KR100773059B1 - Novel microorganism having deglycosylating ability, the probiotics containing the same and the process for preparing them - Google Patents

Abstract

The present invention relates to a novel microorganism, probiotics (probiotics) using the same and a method for preparing the same, and in particular, a probiotics characterized by using a novel Lactobacillus casei strain Hasegawa having glycoside hydrolysability. To provide. The probiotics of the present invention can significantly contribute to health promotion by improving the intestinal environment through glycoside hydrolyzing ability improving effect, intestinal action, ginseng's efficacy improving effect, anti-allergic action, gastric function improvement.

Glycosides, Hydrolysis, Lactobacillus, Probiotics, Antiallergic

Description

Novel microorganism having deglycosylating ability, the probiotics containing the same and the process for preparing them             

1 is a result of TLC analysis of the hydrolyzate of a probiotic product,

Figure 2 shows the results of TLC analysis of glycoside hydrolysis capacity of volunteers.

The present invention relates to a novel microorganism and a probiotic using the same, and more particularly, to a probiotic using a novel microorganism having a glycoside hydrolytic ability, Lactobacillus casei strain Hasegawa, and a preparation method thereof.

Lactobacillus is a combination of lacto (lacto) means milk and bacillus (bacillus) means rod-like bacteria. Among them, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus and the like. Casei means cheese, and acidophilus is a combination of acido, which means sour, and philus, which means it likes acid. It means strong, and bulgaricus means derived from the Bulgarian.

Lactobacillus has a protective effect on intestinal action and constipation (Reuter G, Zentralbl. Bakteriol. Mikrobiol. Hyg ., 187 (4-6) , pp564-577, 1989), immune enhancing effect (Hosoi T. et al, Can. J.). Microbiol ., 46 (10) , pp892-897, 2000), inhibiting lipid peroxidation and increasing antioxidant enzymes (Kullisaar T. et al, Int. J. Food Microbilol ., 72 (3) , pp215-224, 2002) It is known to exert various physiological effects. Recently, the concept of probiotics, which represent living microorganisms and foods containing them, which are beneficial for maintaining the health of the host by reflecting the well-being, has been introduced, and interest in Lactobacillus is increasing. It's going on. The word probiotics was originally defined as a microbial additive that benefits the host animal by improving the balance of the intestinal flora, but is often used in a broad sense as pointed out earlier (Fuller R., J. Appl. Bacteriol ., 66 , pp 365-378, 1989).

Probiotics must utilize microorganisms that reside in the human intestine and must reach the digestive tract in a live state in order for the probiotics to work effectively in the digestive tract. The survival of microorganisms is inhibited by gastric acid or bile acid. Therefore, in order for probiotics to be active, they must be resistant to the above-mentioned growth inhibitory environment and substances. Currently, microorganisms widely used worldwide as probiotics include Lactobacillus genus Lactobacillus, bifidus, etc. present in the human body.

On the other hand, in the herbal medicine constituting the herbal prescription, various glycosides are included as an active ingredient. For example, glycyrrhizin of Glycyrrhizae Radix, paeoniflorin of Peeoniae Radix, saikosaponin of Bupleuri Racis, ginsenoside of Ginseng Radix ginsenoside, baicalin of Scutellariae Radix, sennoside of Rhei Rhizoma, and geniposide of Gardeniae Fructus (Kobashi, K., J.). Trad. Med. , 15 , pp 1-1, 1998). However, humans do not have digestive enzymes that break down glycosides with β bonds, so they are hydrolyzed only by intestinal bacteria and are absorbed from those with high lipophilic properties. For this reason, the bioavailability in vivo is significantly low (Hasegawa, H. et al., Planta Medica, 62 , pp453-457, 1996).

For example, ginsenosides are classified into two types, diol and triol, according to the difference in the structure of aglycone. The carbon of aglycone C3, C6, and C20 There is a sugar chain to which glucose, arabinose and rhamnose bind, respectively, to the hydroxyl group which binds to the position. After oral ingestion, both diol and triol ginsenosides are hardly degraded by biodigestive enzymes, and are characterized by Bacteroides, Fusobacterium, Eucacterium, and Bifidobacterium.

ium) is hydrolyzed and absorbed by the action of enterobacteria (Tawab, MA et al., Drug Metab. Dispos., 8 , pp1063-1071, 2003).

