TWI384990B - Food and pharmaceutical composition with strains of lactic acid bacteria for treatment of gastric ulcer - Google Patents

Description

Food composition and pharmaceutical composition of lactic acid bacteria strain for treating gastric ulcer

The present invention relates to a food composition and a pharmaceutical composition, and more particularly to a food composition and a pharmaceutical composition of a lactic acid bacteria strain for treating gastric ulcer.

In 1982, Helicobacter pylori was found to be associated with ulcerative inflammation of the digestive organs by Marshall and Warren. Ulcerative inflammation of the digestive organs has long been thought to cause excessive secretion of gastric juice due to stress and lifestyle, causing ulcerative inflammation to occur repeatedly. Therefore, the treatment of this disease has been aimed at inhibiting the secretion of gastric juice or neutralizing the acidity of the gastric juice, so that the disease can be alleviated. It was not until 1982 that the presence of Helicobacter pylori was discovered that the development of antibiotics began to be developed, shortening the time course of treatment for peptic ulcer disease. In 2005, Marshall and Warren won the Nobel Medicine with the title "The bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease". The award directly proves that this contribution is very important for human medicine.

Peptic ulcer disease is endless in today's business community. Modern people's stress, lifestyle and diet cannot be quantified as the main cause. In patients with peptic ulcer, most of them are associated with infection with Helicobacter pylori. The patient's treatment of antibiotics must last for a period of time, but it is often overlooked due to busy life, resulting in the possibility of bacterial resistance. Furthermore, the treatment with antibiotics will eliminate the beneficial bacteria in the human body and cause symptoms to the human body.

Drug treatment of Helicobacter pylori usually involves a combination of drugs, such as amoxicillin, metronidazole, bismuth, proton pump blocker (proton) Pump inhibitor), such as losec, takepron. The use of an antibiotic alone is resistant to the disease, and the antibiotics are also allowed to sit up and take no effect. Therefore, two antibiotics are usually used together with a salt or a proton pump blocker. It is "three in one therapy." But the effect of salt must not be as good as that of proton pump blockers, so it is less used in Taiwan. The general course of treatment is two weeks, and the eradication rate of Helicobacter pylori in the stomach can reach 90%. If only one antibiotic is added plus a proton pump blocker, the eradication rate is reduced to 80% to 85%. Currently in Taiwan, health insurance payments are also limited to two weeks, unless there are special cases that require extended treatment or re-treatment. In addition to the above drugs, there are also B-type anti-tissue ammonia drugs to replace proton blockers. How to choose the best drug combination depends on the patient's condition. However, this three-in-one therapy also has its shortcomings, including the occurrence of side effects, such as nausea, diarrhea, flatulence, dizziness, etc., which often affects the patient's compliance with medication.

Generally, the products containing lactic acid bacteria (LAB) only have the health effect of regulating the intestinal tract. Although there are tens of thousands of strains of lactic acid bacteria in nature, only a few lactic acid strains have the trait of inhibiting the activity of Helicobacter pylori. The ability of a few of these strains to have acid and bile salt tolerance, ability to adsorb gastric epithelial cells, and ability to survive after passage through the gastrointestinal tract is an important basis for screening for strains that promote health effects. To date, only a few strains of lactic acid bacteria that have been shown to have an effect of inhibiting the activity of H. pylori have been identified. The function of lactic acid bacteria on health is the specificity of the strain rather than the species. The strain that has special effects on human health is called the guidelines for probiotics in food. Report of joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food; London Ontario, Canada April 30 and May 1, 2002: 1-7).

There are many reports on the reaction between lactic acid bacteria and Helicobacter pylori in the country at home and abroad. In the in vitro study of the ability of lactic acid bacteria to inhibit the activity of Helicobacter pylori, the linoleic acid produced by the probiotics is decomposed by IKK-γ (IκB kinase: IκB kinase) and Hsp90 (Heat shock protein 90; The shock protein 90) complex, which initiates the NF-κB (Nuclear factor-kappaB; nuclear transcription factor-κB) signaling chain, produces anti-inflammatory activity in gastric mucosal epithelial cells infected with Helicobacter pylori and inflamed (Jung M Kim, 2008). The active lactic acid bacteria Lactobacillus plantarum MLBPL1, Lactobacillus rhamnosus GG, Lactococcus lactis can reduce the adhesion of Helicobacter pylori to cells by about 50%, and the active lactic acid bacteria can inhibit the production of IL-8 by cells infected with Helicobacter pylori (Interleukin-8; Interleukin-8), which reduces the concentration of IL-8 secreted by cells, helps to reduce the inflammatory response of cells and the production of CD74 in the Helicobacter pylori receptor (S. Rokka, 2008).

