JP2005160373A - Glycoside-degradative probiotics - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To standardize the ability to hydrolyze glycosides by improving enteric bacterial flora. <P>SOLUTION: A probiotics is provided by using Lactobacillus casei strain Hasegawa: FERM P-19484. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a lactic acid bacterium having the ability to hydrolyze a glycoside: Lactobacillus casei strain Hasegawa (FERM P-19484), probiotics using the same, and a method for producing the same.

Herbal medicines constituting Kampo prescriptions contain various glycosides as active ingredients. For example, glycyrrhizin of licorice (Glycyrrhizae Radix), paeoniflorin of Paeoniae Radix, saikosaponin of Saikos, saikosaponin, carrot (Ginseng: Gins) These include ginsenoside of ginsenoside, baicalin of Scutellariae Radix, sennoside of Rhei Rhizoma, and geniposide of Gardeniae Fructus.
Kobashi, K. , J .; Trad. Med., 15, 1-13, 1998) However, glycosides having β-bonds are not degraded by human digestive enzymes themselves, so their bioavailability is extremely low. Thus, glycosides are only absorbed and exert their medicinal effects when they are metabolized (hydrolyzed) by intestinal bacteria and increase in lipophilicity.

For example, in the case of ginsenoside, there are two types of glycols and triols depending on the structure of aglycone (aglycone). It has a sugar chain. After ingestion, both diol and triol ginsenosides are hardly degraded by living digestive enzymes and are absorbed after being hydrolyzed by the action of intestinal bacteria such as Bacteroides, Fusobacterium, Eubacterium, and Bifidobacterium. .
Hasegawa, H. et al., Planta Medica, 62, 453-457, 1996 Tawab, M.C. A. et al., Drug Metab. Dispos., 8, 1065-1071, 2003

However, the composition of enteric bacteria is easily affected by constitution, diet, antibiotics, and stress, and there are significant individual differences. Therefore, absorption of glycosides causes individual differences and disadvantages. It can be easily imagined that this contributes to individual differences in medicinal effects encountered in the field of Kampo therapy.

In fact, the present inventor has confirmed that individual differences in the ability of human intestinal bacteria to hydrolyze glycosides (hereinafter referred to as “glycoside hydrolytic ability”) are very large.
Hasegawa, H. et al., Planta Medica, 63, 436-440, 1997 In addition, in cancer metastasis experiments using mice, it was clarified that glycolytic hydrolysis of enteric bacteria affects the anti-metastatic effect of ginseng. Hasegawa, H. et al., Planta Medica, 64, 696-700, 1998

On the other hand, lactic acid bacteria play an important role in the production of cheese, pickles, etc., and are taken into the body in the form of fermented milk, etc., and thus based on the functionality of lactic acid bacteria such as intestinal regulation and serum cholesterol lowering It is known to exert various physiological effects. With regard to the effects of lactic acid bacteria, the concept of “probiotics” (live microorganisms and foods containing them that are beneficial for maintaining the health of the host) has been introduced in recent years, reflecting the health orientation of consumers. Has attracted widespread interest and is undergoing a lot of research. The term "probiotics" was originally "a microbial additive that works beneficially for host animals by improving the balance of the gut microbiota"
Fuller, R. , J .; Appl. Bacteriol., 66, 365-378, 1989, but now it is often used in a broad sense as described above.

As a condition of probiotics, it is assumed that the microorganism is a resident microorganism originally in the human large intestine. By the way, in order for probiotics to work effectively in the digestive tract and exert health effects, it is necessary to reach the digestive tract while remaining alive. However, before reaching the digestive tract, there are various growth-inhibiting environments and growth-inhibiting substances for probiotics such as temperature, pH, oxygen, osmotic pressure, gastric acid and bile acid. Therefore, when using probiotics, it is a necessary condition that the probiotics have resistance to growth-inhibiting environments and growth-inhibiting substances. At present, human-derived Lactobacillus genus lactic acid bacteria and bifidobacteria are known as microorganisms widely used worldwide as probiotics.

