JP4766485B2 - New lactic acid bacteria - Google Patents

Description

  The present invention relates to a composition for preventing liver dysfunction comprising lactic acid bacteria as an active ingredient. The present invention also relates to a novel lactic acid bacterium that is effectively used for preparing a composition for preventing liver dysfunction. Furthermore, the present invention relates to a method for suppressing water separation of fermented foods using lactic acid bacteria.

  The liver plays an important role in the body such as detoxification, metabolism and storage of carbohydrates and proteins, and hormonal regulation, and its dysfunction often has fatal consequences for the living body. The causes of hepatic dysfunction represented by hepatitis are based on lifestyle habits such as excessive consumption of alcoholic beverages, overdrinking, overeating, irregular life, stress, etc. ing. In particular, viral hepatitis often shifts to liver cancer through cirrhosis when it becomes chronic, and establishment of a prevention and improvement method is eagerly desired.

  On the other hand, reflecting the recent health orientation, foods are required not only for nutritional reinforcement but also for more active health maintenance functions. In particular, foods containing lactic acid bacteria such as fermented milk are attracting attention as health foods and foods for health use. In fact, foods containing lactic acid bacteria have various effective physiological actions such as intestinal regulation, immunostimulatory action and anticancer action. (For example, refer to Patent Documents 1 to 4 for lactic acid bacteria having antimutagenicity).

However, the effect of lactic acid bacteria on preventing liver dysfunction is not known.
JP-A-5-236945 Japanese Patent Laid-Open No. 5-236872 JP-A-8-56650 JP-A-11-113564

  An object of the present invention is to provide a composition for preventing hepatic dysfunction that is highly safe and can be taken for a long time. In particular, an object of the present invention is to provide the above-mentioned composition having an antimutagenic action and a liver dysfunction preventing action and used for the purpose of preventing canceration and preventing liver dysfunction. Furthermore, an object of the present invention is to provide a novel lactic acid bacterium as a lactic acid bacterium having a liver dysfunction preventing action in addition to an antimutagenic action, and a food prepared using the same. Furthermore, the present invention provides a fermented dairy product with excellent quality in which water separation and whey separation that can occur in the production process and distribution storage process are significantly suppressed by utilizing the viscous polysaccharide-producing ability inherent in the novel lactic acid bacteria. The object is to provide a method of manufacturing.

  The inventors of the present invention have been diligently researched to achieve the above-mentioned object. As a result, fermented milk prepared from lactic acid bacteria, particularly lactic acid bacteria belonging to the genus Lactobacillus and lactic acid bacteria belonging to the genus Streptococcus, significantly prevents liver dysfunction. It was found that there is an action to do. Furthermore, the present inventors have found that a novel strain belonging to the genus Lactobacillus and Streptococcus isolated as a lactic acid bacterium exhibiting an effect of preventing liver dysfunction further has antimutagenicity. Based on excellent physiological effects such as dysfunction prevention action and antimutagenic action (anticancer action), we were convinced that health foods extremely effective for health promotion and health maintenance can be provided. In addition, the present inventors have found that a strain belonging to the genus Lactobacillus among the above-mentioned new strains produces viscous polysaccharides, and by utilizing such strains, water separation and whey that are problematic in the production process and the distribution and storage process are obtained. It was confirmed that a fermented milk product in which separation was significantly suppressed could be prepared.

The present invention has been completed based on such findings and includes the following aspects.
Item 1. A composition for preventing liver dysfunction, comprising lactic acid bacteria or a culture thereof as an active ingredient.
Item 2. Item 2. The composition for preventing liver dysfunction according to Item 1, wherein the lactic acid bacterium has antimutagenicity.
Item 3. Item 3. The composition for preventing liver dysfunction according to Item 1 or 2, wherein the lactic acid bacterium is a lactic acid bacterium belonging to at least one of Lactobacillus helveticus or Streptococcus thermophilus.
Item 4. Item 4. The composition according to any one of Items 1 to 3, comprising at least one lactic acid bacterium of Lactobacillus helveticus SR-1 (FERM P-20600) or Streptococcus thermophilus SC-1 (FERM P-20601) as an active ingredient.
Item 5. Item 5. The composition for preventing liver dysfunction according to any one of Items 1 to 4, which is an oral pharmaceutical or food having a liver dysfunction preventing action.
Item 6. Item 6. The composition for preventing liver dysfunction according to Item 5, wherein the food is fermented milk, a lactic acid bacteria beverage, or a supplement prepared by processing these.

Item 7. A novel lactic acid bacterium belonging to Lactobacillus helveticus: Lactobacillus helveticus SR-1 (FERM P-20600).
Item 8. A novel lactic acid bacterium belonging to Streptococcus thermophilus: Streptococcus thermophilus SC-1 (FERM P-20601).

Item 9. A food composition prepared using at least one lactic acid bacterium of Lactobacillus helveticus SR-1 (FERM P-20600) or Streptococcus thermophilus SC-1 (FERM P-20601).
Item 10. A method for preparing a fermented milk product in which water separation or whey separation is suppressed, wherein Lactobacillus helveticus SR-1 (FERM P-20600) is used as a lactic acid bacterium.
Item 11. A method for suppressing water separation or whey separation of a fermented milk product, characterized in that fermented milk is prepared using Lactobacillus helveticus SR-1 (FERM P-20600) as a lactic acid bacterium.

