JP2018118915A - Nonalcoholic liver injury inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a non-alcoholic liver injury inhibitor containing a cell or culture of bifidobacteria as an active ingredient. The present invention provides a non-alcoholic hepatic disorder inhibitor and a non-alcoholic hepatic disorder suppressant food or drink containing bifidobacteria longum cells or cultures as active ingredients among bifidobacteria. .. Further, the present invention relates to a non-alcoholic fatty liver inhibitor containing a bacterial cell of Bifidobacterium longum or a culture of the bacterial as an active ingredient, a food or drink for suppressing non-alcoholic fatty liver, a liver cancer inhibitor and a liver. Provide food and drink for cancer control. [Selection diagram] None

Description

  The present invention relates to a non-alcoholic liver injury inhibitor. More specifically, the present invention relates to a non-alcoholic liver injury inhibitor containing Bifidobacterium longum or its culture as an active ingredient.

In recent years, the number of nonalcoholic fatty liver disease (NAFLD) patients is increasing worldwide with the increase of obese people. In Japan, the number of Japanese people who are obese is increasing due to the westernization of food. NAFLD resulting from lifestyle is said to be a liver lesion of metabolic syndrome, and is considered to be the most frequently occurring liver disease and corresponds to 10 to 30% of adults (for example, Non-Patent Document 1). NAFLD also includes non-progressive simple fatty liver, cirrhosis, and nonalcoholic steatohepatitis (NASH) that can progress to liver carcinogenesis. NASH is based on fatty liver formation, and pathological conditions are formed by further induction of oxidative stress and inflammatory cytokines.
Although it is thought that the number of NASH patients will continue to increase in the future, no standard treatment has been established at present.

  The liver is an important organ responsible for the detoxification function of the human body, and the protective effect of the liver, that is, the improvement effect of the liver injury index is an important point in supporting health. To improve liver injury index and prevent fatty liver, maintain a good balance between energy intake and consumption by conducting daily health management such as thorough daily nutrition management and appropriate exercise. It is necessary to do. However, in an environment surrounding a busy modern society, it is not easy to break away from irregular life rhythms, biased nutrition intake, and lack of exercise, so it can be practiced easily and safely and can be expected to have sufficient effects. There is a need for a method.

Examples of methods for suppressing fatty liver and liver damage by ingesting lactic acid bacteria and their cultures include the following methods.
In Patent Document 1, when a diet containing ethanol and a treated product of Lactobacillus brevis was administered to mice, AST and ALT in the serum, total cholesterol concentration in the liver and It is described that the triglyceride concentration has decreased. Therefore, it is described that Lactobacillus brevis treated cells can be used as an inhibitor of alcoholic liver injury.
In Patent Document 2, when high fat diet and carbon tetrachloride are administered to mice to induce NASH, AST and ALT in serum are significantly reduced by adding Lactobacillus plantarum to the high fat diet. It is described that Lactobacillus plantarum can be used as a prophylactic / therapeutic agent for nonalcoholic steatohepatitis (NASH) because it has the effect of suppressing inflammation and fibrosis in the lobule of the liver.
In Patent Document 3, when a high-fat diet to which Lactobacillus helveticus was added was administered to rats, cholesterol and triglycerides in the liver decreased compared to the case of administering only the high-fat diet, so Lactobacillus helveticus was It is described that it can be used as an agent for preventing and suppressing fatty liver.
Non-patent document 2 describes that when a high-fat diet supplemented with Enterococcus ferricus was administered to mice, AST and ALT in the serum were lower than when only a high-fat diet was administered. It is described that it can be used as a function improving agent.

Japanese Patent No. 5203393 JP 2014-24776 A Japanese Patent No. 5804542

Reiko Hashimoto, Latest Medicine Vol. 63, No. 9, 1672, 2008 British Journal of Nutrition, 112, p868-875,2014

However, any of the above is an effect of suppressing liver damage of lactic acid bacteria belonging to the genus Lactobacillus or Enterococcus ferricus, and it has not yet been clarified whether Bifidobacterium has such effects. In addition, in Patent Document 1, Bifidobacterium bifidum, which is a bifidobacteria, has been studied, but only alcoholic liver damage has been studied, and the effect on nonalcoholic liver damage is unknown. It is.
This invention makes it a subject to provide the non-alcoholic liver disorder inhibitor which uses the microbial cell or culture of Bifidobacterium as an active ingredient.

