JP4610525B2 - Composition containing equol-producing lactic acid bacteria - Google Patents

Description

  The present invention relates to a lactic acid bacterium capable of producing equol, a composition containing the lactic acid bacterium, and a method for producing equol using the lactic acid bacterium.

Isoflavone (soy isoflavone) contained in soybean has a preventive effect (anti-estrogenic effect) against breast cancer, prostate cancer, etc., and menopause, postmenopausal osteoporosis, hyperlipidemia, hypertension, etc. It has already been reported to have an improvement effect (estrogenic effect) mainly in the West (H. Adlercreutz, et al., (1992) Lancet, 339 , 1233; H. Adlercreutz, et al., (1992 ) Lancet, 342 , 1209-1210; DD Baird, et al., (1995) J. Clin. Endocrinol. Metab., 80, 1685-1690; AL Murkies, et al., (1995) Maturitas., 21, 198 -195; D. Agnusdei, et al., (1995) Bone and Mineral., 19 (Supple), S43-S48 etc.).

  Recently, the clinical effect of soy isoflavone has been questioned, and instead of soy isoflavone, equol, an active metabolite of soy isoflavone, has been reported to hold the key to its effectiveness in clinical applications. That is, various reports have been found that equol, which is a metabolite thereof, is more effective than soy isoflavone for breast cancer, prostate cancer, menopause and postmenopausal osteoporosis (see Non-Patent Documents 1 to 3). .

  In the symposium held in 2001 (4th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease (San Diego, USA, 2001)), there were many presentations on equol, and in December 2002, A review of equol has been reported, and at present, it is scientifically supported that equol is the main body of soy isoflavone effectiveness (Non-Patent Document 4).

  Equol has a very high ability to migrate to tissues such as breast tissue and prostate tissue, compared to soybean isoflavone, and this also supports its physiological significance (Non-patent Document 5).

  In addition, equol is produced by intestinal bacteria, and it has been reported that there are individual differences in its production. It has also been reported that the proportion of Japanese equol producers is about 50% (Non-patent Document 6). It is assumed that humans who cannot produce equol have no equol-producing bacteria in the intestine. In the case of such a human, it is considered that the desired anti-estrogen effect and estrogen-like effect cannot be expected even if the processed soybean food is ingested. In order to express a desired effect in such a human, it is considered that equol-producing bacteria may be ingested or equol itself may be ingested.

As a result of repeated researches from the above idea, the present inventors have previously described that Bacteroides E-23-15 (FERM BP) from human feces as an equol-producing bacterium for exerting an anti-estrogen effect, an estrogen-like effect, etc. -6435), Streptococcus E-23-17 (FERM BP-6436) and Streptococcus A6G225 (FERM BP-6437) were newly isolated and identified, and these equol-producing bacteria and their utilization Patent application for the invention (Patent Document 1).
WO99 / 07392 pamphlet D. Ingram, et al., (1997) Lancet, 350, 990-994; AM Duncan, et al., (2000) Cancer Epidemiology, Biomarkers & Prevention, 9, 581-586 C. Atkinson, et al., (2002) J. Nutr., 32 (3), 595S; H. Akaza, et al., (2002) Jpn. J. Clin. Oncol., 32 (8), 296- 300 S. Uchiyama ,. et al., (2001) Ann. Nutr. Metab., 45, 113 (abs) KDR Settchell, et al., (2002) J. Nutr., 132, 3577-3584 J. Maubach, et al., (2003) J. Chromatography B., 784, 137-144; TE Hedlund, et al., (2003) The Prostate, 154, 68-78 S. Uchiyama., Et al., (2001) Ann. Nutr. Metab., 45, 113 (abs)

  As a result of continued research, the present inventors have demonstrated the ability to assimilate daidzein glycoside, daidzein or dihydrodaidzein and produce equol as a new bacterium that is essentially different from the previously isolated and identified microorganism. We succeeded in isolating and identifying lactic acid bacteria belonging to the genus Lactococcus. The present invention has been completed as a result of further research based on the isolation and identification of lactic acid bacteria.

