Method for improving gastric juice and bile resistance of lactic acid bacteria

Abstract

The present invention addresses the problem of providing a technique for heightening the resistance of lactic acid bacteria to gastric juices / bile, and improving the in vivo survival thereof. Provided is a method that solves said problem is characterized by cultivating lactic acid bacteria in a culture medium that contains oleic acid or a salt thereof.

Description

[Technical Field]

[0001] The present invention relates to a method for improving the gastric juice and bile resistance of lactic acid bacteria, and more specifically to a method for imparting or enhancing the resistance of lactic acid bacteria themselves to gastric acid, bile acid, etc., so that they can reach the intestines alive after ingestion. [Background technology]

[0002] In recent years, microorganisms that improve the balance of intestinal flora and have beneficial effects on the health of the host have been attracting attention as probiotics. Lactic acid bacteria and bifidobacteria are used as such microorganisms, and it has been reported that ingesting these microorganisms has various physiological effects, such as improving constipation and diarrhea, preventing infection and allergies by improving immune function, and preventing arteriosclerosis.

[0003] In recent years, the definition of probiotics has been proposed to include not only live bacteria but also dead bacteria. However, the mechanism of action of probiotics is that beneficial microorganisms proliferate in the intestinal flora and produce various metabolic products that eliminate or inhibit the growth of competing harmful microorganisms, thereby enabling them to exert their functions more effectively. Therefore, it is considered advantageous for probiotics to exist in a live state in the intestine. Thus, although it is desirable for ingested beneficial microorganisms to reach the intestine alive, various factors that inhibit microbial growth, such as temperature, pH, and oxygen, exist before reaching the intestine. In particular, digestive fluids such as gastric juice and bile contain gastric acid, bile acids, and various digestive enzymes. When exposed to these digestive fluids, microorganisms die or, if they survive, lose their colony-forming ability (proliferation ability), resulting in serious damage.

[0004] Therefore, technologies for protecting beneficial microorganisms from digestive fluids and improving their survival in the digestive tract have been investigated. For example, a method for enhancing the microorganism's resistance to gastric and bile acids by disrupting the microorganism's hrcA gene (Patent Document 1) and a method for encapsulating lactic acid bacteria in an enteric capsule to prevent direct contact with gastric fluids and improve survival (Patent Document 2) have been disclosed. However, the former requires complex genetic engineering techniques, and the latter imposes limitations on dosage forms, making these techniques difficult to generalize. On the other hand, a simpler method has been reported in which Lactobacillus lactis is cultured in a medium containing an excess of Tween 80, improving its bile resistance (Non-Patent Document 1). However, when the present applicant applied this technique to Lacticase Bacillus paracasei, no improvement in bile resistance was observed, and in fact, a decrease was confirmed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-160 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-139200 [Non-patent literature]

[0006] [Non-Patent Document 1] Kimoto H, Ohmomo S, Okamoto T. Enhancement of bile tolerance in lactococci by Tween 80. J Appl Microbiol. (2002) 92, 41-46. [Non-patent document 2] Zheng et al., Int. J. Syst. Evol. Microbiol. 2020;70:2782-2858 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, an object of the present invention is to provide a technique for increasing the resistance of lactic acid bacteria to gastric juice and bile, and improving their survival in the body. [Means for solving the problem]

[0008] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that culturing lactic acid bacteria in a medium to which oleic acid or a salt thereof has been added enhances their resistance to gastric juice and bile, improving their survival rate after exposure, and have thus completed the present invention.

[0009] That is, the present invention relates to a method for improving the gastric juice and / or bile tolerance of lactic acid bacteria, which comprises culturing the lactic acid bacteria in a medium containing oleic acid or a salt thereof.

[0010] The present invention also relates to Lacticase Bacillus paracasei, in which the proportion of oleic acid in the bacterial cells is 28 to 38%.

[0011] The present invention also relates to Lacticase Bacillus paracasei YIT9029 (FERM BP-1366), in which the proportion of oleic acid in the bacterial cells is 28 to 38%.

[0012] The present invention also relates to an oral preparation containing Lacticase Bacillus paracasei having an oleic acid content of 28 to 38%.