An object of the present invention is to standardize a glycoside hydrolyzing ability of human intestinal bacterial flora in which individual differences are remarkably recognized, and hence a microorganism having a glycoside hydrolyzing ability (hereinafter referred to as “glycoside hydrolyzing microorganism”). ) As probiotics.

For example, for glycoside hydrolyzing microorganisms for ginsenoside, soil bacteria
Yoshioka, I. et al., Chem. Pharm. Bull., 20, 2418, 1972, Aspergillus niger Kanda et al., Pharmaceutical Journal, 95, 246, 1975, rat intestinal bacteria Takino et al., Ginseng '89, Kyoritsu Publishing Co., Ltd., p. 267, 1989, enteric bacteria derived from human feces Kanaoka et al., Wakahan Journal of Pharmaceutical Sciences, 11, 241, 1994 have been reported. For human intestinal bacterial isolates, Prevotella oris Hasegawa, H. et al., Planta Medica, 63, 436-440, 1997 JP-A-10-99094 US Pat. No. 5,925,537, Bacteroides JY-6, Bacteroides HJ-15, Fusobacterium K-60, Eubacterium A-44 Bae, EA. et al., Biol. Pharm. Bull., 25, 743, 2002 has been reported.

However, none of these microorganisms has been established as safe for the human body. Furthermore, since a by-product including a odor substance peculiar to bacteria is generated, it is not suitable for supply as an edible product without being subjected to a purification treatment. In addition, the Bifidobacterium strains K-103 and K-506 described in the prior document (Non-patent Document 11) are obligate anaerobic bacteria and are unknown whether they satisfy the conditions as probiotics. Is not very high, so there is a limit to commercialization. Furthermore, there is no report example about the hydrolytic ability with respect to ginsenoside by Lactobacillus lactic acid bacteria, as far as the present inventors can know.

In order to solve the above problems, the present inventor is highly safe to the human body, and from various microorganisms (yeast, bacilli, lactic acid bacteria, bifidobacteria, etc.) used for food processing, as well as enteric bacteria. Probiotics with hydrolytic ability for ginsenoside were eagerly searched.

In the process, lactic acid bacteria that satisfy the conditions of probiotics, originally from the traditional fermented foods of the Korean people, are permanently present in the human intestine, are resistant to gastric acid, and reach the human intestine alive: Lactobacillus Kasei Hasegawa strain (FERM P-19484) was successfully isolated. Probiotics using the lactic acid bacterium have been found to improve the intestinal environment and control immune function without producing unpleasant by-products, thus completing the present invention.

That is, the present invention provides lactic acid bacteria having the ability to hydrolyze glycosides: Lactobacillus casei Hasegawa strain (FERM P-19484), probiotics using the same, and methods for producing the same.

The lactic acid bacterium: Lactobacillus casei Hasegawa strain in the present invention is characterized by having glycoside hydrolytic ability.

In the present invention, a glycoside means a series of compounds defined as “acetal derivatives of sugars having a cyclic structure”, that is, a sugar and an organic compound having a hydroxyl group such as other alcohol or phenol sandwiching an oxygen atom. Refers to a combination.

The base sequence of 16S ribosomal DNA of lactic acid bacteria (GenBank / EMB / DDBJ accession number AB126872) in the present invention is the base sequence (GenBank / EMB / DDBJ accession number) of Lactobacillus paracasei subsp. Paracasei reference strain JCM8130T. D79212) and the best homology (99.934%). Therefore, taxonomically, it is considered to belong to Lactobacillus paracasei subspecies paracasei. However, in the reference strain JCM8130T, the hydrolyzate 20S-protopanaxadiol 20-O-β-D-glucopyranoside by utilizing ginsenoside (20S-protopanaxadiol 20-O-β-D-glucopyranoside) (below) , "M1") and 20S-protopanaxatriol 20-O-β-D-glucopyranoside (hereinafter referred to as "M11") No ability was recognized. Therefore, the Lactobacillus casei Hasegawa strain is considered a mutant strain.