The present invention provides the inventions described in the following items 1-13.
Item 1. A lactic acid bacterium belonging to the genus Lactococcus having the ability to assimilate at least one daidzein selected from the group consisting of daidzein glycoside, daidzein and dihydrodaidzein to produce equol as an essential component A composition containing equol-producing lactic acid bacteria.
Item 2. The composition according to item 1, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus garvieae.
Item 3. The composition according to Item 2, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus 20-92 deposited as FERM BP-10036.
Item 4. The composition according to Item 1, further comprising at least one selected from the group consisting of daidzeins and daidzein-containing substances.
Item 5. The composition according to item 4, wherein the daidzein-containing substance is soy flour or soy milk.
Item 6. The composition according to Item 4, which is in the form of a beverage or dairy product.
Item 7. The composition according to Item 4, further comprising equol.
Item 8. The composition according to item 7, which is in the form of a fermented soymilk.
Item 9. An equol characterized by allowing lactic acid bacteria belonging to the genus Lactococcus having the ability to assimilate daidzeins and produce equol to at least one selected from the group consisting of daidzeins and daidzein-containing substances Production method.
Item 10. The method according to Item 9, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus garvieae.
Item 11. The method according to Item 10, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus 20-92 deposited as FERM BP-10036.
Item 12. The method according to Item 9, wherein the daidzein-containing substance is soy flour or soy milk.
Item 13. A lactic acid bacterium belonging to the genus Lactococcus deposited as FERM BP-10036.

Hereinafter, the equol-producing lactic acid bacteria-containing composition of the present invention will be described in detail.
(1) Lactococcus lactic acid bacterium The equol-producing lactic acid bacterium-containing composition of the present invention is capable of producing equol by assimilating at least one daidzein selected from the group consisting of daidzein glycoside, daidzein and dihydrodaidzein (metabolic activity) ) Containing lactic acid bacteria belonging to the genus Lactococcus as an essential component.

  Specific examples of the lactic acid bacteria include Lactococcus 20-92 (FERM BP-10036) newly isolated and identified from human feces by the present inventors.

Hereinafter, the mycological properties of this lactic acid bacterium will be described in detail.
I. Growth condition on medium This strain is EG (Eggerth-Gagnon) agar medium, BL (Blood Liver) agar medium or GAM (Gifu Anaerobic Medium) medium at 37 ° C for 48 hours with a steel wool anaerobic jar. It shows good or normal growth when it is anaerobically cultured or aerobically cultured at 37 ° C for 48 hours. Its colony bulges up in a round shape and a convex circle shape, and the surface and the periphery are smooth, and grayish white on the EG agar medium and brown on the BL agar medium. The fungal form is Gram-positive diuretic. This strain does not form spores.

II. Biochemical properties
(1) Optimal growth temperature: 37 ℃
(2) Optimum pH: 7.0
(3) Liquefaction of gelatin: −
(4) Acetoin production from pyruvic acid: +
(5) Hippuric acid hydrolysis: −
(6) Esculin hydrolysis: +
(7) Pilonidol allylamidase: +
(8) α-Galactosidase: −
(9) β-galactosidase: −
(10) β-glucuronidase: −
(11) Alkaline phosphatase: −
(12) Leucine allylamidase: +
(13) Arginine dihydrase: +
(14) Assimilation of each carbon source:
D-ribose +
L-arabinose −
D-mannitol +
D-sorbitol −
Lactose −
D-trehalose +
Inulin −
D-Raffinose −
Starch +
Glycogen −
(15) Organic acid composition after utilization of peptone or glucose:
This strain is obtained by adding glucose to the final concentration of 0.5% to the PYF (peptone yeast extract field) nutrient solution (containing about 5% peptone), which is a medium for saccharide utilization The organic acid in the culture obtained by culturing at 37 ° C. for 72 hours under an aerobic condition was measured by HPLC. The results (unit: mM) are shown in Table 1 below.

  Based on the above developmental characteristics and biochemical properties, this strain is classified as a gram-positive cocci, Lacrococcus garvieae, but its type strain (Schleifer, KH, Kraus, J., Dvorak). , C., Kilpper-Balz, R., Collins, MD and Fischer, W. Transfer of Streptococcus lactis and related streptococci to the genus Lactococcus gen. Nov. Syst. Appl. Microbiol., 6, 183-195, 1985; ATCC43921 (JCM10343) and ATCC49156 (JCM8735)) differ in the utilization of starch.

  Therefore, the present inventor named this strain Lactococcus 20-92 (Lactococcus 20-92), and on January 23, 2003, the National Institute of Advanced Industrial Science and Technology (Ibaraki Prefecture, Japan) Deposited as FERM P-19189 at Tsukuba City East 1-3 1-3 Central 6 (zip code 305-8566). This strain has now been transferred to an international deposit and the deposit number is FERM BP-10036.