  ADVANTAGE OF THE INVENTION According to this invention, the product aiming at the liver dysfunction prevention which uses the highly safe lactic acid bacteria ingested as a food ingredient as an active ingredient can be provided. The composition for preventing hepatic dysfunction according to the present invention can be taken for a long time without worrying about side effects, and can prevent or reduce hepatic dysfunction such as hepatitis, particularly hepatic dysfunction associated with viral hepatitis. Further, by using a lactic acid bacterium having an antimutagenic action as the lactic acid bacterium, a composition having a cancer preventing action in addition to a liver dysfunction preventing action can be provided.

  Moreover, according to this invention, in addition to the liver dysfunction prevention effect, the novel lactic acid bacteria which exhibit a strong antimutagenic action with respect to various mutagens relevant to canceration can be provided. Ingestion of the lactic acid bacteria in a form of food such as fermented milk or lactic acid bacteria beverages prevents the occurrence of liver dysfunction, removes or inactivates mutagen, or reduces mutagen action to prevent canceration. It becomes possible to prevent. Therefore, the novel lactic acid bacteria of the present invention are useful as health foods (functional foods, foods for specified health use, etc.) or preparation materials thereof.

  Furthermore, the novel lactic acid bacteria belonging to the genus Lactobacillus provided by the present invention have the ability to produce viscous polysaccharides, and by utilizing this property, whey separation and water separation that are problematic in the production, distribution and storage processes are significantly suppressed. Prepared fermented dairy products can be prepared.

(1) Composition for preventing liver dysfunction The composition for preventing liver dysfunction according to the present invention comprises lactic acid bacteria or a culture thereof as an active ingredient.

  Here, the lactic acid bacteria is not particularly limited as long as it is a microorganism that can perform lactic acid fermentation and exerts an effect of preventing liver dysfunction. Microorganisms belonging to the genus Lactobacillus such as Bulgaricus and Lactobacillus acidophilus; Microorganisms belonging to the genus Streptococcus such as Streptococcus thermophilus; Microorganisms belonging to the genus Lactococcus such as Lactococcus lactis and Lactococcus cremoris; Bifidobacteria such as Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve Mention may be made of a microorganism belonging to the Umm genus. Preferred are lactic acid bacteria belonging to the genus Lactobacillus and lactic acid bacteria belonging to the genus Streptococcus. Among them, Lactobacillus helveticus belonging to the genus Lactobacillus and Streptococcus thermophilus belonging to the genus Streptococcus can be preferably mentioned.

  In addition, the liver dysfunction preventing action of lactic acid bacteria may be either an action based on the lactic acid bacteria (bacteria) itself or an action based on the microbial cell product. That is, microorganisms having (demonstrating) a liver dysfunction preventing action include both a microorganism itself having a liver dysfunction preventing action and a microorganism having an ability to produce a substance having a liver dysfunction preventing action. It is.

  The liver dysfunction preventive effect exhibited by lactic acid bacteria can be evaluated by orally administering lactic acid bacteria or lactic acid fermented foods prepared using lactic acid bacteria to hepatic disorder model animals. As liver injury model animals, D-galactosamine-induced liver injury model rats, D-galactosamine and lipopolysaccharide-induced liver injury model rats, and carbon tetrachloride-induced liver injury model animals can be used. The D-galactosamine-induced liver injury model rat is a model animal widely used as a model animal for hepatitis, particularly viral hepatitis, because hepatitis caused by galactosamine is relatively similar to the symptoms of human viral hepatitis. It is.

  The liver dysfunction-preventing action of the present invention is obtained by orally administering the above-mentioned D-galactosamine alone or in combination with lipopolysaccharide to test animals to which lactic acid bacteria or lactic acid fermented foods (for example, fermented milk and lactic acid bacteria beverages) are orally administered for a predetermined period. Serum transamylase (GOT, GPT) activity and lactate dehydrogenase (LDH) activity in the serum as indicators of liver function Alternatively, it can be evaluated based on an inflammatory reaction (cell infiltration or necrotic lesion) of liver tissue. Evaluate that the lactic acid bacterium has an effect of preventing liver dysfunction when administration or intake of lactic acid bacteria or lactic acid fermented foods suppresses the onset or extent of liver dysfunction inherently induced by the above drugs such as galactosamine can do.

  Lactic acid bacteria exhibiting such an effect of preventing liver dysfunction can be isolated and collected in nature or from fermented foods or fermented food starters. A lactic acid bacterium suitably used in the present invention is a lactic acid bacterium exhibiting such an effect of preventing liver dysfunction, and further has an antimutagenic property. More preferably, it is a lactic acid bacterium having antimutagenicity in the cell itself, regardless of whether it is a live cell or a dead cell.

  Lactic acid bacteria having antimutagenicity can be screened using, for example, the Ames method, which is a method for evaluating antimutagenicity. Specifically, first, prepare a test lactic acid bacterium so that it has an appropriate number of bacteria (for example, about 200-300 million / ml), add a mutagen to this, and further add S9-mix as necessary. Add. Thereafter, a mutation tester (for example, Salmonella typhimurium TA100 or TA98, Escherichia coli WP2 uvrA) is added, the mixture is preincubated, and further cultured in a solid medium. After completion of the culture, a reverse mutant colony generated in the mutant test bacterium is detected, the antimutagenic action of each test lactic acid bacterium is evaluated based on the number of colonies, and a lactic acid bacterium having a high antimutagenicity is selected. In such screening, as a comparative control, it is necessary to perform the same test on a system not added with a mutagen and a system not added with a test lactic acid bacterium, and detect a back mutant colony in the same manner as described above.

  From these results, the antimutagenicity of the test lactic acid bacteria can be evaluated by the following formula.

