JP5422200B2 - Liver dysfunction improving agent - Google Patents

Description

  The present invention relates to an agent for improving liver dysfunction using a plant raw material.

  The liver performs various functions essential to the body, such as metabolic regulation and storage of the three major nutrients, carbohydrates (sugars), proteins and lipids, decomposition and detoxification of unnecessary substances, and production and secretion of bile that helps digest food. I'm in charge. The liver is also said to be a silent organ because of its strong reserve, and symptoms such as malaise, jaundice, edema, and ascites are difficult to appear, and finding the disorder often tends to be delayed.

  Usually, liver function tests are performed using plasma and serum ALT (alanine transaminase) and AST (aspartate aminotransferase) as a sensitive indicator of the degree of hepatic cell necrosis. Is done.

  In recent years, the burden on the liver due to westernization of dietary habits, bias in nutritional balance, excessive intake of alcohol, stress, smoking, etc. is increasing, and the number of patients with liver disorders is also increasing. Liver damage causes fibrosis of liver tissue due to repeated destruction and regeneration of hepatocytes over many years, causing serious diseases such as cirrhosis and hepatocellular carcinoma. The causes of liver damage include various hepatitis virus infections, side effects due to drugs, and the like. In the early stage of liver damage, hepatitis, that is, inflammation in the liver occurs. At the stage where inflammation occurs in the liver, abnormal decrease or enhancement of various inflammatory markers such as IL-10, AST, ALT, TNF-α occurs. In addition, it is said that radical reactions are also involved in hepatocyte damage.

Currently, there are no specific medicines for hepatic disorders, mainly treatments such as diet therapy and resting therapy, but glycyrrhizin preparations such as strong neominophagen C (registered trademark; manufactured by Minophagen) for chronic liver diseases. Is known to be used. However, since this drug is used by intravenous administration, it is painful and has been reported to cause side effects such as hypertension and hypokalemia. Therefore, its use is subject to various restrictions. Moreover, since it is expensive, it is economically difficult to use it regularly over a long period of time.
In addition, it is also known to use various amino acid preparations for the purpose of improving hepatic encephalopathy and hypoalbuminemia associated with liver diseases such as cirrhosis and liver failure. It is an improvement.

  In view of this, a method has been proposed for improving liver dysfunction with components derived from natural products that have few side effects and can be taken on a daily basis. For example, the method of using turmeric, Maria thistle, sesame lignan, oyster extract, liver extract, etc. can be mentioned (Non-patent Document 1). In addition to this, a method using lactoperoxidase and / or lactoferrin (Patent Document 1), a method using whey (Patent Document 2), a method using a sweet tea alcohol or a hydrous alcohol extract (Patent Document 3). ), A method of using dietary fiber such as cereal or beans-derived cellulose and hemicellulose (Patent Document 4), and the like.

  On the other hand, it is well known that wort is prepared by saccharifying malt (also referred to as malt) and mainly used as a raw material for producing alcoholic beverages such as beer and whiskey. In addition, fermentation of wort using lactic acid bacteria having physiological functions beneficial to the living body such as intestinal regulation and immunostimulatory action has long been performed. For example, a method for producing a fermented malt beverage (Patent Document 5) in which alcohol fermentation is performed after lactic acid fermentation with lactic acid bacteria having antiallergic activity can be exemplified.

However, the fermentation of wort using lactic acid bacteria that has been reported so far involves alcoholic fermentation, and there are few reports on fermented lactic acid bacteria of wort without alcoholic fermentation. Little consideration has been given.
JP 2001-226289 A International Publication No. 2005/094848 Pamphlet Japanese Patent No. 4104853 Japanese Patent Publication No. 7-121867 JP 2006-50979 A FOOD Style 21, Vol. 12, No. 12, 1998, Food Chemistry Newspaper

  An object of the present invention is to provide a liver dysfunction ameliorating agent that is effective in using plant raw materials and can be used on a daily basis, and is inexpensive and excellent in safety.

  As a result of intensive studies in order to solve the above problems, the present inventors have found that a wort fermented product obtained by inoculating and cultivating a specific lactic acid bacterium of physiological origin in wort has excellent anti- The present invention was completed by finding that it has an oxidative action and an anti-inflammatory action and can improve liver dysfunction.