  In addition, after assimilating glucose, this strain produced lactic acid (L-type lactic acid), which was confirmed to be a homofermentative lactic acid bacterium.

  Furthermore, as a result of analyzing the sequence of 16S rRNA of this strain, the sequence was confirmed to have 99.189% homology with Lactococcus garvier (JCM10343) and 99.375% with Enterococcus seriolicida (JCM8735). .

  The Enterococcus ceriolicida was reclassified as Lactococcus garbier in 1996 because it approximated Lactococcus garbier as a result of DNA-DNA homology. Accordingly, there are two types of strains (JCM8753 strain and 10343 strain) having different origins as described above in Lactococcus garbier. Enterococcus ceriolicida (JCM8735) originates from the infected yellowtail kidney, and the original Lactococcus garbier (JCM10343 strain) originates from bovine mastitis.

  In order to examine which type strains Lactococcus 20-92 approximates, phenotypes were compared. The results are shown in Table 2 below.

  As is apparent from Table 2, the present strain Lactococcus 20-92 matched the phenotype of Lactococcus garvier type strain (JMC10343), but the Enterococcus ceriolicidae type strain (JMC8735) at 40 ° C. They differed in viability and no quinone production. Therefore, this strain was judged to be close to Lactococcus garbier (JCM10343 strain).

  The strain Lactococcus 20-92 is classified as a lactobacilli on GRAS (Generally Recognized As Safe) listed on GRAS (FDA (Food and Drug Administration)). Safety as food is considered high.

  As for Lactococcus garbier, there has been no report on pathogenicity to the human body so far, and no toxins such as toxins are produced, and it is considered as a highly safe species.

In addition, Lactococcus garbier has traditionally been mozzarella cheese, raw milk, processed meat foods that have been cryopreserved, prasomes (Thai fish fermented foods), Toma Piemontese cheese (PDO )). In particular, there is a report that it is detected at a high order of 10 ⁵ pieces / g from Prasom and 10 ⁸ pieces / g from Toma Piemontese cheese. (PM.Christine, et al., (2002) Int. J. Food Microbiol., 73 , 61-70; MG Fortina, et al., (2003), Food Microbial., 20, 379-404).

On the other hand, it is reported that Enterococcus ceriolicida is pathogenic in cultured fish such as yellowtail. For this reason, since Lactococcus 20-92 is also Lactococcus garvier, it is thought that it is systematically related to Enterococcus seriolisida, and there is concern about the possibility of pathogenicity to cultured fish. According to research, the electron micrograph of Lactococcus 20-92 was compared with the pathogenic strain (Enterococcus ceriolicida-KG strain). It was confirmed not to. From this, it is considered that this strain has no pathogenicity and does not have a problem of environmental pollution. This is also supported by the following literature. That is, Yoshida et al., Regarding the pathogenicity of bacterial cells to cultured fish, phagocytosis by macrophages is inhibited when there is a capsule on the surface of the bacterial cells, and as a result, the bacteria are not sterilized and the cultured fish contaminated with the bacteria Describes that it induces systemic sepsis (T. Yoshida, et al., (1996) Dis. Aquat. Org., 25, 81-86).

  Furthermore, the present strain Lactococcus 20-92 maintains the desired equol production ability (activity) even when directly fermented with milk, and does not require a special medium to maintain this equol production ability. By fermenting soy milk as it is, it has a feature that it can assimilate daidzeins in the soy milk to produce equol.

  Conventionally, there has been no reported example of lactic acid bacteria belonging to the genus Lactococcus having such an equol-producing ability. Therefore, the present invention also provides a new lactic acid bacterium having such an equol-producing ability.

(2) Daidzeins and daidzein-containing substances The daidzeins utilized by the present strain Lactococcus 20-92 include daidzein glycosides, daidzein and dihydrodaidzein. Specific examples of daidzein glycosides include, for example, daidzin. The daidzin is an isoflavone glycoside (daidzein glycoside) having daidzein as an aglycon. In the case of the daidzin, it is assimilated by the microorganism to release daidzein, which is further assimilated into dihydrodaidzein, and finally equol is produced.