  The mutagen used in the above is not particularly limited as long as it is known in the art. For example, base-substituted mutagens such as 4-nitroquinoline-N-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 3-amino-1,4-dimethyl- 5H-pyrido [4,3-b] indole (Trp-P-1), 2-amino-6-methylimida-5H-pyrido [1,2-a-3 ', 2'-d] -imidazole (Glu- P-1), and frameshift mutagens such as 2-amino-3,4-dimethylimidazo [4,5-f] quinoline (MeIQ). The lactic acid bacterium targeted by the present invention is not limited, but preferably has a strong antimutagenic activity against a base-substituted mutagen, more preferably 4NQO. In particular, lactic acid bacteria exhibiting an antimutagenic activity of 50% or more, preferably 55% or more, with respect to such base-substituted mutagen are suitable.

  The present invention provides a novel lactic acid bacterium having the above liver dysfunction preventing action and antimutation action. The lactic acid bacterium is obtained by isolation from the natural world as described above. SR-1 lactic acid bacterium (Lactobacillus helveticus SR-1), which is one of the novel lactic acid bacteria of the present invention, has the mycological characteristics shown in FIG.

  As a result of DNA analysis using MicroSeq, the SR-1 lactic acid bacterium has a 16S rDNA base sequence with a high homology rate of 99.93%, compared to 16S rDNA of the known Lactobacillus helveticus (NCIMB11971 strain, Accession No. AY369116). The difference between the two 16S rDNAs was only one base in the mixed base (IUB code Y = C or T). It was also shown that the 16S rDNA of the SR-1 lactic acid bacterium formed a phylogenetic branch with Lactobacillus helveticus 16S rDNA on the molecular phylogenetic tree. As a result of homology search against GenBank / DDBJ / EMBL using BLAST (homology search against international nucleotide sequence database), SR-1 lactic acid bacteria 16S rDNA has a homology of 99.9%, and the above-mentioned L. helveticus (NCIMB11971 strain) It showed the highest homology to 16S rDNA. As described above, from the results of mycological characteristics and 16S rDNA-Full nucleotide sequence analysis, the SR-1 lactic acid bacteria were determined to be lactic acid bacteria belonging to Lactobacillus helveticus belonging to the genus Lactobacillus.

  The strain of lactic acid bacteria is “Lactobacillus helveticus SR-1” (deposited by the depositor) at the National Institute of Advanced Industrial Science and Technology (AIST), which has an address at Tsukuba Center Center 6-1, Tsukuba City, Ibaraki Prefecture. As an indication for identification), it was deposited on July 21, 2005 (Accession Number: FERM P-20600).

  Moreover, another SC-1 lactic acid bacterium (Streptococcus thermophillus SC-1) of the novel lactic acid bacterium of the present invention has the mycological characteristics shown in FIG.

  As a result of analysis using MicroSeq, the SC-1 lactic acid bacterium has a 16S rDNA base sequence of 100% homology with the 16S rDNA of Streptococcus thermophillus (ATCC19258, Accession No. AY188354 and DL1, Accession No. AB200871) did. In the molecular phylogenetic tree, 16S rDNA of SC-1 lactic acid bacteria formed a phylogenetic branch with the 16S rDNA of Streptococcus thermophillus, and was shown to be closely related. As a result of homology search against GenBank / DDBJ / EMBL using BLAST (homology search against international nucleotide sequence database), the 16S rDNA of SC-1 lactic acid bacteria has a homology of 100% and is the reference strain of Streptococcus thermophillus, ATCC It was consistent with 16S rDNA of 19258 strain. As described above, from the results of mycological characteristics and 16S rDNA-Full nucleotide sequence analysis, it was determined that the SC-1 lactic acid bacterium belongs to the genus Streptococcus and is a lactic acid bacterium belonging to Streptococcus thermophillus.

  This strain has been identified as "Streptococcus thermophillus SC-1" (identified by the depositor) at the National Institute of Advanced Industrial Science and Technology (AIST), which has an address at Tsukuba Center Center 6-1, Tsukuba City, Ibaraki Prefecture. As a display for this purpose) was deposited on July 21, 2005 (accession number: FERM P-20601).

  The composition for preventing liver dysfunction targeted by the present invention includes oral pharmaceuticals and foods containing an effective amount of the above lactic acid bacterium or a culture thereof that can prevent liver dysfunction. Only one type of lactic acid bacterium may be used, or a combination of two or more types may be used. A preferred combination is a combination of a lactic acid bacterium belonging to the genus Lactobacillus and a lactic acid bacterium belonging to the genus Streptococcus, and a combination of Lactobacillus helveticus and Streptococcus thermophilus is particularly preferred. The lactic acid bacterium is appropriately cultured and proliferated using a conventional lactic acid bacterium culture medium such as MRS (de Man, Rogosa, Sharpe) medium, and then remains live or sterilized. It can be spray-dried and powdered and used as an active ingredient of the composition of the present invention.

The oral drug targeted by the present invention is a mixture of the above active ingredients with a pharmaceutically acceptable carrier (excipient, binder, disintegrant, disintegration aid, lubricant, etc.) or additive, It can be prepared by formulating it into a desired form such as granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, suspensions and the like. Examples of the pharmaceutically acceptable carrier include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, merylcellulose, carboxymethylcellulose, cocoa butter and the like. Examples of additives include fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, and the like. The dosage of the pharmaceutical composition can be appropriately determined according to various conditions such as the sex, weight, age, and symptoms of the patient. For example, when expressed in terms of the number of lactic acid bacteria, the ratio is such that the daily dose is 1 × 10 ⁷ or more, preferably 1 × 10 ⁸ or more, more preferably 5 × 10 ⁸ or more. Can do.