  That is, the present invention relates to a liver dysfunction improving agent, an antioxidant and an active ingredient containing a wort fermentation product obtained by fermenting wort using Lactobacillus pentosaus YIT10313 strain (FERM AP-21755). An anti-inflammatory agent is provided.

  The fermented wort used in the present invention has an excellent antioxidant action, anti-inflammatory action, and an effect of improving and treating liver dysfunction, and can be used on a daily basis with excellent safety. As a result, it is possible to provide various foods and drinks aimed at improving health.

The liver dysfunction-improving agent of the present invention contains, as an active ingredient, a wort fermentation product obtained by fermenting wort using lactic acid bacteria belonging to Lactobacillus, specifically, Lactobacillus pentosus YIT10313 strain. Is.
The Lactobacillus pentosus YIT10313 strain used for the preparation of this wort fermented product is isolated from fermented tea according to a conventional method. Specifically, a 0.85% NaCl solution was added to the sample (fermented tea) and suspended to obtain a 10-fold serial dilution. The diluted solution was spread on MRS agar containing 0.5% calcium carbonate solution, sodium azide, and cycloheximide at 10 ppm each, and cultured at 30 ° C. for 2 days under anaerobic conditions. Released.

  This bacterium grows well in wort and has high survival even when stored at 10 ° C. for 1 month (4 weeks). In addition, it has been confirmed that it has resistance to acids and bile acids, and has cytokine production ability and lipid micelle insolubilization activity in immune cells, and as FERM AP-21755, an independent administrative agency as of December 25, 2008. Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (postal code 305-8656, 1st, 1st East, 1st Street, Tsukuba, Ibaraki, Japan).

The mycological properties of this YIT10313 strain are as follows.
A) Morphological properties (1) Bacterial shape; Neisseria gonorrhoeae (2) Bacterial size; 1 μm
(3) Bacterial polymorphism; None (4) Motility; None (5) Spore; None B) Culture properties (growth state)
(1) MRS agar plate culture; good (2) MRS liquid culture; good C) physiological properties (1) Gram staining; positive (2) catalase reaction; negative

  In addition, wort fermented using the Lactobacillus pentosaus YIT10313 strain (FERM AP-21755) is, as is well known, germinated by adding water to wheat such as barley and dried (both malt and malt) It is a general product obtained by decomposing (saccharifying) starchy material by the action of an enzyme contained in malt by adding warm water and treating it under certain conditions.

  In the present invention, any wort prepared according to a conventional method can be suitably used without particular limitation. However, wort contains components such as carbohydrates, amino acids, β-glucan, and polyphenols. Since these component content ratios may affect the fermentability of lactic acid bacteria, conditions for preparing wort (saccharification conditions) so that the component content ratio is beneficial to the fermentability of lactic acid bacteria. ) Is more preferably set as appropriate.

  Specifically, when preparing wort, it is preferable to carry out saccharification with an enzyme derived from malt at 60 ° C. to 65 ° C. for about 1 hour, and then at 75 ° C. to 80 ° C. for about 10 minutes. In this case, it is not preferable to perform the saccharification treatment at a temperature higher than 65 ° C. because there may be cases where sufficient assimilable sugars and amino acids necessary for lactic acid bacteria fermentation may not be secured. Since the time required for the saccharification treatment has been confirmed not to affect the component content ratio of the wort, it may be set in consideration of workability and the like.

  Moreover, in preparation of wort of this invention, caramel malt can also be used for a part of malt used as a raw material. Caramel malt is a sweet product obtained by causing the drying temperature during malt preparation to be 100 ° C. or higher (usually about 80 to 85 ° C.) and causing a caramelization reaction (a reaction in which the sugar is heated to turn brown). This means malt (also called colored malt) having a fragrant roasting scent. By using this caramel malt, a unique flavor and flavor can be imparted to the wort, and the palatability of the fermented wort can be enhanced. In the present invention, the amount of caramel malt used may be set as appropriate and is not particularly limited.

  Furthermore, in the present invention, in addition to the above-mentioned wort, a malt extract that is commercially available for fermented malt beverages can be used as it is or by appropriately mixing with the wort. Examples of the malt extract that can be used in the present invention include MALT EXTRACT PASTE (manufactured by DIAMALTERIA ITALIANA Srl).

