TW202214842A - Method for improving resistance of lactic acid bacteria to gastric juices/bile - Google Patents

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

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

The present invention relates to a method for improving the resistance of lactic acid bacteria to gastric juice and bile, more specifically, to a method for imparting or enhancing the tolerance of lactic acid bacteria to gastric acid, bile acid, etc., and after ingestion, it can survive in The method of reaching the intestines in the state.

In recent years, as probiotics, microorganisms that can improve the balance of intestinal flora and can exert a beneficial effect on the health of the host have been attracting attention. These microorganisms use lactic acid bacteria or bifidobacteria. It has been reported that by ingesting these bacteria, various physiological effects such as the effect of improving constipation/diarrhea, the effect of preventing infection and allergy by improving the immune function, and the effect of preventing arteriosclerosis can be obtained. .

Regarding the definition of probiotics, some experts have advocated new thinking in recent years, such as including dead bacteria instead of only live bacteria. However, as far as the mechanism of action of probiotics is concerned, by multiplying useful microorganisms in the intestinal flora, producing various metabolites, and eliminating competing harmful microorganisms or inhibiting their reproduction, it can be said that their functions can be exerted more effectively. Therefore, it is considered that it is more beneficial to exist in a state of survival in the intestine. In this way, the ingested useful microorganisms preferably reach the intestines in a living state. However, before reaching the intestines, there are various factors that hinder the growth of microorganisms such as temperature, pH, oxygen, etc., especially digestive juices such as gastric juice or bile, in addition to gastric acid or bile acid. In addition, it also contains various digestive enzymes. When exposed to these digestive juices, the useful microorganisms will die, or even if they survive, they will suffer serious damage such as loss of colony-forming power (reproductive power).

Therefore, a technique for protecting useful microorganisms from digestive juices and improving their survivability in the digestive tract has been explored. For example, some people have disclosed a method of enhancing the gastric acid and bile acid tolerance of microorganisms by destroying the hrcA gene of microorganisms (Patent Document 1), or a method of encapsulating lactic acid bacteria in enteric capsules to avoid direct contact with gastric juice and the like. A method of improving survivability (refer to Patent Document 2) and the like. However, the former requires complex genetic engineering methods, and the latter is limited by dosage forms, etc., and these technologies can be said to be not universally applicable. On the other hand, as a simpler method, it has been reported that by culturing Lactobacillus lactis in a medium supplemented with Tween 80 in excess, its bile tolerance is improved (Non-Patent Document 1). However, when the applicant of the present application applied this technique to Lactobacillus paracasei, the bile tolerance was not seen to be improved, but it was confirmed that it decreased instead. [Prior Art Literature] [Patent Literature]

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

[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

[The problem to be solved by the invention]

Therefore, the subject of this invention is to provide the technique which improves the tolerance to gastric juice or bile of lactic acid bacteria, and improves the survivability in a living body. [Means of Solving Problems]

As a result of diligent studies to solve the above-mentioned problems, the present inventors found that by culturing lactic acid bacteria in a medium supplemented with oleic acid or a salt thereof, the tolerance to gastric juice and bile can be enhanced, and the survivability after exposure can be improved. , and finally complete the present invention.

That is, the present invention is a method for improving gastric juice and/or bile resistance of lactic acid bacteria, characterized by culturing lactic acid bacteria in a medium containing oleic acid or a salt thereof.

In addition, the present invention is a kind of Lactobacillus paracasei, wherein the ratio of oleic acid to the thalline is 28-38%.

In addition, the present invention is a kind of Lactobacillus paracasei YIT9029 (FERM BP-1366), wherein the proportion of oleic acid in the cells is 28-38%.

Furthermore, the present invention is an oral preparation containing Lactobacillus paracasei in a ratio of 28 to 38% of the above-mentioned oleic acid.

Moreover, this invention is a food-drinks product which contains the ratio of the said oleic acid of Lactobacillus paracasei of 28-38%. [Effect of invention]

According to the present invention, since the lactic acid bacteria can impart or enhance their own tolerance to gastric juice, bile, etc., when ingesting food and beverages containing them, the lactic acid bacteria can reach the intestines in a probiotic state with more bacterial cells maintaining their fecundity. Therefore, with regard to various physiological functions possessed by lactic acid bacteria, more excellent effects can be continuously obtained.