  The food which this invention makes object can be prepared by mix | blending the said active ingredient as one of the manufacturing raw materials of food and drink. The type of food is not particularly limited, and examples include dairy products (including beverages), fermented foods, beverages (including powdered beverages), confectionery, bread, processed fish products, processed meat products, noodles, soups, and prepared dishes. be able to. Preferred are dairy products, fermented products and beverages prepared using lactic acid bacteria having a liver function disorder preventing action. As an example of dairy products and fermented products, fermented milk (non-fat milk solids) such as yogurt prepared by fermenting animal milk such as cow's milk or milk raw materials (mix) with lactic acid bacteria that have liver function disorder preventing action 8% or more, and the number of lactic acid bacteria or yeasts is 10 million or more per ml), fermented liquor such as Kumis or Kefir, and dairy lactic acid bacteria beverages (non-fat milk solid content is 3% to 8% Less). Examples of beverages include lactic acid bacteria beverages (non-fat milk solid content of less than 3%), lactic acid bacteria-containing beverages (lactic acid bacteria) prepared by adding lactic acid bacteria having a liver function disorder preventing action to fruit juice, vegetable juice, etc. Juice) and the like. In preparing the composition of the present invention, the number of lactic acid bacteria to be added is not limited, but a larger number of lactic acid bacteria is preferable because an excellent physiological action can be expected. In this sense, fermented foods that can increase lactic acid bacteria by fermentation are preferred. Such food can be carried out in a usual manner except that the above-mentioned lactic acid bacteria are used, and various products and additives according to the type of food such as milk, dairy products such as milk, dairy products, sugar, fruit juice , Vegetable juice, gelling agent, fragrance and the like can be optionally added.

  In addition, the food targeted by the present invention includes granules, powders, tablets, pills, capsules, or drinks (solutions, emulsions) together with carriers and additives that can be used as such or as foods. Or a supplement prepared in the form of a suspension). As the carrier and additive, those used for oral pharmaceuticals can be used in the same manner.

The amount of food intake can be determined as appropriate according to various conditions such as the sex, weight, age, and symptoms of the user. For example, when expressed in terms of the number of lactic acid bacteria, the ratio is such that the daily intake is 1 × 10 ⁷ or more, preferably 1 × 10 ⁸ or more, more preferably 5 × 10 ⁸ or more. Can do. Usually, it is prepared by blending the above-mentioned ratio of lactic acid bacteria to the amount of food taken daily. For example, in the case of fermented milk such as yogurt, it is set so that lactic acid bacteria having a liver function disorder-preventing action are contained in the above ratio in an adult daily intake of 50 to 500 g, preferably 60 to 200 g.

The food of the present invention can be provided as a health food, a functional food, a food for specified health use, or a food for a sick person having a liver dysfunction prevention function, preferably an antimutagenic function (carcinogenesis prevention function). ,
(2) Novel Lactic Acid Bacteria and Food Composition Utilizing the Same The present invention is a novel lactic acid bacterium having a liver dysfunction-preventing action, and the aforementioned SR-1 lactic acid bacteria belonging to the genus Lactobacillus ("Lactobacillus helveticus SR-1" : FERM P-20600) and SC-1 lactic acid bacteria belonging to the genus Staphylococcus ("Streptococcus thermophillus SC-1", accession number: FERM P-20601).

  In addition to preventing liver dysfunction, the lactic acid bacteria have antimutagenic activity, particularly strong antimutagenic activity against base-substituted mutagens (eg 4NQO and MNNG) regardless of whether they are live or dead. have. For this reason, the lactic acid bacteria or a culture thereof is used as an oral medicine having a liver dysfunction preventing action, preferably an antimutagenic action (cancer prevention action), and a food (health food, functional food, food for specified health use). Or can be effectively used as a food for a sick person). In the preparation of oral pharmaceuticals and foods, these novel lactic acid bacteria may be used alone or in combination. Moreover, as long as the effect of this invention is not impaired, other lactic acid bacteria and yeast can also be used together.

  The type, form, and administration (intake) amount of an oral drug or food can be set according to the description in (1). Preferred examples of the food include dairy products such as fermented milk, lactic acid bacteria beverages, and supplements prepared by processing these.

(3) Method for preparing fermented dairy product with reduced whey separation and water separation SR-1 lactic acid bacteria belonging to the genus Lactobacillus, which is a novel lactic acid bacterium of the present invention ("Lactobacillus helveticus SR-1," accession number: FERM P-20600) Has the ability to produce viscous polysaccharides in addition to the above-mentioned liver dysfunction preventing action and antimutagenic action. Therefore, the present invention provides a method for preparing a fermented milk product with reduced whey separation and water separation, characterized by using the SR-1 lactic acid bacteria. Suitable examples of fermented milk include yogurt (hard yogurt, soft yogurt).

  Such a method for preparing a fermented milk product is characterized in that SR-1 lactic acid bacteria are used as starter bacteria. Except for this point, the rest can be performed according to conventional methods.

  Specifically, skim milk, skim milk powder, and sucrose, and yogurt mix containing a stabilizer as necessary are emulsified, sterilized and cooled, and then starter bacteria containing SR-1 lactic acid bacteria are added to 1 to 3%. Inoculate at a moderate rate and ferment according to conventional methods. Here, the process from fermentation to commercialization differs between hard type and soft type. For example, hard type yogurt is fermented at 30 to 45 ° C. for about 2.5 to 12 hours after filling in a retail container, and 0.5 to 1. Finish with the acidity of about 5% as a guide. On the other hand, soft type yogurt is fermented in the same manner in a tank, cooled, and then filled into a retail container to obtain a product.

