TWI392498B - A novel strain of lactobacillus pentosus lps16 and its use in inhibition of helicobacter pylori - Google Patents

Description

Lactobacillus pentosus LPS 16 and its use in inhibiting Helicobacter pylori

The present invention relates to a lactic acid bacterium, particularly a Lactobacillus pentosus LPS16 having the ability to inhibit Helicobacter pylori .

Helicobacter pylori is a Gram-negative bacterium, one of the most important pathogenic bacteria in clinical medicine development in the past decade. It is known that Helicobacter pylori is the main cause of gastric ulcer and various clinical stomachs. Some diseases such as gastritis, atrophic gastritis, peptic ulcer, and even gastric cancer are directly related. The World Health Organization has classified Helicobacter pylori as the first-grade carcinogen. According to statistics, more than 50% of the global population has been infected with Helicobacter pylori, and in countries with high infection rates of Helicobacter pylori infection in Taiwan, the majority of the population was infected before the age of 1 year, however, only 10-20% The infected person has symptoms of gastric ulcer, digestive tract ulcer or gastric cancer, which is very harmful to the health of modern people.

The clinical treatment of Helicobacter pylori mostly uses triple therapy, which is mainly treated with two antibiotics and a proton-pump inhibitor. For example, the successful hospital in Taiwan is currently attached to the hospital. The digestive tract disease caused by Helicobacter pylori is treated with a combination of amoxicillin and clarithromycin in combination with omeprazole ol; however, the three-in-one therapy is used to treat Helicobacter pylori The success rate of the method is only 80~95%, and most patients still can't avoid the crisis of peptic ulcer. The three-in-one therapy mainly uses antibiotics as the main mechanism of action. After taking it, it is easy to affect the normal bacterial phase in the intestine of patients. The distribution causes side effects such as abdominal pain, diarrhea and flatulence. Furthermore, the long-term use of antibiotics may cause bacterial resistance, making the cure rate of the three-in-one therapy even less optimistic; the cost of triple therapy is higher. It often causes a heavy burden on the patient but does not effectively cure the disease.

In recent years, it has been reported that specific lactic acid bacteria [such as Lactobacillus rhamnosus , Lactobacillus johnsonii , Lactobacillus casei , Lactobacillus gasseri , and Reiter's) have been reported. B bacteria ( Bifidobacterium lactis ), etc., have the function of inhibiting the growth of Helicobacter pylori; lactic acid bacteria having the ability to resist Helicobacter pylori, such as the Republic of China Public Publication No. 200530395 "Lactic acid strains with anti-Helicobacter pylori ability and compositions containing the same" patent The case reveals an Enterococcus faecium which inhibits the growth of Helicobacter pylori for use in the manufacture of a medicinal food resistant to Helicobacter pylori; and Japanese Patent Publication No. JP2001258549A and U.S. Patent No. 6,491, 956 disclose a Lactobacillus acidophilus and a food for preventing Helicobacter pylori infection, which contain Lactobacillus acidophilus HY2177 and Lactobacillus casei HY2743); the The mechanism by which each lactic acid bacteria inhibits the growth of Helicobacter pylori is not fully understood. It may inhibit the pylorus through metabolites secreted by lactic acid bacteria, such as lactic acid, antimicrobial peptides, proteins in the bacteria or competition on gastric cells. Growth of bacilli.

At present, a variety of conventional lactic acid bacteria have been used to prepare products having the effect of inhibiting Helicobacter pylori; however, most products have a low inhibitory effect on Helicobacter pylori, and the selection of conventional lactic acid bacteria which can be used for commercial preparation is limited, and cannot be further improved. The antibacterial effect of various commodities; in view of the high threat and high infection rate caused by Helicobacter pylori in Taiwan population in recent years, the multi-drug resistant strains have a low cure rate for patients with gastric cancer, duodenal ulcer and gastric ulcer, and the medical cost is increasing day by day. For other problems, novel strains with better ability to inhibit Helicobacter pylori should be developed to effectively improve the clinical prevention and treatment of Helicobacter pylori, and avoid the excessive use of antibiotics and the derivation of side effects.

The main object of the present invention is to provide a Lactobacillus pentosus which inhibits Helicobacter pylori, which has the ability to limit the growth of Helicobacter pylori.

A second object of the present invention is to provide a Lactobacillus pentosus which inhibits Helicobacter pylori, which has an inhibitory effect against a clinically resistant strain of Helicobacter pylori.