On the other hand, the composition of the intestinal bacteria is susceptible to the effects of constitution, eating habits, taking antibiotics and stress, so the ability to hydrolyze glycosides shows a remarkable individual difference. Therefore, significant differences occur in glycoside absorption, which is known as one cause of individual differences in drug efficacy (Hasegawa H. et al., Planta Medica , 64 , pp 436-440, 1997). This is supported by the cancer metastasis experiment using mouse, and it can be seen that the glycoside hydrolytic ability has a great influence on the efficacy of ginseng's anti-transduction effect in each mouse according to the glycoside hydrolysis ability of the intestinal bacteria. (Hasegawa H. et al., Planta Medica, 63 , pp696-700, 1998). Therefore, microorganisms having a glycoside hydrolysis ability may be usefully used for enhancing drug efficacy.

Glycoside hydrolysis microorganisms for ginsenosides include soil bacteria (Yoshioka I. et al., Chem. Pharm. Bull. , 20 , p2418, 1972), Aspergillus niga (Kanda et al., Pharmacy Magazine, 95 , p246, 1975), Rat enterobacteria (Takino et al., Medicinal ginseng '89', p267, 1989), Enterobacteriaceae collected from human feces (Kanaoka et al., Ilhan Pharmaceutical Co., 11 , p241, 1994) And the like have been reported. In particular, enterobacterial bacteria collected from human feces include Prevotella oris (U.S. Patent No. 5925537), Bacteroides JY-6, Bacteroid HJ-15, Fuzobacterium K-60, Eukacterium A-44 (Bae EA. Et al., Biol. Pharm. Bull ., 25 , p743, 2002) is reported. However, since all of these microorganisms have not been established for the safety of the human body and produce by-products with a characteristic smell due to the action of the microorganisms, it is not suitable to supply them without food treatment. In addition, there is a limit to commercial commercialization due to the low hydrolysis capacity.

In addition, neither the Lactobacillus casei Hasegawa bacterium of the present invention nor the hydrolytic ability of the Lactobacillus strain against ginsenosides has been taught or described anywhere.

In order to solve the above problem, the present inventors find microorganisms having the same hydrolytic ability as intestinal microorganisms among various microorganisms (yeast, yeast mold, Lactobacillus, bifidus, etc.) which are used in food processing while having high safety in the human body. To separate Lactobacillus casei strain Hasegawa, a lactic acid bacterium that resides in the human intestine and is resistant to stomach acid and lively enters the human intestine from traditional fermented foods of the Korean people through research and development. Succeeded. Probiotics using this, as well as not producing an unpleasant by-product, of course, has completed the present invention by confirming to control the immune function by improving the intestinal environment.

It is an object of the present invention to provide as a probiotic a microorganism having the ability to hydrolyze glycosides in order to standardize the glycoside hydrolysis ability of the intestinal microorganisms of the human body in which individual differences are remarkable.

In order to carry out the above object, the present invention is a Lactobacillus casei strain Hasegawa strain having a glycoside hydrolysing ability (deposited institution: National Institute of Bioscience and Human-technology (NIBH), date of deposit: August 2003 11, Deposit No.:FERM BP-10123.

Lactobacillus casei strain Hasegawa according to the present invention (Lactobacillus casei strain Hasegawa) is a novel strain of the genus Lactobacillus casei isolated and identified from traditional fermented foods.

Examples of the traditional fermented foods include, but are not limited to, kimchi, vegetable fermented products, soybean paste, soy sauce, cheonggukjang, salted fish.

Isolation method, bacteriological properties and uses of Lactobacillus casei Hasegawa strains are as follows and will be described in detail below.

The Lactobacillus casei Hasegawa strain of the present invention was isolated from traditional fermented foods and identified by 16S rDNA analysis, and the base sequence (GenBank / EMB) of 16S ribosomal DNA of Lactobacillus paracasei subsp.paracasei was identified. / DDBJ reg. No. D79212), showing the best homology (99.934%) to taxonomically belong to Lactobacillus paracasei subsp. Paracasei. However, the base strain JCM8130T is a hydrolyzate 20S-protopanaxadiol 20-0-β-D-glucopyranoside (20S-protopanaxadiol 20-O-β-D-glucopyranoside, FGM1) using ginsenosides as a substrate and There is no ability to produce 20S-protopanaxatriol 20-0-β-D-glucopyranoside (FGM11). Therefore, the microorganism of the present invention was regarded as a mutant strain, named as Lactobacillus casei strain Hasegawa, and deposited on August 11, 2003 at the National Institute of Bioscience and Human-technology (NIBH).

In the present invention, a glycoside is a series of compounds defined as acetyl derivatives of sugars having a cyclic structure, that is, sugars and organic compounds having hydroxyl groups such as alcohols and phenols, which are bonded to each other with oxygen atoms therebetween. Means that.