  Thus, fermented dairy products do not use agar, gelatin, carrageenan, guar gum, locust bean gum, and pectin, which are conventionally used as stabilizers, or can be separated from water or separated from whey in a smaller amount than usual. It can be prepared as fermented milk (yogurt) which is suppressed and has a fine texture and a stable tissue.

  The starter bacterium may be any one that contains SR-1 lactic acid bacteria, and SR-1 lactic acid bacteria can be used alone or in combination with other lactic acid bacteria or yeasts. Preferably, a lactic acid bacterium having an effect of preventing liver dysfunction or a lactic acid bacterium having an antimutagenic activity (for example, the SC-1 lactic acid bacterium of the present invention) can be used in combination as a mixed lactic acid bacterium.

  Hereinafter, the present invention will be described in more detail with reference to experimental examples and examples. However, these examples are merely examples, and the present invention is not limited to these examples. In the following experimental examples and examples, “Lactobacillus helveticus SR-1” (accession number: FERM P-20600) (hereinafter simply referred to as “SR-1 lactic acid bacteria”) belonging to the genus Lactobacillus as lactic acid bacteria, and Streptococcus “Streptococcus thermophillus SC-1” (accession number: FERM P-20601) (hereinafter simply referred to as “SC-1 lactic acid bacteria”) belonging to the genus was used.

Experimental Example 1 Liver Function Prevention / Improvement Effect (1) Preparation of Test Samples Yogurt prepared using SR-1 lactic acid bacteria and SC-1 lactic acid bacteria were used as test samples. The prepared yogurt is also referred to as “SR-1 yogurt”, and the yogurt prepared using SC-1 lactic acid bacteria is also referred to as “SC-1 yogurt”). Specifically, yogurt is fermented by inoculating 1% SR-1 lactic acid bacteria or SC-1 lactic acid bacteria as a lactic acid bacteria starter into 12% reduced skim milk prepared by heat sterilization (42 ° C.), and the lactic acidity is 0.80. When it reached ˜0.85%, the fermentation was stopped, and it was prepared by cooling to 10 ° C. or less (viable cell count: 1.0 × 10 ^{8 to} 5.2 × 10 ⁸ , pH about 4.6).

(2) Experimental method
(2-1) Administration of yogurt and induction of liver damage by galactosamine Eighteen 3-week-old Wistar male rats (CrJ: Wistar, Charles River) were used in the experiment. Specifically, after one week of acclimatization, these rats were divided into 3 groups (control group, SR-1 yogurt administration group, SC-1 yogurt administration group), and the control group (n = 6) had a normal diet ( In the SR-1 yogurt administration group and SC-1 yogurt administration group (each n = 6), SR-1 yogurt and SC-1 yogurt prepared above were mixed in the normal feed, respectively. A sufficient amount of feed was given and ad libitum. The mixing ratio of yogurt to normal feed was 15% by weight with the yogurt content in the feed as the dry matter weight. Mixed feed was prepared and changed daily. The body weight of all individuals (n = 18) was measured once a week.

  Two months later, each group of rats was anesthetized with ether, and galactosamine (D-(+) galactosamine hydrochloride) was intraperitoneally administered at a rate of 300 mg / kg body weight. Note that galactosamine was prepared as an injection solution by dissolving at a concentration of 150 mg / ml with a sterilized phosphate buffer.

(2-2) Blood sampling and liver function test 21 hours after administration of galactosamine, rats in each group were anesthetized with ether and Nembutal, 3 ml of blood was collected from the abdominal vena cava, and serum samples were obtained by centrifugation. From the obtained serum specimen, the GOT activity, GPT activity, γ-GTP activity, LDH activity, ALP activity and total bilibin amount in the serum were measured using a clinical test apparatus (SPOTCHEM SP-4430, manufactured by ARKRAY, Inc.). It was measured.

(2-3) Liver tissue specimen At the same time as blood collection, liver tissue was collected and immediately placed in 10% formalin for fixation. This was stained with hematoxylin and eosin to prepare a liver tissue sample, which was observed under a microscope.

(3) Results FIG. 3 shows changes in body weight of each group (control group, SR-1 yogurt administration group, SC-1 yogurt administration group) due to normal feed and yogurt intake. In addition, liver function test results (GOT, GPT, γ-GTP, LDH, ALP activity and total bilibin amount in serum) after galactosamine administration in each group (control group, SR-1 yogurt administration group, SC-1 yogurt administration group) ) Is shown in FIG.

  As can be seen from FIG. 4, the serum GOT activity, GPT activity, LDH activity and ALP activity of the group of rats fed with the normal diet were extremely increased by the administration of galactosamine, and the total bilirubin amount was slightly increased to 0.6 mg / dl. Hepatic injury was induced by galactosamine. On the other hand, in the rats administered the yogurt mixed diet (SR-1 yogurt administration group, SC-1 yogurt administration group), these increases due to galactosamine administration were clearly suppressed (of galactosamine-induced liver injury). Suppression). From this, it was shown that yogurt (fermented food) prepared from lactic acid bacteria has an effect of preventing liver dysfunction. In addition, as shown in FIG. 3, the body weight of the rats of any group increased with the growth, and no effect of SR-1 yogurt administration or SC-1 yogurt administration was observed.

Experimental Example 2 Antimutagenicity test (fermented milk)
The antimutagenicity of SR-1 lactic acid bacteria and SC-1 lactic acid bacteria was evaluated according to the Ames method. Specifically, fermented milk was prepared using SR-1 lactic acid bacteria and SC-1 lactic acid bacteria, respectively, and antimutagenicity was measured for fermented milk, its filtrate, and the cells themselves (live cells, dead cells). did.