Still another object of the present invention is to provide a use of Lactobacillus pentosus which is a method of preparing a food or pharmaceutical compound for inhibiting infection by Helicobacter pylori.

In order to achieve the aforementioned object, the technical means used in the present invention comprises: a lactic acid bacteria inhibiting Helicobacter pylori, belonging to Lactobacillus pentosus , and having the accession number BCRC910467.

The use of a lactic acid bacteria for the preparation of a food or pharmaceutical compound for inhibiting Helicobacter pylori infection by using Lactobacillus pentosus as described above

The above and other objects, features, and advantages of the present invention will become more apparent from the aspects of the invention. Lactobacillus pentosus LPS16 is obtained from a pickled food, and the accession number is BCRC910467. The Lactobacillus pentosus LPS16 has the bactericidal ability of Helicobacter pylori, but the past has never inhibited Helicobacter pylori According to the report of the growing Lactobacillus pentosus, the Lactobacillus pentosus LPS16 has better antibacterial and antibacterial effects than the conventionally developed lactic acid bacteria, and can be applied to the treatment or prevention of digestive ulcer products. preparation.

Referring to Fig. 1, the Lactobacillus pentosus LPS16 of the present invention is obtained by the following various methods, including: culture of lactic acid bacteria S1, screening and identification of lactic acid bacteria S2, and preservation of lactic acid bacteria S3:

The culture of the lactic acid bacteria S1 separates the lactic acid bacteria contained in the commercially available processed food and cultures them. Samples are taken from a pickled food (such as dried bamboo shoots), placed in a culture medium for enrichment culture, and after appropriate incubation time, the culture solution is serially diluted, and further inoculated into a medium to select a growable strain. The proliferation culture of the present embodiment is to inoculate the pickled food in a liquid MRS medium (MRS Broth) at a ratio of 1:50, and culture at 37 ° C for 20 hours to obtain a proliferation culture liquid, and then serially dilute the proliferation culture. In the bacterial solution, the diluted culture medium was inoculated by a smear method into a solid MRS medium (MRS Agar), and the growable strain was selected; the formulation of the liquid MRS medium and the solid MRS medium is shown in the following table.

The lactic acid bacteria were screened and identified as S2 , and strains having inhibitory ability against Helicobacter pylori were selected from the isolated lactic acid bacteria strains. The isolated strains are tested for the bacteriostatic (Helicobacter pylori) ability by the paper ingot diffusion method, and the strain having the significant inhibition ability is selected, that is, the lactic acid bacteria of the present invention; the lactic acid bacteria according to the physical form, physiological characteristics or molecules thereof Characteristics, etc. determine the classification of their species. In this embodiment, the lactic acid bacteria are subjected to Gram staining, catalase reaction, and molecular identification of 16SrDNA, and the staining map of the lactic acid bacteria (gram positive), the 16S rDNA partial sequence, and the catalase reaction test (negative reaction), etc. The identification result determined that the lactic acid bacteria of the present invention is Lactobacillus pentosus , named LPS16, and the accession number is BCRC910467. Among the biochemical test results of the Lactobacillus pentosus, GLY, LARA, RIB, DXYL, GAL, GLU , FRU, MNE, RHA, DUL, MAN, SOR, MDG, NAG, AMY, ARB, ESC, SAL, CEL, MAL, LAC, MEL, SAC, TRE, MLZ, RAF, GEN, TUR, DARL and GNT tests Positive reaction, ERY, DARA, LXYL, ADO, MDX, SBE, INO, MDM, INU, AMD, GLYG, XLT, LYX, TAG, DFUC, LFUC, LARL, 2KG and 5KG tests are negative reactions, and the detailed identification results are as follows Figures 4 and 5 are shown.

The preservation of the lactic acid bacteria S3 enables the Lactobacillus pentosus LPS16 to be stored in a low temperature environment for long-term storage for subsequent use. The Lactobacillus pentosus LPS16 of the present invention was inoculated to the solid MRS medium, and cultured at 37 ° C for 48 hours, and then a single colony was selected and transferred to a BHI culture solution (the formulation is shown in Table 3) for cryopreservation; the BHI Preferably, the culture solution is mixed with 30% antifreeze agent (such as glycerin), and temporarily stored in a constant temperature storage room at -80 ° C for later use.