The present invention provides a non-alcoholic hepatic disorder inhibitor and a food and drink for suppressing non-alcoholic hepatic disorder, comprising a Bifidobacterium longum cell or culture among Bifidobacteria as active ingredients.
The present invention also provides a non-alcoholic fatty liver inhibitor and a non-alcoholic fatty liver-suppressing food / beverage product comprising a Bifidobacterium longum cell or a bacterial culture as an active ingredient.
Moreover, this invention provides the hepatoma inhibitor and the food-drinks for liver cancer suppression which use the microbial cell of Bifidobacterium longum or this fungus culture as an active ingredient.
In addition, the present invention provides a method for suppressing nonalcoholic liver injury, a method for suppressing nonalcoholic fatty liver, or a method for suppressing liver cancer by administering an agent having the above action or a food or drink.

  The present invention provides a non-alcoholic liver injury inhibitor and a non-alcoholic liver injury-suppressing food / beverage product comprising a Bifidobacterium longum cell or culture as an active ingredient. Can be expected to suppress liver damage sufficiently.

It is a photograph which shows the condition of the liver in each individual. (A) It is not fatty liver. (B) Although it is an impression of fatty liver, fat accumulation is small. (C) Obvious fatty liver. (D) shows liver cancer. (E) Obvious fatty liver and liver cancer. It is a graph which shows the measurement result of AST and ALT which are the liver disorder | damage | failure parameter | index of AD group. Group A: Normal food control. Group B: High fat diet control. Group C: Examples. Group D: Comparative example.

The non-alcoholic liver injury inhibitor of the present invention contains Bifidobacterium longum cells and / or cultures thereof as active ingredients.
The Bifidobacterium longum is not particularly limited as long as it has a non-alcoholic liver injury-suppressing effect of the present invention, and examples thereof include SBT2928 (FERM P-10657) and reference strain JCM1217. Bacteria belonging to Bifidobacterium longum of the present invention can be cultured according to a conventional method. As the medium, various media such as a milk medium or a medium containing milk components and a semi-synthetic medium not containing the milk medium can be used. Examples of such a medium include reduced skim milk medium.
The cells isolated from the obtained culture by means of collection such as centrifugation can be used as they are as the active ingredient of the present invention. Concentrated, dried, freeze-dried cells, etc. can be used, or dead cells can be formed by heat drying.
Not only those that are purely isolated as cells, but also cultures, suspensions, other cell-containing materials, and cytoplasm and cell wall fractions obtained by treating cells with enzymes or physical means should be used. it can.
Examples of the culture include not only a culture using a medium generally used for culture of Bifidobacterium longum, such as GAM medium (manufactured by Nissui Pharmaceutical), which is a synthetic medium, and reduced skim milk medium. Examples of dairy products such as cheese, fermented milk, and dairy lactic acid bacteria beverages are not particularly limited.
Furthermore, a culture supernatant prepared by removing milk protein precipitates and bacterial cell components from the obtained culture using methods such as centrifugation and filtration can also be used as the culture of the present invention. Since it is a supernatant with a low solid content, the range of application to foods and drinks is widened. For example, the culture supernatant can be prepared by centrifuging the reduced skim milk culture at 5,000 rpm for 10 minutes.

The non-alcoholic hepatic disorder inhibiting action in the present invention refers to the action of suppressing non-alcoholic hepatic disorders (fatty liver, hepatitis, liver fibrosis, cirrhosis, liver cancer, etc.) excluding alcoholic liver disorders.
That is, it means the action of suppressing the symptoms of fatty liver injury similar to alcoholic liver injury despite not taking alcohol.
Here, “suppression” includes prevention, treatment, alleviation, alleviation and the like. Whether liver damage has been suppressed is, for example, an increase in serum AST (aspartate aminotransferase) or serum ALT (alanine aminotransferase) due to intake of a high fat diet, or accumulation of fat (cholesterol, triglycerides, etc.) in the liver , Can be determined by whether or not. AST and ALT are well-known indicators in health examinations, and these are known to increase due to various factors such as hepatitis virus, alcoholic liver injury, fatty liver, and are indicators of liver diseases.