The glycoside hydrolyzing ability of lactic acid bacteria in the present invention will be described in more detail. The hydrolyzate, that is, M1 and M11 are produced by assimilating ginsenoside, which is an active ingredient of the plant of the genus Panax. And

Here, the ginseng plants include Korean ginseng (Panax ginseng CA Meyer), Sanchi ginseng (Panax notoginseng (Burk.) FH Chen), American ginseng ( American ginseng: Panax quinquefolium L., Chikusetsu ginseng: Panax japonicus C. A. Meyer, Himalayan ginseng: Panax pseudo-ginseng Wall. Subsp. Himalaicus Hara, and Vietnamese ginseng: Panax vietnamensis Ha et Grushv.) and the like.

In addition to ginsenoside, the lactic acid bacterium in the present invention includes licorice glycyrrhizin, saiko psychosaponin, soybean (Glycine max) isoflavone glycoside, and onjisaponin such as Onjisaponin. The body and cell fibers such as bark, leaves and cocoons also have the characteristic of hydrolyzing.

Therefore, probiotics using lactic acid bacteria in the present invention include glycoside-containing crude drugs (carrots, licorice, saiko, and ambition), cereals (rice bran, rice flour, bran, corn, malt, etc.), bark [Tahibu (Tabebuia avellanedae, etc.)], leaves [tea (Camellia sinensis, etc.)], and mosses (Ganoderma lucidum, Agaaricus blazei Murril, Mulberry (Phellinus linteus, etc.)) It can be produced by culturing alone or in combination as a nutrient source (hereinafter referred to as “medium”).

In addition, the production of the probiotics of the present invention may be performed by a conventional method except that lactic acid bacteria: Lactobacillus casei Hasegawa strain is used. For example, after first sterilizing a medium as a nutrient source, inoculating about 0.5% to 10.0% by weight of Lactobacillus casei Hasegawa strain, culturing it, and processing it to produce probiotics Can be obtained.

The conditions for culturing may be a medium having a concentration of 1 to 40% in terms of solid content, and may be cultured at 25 ° C to 37 ° C for about 24 hours to 30 days. In this culture, various saccharides such as glucose, sucrose, fructose, and lactose may be added to the medium at about 0.5 wt% to 10.0 wt%.

The probiotics obtained as described above can be used as they are, but in general, various ingredients are added and blended to improve the flavor or form the necessary shape. Further flavors can be added to make the final product.

Examples of components added to and mixed with the probiotics of the present invention include various sugars, emulsifiers, sweeteners, acidulants, fruit juices, and the like. More specifically, sugars such as glucose, sucrose, fructose, lactose, oligosaccharides and honey, sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, and palatinit, emulsifiers such as sucrose fatty acid ester, glycerin sugar fatty acid ester and lecithin . In addition to this, even if various vitamins such as vitamin A, vitamin B, vitamin C and vitamin E, herbal extracts, cereal ingredients, vegetable ingredients, milk ingredients, etc. are blended, an excellent flavored probiotic is obtained. be able to.

In addition, flavors that can be added to the probiotics of the present invention include yogurt, berry, orange, quince, perilla, citrus, apple, mint, grape, pair, custard cream, Flavors such as peach, melon, banana, tropical, herbal, tea, and coffee are listed, and these can be used alone or in combination of two or more. The amount of flavor added is not particularly limited, but is preferably 0.05 to 2.0% by mass, particularly preferably about 0.1 to 0.5% by mass in terms of flavor.

The probiotic of the present invention described above can be a product in any form such as solid or liquid.

EXAMPLES Next, although an Example and a reference example are given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

(Production of probiotic granules) 9 parts of rice germ, 1 part of ginseng dry powder (from Korea) and 40 parts of water were mixed and sterilized at 95 ° C. 3% of a starter in which the present lactic acid bacterium was previously cultured was added thereto and cultured at 30 ° C. for 2 days to obtain a lactic acid bacterium culture. This culture was freeze-dried, and into 105 parts thereof, 20 parts of oligosaccharide, 8 parts of maltose, 3 parts of vitamin C, 1 part of yogurt flavor and 63 parts of lactose were added and granulated to obtain granules. The said granule was a food with a fermented feeling and a pleasant acidity of lactic acid.