The Lactobacillus pentosus LPS16 system has the property of adsorbing cells in the digestive tract of the host, and mainly achieves the effect of inhibiting Helicobacter pylori through the secreted bactericidal substance. Referring to FIG. 2, the Lactobacillus pentosus LPS16 of the present invention is used to adsorb the two groups of gastric epithelial cells with a cursor and then the fluorescence intensity of the gastric epithelial cells is measured by flow cytometry; The experiment was carried out in accordance with the method published by Sheu et al. in 2007, using a fluorescent treatment kit (PKH2 Green Fluorescence Cell Linker Kit; Sigma, Missouri, USA) for the fluorescence cursor setting of Lactobacillus pentosus LPS16. The calibrated Lactobacillus pentosus LPS16 can produce green fluorescence, and the quantitative Lactobacillus pentosus LPS16 is subjected to adsorption test for two groups of gastric epithelial cells (including human gastric epithelial cell tumor cell lines AGS and MKN45), which are better infected. The concentration system MOI=100, that is, 5×10 ⁵ gastric epithelial cells were infected with 5×10 ⁷ CFU of Lactobacillus pentosus LPS16. The human gastric epithelial cell adenocarcinoma cell lines AGS and MKN45 selected in this example were purchased separately. From the Taiwan Food Industry Development Institute and the Health Science Research Resources Bank, detailed infections are recorded in Table 4:

From the results of Fig. 2, wherein the background value (2C) of the fluorescence measurement is about 3, and the fluorescent intensity of the AGS cells is significantly increased after the ABS cell line is adsorbed by the H. pluvialis LPS16. The enhanced fluorescence intensity peak (Gmean) is about 15, indicating that the Lactobacillus pentosus LPS16 has the ability to adsorb to the AGS cell line. In addition, the MKN45 cell line is adsorbed by the Lactobacillus pentosus LPS16 after adsorbing the MKN45 cell line. The fluorescence intensity of the expression also showed a significant enhancement trend, and the highest fluorescence intensity peak (Gmean) was about 13, indicating that the Lactobacillus pentosus LPS16 has the ability to adsorb to the MKN45 cell line; from the above, the pentose sugar of the present invention is known. Lactobacillus LPS16 does have the property of adsorbing human gastric epithelial cells, and can stay in the human digestive tract for bactericidal action for a long time. Therefore, the Lactobacillus pentosus LPS16 of the present invention can exert its activity of inhibiting Helicobacter pylori on the host digestive tract cells for a long time. ability.

Please refer to Fig. 3, which is a graph of the amount of bacteria after co-culture of the sterile supernatant of Lactobacillus pentosus LPS16 with a Helicobacter pylori; this example is sampled from the overnight culture of Lactobacillus pentosus (i.e., The bacterial liquid of Lactobacillus pentosus LPS16 of the present invention is precipitated by centrifugation (preferably at 3500 rpm) The rotation speed of the cells is precipitated for separation, and the obtained supernatant liquid is collected, and the bacterial portion remaining in the supernatant liquid is removed by filtration, preferably by filtration on a 0.22 micrometer (μm) filter to obtain complete The culture solution containing no cells is preferably adjusted to an appropriate concentration [eg, 300 Ng/μl (ng/μl)] to facilitate subsequent experiments.

In this embodiment, a culture solution having a protein concentration of 300 ng/μl (ng/μl) (ie, a sterile supernatant of Lactobacillus pentosus LPS16) is co-cultured with the Helicobacter pylori, and coated at different times. The survival method of the Helicobacter pylori was analyzed by the counting method, and the results were recorded in Fig. 3 (i.e., 3A); and the control group (3B) was tested by substituting the MRS liquid medium for the culture solution of the present invention. The Helicobacter pylori strain was purchased from the Taiwan Food Industry Development Research Institute, and a certain amount of Helicobacter pylori (such as 1×10 ⁸ CFU) was co-cultured in the culture solution to carry out a counting test.

As a result of Fig. 3, after the culture solution of Lactobacillus pentosus LPS16 of the present invention was co-cultured with the Helicobacter pylori for 15 minutes, the amount of the bacterium of the Helicobacter pylori was greatly reduced, from 1 × 10 ⁸ CFU to 1 × 10 ^1.5 CFU. (Results from 3A), and decreased to 1 × 10 ⁰ CFU in 30 to 45 minutes; compared with the control group (3B), the amount of Helicobacter pylori did not decrease at all after 15 to 45 minutes of co-culture. The result shows that the Lactobacillus pentosus LPS16 can produce a bactericidal substance and kill the Helicobacter pylori during the culture; therefore, the Lactobacillus pentosus LPS16 of the present invention is adsorbed to the host digestive tract cells for a long time to secrete the bactericidal substance. The effect of inhibition by Helicobacter pylori is achieved.