  In formulating the non-alcoholic liver injury inhibitor of the present invention, excipients, stabilizers, corrigents and the like that are permitted in the formulation are mixed as appropriate, concentrated, freeze-dried, and heat-dried to kill dead cells. It may be. These dried products, concentrates, and pastes are also included. In addition, as long as it does not interfere with the non-alcoholic liver injury inhibitory action of bacteria belonging to Bifidobacterium longum, excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, It can also be formulated by mixing other arbitrary drugs. The dosage form can be tablets, capsules, granules, powders, powders, syrups, etc., and these are preferably administered orally.

  The non-alcoholic hepatic disorder inhibitor of the present invention may be added to any food or drink, and may be added to the raw material during the production process of the food or drink. Examples of food and drink include dairy products such as cheese, fermented milk, dairy lactic acid bacteria beverages, lactic acid bacteria beverages, butter and margarine, beverages such as dairy beverages, fruit juice beverages and soft drinks, eggs such as jelly, candy, pudding and mayonnaise Processed products, confectionery and breads such as butter cake, and various types of powdered milk, infant foods, nutritional compositions and the like can be mentioned, but are not particularly limited.

  The non-alcoholic liver injury inhibitor of the present invention can be blended in feed. Like the said food-drinks, you may mix | blend with what kind of feed, and may add to a raw material during the manufacturing process of a feed.

A non-alcoholic liver injury inhibitor, a non-alcoholic liver injury-suppressing food / drink, a nutritional composition, a feed, or a processed product of these materials, containing Bifidobacterium longum cells or culture When used in combination with Bifidobacterium longum, the proportion of lactic acid bacteria belonging to Bifidobacterium longum or the culture is not particularly limited, and may be appropriately adjusted according to the ease of production and the preferred daily dose. . It is determined individually in consideration of the symptom, age, etc. of the administration subject, but in the case of an adult, 10 to 200 g of a culture containing 0.1 to 5,000 mg of Bifidobacterium longum cells. Alternatively, the blending amount and the like may be adjusted so that 0.1 to 5,000 mg of the bacterial cells themselves can be ingested. A desired effect can be exhibited by ingesting in this way.
Hereinafter, the present invention will be described in more detail based on examples, but the present invention should not be construed as being limited to such examples.

A test was conducted to confirm the effect of Bifidobacterium longum on inhibiting liver damage.
1. Test Method (1) Preparation of Bifidobacteria Cell Powder Bifidobacterium longum SBT2928 and B. pseudolongum SBT2908 were selected as test bacteria. Skim milk medium (medium composition: 11.55% (w / w) skim milk powder, 0.5% (w / w) yeast extract, 0.1% (w / w) sodium ascorbate, 0.04% (w / V) L-cysteine) was inoculated with 3% (v / v) of the preculture and cultured at 37 ° C. for 16-18 hours. A jar fermenter was used for the culture. In a nitrogen atmosphere, the cells were cultured under the condition of maintaining pH 6 with 20% (w / w) ammonium carbonate. After completion of the culture, the culture was lyophilized to prepare a cell powder.

(2) Preparation of Bifidobacterium longum-containing feed D12492 high-fat diet (60% kcal fat, 20% kcal protein, 20% kcal carbohydrate) and 1.5% (w / w) bacteria purchased from Research Diet Body powder, 0.5% (w / w) lactose was mixed. Moreover, the food which mixed 1.5% (w / w) lyophilized skim milk culture medium and 0.5% (w / w) lactose in D12492 was prepared as a food for the control group of high fat diet intake. In addition, CE-2 (purchased from CLEA Japan) was prepared as a bait for the control group for normal food intake.