  In the present invention, the above daidzeins are used as a substrate. Further, the substrate is not limited to daidzein, and various substances containing it can be used. As a representative example of the substance containing daidzein (daidzein-containing substance), soybean isoflavone can be exemplified. Soy isoflavones are already commercially available, and in the present invention, such commercially available products such as “Fujiflavone P10” (registered trademark) manufactured by Fujicco Corporation can also be used. Moreover, not only soybean isoflavones, but also plants such as kudzu, kudzu, red clove, alfalfa, and isoflavone derivatives originating from these are also included in daidzein-containing substances.

  Furthermore, as other specific examples of substances containing daidzein, in addition to the above-mentioned food materials such as soybean, kuzu, kudzu, red glove and alfalfa, processed products such as soybean flour, boiled soybean, tofu Fried, soy milk, soybean hypocotyl extract and the like, and fermented preparations thereof such as natto, soy sauce, miso, tempeh, fermented soy beverage, and the like. These all contain daidzeins. In addition to daidzeins, isoflavones having an estrogenic action such as genistein and its glycosides (genistin etc.); glycitein and its glycosides (glycitin etc.); daidzein and part of genistein are methyl Biochain A, Formonetin and the like, which are converted precursors, can be suitably used in the present invention.

(3) Composition of the present invention
(3-1) Equol-producing lactic acid bacteria-containing composition The equol-producing lactic acid bacteria-containing composition of the present invention produces equol by acting on the daidzein or daidzein-containing substance as a substrate, starting with the aforementioned lactococcus 20-92. A lactic acid bacterium belonging to the genus Lactococcus having an ability is contained as an essential component. The lactic acid bacterium as the essential component is generally a living bacterium itself, but is not particularly limited thereto. For example, a crude solution of the culture solution containing the microbial cell isolated from the culture solution or the culture solution is used. It may be a product or a refined product or a freeze-dried product thereof.

  For example, the microorganism culture solution can be typically obtained by culturing the microorganism at 37 ° C. for about 48 hours using a medium suitable for culturing the microorganism, such as an MRS medium. The cells can be obtained, for example, by centrifuging the culture solution after centrifugation for example by centrifuging the culture solution at 3000 rpm for 4 minutes at 4 ° C. These can be purified according to conventional methods. Moreover, the said microbial cell can also be freeze-dried. The freeze-dried microbial cell thus obtained can also be used as an active ingredient of the composition of the present invention.

  The composition of the present invention only needs to contain a microorganism (such as a fungus body) as the active ingredient, and is not particularly necessary, but is suitable for maintenance (or growth) of the microorganism as the active ingredient as desired. Nutritional components can also be included. Specific examples of the nutrient component include a nutrient medium for culturing each microorganism as described above, such as BHI, EG, BL, and GAM medium.

  Examples of other nutritional components include various oligosaccharides such as dairy oligosaccharide, soybean oligosaccharide, lactulose, lactitol, fructooligosaccharide, and galactooligosaccharide. The amount of these oligosaccharides to be blended is not particularly limited, but it is preferably selected from an amount range which is usually about 1-3% by weight in the composition of the present invention.

  The above-mentioned composition of the present invention exhibits a desired equol-producing ability in the body of the ingestor when ingested. In general, Japanese people have a habit of eating foods containing daidzeins, typically food materials such as soybeans, processed products thereof, fermented preparations thereof, and the like. For example, equol is produced in vivo.

  Moreover, various vitamins, trace metal elements, etc. can also be suitably added and mix | blended with this invention composition as needed. Examples of the vitamins include vitamin B, vitamin D, vitamin C, vitamin E, vitamin K (particularly MK-7 (menaquinone-7) derived from Bacillus natto) and the like. Examples of trace metal elements include zinc, selenium, iron, and manganese.

The amount of microorganisms to be blended in the composition of the present invention can be appropriately determined according to the type of lactic acid bacteria used. For example, in Lactococcus 20-92, it is preferably prepared in an amount of cell count (viable cell count) is 10 ⁸ to 10 ⁹ / 100g composition before and after. The number of bacteria is calculated by applying a diluted sample to an agar medium for culturing bacteria, performing aerobic culture at 37 ° C., and counting the number of grown colonies. The blending amount of the microorganism can be appropriately changed according to the form of the composition of the present invention to be prepared using the above amount as a guide.