In order to confirm that the Lactobacillus pentosus LPS16 strain of the present invention has better antibacterial ability, the bactericidal ability of the Lactobacillus pentosus LPS16 compared to other conventional lactic acid bacteria is tested, and the Lactobacillus pentosus LPS16 is directed against Helicobacter pylori. The inhibition effect of the clinically resistant strains was observed, and the sterilization results were observed.

Please refer to Table 5 below. This example is to test a number of commercially available yoghurt strains from the lactic acid bacteria for the test. The yoghurt strain contains Bifidobacterium lactis . Lactobacillus acidophilus and Lactobacillus bulgaricus are obtained by quantitatively diluting the concentrated bacterial liquid (containing the conventional lactic acid bacteria and the bacterial liquid of Lactobacillus pentosus LPS16 of the present invention) by a paper ingot diffusion test. Comparison of bactericidal effects against several strains of Helicobacter pylori. The Helicobacter pylori (No. 1 to 3) used in the present example was purchased from the Taiwan Food Industry Development Research Institute and the like.

In the present embodiment, the lactic acid bacteria liquid (containing the conventional lactic acid bacteria and the bacterial liquid of the Lactobacillus pentosus LPS16 of the present invention) was sampled, and the cells were precipitated and separated at a rotational speed of 3500 rpm, and the supernatant liquid was further obtained. 0.22 micron (μm) filter was filtered to obtain a completely free culture medium, which was concentrated by freeze-drying to a protein concentration of 2500 Ng/μl, and dropped into commercial paper ingots. (BBL, Becton, Dickinson System) was used to make a number of test paper ingots. The test paper ingot was applied to a culture medium of Helicobacter pylori and cultured in an appropriate environment for 2 days, and the size of the inhibition zone exhibited by the test paper ingot was observed; wherein the culture environment was according to a conventional lactic acid bacteria or the Lactobacillus pentosus LPS 16 of the present invention. The culture conditions are different. As described above, the test paper ingot prepared by the Lactobacillus pentosus LPS16 of the present invention is preferably cultured in a microaerobic environment at 37 °C.

^a The size of the suppression ring recorded in this example is the average zone of inhibition obtained after the three-dimensional diffusion test.

As a result of the fifth table, the Lactobacillus pentosus LPS16 of the present invention has a better inhibitory effect on Helicobacter pylori than other conventional lactic acid bacteria, and the inhibition ring is about 14.7 to 16 mm, which is suppressed compared with other lactic acid bacteria. The circle (only about 13 to 7 mm) was significantly large; thus, it was confirmed that the Lactobacillus pentosus LPS16 of the present invention has an ability to inhibit Helicobacter pylori and has a bacteriostatic effect.

Please refer to the sixth table, which is the bacteriostatic effect of the Lactobacillus pentosus LPS16 applied to several clinical pathogenic strains of Helicobacter pylori; the Helicobacter pylori pathogenic strain is screened from a medical unit in Taiwan, and is selected to have multiple drug resistance or A total of 20 pathogenic strains capable of causing digestive diseases such as gastric cancer and duodenal ulcer were subjected to a paper ingot diffusion test. In this embodiment, the Helicobacter pylori isolated from the large hospital is collected, and the Helicobacter pylori is divided according to the medical records of the separated patients, such as Gastritis as the G group, Gastric ulcer as the GU group, and Gastric cancer (Gastric). Cancer) is the GC group, and duodenal ulcer is classified as the DU group; the Helicobacter pylori operates according to the standard The procedure of the Clinical and Laboratory Standards Institute (CLSI) was conducted to test the antibiotic resistance, and the Helicobacter pylori was tested for the antibiotics (amoxicillin), clarithromycin, metronidazole, and fluoropropane. The minimum inhibitory concentration of antibiotics such as ciprofloxacin, if its minimum inhibitory concentration exceeds the critical concentration of antibiotics, is defined as resistance. For example, if the minimum inhibitory concentration of the Helicobacter pylori against penicillin series antibiotics exceeds 0.5 Micrograms per milliliter (μg/ml) is defined as a penicillin series antibiotic (amoxicillin) resistant type, while resistance to more than one type of antibiotic is defined as a multiple drug-resistant strain, the details of the Helicobacter pylori The classification results are shown in Table 6.

^a R means that the Helicobacter pylori is a multi-drug resistant strain ^b. The size of the inhibition zone recorded in this example is the average zone of inhibition obtained after the three-dimensional diffusion test.