(Production of probiotic beverage) 80 parts of water was added to 20 parts of malt (Germany) and saccharified at 63 ° C to 67 ° C, and then the residue was filtered and sterilized by boiling. Ten parts of hops were added 10 minutes before the end of heating. To this was added 6% of a starter in which the present lactic acid bacteria were cultured in advance, and sealed culture was performed at 30 ° C. for 3 days. The culture solution was filtered and filled with carbon dioxide gas to obtain a malt lactic acid foam beverage. The beverage was a beer-taste soft drink with pleasant acidity and carbonic acid.

(Production of probiotic yogurt) 2 parts of ginseng extract (from Korea) and 98 parts of raw milk were mixed and sterilized at 95 ° C. 1% of a starter in which the present lactic acid bacterium was previously cultured was added thereto and cultured at 30 ° C. for 3 days to obtain ginseng yogurt. The yogurt was a food with a pleasant sour taste with a carrot fermentation feeling.

(Production of solid probiotics) 1 part of fresh ginseng root (6-year-old Korean root), 1 part of rice bran, and 4 parts of water were mixed and sterilized at 95 ° C. 1% of a starter previously cultured with the lactic acid bacteria was added thereto and cultured at 30 ° C. for 30 days to obtain a lactic acid bacteria culture. Ginseng root was washed with water and rice bran was removed. When this was chopped and eaten, it was a crunchy food with a pleasant fermentation and moderate acidity.

(Production of gel-like probiotics) 5 parts of agaricus dry powder (from Yamanashi Prefecture), 4 parts of ginseng dry powder (from Korea), 1 part of rice bran, and 20 parts of water were mixed and sterilized at 95 ° C. 1% of a starter previously cultured with the lactic acid bacterium was added thereto and cultured at 30 ° C. for 7 days to obtain a lactic acid bacterium culture. To 30 parts of the culture, 69 parts of a gel solution dissolved in advance and 1 part of yogurt flavor were added and cooled to obtain a gel. The gel was a food with a pleasant fermentation feeling of Agaricus and a sour taste of lactic acid.

(Production of Probiotic Tablets) 4 parts of Toshi dry powder (from China), 4 parts of ginseng dry powder (from Korea), 2 parts of rice bran, and 20 parts of water were mixed and sterilized at 95 ° C. 3% of a starter in which the present lactic acid bacterium was previously cultured was added thereto and cultured at 30 ° C. for 7 days to obtain a lactic acid bacterium culture. The culture was freeze-dried, and to 80 parts thereof, 16 parts of lactose, 2 parts of eggshell calcium and 2 parts of silicon dioxide were added and granulated to obtain tablets. As a result of analyzing the tablet, the presence of trimethoxycinnamic acid generated by hydrolysis of onjisaponin was confirmed. Trimethoxycinnamic acid has been reported to be capable of increasing the decrease in expression of cerebral cortical acetylcholine synthase (choline acetyltransferase) at the mRNA level, which is considered to be one of the causes of Alzheimer's disease.
Takeshi Yabe, Japanese Journal of Oriental Medicine, 50, 776-782, 2000

(Manufacture of soy isoflavone aglycone-containing probiotics) 98 parts of soy milk (12% solids) prepared by a conventional method was mixed with 1 part of Agaricus dry powder (Yamanashi) and 1 part of ginseng dry powder (Korea) And sterilized at 95 ° C. To this, 6% of a starter in which the present lactic acid bacterium was cultured in advance was added and cultured at 30 ° C. for 3 days to obtain a gel-like fermentation product. The fermented product was a food product in which the blue odor and bitterness of soy milk disappeared, and the acidity containing isoflavone aglycone was pleasant.