The glycoside hydrolytic ability of the Lactobacillus casei Hasegawa bacterium of the present invention is described in more detail, using a hydrolyzate, ie, FGM1 and FGM11, based on ginsenoside, which is an active ingredient of the plant of Panax genus. It characterized in that to generate.

The ginseng plants include Korean ginseng (Panax ginseng CA Meyer), Sanchi ginseng (Panax notoginseng (Burk.) FH Chen), American ginseng (Panax quinquefolium L.), Chok ginseng (Chikusetsu ginseng: Panax Japonicus CA Meyer, Himalayan ginseng: Panax pseudo-ginseng Wall.subsp.himalaicus Hara, and Vietnamese ginseng (Vietnamese ginseng: Panax Vietnamensis Ha et Grushv.).

In addition to the ginsenoside of Ginseng Radix, the microorganism of the present invention is glycyrrhizin of licorice, paeoniflorin of Paeoniae Radix, saikosaponin of Shiho, and gold ( Baicalin of Scutellariae Radix, sennoside of Rhei Rhizoma, geniposide of Gardeniae Fructus, isoflavone of soybean, polysulfonic acid of Polygalae Radix Glycosides such as (onjisaponin) and cell fibers such as bark, leaves and mushrooms are also hydrolyzed.

The present invention also provides a probiotic and a method for producing the same, characterized by using the Lactobacillus casei Hasegawa strain.

Probiotics of the present invention is a herbal medicine such as ginseng, licorice, peony, raw grass, glycoside, brown rice, rice bran, rice flour, cecum, corn, malt and other grains, bark such as Tabebuia avellanedae, tea The microorganisms of the present invention are cultured using a single leaf or a mushroom such as ganoderma lucidum and agar, as a medium alone or in combination of two or more kinds, and carried out according to a commercial method. For example, first, the above medium is sterilized, and then inoculated with 0.1% to 20.0% by weight, preferably 0.5% to 10.0% by weight of the Lactobacillus casei Hasegawa of the present invention, followed by incubation and processing. Probiotics can be obtained. At this time, the culture conditions are 1% to 60% in terms of solid content, preferably 1% to 40% medium, 20˚C ~ 40˚C, preferably 18 hours at a temperature of 25˚C ~ 37˚C ~ Incubate for 40 days, preferably 24 hours to 30 days.

In addition, the probiotics of the present invention can be prepared as described above to be a product, but in accordance with conventional methods, in order to improve the taste or to make the required shape, various ingredients and flavors can be added and blended into the final product. .

As mentioned above, the component which can be added and mix | blended with the probiotics of this invention is various sugars, an emulsifier, a sweetener, an acidulant, fruit juice, a fragrance | flavor, etc. are mentioned. Specifically, glucose, sucrose, fructose, fructose, sugar such as beeswax, sucrose fatty acid ester, glycerin fatty acid ester, emulsifier such as lecithin, sorbitol, xylitol, lecithin ( lecithin), sweeteners such as lactitol, citric acid, acetic acid, tartaric acid, lactic acid, fumaric acid, malic acid, succinic acid, acidulants such as gluconodelta-lactone, yogurt, berry, orange, quince tree, tea plant, Spices such as citrus, tortilla, mint, grape, fair, custard cream, peach, melon, banana, tropical fruit juice, tea, coffee, herbal extract, plant extract, grains, vegetable ingredients, milk ingredients , Vitamin A, vitamin B, vitamin C, vitamin E, and the like, etc. There may be used one or two or more of the components selected from the group. Although the addition amount of the said addition and a compounding component is not limited, About 0.1%-1.0%, Especially about 0.1%-0.5% are preferable.

In addition, the probiotics of this invention can be a product of any form, such as a solid and a liquid.

The present invention will be described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Example 1 Acquisition of Lactobacillus kasei Hasegawa Strain and Preparation of Starter

NIBH accept pre-sale an accession number of Lactobacillus casei Hasegawa strain FERM BP-10123 deposited on August 11, 2003 (National Institute of Bioscience and Human- technology) into a sterile and degassed saline ^{10 6 ~} 10 CFU / After adjusting to ml, 9 ml of GAM broth medium was added to 1 ml of this solution, and cultured for 1 day under anaerobic culture conditions while maintaining 30 to 35 ° C, and used as a starter.

Example 2. Preparation of Probiotic Granules

100 g of embryonic powder, 1000 g of Korean ginseng dry powder (Korean), and 10 L of water were mixed and sterilized at 95 ° C. Then, 3% of the starter of Example 1 was added thereto, followed by incubation at 30˚C for 2 days. Lactobacillus culture dir 10 L was obtained. The culture was lyophilized to obtain about 1000 g, and 95 g of oligosaccharide, 38 g of malt sugar, 14 g of vitamin C, 5 g of yogurt flavor, and 300 g of lactose were added. Water was added 30-40% of the total weight, and then granulated and dried to give 1400 g of granules. Obtained.