The invention broadly comprises Lactobacillus salivarius AP-32 strain, Food Industry Development Institute, accession number BCRC 910437, or a combination of Lactobacillus salivarius AP-32 strain, Food Industry Development Institute, registration number BCRC 910437 and Lactobacillus johnsonii MH-68 strain, Food Industry Development Institute, a biological culture of accession number BCRC 910438, and a food composition or pharmaceutical composition consisting of a physiologically acceptable excipient or diluent.

In another specific embodiment, the physiologically acceptable Lactobacillus salivarius AP-32 strain that enhances the ability of anti-Helicobacter pylori, the Food Industry Development Institute registration number BCRC 910437, or the combination of Lactobacillus salivarius AP-32 strain, The Food Industry Development Institute stores the number BCRC 910437 and the Lactobacillus johnsonii MH-68 strain, and the Food Industry Development Institute registers the biological culture of the number BCRC 910438.

Further, in another specific embodiment, the present invention can broadly include any of the aforementioned biological properties which are inhibited by the lactic acid bacteria strain and the Helicobacter pylori competition, inhibition analysis, animal test, human clinical trial and have the activity of inhibiting the activity of H. pylori. Cultures.

The invention may also be broadly construed as including a part or a combination of elements and features in the description of the application, and any or all combinations of any one or more of those parts, constituent elements and features. And where well-known and equivalents are described in the art to which the present invention pertains, such known equivalents will be incorporated herein.

The present invention broadly encompasses a lactic acid bacteria strain selected from any one or more of the following biological cultures: Lactobacillus salivarius AP-32 strain, Food Industry Development Institute registration number BCRC 910437 and Lactobacillus johnsonii MH-68 strain, Food Industry Development Institute, accession number BCRC 910438.

Many literatures indicate that probiotics can secrete large amounts of acidic substances, help patients with infected Helicobacter pylori to balance the pH of gastric juice in the stomach, destroy the ammonia cloud formed by Helicobacter pylori eroding cells, and compete with the Helicobacter pylori. Space to achieve the effect of inhibiting the activity of H. pylori.

The freeze-dried culture of the lactic acid bacteria strain of the present invention has been deposited at the Taiwan Food Industry Development Research Institute, and the address is No. 331, Food Road, Hsinchu City, Republic of China. The details of the deposit are shown in Table 1:

The two strains of lactic acid bacteria listed in Table 1 have been found to have anti-Helicobacter pylori ability, including inhibition of the activity of Helicobacter pylori, side effects caused by antibiotic treatment of Helicobacter pylori (such as diarrhea, etc.) and stomach Uncomfortable symptoms (such as flatulence, acid reflux, etc.) have the function of slowing down and treating.

Example 1: Morphology and general properties of lactic acid bacteria against Helicobacter pylori.

The taxonomic characteristics of the strains were confirmed based on the 16S rDNA sequence analysis and the results of the API bacterial identification system analysis. The two strains of lactic acid bacteria described in the present invention were found to be Lactobacillus saliva and Lactobacillus johnsonii, respectively. The morphological and general characteristics of the above strains are detailed in Table 2:

Example 2: Anti-Helicobacter pylori lactic acid bacteria inhibit the activity of gastric Helicobacter pylori to decompose urea to produce ammonia and carbon dioxide, mainly to detect the concentration of ammonia and inhibit the percentage of Helicobacter pylori (the urease activity in liquid culture) The analytical test is an in vitro efficacy verification platform).