[Form of implementing the invention]

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), for the lactic acid bacteria belonging to the genus Lactobacillus in the past, the bacterial genus has been subdivided, and some bacterial species and their genus names have been change. In this specification, it shows by the new classification notation after reclassification. In addition, among the lactic acid bacteria classified as Lactobacillus casei or Lactobacillus paracasei in the old classification, those newly classified as Lacticaseibacillus paracasei are included in the paracasei milk of this case. Bacillus (Lacticaseibacillus paracasei). The lactic acid bacteria used in the present invention are not particularly limited, and examples thereof include Lactobacillus bacteria such as Lacticaseibacillus paracasei, Lacticaseibacillus casei, Lactobacillus gasseri, Lactobacillus acidophilus ( Lactobacillus acidophilus), Lactobacillus helveticus, Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. delbrueckii Lactobacillus bacteria such as Lactobacillus johnsonii, Streptococcus bacteria such as Streptococcus salivarius, Lactobacillus silicate bacteria such as Limosilactobacillus fermentum, and Liquorilactobacillus such as Liquorilactobacillus mali Bacteria, Streptococcus bacteria such as Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus plantarum), Lactococcus raffinolactis and other Lactococcus bacteria, Enterococcus faecalis (Enterococcus faecalis), Enterococcus faecium (Enterococcus faecium) and other bacteria belonging to the genus Enterococcus, these lactic acid bacteria can be used 1 type or 2 or more. In addition, according to the reclassification of Lactobacillus by Zheng et al. (Non-Patent Document 2), Lacticaseibacillus casei was classified as Lactobacillus casei in the old classification; Streptococcus salivarius was classified in the old classification. It is Lactobacillus salivarius; Lactobacillus fermentum is Lactobacillus fermentum in the old classification; Liquorilactobacillus mali is Lactobacillus mali in the old classification.

Among the above-mentioned lactic acid bacteria, Lactobacillus paracasei (Lacticaseibacillus paracasei) is preferred because of its excellent tolerance to gastric juice and bile. Lactobacillus paracasei is not particularly limited as long as it is classified as such, and commercially available products, those deposited in depository institutions such as ATCC, newly discovered ones, or mutants thereof can be used. Examples of such Lactobacillus paracasei include Lactobacillus paracasei YIT9029, Lactobacillus paracasei YIT0209 ^T , and the like. Among these Lactobacillus paracasei, Lactobacillus paracasei YIT9029 is preferable in terms of better tolerance to gastric juice and bile. Lactobacillus paracasei YIT9029, the applicant in this case is an independent administrative legal person product evaluated by the international depositary authority of the Budapest Treaty under the name of Lactobacillus casein YIT9029 (FERM BP-1366, deposit date: May 18, 1987). Patent Bio-Deposit Center, Technical Infrastructure Agency (Room 120, Kazusa 2-5-8 Kazusa, Kisarazu City, Chiba Prefecture, Japan 292-0818). In addition, Lactobacillus paracasei YIT9029 was previously classified as Lactobacillus casei, but is classified as Lactobacillus paracasei according to the reclassification of the Lactobacillus genus.

The culture medium for culturing the above-mentioned lactic acid bacteria is not particularly limited, and examples thereof include animal milk culture media and various synthetic media consisting of raw milk such as cow's milk, goat's milk, horse's milk, and goat's milk, or dairy products such as skimmed milk powder, whole milk powder, and fresh cream. Components used in general lactic acid bacteria culture media can be added to the culture medium. Examples of such components include vitamins such as vitamin A, vitamin B, vitamin C, and vitamin E, various peptides, amino acids, calcium, magnesium, and the like. of salts. The synthetic medium may be either a liquid medium or a solid medium, and preferably one containing a nitrogen source and a carbon source. As the nitrogen source, meat extract, protein gluten, gluten, casein, yeast extract, amino acid, etc. can be used; and as the carbon source, glucose, xylose, fructose, inositol, maltose, jelly, koji juice, starch, sugar cane can be used Residue, bran, molasses, glycerin, etc. Moreover, ammonium sulfate, potassium phosphate, magnesium chloride, salt, iron, manganese, molybdenum, etc. can be added as inorganic substances, and even vitamins and the like can be added. Suitable synthetic mediums include MRS medium, LBS medium, Rogosa medium, WYP medium, GYP medium and the like.

In the present invention, by adding oleic acid or its salt to the above-mentioned medium, the tolerance of the lactic acid bacteria after culture to gastric juice and bile can be improved. The salt of oleic acid includes sodium oleate, potassium oleate, and the like, and one or two of these can be used. Among these, sodium oleate is preferable from the viewpoint of the effect of improving gastric juice and bile tolerance.