[0013] The present invention also relates to a food or drink containing Lacticase Bacillus paracasei having an oleic acid content of 28 to 38%. [Effects of the Invention]

[0014] The present invention can confer or enhance the resistance of lactic acid bacteria to gastric juice, bile, etc., and therefore, when a food or drink containing the bacteria is ingested, a larger number of live bacteria can reach the intestines while maintaining their proliferation ability, thereby enabling more sustained effects to be obtained in the various physiological functions of lactic acid bacteria. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph showing the survival rate of Lacticase Bacillus paracasei after continuous exposure to gastric bile in Example 1. [Figure 2] 1 is a graph showing the fatty acid composition of Lacticase Bacillus paracasei cells after continuous exposure to gastric bile in Example 1. [Figure 3] 1 is a graph showing the survival rate of Lacticase Bacillus paracasei after continuous exposure to gastric juice and bile in Comparative Example 1. [Figure 4] 1 is a graph showing the constituent fatty acid composition of Lacticase Bacillus paracasei cells after continuous exposure to gastric bile in Comparative Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0016] Regarding the lactic acid bacteria used in the present invention, according to the reclassification of lactic acid bacteria by Zheng et al. (Non-Patent Document 2), the genus of lactic acid bacteria that previously belonged to the genus Lactobacillus has been subdivided, and the genus names of some of the bacterial species have been changed. In this specification, the lactic acid bacteria used in the present invention will be referred to by the new classification notation after the reclassification. In addition, among the lactic acid bacteria classified as Lactobacillus casei or Lactobacillus paracasei in the old classification, those that can now be classified as Lacticaseibacillus paracasei are included in Lacticaseibacillus paracasei in this application. The lactic acid bacteria used in the present invention are not particularly limited, and examples thereof include bacteria of the genus Lacticaseibacillus, such as Lacticaseibacillus paracasei and Lacticaseibacillus casei, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus johnsonii, and the like. Lactobacillus bacteria such as Lactobacillus johnsonii, Ligilactobacillus bacteria such as Ligilactobacillus salivarius, Limosilactobacillus fermentum, Liquorilactobacillus mali, Streptococcus bacteria such as Streptococcus thermophilus, Lactococcus lactis subsp. lactis, etc.lactis, Lactococcus lactis subsp. cremoris, Lactococcus plantarum, Lactococcus raffinolactis, and other Lactococcus bacteria; and Enterococcus faecalis, Enterococcus faecium, and other Enterococcus bacteria. One or more of these lactic acid bacteria can be used. According to the reclassification of lactic acid bacteria by Zheng et al. (Non-Patent Document 2), Lacticaseibacillus casei was classified as Lactobacillus casei in the old classification, Ligilactobacillus salivarius was classified as Lactobacillus salivarius in the old classification, Limosilactobacillus fermentum was classified as Lactobacillus fermentum in the old classification, and Liquorilactobacillus mali was classified as Lactobacillus mali in the old classification.

[0017] Among the above lactic acid bacteria, Lacticaseibacillus paracasei is preferred due to its excellent resistance to gastric juice and bile. Lacticaseibacillus paracasei is not particularly limited as long as it is classified into this species, and commercially available strains, strains deposited in depositories such as ATCC, newly discovered strains, or mutant strains thereof can be used. Examples of such Lacticaseibacillus paracasei include Lacticaseibacillus paracasei YIT9029 and Lacticaseibacillus paracasei YIT0209. TAmong these Lacticaceae Bacillus paracasei, Lacticaceae Bacillus paracasei YIT9029 is preferred because of its superior resistance to gastric juice and bile. Lacticaceae Bacillus paracasei YIT9029 has been internationally deposited by the present applicant as Lacticaceae Bacillus casei YIT9029 (FERM BP-1366, deposit date: May 18, 1987) with the International Patent Organism Depositary of the National Institute of Technology and Evaluation (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), the international depositary authority under the Budapest Treaty. Lacticaseibacillus paracasei YIT9029 was previously classified as Lactobacillus casei, but was reclassified as Lacticaseibacillus paracasei due to a reclassification of the Lactobacillus genus.