  The preparation of the fermented wort of the present invention may be carried out by an ordinary production method, as in the case of fermented foods using lactic acid bacteria, and is not particularly limited. That is, it can be performed by inoculating the Lactobacillus pentosasus YIT10313 strain (FERM AP-21755) into the wort prepared as described above and culturing it according to a conventional method.

  At this time, the wort may be appropriately sterilized to be a fermentation substrate for lactic acid bacteria, and the sterilization conditions and the like are not particularly limited. The wort concentration is preferably 8 to 30% in terms of solid content from the viewpoint of fermentability and the like. Also, wort alone contains a sufficient amount of nutrients necessary for lactic acid bacteria fermentation, so it can be used as a fermentation substrate as it is, but for the purpose of further improving fermentability, A component used as a secondary agent can be blended. Examples of such components include yeast extracts, chlorella extracts, vitamins A, vitamins B, vitamins C and vitamin E, proteolysates containing various peptides, amino acids, potassium, calcium, Mention may be made of salts such as magnesium and manganese. In addition, the usage-amount of these components mix | blended separately is not restrict | limited, What is necessary is just to use it according to the objective.

Moreover, it is preferable to inoculate wort Lactobacillus pentosasus YIT10313 strain (FERM AP-21755) as a preculture liquid prepared by preculture. This pre-culture solution is cultured at 30 ° C. to 37 ° C. for about 16 to 24 hours using an edible medium on which lactic acid bacteria belonging to Lactobacillus pentosas can grow, for example, Lactobacilli MRS broth (manufactured by Difco). The one prepared by doing.
Then, about 0.01 wt% to 1.0 wt% of the preculture solution is inoculated to the wort and cultured. The culture conditions at this time are not particularly limited, and may be performed in accordance with general culture conditions for lactic acid bacteria. For example, as culture conditions, the culture temperature is 25 ° C. to 40 ° C., preferably 30 ° C. to 37 ° C., and the culture time is 16 hours to 50 hours, preferably about 18 hours to 36 hours. Good. In addition, as a culture method, a method suitable for culturing lactic acid bacteria belonging to Lactobacillus pentosas may be appropriately selected from stationary culture, stirring culture, shaking culture, aeration culture, and the like.

  In addition, in the preparation of the wort fermented product of the present invention, if it does not affect the fermentability of Lactobacillus pentosaus YIT10313 strain (FERM AP-21755), the functionality of the wort fermented product, etc. Other animal and plant-derived lactic acid bacteria useful for the living body, such as environmental improvement, can also be used in combination. Examples of such lactic acid bacteria include Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus fermentum, and the like.

  The wort fermented product has excellent antioxidant action, anti-inflammatory action and liver dysfunction improving action, as shown in the examples below, and therefore a pharmaceutical product for improving liver function deterioration due to various causes, Useful as food. Here, liver dysfunction includes viral hepatitis, fatty liver, alcoholic hepatitis, non-alcoholic fatty liver, drug-induced liver injury, a state in which liver function is impaired or decreased due to fatigue, stress, or the like. Whether liver function is reduced or impaired is determined by ALT, AST, direct bilirubin, indirect bilirubin, ALP, γ-GTP, cholinesterase (CHE), serum albumin level in plasma or serum as liver disease markers Although it can be determined by (ALB) or the like, it is preferable to determine by ALT or AST. Moreover, you may determine by IL-10, TNF- (alpha), INF- (gamma), ST6GalI, etc. which change with hepatitis.

  Antioxidants, anti-inflammatory agents, and liver dysfunction-improving agents (hereinafter referred to as liver dysfunction-improving agents, etc.) of the present invention are obtained by converting the wort fermented product obtained as described above into appropriate forms or liquids such as pharmaceuticals and foods. It is prepared by making it into an appropriate shape such as a paste or a solid. In addition, since the above-mentioned fermented wort is excellent in safety and can be used on a daily basis, it can be used as it is or in a state in which the processed product is contained alive with lactic acid bacteria. , One that has been subjected to heat sterilization treatment, one in which the cells have been removed from the fermented product by filtration, centrifugation, etc., and one that has been pulverized by an existing method such as freeze drying, spray drying, hot air drying or spray drying Can be used.