(3) Liver cancer model mouse and rearing conditions In the mouse animal test, the test system of Yoshimoto (Reference 1) et al. Was used. This is a technique for inducing NASH liver cancer by continuously ingesting a high fat diet in mice. Yoshimoto et al. Have reported that fat accumulates in the liver of mice and the liver injury index (AST, ALT) increases (Reference 1, Supplementary Fig. 8).
Use C57BL / 6J mice (male) for the test, and apply dimethylbenzanthracene (DMBA), a carcinogen, once to the back of the mice used for liver cancer models at the stage of 4-5 days after birth. Went.
The test was conducted in 4 groups as shown in the table below, and N = 10 in each group.
Group A is a normal diet control fed “normal diet”, Group B is a high fat diet control fed “high fat diet + skim milk medium”, and Group C is “high fat diet + Bifidobacterium longum” Feeded Example of the present application, Group D is a comparative example fed with “high fat diet + Bifidobacterium pseudolongum”. The diet provided 5 g daily, and each group was bred for 30 weeks with a private plastic gauge. Water and food were ad libitum.
<Reference 1>
Yoshimoto S, Loo TM, Atarashi K, Kanda H, Sato S, Oyadomari S, Iwakura Y, Oshima K, Morita H, Hattori M, Honda K, Ishikawa Y, Hara E, Ohtani N. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature. 2013 Jul 4; 499 (7456): 97-101.

(4) Preparation and Storage of Biological Samples Individuals with a body weight of 45 g or more were adopted as obese mice for the B, C and D group high-fat diet intake mice. Individuals with abnormalities during the breeding period and individuals whose body weight was less than 45 g on the day of dissection were excluded from the subjects. Therefore, the effective number of N was 10 in the A group, 8 in the B group, 9 in the C group, and 9 in the D group. It should be noted that the amount of food in the groups C and D was maintained at about 3 g during the breeding period, and the live bacteria corresponding to 1 × 10 ⁸ cells / day were ingested at the minimum.
Each mouse after breeding was anesthetized under an isoflurane atmosphere, and after blood collection, it was euthanized by cervical dislocation. The situation at each site was observed and sampled and stored as necessary.

(5) Measurement of liver injury index Liver injury index (AST, ALT) in serum was analyzed according to the attached manual using a dry chem apparatus (manufactured by Fuji Film).

2. Findings of mice in each group and measurement results of liver injury index The test results are shown in Table 2 and FIG.
Regarding the weight (average value) of the liver, 1.4 g in group A (normal diet control), 2.6 g in group B (high fat diet control), and group C (Bifidobacterium longum intake group: Examples) 1.9 g and 2.3 g in Group D (Bifidobacterium pseudolongum: Comparative Example), and Group C showed a lower tendency than Group B.
Moreover, as for the findings of the liver, the ratio to the effective N number of individuals exhibiting a clear fatty liver state (the state (C) or (E) in FIG. 1) was 0% in the A group and 88% in the B group. %, 44% in Group C and 67% in Group D, which was again lower than Group B. Moreover, the ratio with respect to the effective N number of the individual who exhibited the state of liver cancer (the state of (D) or (E) in FIG. 1) is 0% in the A group, 63% in the B group, and 56% in the C group. , 78% in the D group, again the C group was lower than the B group.
In addition, all the groups that ingested the high fat diet showed the characteristics observed in fatty liver, such as increased AST and ALT values, and AST / ALT ratio was less than 1 in many individuals.
When compared in the high fat diet intake group (B, C, D group), the AST and ALT values in the C group were significantly lower than those in the B group (control group) (FIG. 2, t -Test, p <0.05), D group did not show a significant decrease (FIG. 2).
From the above, it was shown that the Bifidobacterium longum of the present invention has a protective effect on the liver such as suppressing fatty liver and liver cancer in the NASH model.

1. Preparation of Bifidobacteria (1)
Bifidobacterium longum SBT2928 was inoculated into a GAM (1% Glc added) medium, and statically cultured at 37 ° C. for 16 hours. After centrifuging the liquid culture at 4 ° C. and 7000 rpm for 15 minutes, washing with sterile water and centrifugation were repeated three times to obtain washed cells. Then, Bifidobacterium longum SBT2928 powder was obtained by freeze-drying.
2. Preparation of bifidobacteria cells (2)
Bifidobacterium longum SBT2928 was defatted milk medium (medium composition: 11.55% (w / w) nonfat dry milk, 0.5% (w / w) yeast extract, 0.1% (w / w) ascorbic acid Sodium, 0.04% (w / v) L-cysteine) and static culture at 37 ° C. for 16 hours. The obtained culture was lyophilized as it was to obtain Bifidobacterium longum SBT2928 powder.
The bacterial cell powders of Bifidobacterium longum obtained in 1 and 2 above can be used equally in Examples 3, 5, 6, and 7 below.