(1) Experimental method
(1-1) Preparation of test sample
(i) Fermented milk
SR-1 lactic acid bacteria and SC-1 lactic acid bacteria are subcultured in TYLG liquid medium (containing 1% tryptone, 0.5% yeast extract, 0.5% lactose, 0.5% glucose, 0.1% Tween80, 0.01% L-cysteine hydrochloride) The lactic acid bacteria culture solution (fermented milk starter) thus prepared was inoculated at a rate of 1% into the sterilized 10% reduced skim milk and cultured at 37 ° C. for 24 hours to prepare fermented milk. Fermented milk prepared from SR-1 lactic acid bacteria is referred to as “SR-1 fermented milk”, and fermented milk prepared from SC-1 lactic acid bacteria is referred to as “SC-1 fermented milk”.

(ii) Filtrate of fermented milk The fermented milk obtained above (SR-1 fermented milk, SC-1 fermented milk) is filtered through filter paper, and then filtered through a filter (0,2 μm) to completely remove the cells. To obtain a filtrate.

(1-2) Measurement of antimutagenicity Table 1 shows the mutagenic substances used in the measurement of antimutagenicity. For the measurement, each mutagen was dissolved in DMSO (dimethyl sulfoxide) and used as a mutagen solution.

  The antimutagenicity was measured according to the following procedure according to the Ames method using Salmonella typhimurium strains TA98 and TA100 as the indicator bacteria.

  (a) To a sterile test tube, add 100 μl of the test sample, 100 μl of the mutagen solution, 700 μl of 0.1 M phosphate buffer (pH 7.0), and 100 μl of the indicator bacteria culture solution in order and mix. However, when Trp-P1, MeIQ or Glu-P1 is used as a mutagen, in a sterile test tube, 100 μl of test sample, 100 μl of mutagen solution, 600 μl of 0.1M phosphate buffer (pH 7.0), S9-mix Sequentially add 100 μl and 100 μl of indicator medium culture solution.

  (b) Shake culture at 37 ° C. for 20 minutes, then add 2 ml of top agar (soft agar containing about 0.05 mM of biotin and L-histidine, respectively) to this, mix well, then overlay the minimum glucose solid medium static To do. This is incubated at 37 ° C. for 48 hours.

  (c) After completion of the culture, histidine-unnecessary colonies formed on the medium are detected, and the antimutagenicity of each test sample is evaluated by the following formula based on the number of colonies. In the evaluation of the antimutagenicity of each test sample, in parallel with the above, the same test was conducted for the system not containing the test sample and the system not containing both the test sample and the mutagen solution. Was measured.

(2) Results
(2-1) Table 2 shows the antimutagenicity of each fermented milk (SR-1 fermented milk, SC-1 fermented milk) against each mutagen.

  As can be seen from the table, the SR-1 fermented milk prepared from SR-1 lactic acid bacteria and the SC-1 fermented milk prepared from SC-1 lactic acid bacteria are more base-substituted than mutagen of frameshift type. High antimutagenicity against mutagens.

  (2-2) Antimutagenicity of each fermented milk (SR-1 fermented milk, SC-1 fermented milk) and its filtrate using 4NQO, which showed the highest antimutagenic activity above, as an indicator mutagen The results of evaluation are shown in FIG. As can be seen, the antimutagenicity of the filtrate was significantly reduced compared to fermented milk (p <0.05). From these results, it was suggested that the bacterial body was greatly involved in the antimutagenicity of fermented milk.

Experimental Example 3 Antimutagenicity test (bacteria)
As described above, since the results of Experimental Example 2 suggested that the bacterial body was greatly involved in the antimutagenicity of fermented milk, the antimutagenicity of the bacterial body itself was evaluated here. As test cells, cell powders (live cell powder and dead cell powder) prepared by freeze-drying SR-1 lactic acid bacteria and SC-1 lactic acid bacteria were used. The viable cell powder was obtained by subculturing SR-1 lactic acid bacteria or SC-1 lactic acid bacteria several times in a TYLG liquid medium, and then centrifuging at 5000 rpm for 10 minutes to collect the cells, which was then washed with phosphate buffer. It was prepared by lyophilization after two washes. The dead cell powder was prepared by washing with a phosphate buffer solution as described above, treating with autoclave at 121 ° C. for 15 minutes, and then freeze-drying.

  These lactic acid bacteria powders (live cell powders, dead cell powders) were dissolved in 4NQO solution (2.5 μg / ml) to 5 mg / ml, and this was cultured with shaking at 37 ° C. for 24 hours. The culture solution was centrifuged at 3000 rpm for 10 minutes, and the supernatant was further filtered. The mutagenicity of the filtrate was measured according to the method of Experimental Example 2 (Ames method). As a control, the mutagenicity of the 4NQO solution without addition of bacterial cells was measured, and the antimutagenicity of the bacterial cells was calculated according to the following formula.

  The results are shown in FIG. As can be seen from the figure, anti-mutagenic activity was observed in both SR-1 lactic acid bacteria and SC-1 lactic acid bacteria. It is said that the antimutagenicity due to these cells is due to the mutagen binding action of the cells (Rajam Rajendran and Yoshiyuki Ohta, Canadian Journal of Microbiology Vol.44: 109-115 (1998); Taku Miyamoto et al. Japanese Journal of Dairy and Food Science Vol.40, No.3 (1991)).