(3-2) Composition of the Present Invention Containing Daidzein The composition of the present invention can further contain at least one selected from the group consisting of the aforementioned daidzeins and daidzein-containing substances, if desired. Among these daidzeins and daidzein-containing substances, soybean hypocotyls and food materials prepared from these are preferred, and water-soluble or emulsified food materials are more preferred. Examples of other preferred daidzein-containing substances include soy flour or soy milk.

  This composition is based on the inclusion of a substrate in the composition, and microorganisms assimilate the substrate in the composition in vivo even when people who have no habit of eating food ingredients such as soybeans ingest it. Thus, the desired equol can be produced.

  The amount of daidzein and / or daidzein-containing substance in the composition is not particularly limited, but it is appropriate to set it to about 10-25 mg, which is usually taken by Japanese per day.

(3-3) Equol-containing composition of the present invention The composition of the present invention may further contain equol.

  Generally, food palatability is improved by, for example, lactic acid fermentation of a food material. In addition, Lactococcus 20-92, which is a representative example of the microorganism used in the composition of the present invention, has an excellent equol production ability (activity). The present invention also provides a composition containing equol, such as fermented soymilk, which is produced by reacting daidzein-containing substances such as soymilk with the above microorganisms to assimilate daidzeins in soymilk to produce equol. To do.

  Here, as the substrate, the aforementioned various daidzeins and daidzein-containing substances can be used. Among these, a solution or an emulsion prepared from soy milk, soybean powder, or the like is preferable.

As a preferred specific example of the equol-containing composition of the present invention, soybean isoflavone or a food material containing the same is added to a suitable medium, and the microorganism of the present invention, preferably Lactococcus 20-92, is fermented in the medium. Mention may be made of the fermentation products obtained. Here, more specifically, fermentation is performed by, for example, sterilizing a substrate in a solution state, and then growing a nutrient medium capable of growing the microorganism of the present invention, such as BHI, EG, BL, GAM medium, or milk that can be used as a food, Add a certain amount of the microorganism of the present invention to soymilk, vegetable juice, etc., and ferment for 48-96 hours at 37 ° C in anaerobic or aerobic standing (pH adjuster, reducing substance if necessary) (e.g. yeast extract, vitamin K _1, etc.) can be implemented by may be added). In the above, the base mass can be about 0.01-0.5 mg / mL, and the inoculum of the microorganism can be selected from the range of about 1-5%.

  Thus, the equol-containing composition of the present invention can be produced. The composition can be a product suitable as a food product, a pharmaceutical product or the like in the form of the fermentation product itself. In addition, equol can be isolated and purified from the obtained culture according to a conventional method, and if necessary, appropriate other food ingredients can be appropriately blended to prepare an appropriate food form or pharmaceutical form. .

  The isolation and purification can be performed, for example, by a method in which the fermentation culture is adsorbed on an ion exchange resin (DIAION HP20, manufactured by Mitsubishi Kasei Co., Ltd.), then eluted with methanol and dried.

  The amount of equol in the composition of the present invention is determined according to the food form and pharmaceutical form to be prepared, and is not particularly limited, but is usually about 2-5 mg of equol in 100 g of the total composition. It is preferable to be in the range.

  It can be confirmed, for example, by the method shown in Test Example 1 described later that the resulting composition of the present invention contains equol.

  The equol-containing composition of the present invention is excellent in safety because equol as its active ingredient is a natural product, and is derived from the manufacturing process based on being prepared using lactic acid bacteria. There is an advantage that there is no risk of mixing chemicals, etc., high yield, low production cost, and excellent flavor as a food.

(3-4) Food Form The equol-producing lactic acid bacterium-containing composition of the present invention is generally prepared in a food form containing the specific lactic acid bacterium as an essential component together with a suitable edible carrier.

  Specific examples of the food composition of the present invention include beverage forms, dairy product forms (including fermented milk) other than beverage forms, solid food forms, and fungus-containing microcapsule forms. The beverage composition of the present invention includes lactic acid bacteria beverages and beverages containing lactic acid bacteria.

  Here, the terms “fermented milk” and “lactic acid bacteria beverage” shall conform to the definitions of the former Ministry of Health and Welfare “Ministerial Ordinance on Components of Milk and Dairy Products”, Article 2 37 “Hatsu Fermented Milk” and 38 “Lactic Acid Beverages”. That is, “fermented milk” refers to a milk or dairy product made into a paste or liquid obtained by fermentation with lactic acid bacteria or yeast. Accordingly, the fermented milk includes a yogurt form as well as a beverage form. The “lactic acid bacteria beverage” refers to a beverage obtained by diluting milk or a dairy product into a paste or liquid obtained by fermenting with lactic acid bacteria or yeast as a main ingredient.