(Manufacture of tablets)
4 parts of skimmed milk powder was mixed with 1 part of Bifidobacterium longum cell powder prepared in Example 2, and this mixed powder was tableted 1 g by a conventional method with a tableting machine. Tablets each containing 200 mg of bacterial longum were prepared.

(Manufacture of powder)
Bifidobacterium longum SBT2928 was ingested in 5 L of GAM liquid medium (manufactured by Nissui Pharmaceutical Co., Ltd.), followed by stationary culture at 37 ° C. for 18 hours. After completion of the culture, centrifugation was performed at 7000 rpm for 15 minutes to obtain 1/50 amount of concentrated bacterial cells of the culture solution. Next, the concentrated cells were mixed with the same amount of a dispersion medium containing 10% by weight of skim milk powder and 1% by weight of sodium glutamate, adjusted to pH 7, and then freeze-dried. The obtained freeze-dried product was sized with a 60-mesh sieve to produce freeze-dried bacterial powder. In accordance with the provisions of the 13th revised Japanese Pharmacopoeia General Rules for Preparations, “Powder”, add 1 g of this freeze-dried bacterium to 400 g of lactose (JP) and 600 g of potato starch (JP) and mix evenly. Obtained.

(Manufacture of capsules)
The raw materials were mixed according to the formulation shown in Table 3, granulated by granulation, and then filled into empty capsules by 10 mg to produce capsules.

(Manufacture of stick health food)
To 30 g of Bifidobacterium longum powder obtained in Example 2, 40 g of an equal mixture of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of an equal mixture of corn starch and lactose were added and mixed. The mixture was packed in stick bags to produce stick health food.

(Manufacture of beverages)
The raw materials were mixed according to the formulation shown in Table 4, filled into a container, and then heat sterilized to produce a fruit juice drink.

(Manufacturing fermented milk)
Bifidobacterium longum SBT2928 was cultured in a GAM (1% Glc added) liquid medium. The culture solution in the logarithmic growth phase was inoculated with 1% in 10% reduced skim milk (115 ° C., sterilized for 20 minutes) supplemented with 0.3% yeast extract to prepare a mother culture.
2.5% of this was added to the yogurt mix (10% reduced skim milk was added and heated at 100 ° C. for 10 minutes), and fermentation was carried out at 37 ° C. with a commercially available starter culture. When the lactic acid acidity reached 0.85, the mixture was cooled to terminate the fermentation, and fermented milk having a non-alcoholic liver injury inhibitory action of the present invention was prepared.

  ADVANTAGE OF THE INVENTION According to this invention, the nonalcoholic liver disorder inhibitor and the food-drinks for nonalcoholic liver disorder suppression which use the microbial cell or culture of Bifidobacterium longum as an active ingredient can be provided. Therefore, by ingesting the non-alcoholic hepatic disorder inhibitor and the non-alcoholic hepatic disorder-suppressing food and drink of the present application, a sufficient hepatic disorder suppressing effect can be expected by an easy and safe method.

FERM P-10657
[Reference to deposited biological materials]
Bifidobacterium longum SBT2928
The name and address of the depositary that deposited the biomaterials National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center 1-chome, East 1-chome, Tsukuba City, Ibaraki Prefecture, Japan 6 (zip code 305-8666)
Date of deposit of biological materials at the depository of Loi April 13, 1989 Deposit number FERM P-10657 attached by the depository of hi

Claims (2)

A non-alcoholic hepatic disorder inhibitor comprising Bifidobacterium longum or a bacterial culture as an active ingredient. A non-alcoholic liver injury-suppressing food or drink comprising an active ingredient of Bifidobacterium longum or a bacterial culture.