Example 3 Preparation of Probiotic Beverage

After adding 80L of water to 20 liters of malt (German) and saccharifying at 63˚C, the residue was filtered and sterilized, and then 0.1 kg of hoff was added and 6% of the starter of Example 1 was added to 30˚C. Sealed culture for 3 days. The culture solution was filled with a carbon dioxide gas in a filtrate filtered through a 100 mesh sieve or a supernatant made by centrifugation at 500 rpm or more to obtain 100 L of malt lactic fermented beverage.

Example 4 Preparation of Probiotics Yogurt

After mixing 2 kg of Korean ginseng extract (from Korea) and 98 kg of raw milk and sterilizing at 95 ° C., 1% of the starter of Example 1 was added thereto, followed by incubation at 30 ° C. for 3 days to obtain about 95 kg of ginseng yoghurt.

Example 5 Preparation of Solid Probiotics

Mix 1kg fresh Korean ginseng root (Korean) or powder 1kg, 0.1kg rice bran, 4kg water, sterilize at 95˚C, add 1% of starter of Example 1, incubate at 30˚C for 30 days, and dry To yield about 0.7 kg of dry matter.

Example 6 Preparation of Gel Probiotics

5 kg of Agaricus dry powder (from Yamanashi Prefecture), Korean ginseng dried powder (from Korea) kg, 0.1 kg of rice bran, and 20 kg of water are mixed and sterilized at 95 ° C., followed by adding 1% of the starter of Example 1 to 30 ° Incubated with C for 7 days to obtain a Lactobacillus culture. To 30 kg of the culture, 69 kg of a pre-dissolved gel solution and 0.1 kg of yoghurt flavor were added, followed by cooling to obtain gel probiotics.

Example 7 Preparation of Aglycone-Containing Probiotics

98 kg of soy milk prepared by the commercial method (12% solids), 1 kg of Agaricus dry powder (from Yamanashi Prefecture), 1 kg of Korean ginseng dry powder (from Korea), and sterilized at 95 ° C., followed by Example 1 6% of the starter was added and incubated at 30 ° C. for 3 days to obtain 90 kg of gel fermented product.

Example 8 Preparation of Probiotic Tablets

4 kg of Chinese dry paper powder, 4 kg of Korean ginseng dry powder (from Korea), 2 kg of rice bran, 20 kg of water are mixed, and after sterilizing at 95 ° C, 3% of the starter of Example 1 is added and 30 ° C. The culture was carried out for 7 days to obtain a Lactobacillus culture. The culture was lyophilized to add 80 kg of lactose, 16 kg of lactose, 2 kg of eggshell calcium and 2 kg of silicon dioxide to granulate to obtain 99 kg of tablets. As a result of analysis of the purification, the presence of trimethoxy cinnamic acid (3,4,5-trimethoxycinnamic acid) produced by hydrolysis of onjisaponin was confirmed. Trimethoxy cinnamic acid has been reported to increase and decrease the expression of choline acetyltransferase in the cerebral cortex, which is thought to be one of the causes of Alzheimer's disease to mRNA levels (Yabe Musa, Japanese Oriental Medicine, 50). , pp776-782, 2000).

Experimental Example 1. Evaluation of Probiotic Products

Taste of the probiotics products prepared in Examples 2, 3, 4, 5, 6, and 7 of 30 healthy men and women (13 males and 17 females) randomly selected from 20 to 50 years old. See Table 1) and preferences (see Table 2).

Strong crush Crush Dislike Strong dislike Remarks Probiotic granules 4 24 2 Fermented sour taste Probiotic Drinks 8 17 4 One Sour beer flavor Probiotics Yogurt 3 25 2 Fermented sour taste Solid State Probiotics 3 22 5 Chewing Fermentation Sour Gel Probiotics 5 22 3 Fermented sour taste Alicon-containing probiotics 2 23 5 Sour taste

Probiotic granules Probiotic Drinks Probiotics Yogurt Solid State Probiotics Gel Probiotic Alicon-containing probiotics system south 6 5 One One 13 female One 4 8 3 One 17 system One 10 13 One 3 2 30

Experimental Example 2 Determination of Glycoside Hydrolysates in Probiotic Products

The thin layer chromatography described in Hasegawa, H. et al., Planta Medica , 62 , pp453-457, 1996, contains the components of the probiotics prepared in Examples 2, 3, 4, 5, 6, 7, and 8. As a result of analyzing the modified method, the presence of the hydrolyzate FGM1 and FGM11 of ginsenoside was commonly confirmed (see FIG. 1).