The concentration in the medium of oleic acid or its salt is preferably 0.001 to 0.3 mass % from the viewpoint of the effect of improving gastric juice and bile tolerance (hereinafter, "%" means mass % unless otherwise specified) , more preferably 0.005~0.2%. The timing of adding oleic acid or its salt to the medium is not particularly limited, but it is preferably added before culturing the lactic acid bacteria.

The culture conditions are not particularly limited. For example, the culture temperature is usually 20 to 50°C, preferably 25 to 40°C, and more preferably 35 to 38°C. The culturing 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 3-8, preferably 4-7, more preferably 6-7. The culture can be performed in an incubator, and aeration and shaking can be performed during the culture.

The proportion of oleic acid accounting for the constitutive fatty acid of the above-mentioned lactic acid bacteria cells varies with the lactic acid bacteria species, etc.; for example, when Lactobacillus paracasei, the proportion of oleic acid accounting for constituting fatty acid is preferably 28 to 38%, more preferably 34%. ~36%. The ratio of oleic acid to the constituent fatty acids of the cells is the value measured according to the fatty acid composition analysis described in the Examples.

The form of adding the lactic acid bacteria cultured as described above to oral preparations, food and beverages, etc. is not particularly limited, and for example, a method of directly adding a culture obtained by culturing the above-mentioned various culture media such as animal milk culture medium, and adding lactic acid bacteria from a culture solution can be mentioned. Centrifugal separation, and adding a method of collecting bacteria collected by membrane separation, etc., a method of adding a dried culture solution obtained by drying the culture solution, or a method of drying the dried bacteria after collecting the collected bacteria. A known method can be used for the method for collecting bacteria of the lactic acid bacteria or the method for drying the culture solution or the collected bacteria. In addition, the collected bacteria can also be washed by centrifugal washing or the like as required.

The cultured lactic acid bacteria can be formulated into oral preparations directly or in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, and polyoxyethylene sorbitan fat. acid esters, amino acids, gelatin, albumin, water, physiological saline, etc. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, and excipients can also be appropriately added according to requirements. The dosage form thereof is not particularly limited as long as it is an oral preparation, and examples thereof include liquids, powders, granules, capsules, lozenges, chewing lozenges, and the like, which can be prepared according to conventional methods.

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 liquid, the number of lactic acid bacteria is preferably 10 ⁶ cfu/mL ~ 10 ⁸ cfu/mL; and when it is solid, it is preferably 10 ⁷ cfu/g~10 ¹⁰ cfu/g. The dosage is not particularly limited, but is preferably 10 ³ cfu or more per day, more preferably 10 ⁶ cfu or more per day in terms of the number of viable bacteria.

On the other hand, the food-drinks of this invention can mix well-known food additives and/or food materials with the lactic acid bacteria cultured as mentioned above, and can prepare it according to a usual method. The form is not particularly limited, and for example, liquid, tablet, capsule, paste, granule, etc. can be prepared. Foodstuffs include fermented milk food and drink, fermented soymilk, fermented fruit juice, fermented vegetable juice, and other fermented food and drink, bread, biscuits, American muffins, noodles, candy, and other starch-based foods, chewing gum, candy, Sweets such as Japanese desserts, livestock food such as ham and sausage, fish food such as chikuwa and fish plate, seafood food, salad dressing, soy sauce, jam, spices such as pine, tea, juice, soft drinks, Alcoholic beverages, etc.

The said fermented milk food-drinks can be manufactured according to a usual method. For example, a fermented milk base is obtained by inoculating lactic acid bacteria into an animal milk medium composed of dairy products such as raw milk, skimmed milk powder, whole milk powder, and fresh cream, such as cow's milk, goat's milk, etc. The milk base is added, mixed with a syrup solution, and further added with flavors or food materials to make the final product.