  Therefore, the lactic acid bacteria of the present invention (SR-1 lactic acid bacteria and SC-1 lactic acid bacteria) exhibit antimutagenic activity based on their mutagen binding properties (particularly mutagen binding properties to base-substituted mutagens). it is conceivable that. Further, as shown in the figure, antimutagenicity was observed not only in living cells but also in dead cells. Therefore, according to the lactic acid bacteria of the present invention (SR-1 lactic acid bacteria and SC-1 lactic acid bacteria), regardless of their survival, even if the bacteria die due to the effects of gastric acid or bile acid in the digestive tract, mutagen in the intestine. It seems that substances can be combined and discharged out of the body.

Experimental Example 4 Antimutagenicity test (viscous polysaccharide)
SR-1 lactic acid bacteria have the ability to produce viscous polysaccharide substances. Therefore, the antimutagenic action of the viscous polysaccharide produced by the lactic acid bacteria was examined according to Experimental Example 2.

(1) Preparation of viscous polysaccharide
1% SR-1 lactic acid bacteria culture solution was added to 10% reduced skim milk and cultured at 37 ° C. for 24 hours. After culturing, the supernatant was collected by centrifugation at 10000 rpm for 30 minutes, and then concentrated by filtration under reduced pressure. Then, an equal amount of 10% trichloroacetic acid was added, and the mixture was allowed to stand overnight under ice cooling. This was centrifuged at 10000 rpm for 30 minutes, the protein was precipitated and the supernatant was collected, and ethanol was further added thereto to collect the precipitate (polysaccharide). This was again dissolved in distilled water, and ethanol precipitation was repeatedly washed. The resulting washed product was lyophilized to prepare a viscous polysaccharide substance as a white lyophilized product.

(2) Antimutagenicity test In the same manner as in Experimental Example 2 using a polysaccharide aqueous solution prepared by dissolving the lyophilized product in distilled water to 50 mg / ml, 4NQO was used as a mutagen. Antimutagenicity was evaluated. The results are shown in FIG. From the figure, the antimutagenic effect was also observed in the viscous polysaccharide produced by SR-1 lactic acid bacteria.

Experimental Example 4 Water separation inhibition test (SR-1 lactic acid bacteria)
Sterilized at 85 ° C, milk was inoculated with 1% SR-1 lactic acid bacteria as starter bacteria, and dispensed into a sterilized graduated pitch centrifuge tube. This was fermented at 42 ° C., and when the lactic acid acidity reached 0.8%, the fermentation was stopped and cooled overnight to measure the rate of water separation (water separation rate). The water separation rate was determined by centrifuging the fermented milk that entered the spitch centrifuge tube and calculating the ratio of water separation relative to the total amount of fermented milk. As a comparative test, the water separation rate was similarly evaluated using a standard strain Lactobacillus helveticus NBRC 15019 belonging to the same Lactobacillus helvetica instead of SR-1 lactic acid bacteria.

  The results are shown in FIG. As can be seen from the results, fermented milk prepared using SR-1 lactic acid bacteria showed superior water separation prevention effect. Since SR-1 lactic acid bacteria have the ability to produce viscous polysaccharides, it was considered that the tissue of fermented milk was stabilized by the viscous polysaccharide substances produced by SR-1 lactic acid bacteria. For this reason, by using SR-1 lactic acid bacteria as starter bacteria for fermented milk, it is possible to prevent water separation and whey separation that may occur in the fermented milk production process or storage / distribution process.

  Hereinafter, as an example of the food targeted by the present invention, a method for producing a supplement prepared by processing fermented milk, fermented milk (drink type), lactic acid bacteria beverage, fermented milk, and the like will be given. For the preparation of these foods, the SR-1 lactic acid bacteria and SC-1 lactic acid bacteria of the present invention can be used alone or in combination as starter bacteria.

Example 1 Fermented milk The preparation process of fermented milk is shown in FIG. Specifically, the yogurt mix prepared by blending raw materials (milk, skim milk, skim milk powder, and sucrose, and, if necessary, fragrance and stabilizer) is homogenized at a homogeneous pressure of 130 kg · f / cm ² . Sterilize at 122 ° C for 2 seconds. After cooling to 45 ° C., SR-1 lactic acid bacteria and SC-1 lactic acid bacteria are inoculated alone or in combination as a lactic acid bacteria starter and fermented at 42 ° C. When the lactic acid acidity reaches 0.85%, the fermentation is stopped and cooled to 10 ° C. or lower to prepare fermented milk.

Example 2 Fermented milk (drink type)
The preparation process of fermented milk (drink type) is shown in FIG. Specifically, the yogurt mix prepared by blending raw materials (milk, skim milk, skim milk powder, and sucrose, and, if necessary, fragrance and stabilizer) is homogenized at a homogeneous pressure of 130 kg · f / cm ² . Sterilize at 122 ° C for 2 seconds. After cooling this to 45 ° C., SR-1 lactic acid bacteria and SC-1 lactic acid bacteria are inoculated alone or in combination as a lactic acid bacteria starter and fermented at 42 ° C. The fermentation is stopped when the lactic acidity reaches 0.60%. This is homogenized at a homogenous pressure of 100 kg · f / cm ² , filled into a container, cooled at 10 ° C. or lower to obtain fermented milk (drink type).