Clinical Example 1. Standardization of Glycoside Hydrolysis Capacity

120 volunteers were given 3 g of probiotics prepared in Example 2 per day. Seventeen randomly selected glycoside hydrolytic abilities were assessed prior to dosing. As a result of measuring glycoside hydrolysis ability of these 8 people after 3 weeks of administration, all 8 patients were remarkably improved to recognize the glycoside hydrolysis capacity (see FIG. 2). Therefore, it was confirmed that the effective rate of the probiotics of the present invention is 100%. The hydrolysis capacity of the glycoside was carried out according to the same thin layer chromatography as in Experimental Example 1.

Clinical Example 2. Intestinal Action

In 120 volunteers who took the probiotics of Example 2 for 3 days a day for 4 weeks, the effect of 52 days without bowel movement on two or more days a week was observed. The number of patients (21%), 36 (69%) decreased (improved) and 5 (10%) increased (retracted) (see Table 3).

Improving No change worse sum Number of people 36 people 11 people 5 people 52 people ratio 69% 21% 10% 100%

Clinical Example 3. Improvement Effect of Ginseng

Of 120 volunteers who took the probiotics of Example 2 for 3 weeks at 3 g per day for 60 subjects who took Korean ginseng, the efficacy of ginseng did not change in 18 patients (30.0%), and 40 improved patients. (66.5%) and 2 patients (3.5%) were worsened (see Table 4).

Improving No change worse sum Number of people 40 persons 18 people 2 people 60 people ratio 66.5% 30.0% 3.5% 100%

Clinical Example 4. Antiallergic Action

Among 120 volunteers who took the probiotics of Example 2 for 3 weeks at 3 g per day, the effects on 34 subjects with allergic symptoms such as asthma, hay fever, and atopic dermatitis were 13 (38.3%) In addition, 19 (55.9%) improved and 2 (5.9%) worsened (see Table 5).

Improving No change worse sum Number of people 19 people 13 people 2 people 34 people ratio 55.9% 38.3% 5.9% 100%

Immediate allergic reactions such as asthma, hay fever, and atopic dermatitis are degranulation enzyme β-hexamimin by degranulation reactions by IgE antibody-antigen reactions that bind to immunoglobulin E (IgE) receptors on mast cells or basophils It is a series of reactions in which chemical carriers such as β-hexosaminidase are released. Therefore, it was confirmed that the probiotics of the present invention have an effect of inhibiting the production of IgE antibodies by the cell components of lactic acid bacteria as anti-allergic agents (Shida, K. et al., Int. Arch.Allergy Immuno1. , 115 , pp278-287). , 1998).

Clinical Example 4. Gastric Function Improvement

Among 120 volunteers who took the probiotics of Example 2 for 3 weeks at 3 g per day, the effects on 39 subjects complaining of weak stomach were 18 (46.1%) unchanged (impact) and 20 improved ( 51.3%) and one deteriorated person (2.6%) (see Table 6).

Improving No change worse sum Number of people 20 people 18 people 1 person 39 persons ratio 51.3% 46.1% 2.6% 100%

According to the results of the clinical examples 1, 2, 3, 4, the glycoside hydrolyzing effect of the probiotics obtained by culturing the Lactobacillus casei Hasegawa strain of the present invention, intestinal action, potency improvement effect, anti-allergic action, Gastric function improvement was confirmed. As a result, it can be seen that the probiotics of the present invention can greatly contribute to health promotion by improving the intestinal environment.

In order to standardize the glycoside hydrolysis capacity of intestinal microorganisms in which individual differences are remarkable, microorganisms having glycosides as hydrolysable enzymes are provided as probiotics to improve the intestinal environment, improving the intestinal action, improving the efficacy of ginseng, anti-allergic and gastric function It can contribute greatly to health promotion through action.

Claims (4)

Novel Lactobacillus casei strain Hasegawa (Accessory: National Institute of Bioscience and Human-technology (NIBH), Accession No .: FERM BP-10123), characterized by glycoside hydrolysis. Probiotics (probiotics) characterized by using the novel Lactobacillus casei Hasegawa strain of claim 1. The probiotics of claim 2 having any one form selected from the group consisting of granules, beverages, yogurts, solids, gels and tablets. A method for producing probiotics, comprising the novel Lactobacillus casei Hasegawa strain of claim 1.

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