mucin was scraped off with a spatula and suspended in PBS containing 10 mM sodium azide. After vigorous stirring for 5 minutes, centrifugation was performed at 10,000 g for 20 minutes, and the resulting supernatant was dialyzed with a dialysis tube having a MW of 10,000 to 12,000. The dialyzed solution was treated with a 0.45 μm filter and then freeze-dried, and the powder obtained was used as a mouse stomach crude mucin.

  In addition, the Hp suspension was obtained by collecting Hp colonies cultured for 96 hours on the above medium with a cotton swab and suspending them in PBS so that the absorbance at 660 nm was 0.4 ± 0.01. Prepared solution.

(4) Statistical processing Adhesive comparison was performed by t-test of two independent samples, and p <0.05 was considered statistically significant. The statistical analysis software used was STATISTICA (Design Technologies).

Results The adhesiveness of various protease-treated BM hydrolysates to ATCC 43504 ^T and ATCC 43579 is shown in FIG. 1 as the average number of adherent cells ± standard error (average of 4 times).

As for the adhesion to ATCC 43504 ^T , trypsin, papain and pancreatin-degraded BM showed significantly strong (p <0.05) adhesion as 6 to 12 times that of untreated BM.

  In terms of adhesion to ATCC 43579, trypsin (p <0.01), papain (p <0.001) and pancreatin (p <0.05) -decomposed BM was 10-15 times that of untreated BM. Significantly strong adhesion was observed. This result shows that Hp adhesion is increased by treating BM with protease.

As a result of comparison with mouse gastric mucin and porcine gastric mucin adhesion, trypsin-degraded BM showed significantly (p <0.05) stronger adhesion with ATCC 43504 ^T. Against ATCC 43579, trypsin (p <0.001), papain (p <0.001) and pancreatin (p <0.05) -degraded BM showed significantly stronger adhesion. On the other hand, untreated BM was confirmed to be significantly weaker (p <0.05) than both gastric mucins in ATCC 43579, and the adhesion to ATCC 43504 ^T also tended to be weak at about 50% of gastric mucins. It was. These results suggest that by treating BM with protease, an affinity for Hp that exceeds the affinity between Hp and gastric mucin was obtained.

Hp Peeling Test Since it was confirmed that a degradation product exhibiting strong adhesion to Hp was obtained by treating BM with protease in Example 1, Hp peeling activity was confirmed using this BM degradation product.

Materials and Methods (1) Strain used Helicobacter pylori ATCC 43504 ^T and ATCC 43579 were purchased from the American Type Culture Collection. Hp LKM HP-2, LKM HP-3, LKM HP-4, LKM HP-5, and LKM HP-7 were isolated from the main body of the stomach of a stomach ulcer patient who visited Osaka Medical University Second Internal Medicine. is there. In addition, Helicobacter pylori was separated in accordance with the spirit of the Helsinki Declaration, and the purpose and contents of the test were explained in advance and carried out based on the consent of the carriers. These strains were smeared on a Tryptic soy agar medium (MERCK) supplemented with 5% sheep defibrinated blood with platinum ears, and subcultured every 96 hours in microaerobic culture using Aneropac Helico (Mitsubishi Gas). . In addition, it was confirmed that pure culture was performed by morphology observation by Gram staining and catalase, urease, and oxidase tests at each passage.

(2) Preparation of BM hydrolyzate BM hydrolyzate was prepared in the same manner as in Example 1. As the protease, trypsin and pancreatin in which high adhesion activity to Hp was expressed in Example 1 were used.

(3) Test method Porcine gastric mucin was suspended in 0.1 M carbonate buffer so as to be 10 mg / mL, and 75 μL / well was added to a 96-well microplate and coated at 37 ° C. for 2 hours. Each well was washed 3 times with 250 μL of sterile PBS (pH 7.2), Hp suspension was added at 50 μL / well, and gently shaken under microaerobic conditions (30 rpm) at 37 ° C. for 2 hours. Cultured. After washing twice with sterile PBS, BM hydrolyzate (0.0625, 0.125, 0.25, 0.5, 1.0, 10 mg / mL) suspended / dissolved in PBS at various concentrations 50 μL / well was added, and the mixture was cultured at 37 ° C. for 1 hour while gently shaking (30 rpm) under microaerobic conditions. After washing twice with sterile PBS, add 100 μL of PBS, peel off the adherent cells by vigorous pipetting, smear 50 μL on the above medium, and count the number of colonies after culturing under microaerobic conditions did. As a control, PBS was used instead of BM hydrolyzate. The peeling rate at each concentration was calculated by the following formula.