The components of the probiotics produced in Examples 1, 3, 4, 5, 6, and 7 were analyzed by thin layer chromatography. As a result, the presence of ginsenoside hydrolysates M1 and M11 was confirmed in common. It was.

(Reference Example 1: Standardization of glycoside hydrolyzing ability) 120 g volunteers were allowed to take 3 g of the probiotic produced in Example 1 per day. As a result of investigating the glycoside hydrolyzing ability of 17 people randomly selected before taking, no glycoside hydrolyzing ability was found in 8 people. As a result of measuring glycoside hydrolyzing ability again after taking 3 weeks for these 8 people, glycolytic hydrolyzing ability was recognized by all 8 people, and the effectiveness rate was 100%. The glycoside hydrolyzing ability is measured by the method of the present inventor.
Planta Medica, 62, 453-457, 1996 Microb. Ecol. Health Dis. 12, 85-91, 2000 by thin layer chromatography.

(Intestinal regulating action) Among 120 volunteers who took the probiotic produced in Example 1 for 3 weeks a day for 4 weeks, the effect on 52 subjects whose days of no bowel movement were 2 days or more per week was the same as the days of no bowel movement There were 11 (21%) who were affected (no effect), 36 (69%) who decreased (improved), and 5 (10%) who increased (retreated). The ratio of the number of improvement cases to the effect was 88%, and the number of defecations for constipation was significantly improved by the probiotic according to the present invention.

(Ginseng sensation improvement effect) Also, among 120 volunteers who took 3 g / day for 4 weeks of probiotics produced in Example 1, the effect on ginseng sensation is the same as that of ginseng sensation. There were 18 people (30.0%) who were not affected, 40 people (66.5%) who improved, and 2 people (3.5%) who got worse.

Among them, the effect on 34 subjects with allergic symptoms such as asthma, hay fever and atopic dermatitis was 13 (38.2%) without change (no effect), and 19 with improvement (55) 0.9%) and 2 (5.9%) were worse (antiallergic effect). In addition, the effect on 39 subjects complaining of stomach weakness was 18 (46.1%) without change (no effect), 20 (51.3%) with improvement, and 1 with deterioration (1) 2.6%) (gastric weakness improving effect).

Immediate allergic reactions such as asthma, hay fever, and atopic dermatitis are caused by degranulation by the reaction of IgE antibodies bound to immunoglobulin E (IgE) receptors on mast cells or basophil cell membranes and antigens. It is a series of reactions in which a chemical transmitter (intragranular enzyme β-hexosaminidase is one of them) is released. As an action mechanism of the antiallergic action by the probiotic produced in Example 1, the inhibitory action on IgE antibody production by the bacterial components of lactic acid bacteria
Shida, K. et al. , Int. Arch. Allergy Immunol. , 115, 278-287, 1998, and β-hexosaminidase release inhibition by hydrolyzate M1 Bae, E .; A. et al., Biol. Pharm. Bull., 25, 743-747, 2002.

As can be seen from these examples, ingestion of probiotics obtained by culturing Lactobacillus casei Hasegawa strain (FERM P-19484) normalizes glycoside hydrolytic ability of human intestinal flora. In addition to the above, it was confirmed that the intestinal regulating action, the ginseng bodily sensation improving action, the antiallergic action, the gastric weakness improving action, and the like. From this, it was revealed that the probiotics according to the present invention can improve the intestinal environment and greatly contribute to the promotion of health.

According to the present invention, probiotics that standardize glycolytic hydrolytic ability of human intestinal flora can be obtained by culturing lactic acid bacteria: Lactobacillus casei Hasegawa strain (FERM P-19484). The intestinal environment was improved by taking the probiotics of the present invention. Thus, the probiotic of the present invention is extremely useful as a health food.

Claims (3)

Lactic acid bacteria having the ability to hydrolyze glycosides: Lactobacillus casei strain Hasegawa (FERM P-19484). A probiotic using the lactic acid bacterium according to claim 1. A method for producing probiotics using the lactic acid bacteria according to claim 1.