Example 3 Lactic Acid Beverage Beverage The lactic acid bacteria beverage preparation process is shown in FIG. Specifically, the prepared ingredients (fat milk, skim milk powder) are sterilized at 95 ° C. for 90 minutes and cooled to 36.5 ° C. Add and ferment SR-1 lactic acid bacteria and SC-1 lactic acid bacteria alone or in combination as lactic acid bacteria, and cool (less than 10 ° C) to homogenize (150 kg · f / cm ² ), and use this as a card. Separately prepared syrup (fructose glucose liquid sugar, sucrose sugar) is mixed with curd, and after adding a fragrance, homogenized (150 kg · f / cm ² ) is used as a lactic acid bacteria beverage stock solution. This is mixed with sterilized water, homogenized again (120 kg · f / cm ² ), and after filling, refrigerated at 10 ° C. or lower to prepare a lactic acid bacteria beverage.

Example 4 Supplement (Freeze Dry Powder)
The preparation process of a supplement (powder form) is shown in FIG. Specifically, for example, 4 kg of the fermented milk (raw material) prepared in Example 1 is pre-frozen at -20 ° C or lower for 8 hours or more per tray. Subsequently, vacuum freeze-drying is performed for 24 hours under the conditions of a maximum shelf temperature of 80 ° C, a final shelf temperature of 50 ° C, and a degree of vacuum of 0.5 torr or less with a vacuum freeze dryer, then pulverized with a power mill and screened with a screen of 0.8 mm. Powderize. The thus-prepared supplement (freeze-dried powder) is subjected to metal detection and then used in aluminum packaging containing a desiccant to obtain a product.

Example 5 Supplement (Tablets)
The preparation process of a supplement (tablet form) is shown in FIG. Specifically, for example, freeze-dried fermented milk (raw material) prepared in Example 4 is mixed with other raw materials (maltitol). Using this as a binder, an aqueous solution of thickening polysaccharide (pullulan) is allowed to flow while spraying, and simultaneously dried with hot air and granulated. The remaining ingredients (fragrance, milk calcium, sucrose fatty acid ester) are mixed and tableted with a tableting machine. The supplement (tablet) thus prepared is filled and packaged in a container to obtain a product.

BRIEF DESCRIPTION OF THE DRAWINGS It is the figure which put together the mycological characteristics of the novel lactic acid bacterium "Lactobacillus helveticus SR-1" (accession number: FERM P-20600) of this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is the figure which put together the mycological characteristics of the novel lactic acid bacterium "Streptococcus thermophillus SC-1" (accession number: FERM P-20601) of this invention. Change in body weight (g) of rats administered with SR-1 yogurt (labeled L in the figure) and SC-1 yogurt (labeled S in the figure), and change in body weight of control group (labeled Control in the figure) It is a result shown with (Experimental example 1). Liver function results of rats administered SR-1 yogurt (labeled L in the figure) and SC-1 yogurt (marked S in the figure) [serum GOT activity (GOT), GPT activity (GPT), γ -GTP activity (GGT), LDH activity (LDH), ALP activity (ALP) and total amount of bilibin (T-Bil)] are shown together with liver function results of the control group (labeled Control in the figure) (Experimental Example 1) ). Results of measurement of antimutagenicity (%) of fermented milk and its filtrate prepared using Lactobacillus helveticus SR-1 (FERM P-20600) or Streptococcus thermophilus SC-1 (FERM P-20601) 2). Results of measurement of antimutagenicity (%) against 4NQO of Lactobacillus helveticus SR-1 (FERM P-20600) or Streptococcus thermophilus SC-1 (FERM P-20601) cells (live cells, dead cells) This is shown (Experimental Example 3). The result of having measured the antimutagenicity (%) with respect to 4NQO about the viscous polysaccharide substance which Lactobacillus helveticus SR-1 (FERM P-20600) produces is shown (Experimental example 4). The result of having compared the water separation rate (%) of fermented milk by Lactobacillus helveticus SR-1 (FERM P-20600) with the standard strain Lactobacillus helveticus NBRC 15019 strain which belongs to Lactobacillus helveticus is shown (Experimental example 5). It is a figure which shows the manufacturing process of fermented milk. It is a figure which shows the manufacturing process of fermented milk (drink type). It is a figure which shows the manufacturing process of a lactic acid bacteria drink. It is a figure which shows the manufacturing process of the supplement (freeze-dried powder) which processed fermented milk. It is a figure which shows the manufacturing process of the supplement (tablet) which processed fermented milk.

Claims (8)

A composition for preventing hepatic dysfunction comprising an anti-mutagenic lactic acid bacterium or its culture as an active ingredient, which can be obtained only by lactic acid bacteria, and as an active ingredient Lactobacillus helveticus SR-1 (FERM P-20600 ) And Streptococcus thermophilus SC-1 (FERM P-20601) . The composition for preventing liver dysfunction according to claim 1, which is an oral pharmaceutical or food having a liver dysfunction preventing action. The composition for preventing liver dysfunction according to claim 2 , wherein the food is fermented milk, a lactic acid bacteria beverage, or a supplement prepared by processing these. A novel lactic acid bacterium belonging to Lactobacillus helveticus: Lactobacillus helveticus SR-1 (FERM P-20600). A novel lactic acid bacterium belonging to Streptococcus thermophilus: Streptococcus thermophilus SC-1 (FERM P-20601). A food composition prepared using at least one lactic acid bacterium of Lactobacillus helveticus SR-1 (FERM P-20600) or Streptococcus thermophilus SC-1 (FERM P-20601). A method for preparing a fermented milk product in which water separation or whey separation is suppressed, wherein Lactobacillus helveticus SR-1 (FERM P-20600) is used as a lactic acid bacterium. A method for suppressing water separation or whey separation of a fermented milk product, characterized in that fermented milk is prepared using Lactobacillus helveticus SR-1 (FERM P-20600) as a lactic acid bacterium.

Images