  For example, the liver dysfunction-improving agent of the present invention can be used in the form of a conventional pharmaceutical preparation by mixing it with a pharmacologically acceptable carrier as it is or after appropriately applying a treatment such as heat sterilization or lyophilization. Can be administered as Examples of such preparations include solid preparations such as tablets, granules, powders and capsules; liquid preparations such as solutions, suspensions and emulsions; lyophilized preparations and the like. These preparations can be prepared by conventional means on preparations. Non-toxic pharmaceutical carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water, physiological Examples include saline. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent can be appropriately added.

  Moreover, the liver dysfunction-improving agent and the like of the present invention can be used not only as the above-mentioned pharmaceutical preparation but also as a food or drink. In this case, the wort fermented product or processed product that has been heat sterilized or pulverized as it is or contained in various foods can be ingested as a food material useful for improving liver dysfunction or a food for health use. can do. For example, various foods, processed meat products such as ham and sausage; processed fish foods such as kamaboko and chikuwa; added to bread, confectionery, butter, milk powder, fermented dairy products, water, fruit juice, milk, refreshing You may add and use for drinks, such as a drink.

Furthermore, various food materials can be blended in the wort fermented product as it is or in its processed product, and can be ingested as a form of food or drink such as a beverage or a tablet according to the consumer's preference. These foods and drinks can be manufactured by a normal method.
Food materials that can be blended in this case include sucrose, glucose, fructose, palatinose, trehalose, lactose, xylose, maltose, glucose fructose liquid sugar, various saccharides such as honey, sorbitol, xylitol, erythritol, lactitol, palatitol, reduced Various sugar alcohols such as syrup, reduced maltose syrup, various high-potency sweeteners such as aspartame, sucralose, and acesulfame K, various natural sweeteners such as licorice, stevia, and glycyrrhizic acid glycosides, sucrose fatty acid esters, glycerin fatty acid esters And various emulsifiers such as lecithin, agar, gelatin, carrageenan, guar gum, xanthan gum, pectin, locust bean gum, gellan gum, carboxymethylcellulose, soybean polysaccharide, propylene glycol alginate, etc. (Stabilizing) agent, polydextrose, β-glucan, mannan, galactan, arabinogalactan, glucomannan, galactomannan, wheat fiber, corn fiber, apple fiber, pine fiber and other dietary fibers or their hydrolysates, orange Various juices such as fruit juice, lemon juice, grape juice, grapefruit juice, apple juice, pineapple juice, various acidulants such as citric acid, lactic acid, acetic acid, malic acid, tartaric acid, gluconic acid, vitamin A, vitamin B, vitamin C , Various vitamins such as vitamin E, various minerals such as calcium, magnesium, zinc, iron, manganese,
Various flavors such as berry, orange, citrus, apple, mint and grape can be mentioned.

  Further, in the present invention, there is no strict limitation on the amount of wort fermented product used (ingestion) necessary for obtaining a desired antioxidant effect, anti-inflammatory effect, and liver dysfunction improving effect. , 5g-30g per day, preferably 10g-20g.

  EXAMPLES Hereinafter, although an Example, a test example, and a reference example are given and this invention is demonstrated further in detail, this invention is not restrict | limited at all.

Example 1
<Preparation of fermented wort>
Impurities were removed from the wort prepared according to a conventional method using centrifugation (8,000 rpm, 10 minutes), and then autoclaved at 121 ° C. for 15 minutes. 1% of this wort (12 ° Bx) was inoculated with 1% of a pre-culture solution of Lactobacillus pentosaus YIT10313 (FERM AP-21755) prepared by pre-culturing using Lactobacilli MRS broth (manufactured by Difco), Culturing was carried out at 37 ° C. for 48 hours to obtain a wort fermented product (Example product 1). In addition, fermented wort (samples 1 to 3) was prepared in the same manner as described above using Lactobacillus plantarum YIT0102 strain, Lactobacillus plantarum YIT11201 strain and Lactobacillus plantarum YIT11203 strain.

(Test Example 1)
<Examination of antioxidant capacity>
Put 0, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100 μL of the fermented wort (Example 1 and Samples 1 to 3) prepared in Example 1 into a plate, and the total amount is 100 μL. 50% ethanol was added so that To this sample solution, 50 μL each of 0.1 M MES buffer (pH 6.0) and 0.1 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added and allowed to react at room temperature in the dark. It was. After 30 minutes from the start of the reaction, the absorbance at 515 nm was measured with a plate reader, and the amount of sample solution added when the reduction rate was 50% was determined. The result is shown in FIG.
In FIG. 1, the antioxidant activity value is represented by the amount of sample solution added corresponding to the activity of 34 μM butylhydroxyanisole (BHA).