From the results of the sixth table, the Lactobacillus pentosus LPS16 of the present invention has a good inhibitory effect on 20 clinical pathogenic strains of Helicobacter pylori, and can exhibit a suppression circle of about 10 to 15 mm; wherein the Lactobacillus pentosus LPS16 is respectively Clinically caused Helicobacter pylori with duodenal ulcer (6-16~6-20,6-12,6-14), gastritis (6-15) and gastric cancer (6-1~6-5) have different degrees of antibacterial effect The size of the inhibition zone is 13.4~16mm, 10.5mm and 12~13.7mm. In addition, for the multi-drug resistant Helicobacter pylori, the Lactobacillus pentosus LPS16 can still exhibit significant bactericidal effect (by 6- The 6~6-15 results show that the inhibition zone size is 9.7~16mm; therefore, the Lactobacillus pentosus LPS16 of the present invention has the ability to inhibit Helicobacter pylori even for the Helicobacter pylori strain having multiple drug resistance types. Or common cause Clinical strains of digestive tract diseases such as duodenal ulcer and gastric cancer have good bactericidal effects.

As described above, the Lactobacillus pentosus LPS16 of the present invention can adsorb to the cells of the host digestive tract and release the bactericidal substance in the host to produce an antibacterial ability; the Lactobacillus pentosus LPS16 has an antibacterial effect superior to that of the commercially available fermented dairy product. It can effectively inhibit the clinical multi-drug resistant type of Helicobacter pylori, or other common cloacae causing peptic ulcer; therefore, the Lactobacillus pentosus LPS16 can be applied to various commercial products for the treatment or prevention of Helicobacter pylori infection or For the preparation of foods, such as health foods, medical preparations or processed foods, the Lactobacillus pentosus LPS16 can be used alone or in combination with other desired ingredients (such as carriers, adjuvants, solvents and other nutrients). In various product forms, such as tablets, capsules, drinks, powders or drops, etc., can be administered to individuals in various ways according to the form of the product, such as oral, injection, inhalation or application, in order to effectively control the infection of Helicobacter pylori The digestive tract disease caused.

The invention relates to a Helicobacter pumilus LPS16 which inhibits Helicobacter pylori, which can secrete bactericidal substances in the host stomach wall cells for a long time, restricts the growth of Helicobacter pylori in the host digestive tract, and has the effect of killing Helicobacter pylori.

The invention relates to a Helicobacter pylori LPS16 which inhibits Helicobacter pylori, which can effectively inhibit a clinically resistant strain of Helicobacter pylori, and a pathogenic strain which clinically induces duodenal ulcer, gastric cancer and the like, and has good Helicobacter pylori resistance, which is the present invention. efficacy.

The use of a Lactobacillus pentosus LPS16 according to the present invention is to apply a Helicobacter pylori-inhibiting LPS16 as described above to the preparation of a commercial antibacterial product to further control the infection of Helicobacter pylori, which is a merit of the present invention. effect.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

Fig. 1 is a flow chart showing the treatment of Lactobacillus pentosus LPS16 of the present invention.

Fig. 2 is a schematic view showing the gastric cell adsorption ability of Lactobacillus pentosus LPS16 of the present invention.

Fig. 3 is a schematic view showing the bactericidal effect of Lactobacillus pentosus LPS16 of the present invention.

Figure 4: Identification results of strains of Lactobacillus pentosus LPS16 of the present invention.

Figure 5: Identification results of strains of Lactobacillus pentosus LPS16 of the present invention.

Claims (2)

A lactic acid bacteria inhibiting Helicobacter pylori, an isolated Lactobacillus pentosus , registered as BCRC910467, in the biochemical test results of the Lactobacillus pentosus , GLY, LARA, RIB, DXYL, GAL, GLU, FRU , MNE, RHA, DUL, MAN, SOR, MDG, NAG, AMY, ARB, ESC, SAL, CEL, MAL, LAC, MEL, SAC, TRE, MLZ, RAF, GEN, TUR, DARL and GNT tests are positive reactions , ERY, DARA, LXYL, ADO, MDX, SBE, INO, MDM, INU, AMD, GLYG, XLT, LYX, TAG, DFUC, LFUC, LARL, 2KG and 5KG tests were negative reactions. The use of a lactic acid bacterium for the preparation of a food or pharmaceutical compound for inhibiting Helicobacter pylori infection according to the Lactobacillus pentosus or its metabolite according to item 1 of the patent application.