[0018] The medium for culturing the lactic acid bacteria is not particularly limited, and examples thereof include animal milk media containing raw milk such as cow's milk, goat's milk, mare's milk, and sheep's milk, and dairy products such as skim milk powder, whole milk powder, and fresh cream, as well as various synthetic media. Components commonly used in lactic acid bacteria culture media may be added to the medium, including vitamins such as vitamin A, B vitamins, vitamin C, and vitamin E, various peptides, amino acids, and salts such as calcium and magnesium. Synthetic media may be either liquid or solid, but preferably contain a nitrogen source and a carbon source. Examples of nitrogen sources that can be used include meat extract, peptone, gluten, casein, yeast extract, and amino acids. Examples of carbon sources that can be used include glucose, xylose, fructose, inositol, maltose, starch syrup, koji soup, starch, bacasu, wheat bran, molasses, and glycerin. In addition, minerals such as ammonium sulfate, potassium phosphate, magnesium chloride, salt, iron, manganese, and molybdenum can be added, and vitamins can also be added. Suitable synthetic media include MRS medium, LBS medium, Rogosa medium, WYP medium, and GYP medium.

[0019] In the present invention, the gastric juice and bile resistance of lactic acid bacteria after culture can be improved by adding oleic acid or a salt thereof to the above-mentioned medium. Examples of salts of oleic acid include sodium oleate and potassium oleate, and one or two of these can be used. Of these, sodium oleate is preferred from the viewpoint of the effect of improving gastric juice and bile resistance.

[0020] From the viewpoint of improving gastric juice and bile resistance, the concentration of oleic acid or a salt thereof in the medium is preferably 0.001 to 0.3% by mass (hereinafter, "%" means % by mass unless otherwise specified), and more preferably 0.005 to 0.2%. There are no particular restrictions on the timing of adding oleic acid or a salt thereof to the medium, but it is preferable to add it before culturing lactic acid bacteria.

[0021] The culture conditions are not particularly limited, but for example, the culture temperature is usually 20 to 50°C, preferably 25 to 40°C, and more preferably 35 to 38°C. The culture time is usually 6 to 62 hours, preferably 12 to 48 hours, and more preferably 15 to 30 hours. The pH of the medium is usually 3 to 8, preferably 4 to 7, and more preferably 6 to 7. The culture may be carried out in an incubator, and may be aerated and shaken during culture.

[0022] The proportion of oleic acid in the constituent fatty acids of the lactic acid bacteria cells cultured as described above varies depending on the species of lactic acid bacteria, but for example, in the case of Lacticaceae Bacillus paracasei, the proportion of oleic acid in the constituent fatty acids is preferably 28 to 38%, more preferably 34 to 36%. The proportion of oleic acid in the constituent fatty acids of the cells is a value measured by fatty acid composition analysis described in the Examples.

[0023] The form in which the lactic acid bacteria cultured as described above are added to oral preparations, foods, beverages, etc. is not particularly limited, and examples include a method in which a culture obtained by culturing in the above-mentioned various media such as animal milk medium is added as is, a method in which lactic acid bacteria are collected from the culture medium by centrifugation, membrane separation, etc., and a method in which a dried culture medium obtained by drying the culture medium or dried bacterial cells obtained by drying the collected bacteria are added, etc. Known methods can be used to collect the lactic acid bacteria and to dry the culture medium or the collected bacteria. If necessary, the collected bacteria may be washed by centrifugal washing, etc.

[0024] The cultured lactic acid bacteria can be formulated as an oral preparation either directly or in combination with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and physiological saline. Conventional additives such as stabilizers, humectants, emulsifiers, binders, isotonicity agents, and excipients can also be added as needed. The dosage form is not particularly limited as long as it is an oral preparation, and examples include liquids, powders, granules, capsules, tablets, and the like, which are prepared according to conventional methods.

[0025] The content of lactic acid bacteria in the oral preparation of the present invention is not particularly limited. For example, when the dosage form is a liquid, the viable count of lactic acid bacteria is 10 6 cfu / mL ~10 8 cfu / mL is preferred, and for solids, 10 7 cfu / g ~10 10 The dosage is not particularly limited, but preferably the number of live bacteria is 10 3 cfu or more, preferably 10 6 cfu or more.

[0026] Meanwhile, the food and beverage products of the present invention can be prepared by blending the lactic acid bacteria cultured as described above with known food additives and / or food ingredients according to conventional methods. The form of the product is not particularly limited, and can be, for example, liquid, tablet, capsule, paste, granule, etc. Examples of the food and beverage products include fermented foods and beverages such as fermented milk foods and beverages, fermented soy milk, fermented fruit juice, and fermented vegetable juice; starch-based foods such as bread, biscuits, pancakes, noodles, and tablet candy; confectioneries such as gum, candy, and Japanese sweets; meat foods such as ham and sausage; fish foods such as chikuwa (fish cakes) and kamaboko (fish paste), seafood foods; seasonings such as dressings, soy sauce, jam, and furikake (rice seasoning); and beverages such as tea, juice, soft drinks, and alcoholic beverages.