Peeling rate (%) = [(A−B) / A] × 100
A: Number of adherent cells per microplate in control sample B: Number of adherent cells per microplate in sample added with BM degradation product (4) Statistical treatment Adhesive comparison was performed by t-test of two independent specimens. P <0.05 was considered statistically significant. The statistical analysis software used was STATISTICA (Design Technologies).

Results Table 2 shows the results of a peel test of trypsin and pancreatin-treated BM (10 mg / ml) that had strong adhesion to ATCC 43504 ^T and ATCC 43579.

  Most of them showed a strong peeling action of 95% or more not only on ATCC strain but also on clinical isolates.

Property analysis In Examples 1 and 2, the BM degradation product was confirmed to have a strong adhesive action against Hp and an exfoliating action, but milk mucin has already been reported as a similar substance showing an inhibitory action against H. pylori (patent) Reference 1). In this example, the following examination was conducted in order to clarify the difference between the BM degradation product and milk mucin.

Materials and Methods (1) Strain used Helicobacter pylori ATCC 43504 ^T was purchased from the American Type Culture Collection and used.

(2) BM hydrolyzate From BM powder (BM spray-dried product), BM was trypsinized according to the method of Example 1 and the enzyme was inactivated by heating. Separated. Each was freeze-dried to obtain a BM degradation product supernatant and a BM degradation product precipitate. Furthermore, the supernatant was separated and purified by gel filtration to obtain a high molecular component and a low molecular component.

(3) Test method The protein and carbohydrate content and molecular weight distribution of the BM degradation product were measured by Kjeldahl method, phenol sulfate method, SDS polyacrylamide gel electrophoresis method, and gel filtration method, respectively. It was compared with the component composition and molecular weight of milk mucin.

The stripping activity was determined by comparing the stripping activity at a concentration of 1 mg / mL according to the method described in Example 2.
Results As shown in Table 3, it was confirmed that the BM degradation product had a higher protein content and less carbohydrates than milk mucin. Moreover, it is a substance different in molecular weight and component composition from milk mucin, and higher activity was recognized compared with milk mucin.

  In particular, regarding the fraction having a molecular weight of 550 to 750 kDa, the yield per solid content is 20.2%, the protein content is 40 to 50%, the sugar content is 10 to 20%, and the peeling activity is 90.5%. there were.

  From the above, it is clear that the BM hydrolyzate has strong Hp adhesion and peeling activity. The polypeptide of the present invention contained in this BM degradation product has high productivity and heat stability as well as exfoliation activity as well as existing anti-pylori food materials such as milk mucin. It can be greatly expected as a functional material that can be preserved.

It is a figure which shows the adhesiveness with respect to Hp about BM degradation product and BALB / c mouse | mouth stomach mucin by various enzymes.

Claims (8)

Helicobacter pylori comprising as an active ingredient a liquid obtained by removing a precipitate from a hydrolyzate obtained by protease treatment of a solution of buttermilk or a dried product thereof or a dry powder thereof, or a precipitate obtained from the hydrolyzate Binder for the surface layer . The binder for the surface layer of Helicobacter pylori according to claim 1, which is used as a food additive. Helicobacter pylori comprising as an active ingredient a liquid obtained by removing a precipitate from a hydrolyzate obtained by protease treatment of a solution of buttermilk or a dried product thereof or a dry powder thereof, or a precipitate obtained from the hydrolyzate Release agent . The Helicobacter pylori remover according to claim 1, which is used as a food additive. Gastrointestinal ulcer comprising, as an active ingredient, a liquid obtained by removing a precipitate from a hydrolyzate obtained by protease treatment of a solution of buttermilk or a dried product thereof or a dry powder thereof, or a precipitate obtained from the hydrolyzate Preventive agent . Helicobacter pylori comprising as an active ingredient a liquid obtained by removing a precipitate from a hydrolyzate obtained by protease treatment of a solution of buttermilk or a dried product thereof or a dry powder thereof, or a precipitate obtained from the hydrolyzate Infection preventive agent . Gastrointestinal ulcer comprising, as an active ingredient, a liquid obtained by removing a precipitate from a hydrolyzate obtained by protease treatment of a solution of buttermilk or a dried product thereof or a dry powder thereof, or a precipitate obtained from the hydrolyzate Therapeutic agent . Helicobacter pylori comprising as an active ingredient a liquid obtained by removing a precipitate from a hydrolyzate obtained by protease treatment of a solution of buttermilk or a dried product thereof or a dry powder thereof, or a precipitate obtained from the hydrolyzate Infection treatment .

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