The inhibitory effect of the lactic acid strain of Lactobacillus licheniformis AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 of the present invention on urease was examined, and the Helicobacter pylori was resistant to the above-mentioned antibiotic The lactic acid bacteria of Helicobacter pylori were co-cultured and the concentration of ammonia in the medium was measured, thereby verifying the ability of the above strains against H. pylori. Use the following experimental steps:

1. The bacterial culture of Helicobacter pylori is cultured to reach an appropriate amount of bacteria.

2. Take appropriate amount of Helicobacter pylori bacteria solution and add appropriate amount of lactic acid bacteria culture solution or appropriate amount of Helicobacter pylori to the appropriate amount of Amoxicillin (positive control group for inhibiting the growth of Helicobacter pylori).

3. After mixing uniformly, 1 mL of liquid was added to the slant medium for biphasic culture, and cultured at 37 ° C for 24 hours in an anaerobic environment.

4. The bacterial liquid of step 3 was centrifuged at 3000 rpm for 10 minutes, and the supernatant was taken for urease activity analysis test.

5. Prepare urease reaction buffer: 20% urea + 0.012% phenol red within PBS, pH 6.5.

6. Take 10 μL of the supernatant solution and add 300 μL of urease reaction buffer to an enzyme immunoassay (ELISA) culture dish and react at 37 ° C for 1 hour.

7. To measure the absorbance at 550 nm, urease reaction buffer was used as a blank control group. The higher the absorbance value, the more red and alkali the liquid, which means that the activity of Helicobacter pylori is better, and the more urease is secreted.

The statistical analysis of the data is shown in Table 3, Table 4, and Figures 1 and 2, and is expressed as mean ± standard deviation (Mean ± SD). As shown in FIGS. 1 and 2, respectively, that when 10 ⁸ to 10 ⁹ cfu (Colony-Forming Units) saliva AP-32 Lactobacillus strain BCRC 910437, or about L. reuteri BCRC 910438 MH-68 strain and ¹⁰⁸ to ¹⁰⁹ A cfu (Colony-Forming Units) gastric Helicobacter pylori was co-cultured at 37 ° C for 24 hours under anaerobic conditions, and the supernatant was taken for urease activity analysis test. The positive control group was Amoxicillin antibiotic and the negative control group was E.coli HB101.

As shown in Figure 1, when the antibiotic concentration is 240 μg/mL, it does not inhibit the ability of gastric Helicobacter pylori to secrete urease to produce ammonia. Therefore, the supernatant is alkaline, indicating that the supernatant contains a high concentration. ammonia. L.gasseri , L.rhamnnosus and 240 μg/mL antibiotics are nearly five times more ammonia than Lactobacillus saliva AP-32 strain BCRC 910437, Lactobacillus johnsonii MH-68 strain BCRC 910438 and 1200 μg/mL antibiotics , showing that L. gasseri , L. rhamnnosus , and 240 μg/mL antibiotics did not cause the loss of biological activity of H. pylori.

Figure 2 uses E. coli HB101 as a negative control group to calculate the inhibitory capacity of lactic acid bacteria strains and antibiotics. The calculation method is as follows: ( E.coli HB101 and the Helicobacter pylori co-culture after the supernatant absorbance value - the lactic acid bacteria and the Helicobacter pylori co-culture after the supernatant absorbance) / E.coli HB101 and Helicobacter pylori After the co-cultivation, the supernatant absorbance value × 100%. The calculated value is the percentage of the range in which the strain inhibits the biological activity of H. pylori. As shown in Fig. 2, the ability of Lactobacillus salivarius AP-32 strain BCRC 910437, Lactobacillus johnsonii MH-68 strain BCRC 910438 and 1200 μg/mL antibiotic to inhibit gastric Helicobacter pylori was more than 76% higher than the other three groups. Lactobacillus saliva AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 were shown to help reduce the biological activity of H. pylori and further inhibit the growth of H. pylori. Table 3 and Table 4 are the strains and control groups of the present invention, respectively, cultured with Helicobacter pylori, the absorbance of the supernatant and the percentage of inhibition range (means ± SD):

Example 3; lactic acid bacteria against Helicobacter pylori in house human clinical trials.