[0027] The fermented milk food and drink products can be produced by a conventional method, for example, by inoculating and culturing lactic acid bacteria in an animal milk medium consisting of raw milk such as cow's milk or goat's milk, dairy products such as skim milk powder, whole milk powder, and fresh cream, and then homogenizing the medium to obtain a fermented milk base, adding and mixing a syrup solution to the fermented milk base, and further adding flavors and food ingredients to produce a final product.

[0028] These fermented milk foods and drinks can be blended with various food ingredients, as needed, for example, various carbohydrates, thickeners, emulsifiers, various vitamins, etc. Specific examples of these food ingredients include carbohydrates such as sucrose, glucose, fructose, palatinose, trehalose, lactose, xylose, maltose, etc., sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, and reduced maltose syrup, high-intensity sweeteners such as aspartame, thaumatin, sucralose, acesulfame K, and stevia, thickeners (stabilizers) such as agar, gelatin, carrageenan, guar gum, xanthan gum, pectin, locust bean gum, gellan gum, carboxymethylcellulose, soybean polysaccharides, and propylene glycol alginate, sucrose fatty acid esters, and glycerin. Examples of suitable flavorings include emulsifiers such as fatty acid esters, polyglycerol fatty acid esters, sorbitan fatty acid esters, and lecithin; milk fats such as cream, butter, and sour cream; acidulants such as citric acid, lactic acid, acetic acid, malic acid, tartaric acid, and gluconic acid; various vitamins such as vitamin A, B vitamins, vitamin C, and E; minerals such as calcium, magnesium, zinc, iron, and manganese; and flavors such as yogurt, berry, orange, quince, shiso, citrus, apple, mint, grape, apricot, pear, custard cream, peach, melon, banana, tropical, herb, black tea, and coffee.

[0029] The content of lactic acid bacteria in the food and drink of the present invention is not particularly limited. For example, when the food and drink is liquid, the viable cell count of lactic acid bacteria is 10 6 cfu / mL ~10 8 cfu / mL is preferred, and for solids, 10 7 cfu / g ~10 10 The dosage is not particularly limited, but preferably the number of live bacteria is 10 3 cfu or more, preferably 10 6 cfu or more.

[0030] The lactic acid bacteria contained in the oral formulation or food or beverage of the present invention are highly resistant to acids such as gastric acid and bile acids, and therefore highly resistant to digestive fluids such as gastric juice and bile. Therefore, even when orally ingested and exposed to digestive fluids, many of the lactic acid bacteria pass through the digestive tract as live bacteria and reach the intestine. For example, compared to bacteria cultured in a medium free of oleic acid or its salts, the survival rate after continuous exposure to gastric juice and bile is preferably at least 5 times, more preferably at least 20 times. In the present invention, the survival rate of lactic acid bacteria after continuous exposure to gastric juice and bile refers to the value determined by the method described in the Examples. Thus, the oral formulation or food or beverage of the present invention has high survivability in the digestive tract or high intestinal reachability. Furthermore, lactic acid bacteria may lose their colony-forming ability when exposed to gastric juice or bile, even if they are not killed. However, the lactic acid bacteria contained in the oral formulation or food or beverage of the present invention can reach the intestine in a live state while maintaining their colony-forming ability, thereby enabling the attainment of a more sustained physiological effect.

[0031] The food and drink of the present invention can be explicitly or implicitly labeled with the effects or functions of the lactic acid bacteria related to high survival rate in the digestive tract or high intestinal reachability. Such labels are not particularly limited, but include "high ability to reach the intestine alive," "high ability to proliferate in the intestine," or labels that can be considered equivalent to these. Such labels may be attached to the food and drink itself, or to the container or packaging thereof, depending on the form of the food and drink of the present invention. [Example]