In these fermented milk food-drinks, various food materials, for example, arbitrary components such as various sugars, thickeners, emulsifiers, and various vitamins are blended as required. Specific examples of such food materials include saccharides such as sucrose, glucose, fructose, palatinose, trehalose, lactose, xylose, and maltose, sorbitol, xylitol, and erythritol. Sugar alcohols such as alcohol, lactitol, reduced palatinose, reduced maltose, reduced maltose and other sugar alcohols, aspartame, thaumatin, sucralose, acesulfame potassium, stevia and other high-intensity sweeteners Flavoring, agar, gelatin, carrageenan, guar gum, sanxian gum, pectin, locust bean gum, gellan gum, carboxymethyl cellulose, soybean polysaccharides, propylene glycol alginate, etc. (stabilizers), sucrose fatty acid esters, glycerin fatty acid esters, polyglycerol fatty acid esters, sorbitan fatty acid esters, emulsifiers such as lecithin, milk fats such as cheese (cream), butter, and sour cream, Acids such as citric acid, lactic acid, acetic acid, malic acid, tartaric acid, gluconic acid, various vitamins such as vitamin A, vitamin B, vitamin C, vitamin E, etc., calcium, magnesium, zinc, iron, manganese, etc. Mineral, yogurt, berry, orange, pear, perilla, citrus, apple, mint, grape, almond, pear, custard paste, peach, cantaloupe, banana, tropical (fruit), herbal, black tea, coffee and other spices.

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 number of lactic acid bacteria is preferably 10 ⁶ cfu/mL to 10 ⁸ cfu/mL; if it is solid, it is preferably 10 ⁷ cfu/g~10 ¹⁰ cfu/g. The dosage is not particularly limited, but is preferably 10 ³ cfu or more per day, more preferably 10 ⁶ cfu or more per day in terms of the number of viable bacteria.

The lactic acid bacteria contained in the oral preparation or food and drink of the present invention have high tolerance to acids such as gastric acid and bile acid, and have high tolerance to digestive juices such as gastric juice and bile. Therefore, after the above-mentioned lactic acid bacteria are orally ingested, even if they are exposed to digestive juices, most of the bacterial cells can still pass through the digestive tract in a viable state and reach the intestines. In contrast, the survival rate after continuous exposure to gastric bile is preferably 5 times or more, and more preferably 20 times or more. In the present invention, the survival rate of lactic acid bacteria after continuous exposure to gastric bile refers to a value obtained by the method described in the Examples. In this way, the oral preparation or food-drinks of the present invention are those with high survivability in the digestive tract of lactic acid bacteria or high intestinal reachability. Moreover, when the lactic acid bacteria are exposed to gastric juice or bile, although they will not die, they will still lose their colony-forming ability; and the lactic acid bacteria contained in the oral preparation or food and drink of the present invention can reach the intestines in a probiotic state while maintaining the colony-forming ability. , and can continue to obtain higher physiological effects.

The food-drinks of the present invention may expressly or implicitly indicate the effect or function related to the high survivability in the digestive tract or the high intestinal reachability of lactic acid bacteria. Such labels are not particularly limited, and examples include "high ability to reach the intestine in a viable state", "high ability to reproduce in the intestine", or labels that can be regarded as equivalent to these. Such labels can be attached to the food and drink itself according to the form of the food and drink of the present invention, and can also be attached to such containers or packages. [Example]

Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited in any way by these examples and the like.

Example 1 (Cultivation of Lactobacillus paracasei) The cryopreserved Lactobacillus paracasei YIT9029 was inoculated into MRS liquid medium at 1% (v/v) and cultured under aerobic conditions at 37°C for 24 hours (pre-culture). Next, MRS liquid medium without or containing 0.01%, 0.1% sodium oleate (Tokyo Chemical Industry) was inoculated with 1% (v/v) pre-culture medium and incubated at 37°C under aerobic conditions for 24 hours ( Formal culture), and made into cultured bacteria liquid. (continuous exposure to artificial gastric juice bile) The above 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 physiological saline (Otsuka Pharmaceutical) (centrifugal washing operation). This centrifugal washing operation was carried out a total of 2 times. The particles were resuspended in 15 ml of sterile physiological saline and a portion was sampled. Next, centrifugation was performed at 7,000×g, 8 minutes, and 4°C. After removing all the supernatant, the pellets were suspended in 150 ml of artificial gastric juice (adjusted to pH 3.0) with the composition shown in Table 1 below. Incubate at 37°C for 2 hours. After exposure to gastric juice, a portion of the bacterial fluid was sampled.

After exposure to gastric juice, the pH of the bacterial solution was adjusted to 4.4 with 1 M NaHCO ₃ , and oxgall (manufactured by BD), an artificial bile, was added so that the final concentration was 0.10%, and the cells were exposed to 37° C. for 10 minutes (exposed to bile). Some bacterial fluids were sampled after exposure to bile. Before exposure to gastric juice, after exposure to gastric juice and after continuous exposure to gastric juice bile, the sampled bacterial fluid was appropriately diluted with PBS, and 50 μl of each was smeared on MRS plate medium. The cells were cultured under aerobic conditions at 37°C for 3 days and the colonies were counted to determine the number of viable bacteria (log ₁₀ cfu/ml) in the bacterial solution. The survival rate of Lactobacillus paracasei after exposure to gastric juice and after continuous exposure to gastric juice bile was calculated by taking the number of living bacteria (cfu/ml) before exposure to gastric juice as 100%. In addition, in this test, in order to confirm the reproducibility, the same test was performed three times with different test days. The results after continuous exposure to gastric bile are shown in FIG. 1 .