Helicobacter pylori secretes urease, which decomposes urea to produce ammonia and carbon dioxide. Therefore, the ¹³ C breath test was developed using this feature. The principle of this method is that Helicobacter pylori produces urease in the body, and the ¹³ C or ¹⁴ C-labeled urea is taken down by the subject, ie, decomposed. Produce carbon dioxide with isotope labeling, collect exhaled specimens, use liquid scintillation counter or gas nuclear mass spectrometer to mark the carbon dioxide, high sensitivity, quantifiable, patient painless, simple and rapid method, whether to detect gastric Helicobacter pylori The radical cure is very reliable. However, due to the small amount of radioactive material in ¹⁴ C, the ¹³ C urea breath test method is currently used. Sixteen patients who had been infected with Helicobacter pylori were identified by blood test, and ¹³ C breath test was performed. Eight patients were identified as infected with Helicobacter pylori:

1. Determine that the patient has no major symptoms of gastric ulcer or gastric perforation until now.

2. Stop taking any products related to lactic acid bacteria four weeks before the test.

3. Taking Lactobacillus saliva AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsules, each capsule containing 2 x 10 ⁹ cfu/capsule (Colony-Forming Units/capsule). One in the morning and evening, continue to take 4 weeks.

4. After taking it for four weeks, the ¹³ C breath test was performed after two weeks of inactivity.

5. Observe the change in the value of the ¹³ C breath test before and after taking.

The statistical analysis of the data is shown in Table 5 and Figures 3 and 4. Figure 3 shows the numerical distribution of the patient's ¹³ C breath test; Figure 4 shows the value of the ¹³ C breath test, expressed as Mean ± SD. Figure 3 shows the distribution and changes of the ¹³ C breath test values before and after administration of Lactobacillus salivarius AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsules. As shown in Fig. 4, the mean value of the ¹³ C breath test value was ± standard deviation of the ¹³ C breath test values before and after administration of Lactobacillus salivarius AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsule. After taking Lactobacillus saliva AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsule, the ¹³ C breath test value was significantly reduced, clearly showing Lactobacillus saliva AP-32 strain BCRC 910437 and Yogurt Bacillus MH-68 strain BCRC 910438 inhibits the growth and physiological activity of Helicobacter pylori in humans. Table 5 shows the ¹³ C breath test values (means ± SD) before and after the patient took Lactobacillus saliva AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438: Table 5 Human clinical trial pretest, ¹³ C breath test values before and after taking Lactobacillus saliva AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsule.

In addition to the acid-producing ability of lactic acid bacteria, in addition to the ability to produce Helicobacter pylori, it is necessary to confirm that the strain can exert its competitive inhibition with Helicobacter pylori in the human body. Therefore, it is necessary to conduct human clinical trials. Through the human clinical trial pretest results, this The invented anti-Helicobacter pylori lactic acid bacteria can provide medical use for treating or relieving symptoms caused by Helicobacter pylori infection. The anti-Helicobacter pylori lactic acid bacteria described in the present invention can simultaneously reduce the physiological activity of H. pylori and inhibit its growth. It is an object of the present invention to continue to achieve these desires or at least to provide the public with new options for the elimination of antibiotics in the treatment of Helicobacter pylori. The present invention finds Helicobacter pylori lactic acid bacteria which have no side effects and are beneficial to the human body as a stomach. New options for the treatment of Helicobacter pylori.

The embodiments described above are only intended to illustrate the technical idea and the features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the contents of the present invention and to implement the present invention. That is, the equivalent variations or modifications made by the spirit of the present invention should still be included in the scope of the present invention.

Figure 1 shows, respectively, of 10 ⁸ to 10 ⁹ cfu (Colony-Forming Units) saliva AP-32 Lactobacillus strain BCRC 910437, or about L. reuteri BCRC 910438 MH-68 strain and ¹⁰⁸ to ¹⁰⁹ cfu (Colony- Forming Units) Helicobacter pylori was co-cultured at 37 ° C in an anaerobic environment. After 24 hours, the supernatant was taken for urease activity assay. The positive control group was Amoxicillin antibiotic. When the antibiotic concentration was 240 μg/mL, it could not inhibit the ability of gastric Helicobacter pylori to secrete urease to produce ammonia. Therefore, the supernatant was alkaline, indicating that the supernatant contained a high concentration of ammonia. L.gasseri , L.rhamnnosus and 240 μg/mL antibiotics are nearly five times more ammonia than Lactobacillus saliva AP-32 strain BCRC 910437, Lactobacillus johnsonii MH-68 strain BCRC 910438 and 1200 μg/mL antibiotics .