[0032] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0033] Example 1 (Cultivation of Lacticase Bacillus paracasei) Cryopreserved Lacticase Bacillus paracasei YIT9029 was inoculated into MRS liquid medium at 1% (v / v) and cultured aerobically at 37°C for 24 hours (preculture). Next, the preculture was inoculated into MRS liquid medium containing no additives or 0.01% or 0.1% sodium oleate (Tokyo Chemical Industry Co., Ltd.) at 1% (v / v) and cultured aerobically at 37°C for 24 hours (main culture). (Continuous exposure to artificial gastric juice and bile) The cultured bacterial solution was centrifuged (7,000 × g, 8 minutes, 4°C). After removing the supernatant, the pellet was resuspended in 15 ml of sterile saline (Otsuka Pharmaceutical) (centrifugal washing procedure). This centrifugal washing procedure was performed twice in total. The pellet was again suspended in 15 ml of sterile saline, and a portion was sampled. Subsequently, the mixture was centrifuged at 7,000 × g, 8 minutes, and 4°C. After removing the entire supernatant, the pellet was suspended in 150 ml of artificial gastric juice (adjusted to pH 3.0) with the composition shown in Table 1 below and incubated at 37°C for 2 hours. After exposure to gastric juice, a portion of the bacterial solution was sampled.

[0034] [Table 1]

[0035] After exposure to gastric juice, the pH of the bacterial solution was adjusted to 4.4 with 1 M NaHCO3, and artificial bile, oxgall (manufactured by BD), was added to a final concentration of 0.10%, followed by exposure at 37°C for 10 minutes (bile exposure). After bile exposure, a portion of the bacterial solution was sampled. The bacterial solutions sampled before gastric juice exposure, after gastric juice exposure, and after continuous gastric juice and bile exposure were appropriately diluted with PBS, and 50 μl of each was smeared on MRS plates. After culturing at 37°C aerobically for 3 days, colonies were counted, and the viable bacterial count (log 10 The viable cell count (cfu / ml) before gastric juice exposure was set at 100%, and the survival rate of Lacticase Bacillus paracasei after gastric juice exposure and after continuous gastric juice and bile exposure was calculated. To confirm the reproducibility of this test, the same test was performed three times on different test days. The results after continuous gastric juice and bile exposure are shown in Figure 1.

[0036] As shown in Figure 1, it was confirmed that culturing Lacticase Bacillus paracasei in a medium containing sodium oleate increased its resistance to gastric juice and bile. After continuous exposure to gastric juice and bile, the survival rate was significantly reduced without the addition of sodium oleate, whereas the addition of sodium oleate to the medium significantly improved the survival rate after exposure.

[0037] (Bacterial cell fatty acid composition analysis) The fatty acid composition of the bacterial cells was analyzed for each bacterial solution using a bacterial fatty acid composition analysis system called the "Sherlock Microbial Identification System" (manufactured by MIDI, gas chromatography). The results are shown in Figure 2.

[0038] From FIG. 2, it was confirmed that the proportion of oleic acid in the cells cultured in the sodium oleate-added medium was increased.

[0039] Comparative Example 1 Analysis of survival rate and fatty acid composition after exposure to gastric juice and continuous exposure to gastric juice and bile was carried out in the same manner as in Example 1, except that Tween 80 was added to the medium at 0.1%, 1%, or 5% instead of 0.01% or 0.1% sodium oleate. The results after continuous exposure to gastric juice and bile are shown in Figures 3 and 4.

[0040] When 0.1% Tween 80 was added, the survival rate after exposure to gastric juice was 87%, which was lower than when 0.1% sodium oleate was added (survival rate 115%). Furthermore, as is clear from Figures 3 and 4, when 0.1% Tween 80 was added, the survival rate after continuous exposure to gastric juice and bile was lower than when 0.1% sodium oleate was added. Furthermore, as the Tween 80 concentration increased to 1% and 5%, the survival rate of Bacillus paracasei decreased and the oleic acid content in the cells also decreased. [Industrial Applicability]

[0041] The present invention is useful as a method for producing health functional foods containing lactic acid bacteria, etc., because it increases the resistance of lactic acid bacteria themselves to gastric juices, bile, etc., allowing them to reach the intestines alive and exert their functions.

Claims

1. A method for improving the gastric juice and bile resistance of Lacticaseibacillus paracasei, characterized by culturing Lacticaseibacillus paracasei in a medium containing an oleate, wherein the content of the oleate in the medium is 0.005 to 0.2% by mass.

2. 2. The method for improving resistance to gastric juice and bile according to claim 1, wherein the Lacticase Bacillus paracasei is Lacticase Bacillus paracasei YIT9029 (FERM BP-1366).

3. 3. The method for improving resistance to gastric juice and bile according to claim 1, wherein the oleate is one or two salts selected from the group consisting of sodium oleate and potassium oleate.

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