As can be seen from FIG. 1 , the tolerance of Lactobacillus paracasei to gastric juice and bile was improved by culturing in the medium supplemented with sodium oleate. The survival rate after exposure was shown to be significantly improved by adding sodium oleate to the medium, compared to the dramatic decrease in survival rate without the addition of sodium oleate after continuous exposure to gastric bile.

(Bacterial fatty acid composition analysis) For each bacterial solution, the constituent fatty acid composition of the bacterial cells was analyzed based on the bacterial cell fatty acid composition analysis system "Sherlock Microbial Identification System" (manufactured by MIDI Corporation, gas chromatograph). The results are shown in FIG. 2 .

It was confirmed from FIG. 2 that the ratio of oleic acid in the bacterial cells increased in the bacterial cells cultured in the medium supplemented with sodium oleate.

Comparative Example 1 In place of adding 0.01% or 0.1% sodium oleate to the medium, and adding 0.1%, 1% or 5% of Tween80, in the same manner as in Example 1, after exposure to gastric juice and continuous exposure to gastric juice bile were performed. Survival and fatty acid composition analysis. The results after continuous exposure to gastric bile are shown in FIGS. 3 and 4 .

When 0.1% Tween80 was added, survival after gastric fluid exposure was 87%, which was lower than when 0.1% sodium oleate was added (115% survival). 3 and 4, when 0.1% of Tween80 was added, the survival rate after continuous exposure to gastric bile was lower than when 0.1% of sodium oleate was added. Moreover, as the concentration of Tween80 was increased to 1% and 5%, the survival rate of Lactobacillus paracasei decreased, and the oleic acid content in the cells decreased. [Industrial availability]

The present invention can improve the tolerance of lactic acid bacteria itself to gastric juice, bile, etc., and can make it reach the intestine in a living state to exert its function, so it is useful as a production method of lactic acid bacteria-containing health functional food and the like.

1 is a graph showing the survival rate of Lactobacillus paracasei after continuous exposure to gastric bile in Example 1. FIG. FIG. 2 is a graph showing the constituent fatty acid composition of Lactobacillus paracasei cells after continuous exposure to gastric bile in Example 1. FIG. [ Fig. 3] Fig. 3 is a graph showing the survival rate of Lactobacillus paracasei after continuous exposure to gastric bile in Comparative Example 1. [Fig. [ Fig. 4] Fig. 4 is a graph showing the constituent fatty acid composition of Lactobacillus paracasei cells after continuous exposure to gastric bile in Comparative Example 1. [Fig.

Claims (11)

A method for improving gastric juice and/or bile resistance of lactic acid bacteria, characterized by culturing lactic acid bacteria in a medium containing oleic acid or a salt thereof. The method for improving gastric juice and/or bile resistance according to claim 1, wherein the lactic acid bacteria are Lacticaseibacillus paracasei. The method for improving gastric juice and/or bile resistance according to claim 2, wherein the aforementioned Lactobacillus paracasei is Lactobacillus paracasei YIT9029 (FERM BP-1366). The method for improving gastric juice and/or bile resistance according to any one of claims 1 to 3, wherein the salt of oleic acid is one or two selected from the group consisting of sodium oleate and potassium oleate. The method for improving gastric juice and/or bile resistance according to any one of claims 1 to 4, wherein the content of oleic acid or its salt in the medium is 0.005 to 0.2 mass %. A kind of Lactobacillus paracasei, wherein oleic acid accounts for 28-38% of the constitutive fatty acid of bacterial cells. A kind of Lactobacillus paracasei YIT9029 (FERM BP-1366), wherein oleic acid accounts for 28-38% of the constitutive fatty acids of bacteria. An oral preparation containing one or more Lactobacillus paracasei as claimed in claim 6. An oral preparation containing Lactobacillus paracasei YIT9029 (FERM BP-1366) as claimed in item 7. A food-drinks product containing 1 type or 2 or more types of Lactobacillus paracasei as claimed in claim 6. A food and drink product containing Lactobacillus paracasei YIT9029 (FERM BP-1366) according to claim 7.

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