2 shows, each of 10 ⁸ to 10 ⁹ cfu (Colony-Forming Units) saliva AP-32 Lactobacillus strain BCRC 910437, or about L. reuteri BCRC 910438 MH-68 strain and ¹⁰⁸ to ¹⁰⁹ cfu (Colony- Forming Units) Helicobacter pylori was co-cultured at 37 ° C in an anaerobic environment. After 24 hours, the supernatant was taken for urease activity assay. The positive control group was Amoxicillin antibiotics, and the negative control group was E.coli HB101, which calculated the inhibition ability of lactic acid bacteria strains and antibiotics. The calculation method is as follows: ( E.coli HB101 and the Helicobacter pylori co-culture after the supernatant absorbance value - the lactic acid bacteria and the Helicobacter pylori co-culture after the supernatant absorbance) / E.coli HB101 and Helicobacter pylori After the co-cultivation, the supernatant absorbance value × 100%. The calculated value is the percentage of the range in which the strain inhibits the biological activity of H. pylori. The ability of Lactobacillus saliva AP-32 strain BCRC 910437, Lactobacillus johnsonii MH-68 strain BCRC 910438 and 1200 μg/mL antibiotic to inhibit Helicobacter pylori was more than 76% higher than the other three groups.

Figure 3 shows the distribution and changes of the ¹³ C breath test values before and after administration of Lactobacillus salivarius AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsules. Before taking the Lactobacillus saliva AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsule, the ¹³ C breath test value was significantly reduced, clearly showing the Lactobacillus saliva AP-32 strain BCRC 910437 and Lactobacillus MH-68 strain BCRC 910438 inhibits the growth and physiological activity of Helicobacter pylori in humans.

Figure 4 shows the mean ± standard deviation of the ¹³ C breath test values before and after administration of Lactobacillus salivarius AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsules. Before taking the Lactobacillus salivarius AP-32 strain BCRC 910437 and Lactobacillus johnsonii MH-68 strain BCRC 910438 capsule, the ¹³ C breath test value was significantly reduced, and the Lactobacillus saliva AP-32 strain BCRC 910437 and Lactobacillus MH-68 strain BCRC 910438 inhibits the growth and physiological activity of Helicobacter pylori in humans.

Claims (6)

A food composition comprising: a lactic acid bacteria strain for treating gastric ulcer, the strain comprising Lactobacillus salivarius AP-32 strain, accession number BCRC 910437, or a combination of Lactobacillus salivarius AP-32 strain, accession number BCRC 910437 and Lactobacillus johnsonii MH-68 strain, a biological culture of the number BCRC 910438, deposited in the Hsinchu Food Industry Development Research Institute (FIRDI), Taiwan; and physiologically acceptable excipients or dilutions Agent. The food composition of claim 1, wherein the excipient or diluent is a food product. The food composition of claim 2, wherein the food product comprises fermented milk, yogurt, cheese, milk powder for dairy beverages, tea, coffee or a combination thereof. The food composition of claim 1, wherein the lactic acid bacteria strain is an active strain. A pharmaceutical composition for treating gastric ulcer, comprising: a lactic acid bacteria strain for treating gastric ulcer, the strain comprising Lactobacillus salivarius AP-32 strain, accession number BCRC 910437, or a combination of Lactobacillus salivarius AP-32 strain, registration number BCRC 910437 and Lactobacillus johnsonii MH-68 strain, hosted the biological culture of BCRC 910438, all of which are deposited in the Hsinchu Food Industry Development Research Institute (FIRDI), Taiwan; and pharmaceutically acceptable forming Agent or diluent. The pharmaceutical composition according to claim 5, wherein the lactic acid bacteria strain is an active strain.