  From FIG. 1, the wort fermented product has higher DPPH radical scavenging activity than wort, and the wort fermented product using Lactobacillus pentosus YIT10313 strain (FERM AP-21755) has particularly high activity. confirmed.

(Test Example 2)
<Examination of anti-inflammatory ability>
2 mL of 4% (w / v) thioglycolate solution was administered intraperitoneally to BALB / c (ｃ, 7-8 weeks old) mice, and cells induced into the peritoneal cavity after 4 days It collected using 5 mL of Hanks's solution, and was set as the intraperitoneal macrophage. Intraperitoneal macrophages were washed with Hank's solution and suspended in RPMI 1640 medium (10% FBS (Fatal Bovine Serum), 1% penicillin / streptomycin, 0.05 mM 2-Mercaptoethanol). A 96-well culture plate was seeded with 5 × 10 ⁵ cells / mL of peritoneal macrophages, and the wort fermented product prepared in Example 1 (Example 1 and Samples 1 to 3) was added so as to be 10%. In culture. After 24 hours, the culture supernatant was collected and centrifuged (1800 rpm × 5 minutes), and the concentrations of IL-10 and TNFα were quantified by ELISA. The results are shown in Table 1.

<ELISA method>
The ELISA method was measured using an eBioScience kit (Mouse TNFα ELISA Ready-SET-Go!) According to the instructions.

  From Table 1, the fermented wort using Lactobacillus pentosaus YIT10313 strain (FERM AP-21755) has lower TNFα production ability and higher IL-10 production ability than other wort fermentation products. It was confirmed and shown to have an anti-inflammatory effect.

(Example 2)
<Examination of liver dysfunction improvement effect 1>
After acclimating 5 weeks old C57BL / 6N mice (♂) for 2 weeks, they were bred for 8 weeks using the feed composition shown in Table 2, blank group (n = 10), control group (n = 10), wheat The juice was divided into fermented product administration groups (n = 10). The animals were fasted for 24 hours, 8 hours after the start of fasting, the samples were administered as shown in Table 3, and after 16 hours, dissected under ether anesthesia, whole blood was collected from the abdominal aorta, and centrifuged (3,000 rpm, 20 min). Serum was prepared. For measurement of serum AST and ALT activity, transaminase CII test Wako (Wako Pure Chemical Industries) was used. The results are shown in Table 4.

  From Table 4, serum AST and ALT activities were increased in the control group compared to the blank, and it was confirmed that liver injury was caused by carbon tetrachloride. And by administering wort fermented product, AST shows a significant tendency (P = 0.08), ALT shows a significantly lower value (P = 0.04) compared with the control group, and hepatic dysfunction It was confirmed to improve.

(Example 3)
<Examination of liver dysfunction improvement effect 2>
Six-week-old Wistar rats (♂) were acclimated for two weeks and divided into a control group (n = 8) and a wort fermented product administration group (n = 7). Each group was fed a feed having the composition shown in Table 5 and reared for 10 days. Thereafter, GalN (D-galactosamine) was injected into the abdominal cavity of a rat at a rate of 500 mg / kg body weight and LPS at a rate of 20 μg / kg body weight to induce liver damage. Twenty-four hours later, it was dissected under ether anesthesia, whole blood was collected from the abdominal aorta, and centrifuged (3,000 rpm, 20 min) to prepare serum. For measurement of serum AST and ALT activity, transaminase CII test Wako (Wako Pure Chemical Industries) was used. The results are shown in Table 6.

  From Table 6, the liver damage caused by galactosamine and LPS was improved by administration of the wort fermented product.

It is a figure which shows DPPH radical scavenging activity by wort fermented material.

Claims (3)

  The liver dysfunction improving agent which contains as an active ingredient the wort fermented material obtained by fermenting wort using Lactobacillus pentosus YIT10313 strain (FERM AP-21755).   An antioxidant containing, as an active ingredient, a wort fermentation product obtained by fermenting wort using Lactobacillus pentosus YIT10313 strain (FERM AP-21755).   An anti-inflammatory agent containing, as an active ingredient, a wort fermentation product obtained by fermenting wort using Lactobacillus pentosus YIT10313 strain (FERM AP-21755).

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