  Examples of beverages containing lactic acid bacteria include fermented vegetable beverages, fermented fruit beverages, and fermented soymilk beverages. Dairy product forms other than beverage forms include card-like forms such as yogurt. Solid food forms include forms such as granules, powders (including fermented milk freeze-dried powders, etc.), tablets, effervescent preparations, gums, gummies, puddings and the like.

  The preparation to each of these forms can follow conventional methods. In addition, the carrier used in the preparation of each of these forms may be any edible carrier. In particular, a carrier having a palatable taste improving effect is preferable. Specific examples of the carrier having a good taste-improving effect include artificial sweeteners, sorbitol, xylitol and the like. Other preferred carriers include masking agents such as trehalose (Hayashibara), cyclodextrin, Benecoat BMI (Kao).

  If beverages containing lactic acid bacteria, which are preferable specific examples of food forms, are described in detail, preparations for these beverage forms can be made by using appropriate fermentation raw materials including nutrient sources for microorganisms such as vegetables, fruits, soy milk (soy emulsification). For example, by culturing microorganisms and fermenting the raw material. Vegetables and fruits as raw materials for fermentation include cuts, crushed products, ground products, juices, enzyme-treated products, diluted products and concentrates of various vegetables and fruits. Vegetables include pumpkins, carrots, tomatoes, peppers, celery, spinach, colored sweet potatoes, corn, beet, kale, parsley, cabbage, broccoli and the like. Fruits include apples, peaches, bananas, strawberries, grapes, watermelons, oranges and mandarin oranges.

  Cut and crushed vegetables and fruits, and grinds, for example after washing the above vegetables or fruits and blanching them into hot water if necessary, crusher, mixer, food processor, pulper fisher It can be obtained by cutting, crushing, and grinding using a mycoloider or the like. Juice can be prepared using a filter press, a juicer mixer, etc., for example. The juice can also be prepared by filtering the ground product using a filter cloth or the like. The enzyme-treated product can be prepared by allowing cellulase, pectinase, protopectin degrading enzyme or the like to act on the cut product, crushed product, ground product, or juice. Dilutions include those diluted 1-50 times with water. Concentrates include those concentrated 1 to 100 times by means such as freeze concentration and vacuum concentration.

  Soymilk, which is another specific example of the raw material for fermentation, can be prepared from a soybean raw material according to a conventional method. In the soy milk, for example, moistened soybeans are immersed in water, wet milled using a suitable mill such as a colloid mill, and then homogenized according to a conventional method, water-soluble soybean protein is dissolved in water. A solution and the like are also included.

  Fermentation using microorganisms can be carried out by inoculating the raw material for fermentation with the microorganism of the present invention and performing stationary culture. In the medium, if necessary, fermentation promoting substances for good growth of the microorganism of the present invention, for example, carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, fructose, nitrogen sources such as yeast extract, peptone, Vitamins and minerals can be added.

Inoculum of microorganisms, generally bacteria during fermentation material containing solution 1cc of about 1 × 10 ⁶ or more is suitable be selected from amounts that preferably is intended to include 1 × 10 ⁷ cells before and after . The culture conditions are generally selected from a fermentation temperature of about 20-40 ° C, preferably about 25-37 ° C, more preferably 37 ° C, and a fermentation time of about 8-24 hours.

In order to perform stable fermentation, a method of preparing a starter in advance, inoculating the raw material for fermentation and fermenting it is recommended. Here, as a starter, for example, typically, the bacterial cell of the present invention is applied to a raw material for fermentation that has been normally sterilized at 90 to 121 ° C. for 5 to 20 minutes, 10% nonfat dry milk added with yeast extract, and the like. Inoculated and cultured under the same conditions. The starter thus obtained usually contains about 10 ⁷ -10 ⁹ cells / g culture of the microorganism of the present invention.

Note that the lactic acid fermented product obtained as described above may have a card-like form (yogurt-like or pudding form), which can be taken as food as it is. The lactic acid fermented product in the form of a card can be made into a desired beverage form (for example, fermented soymilk drink) by further homogenizing it. This homogenization can be carried out using a general emulsifier (homogenizer). Specifically, the homogeneous structure formation, for example using a Gaulin (GAULIN) manufactured by a high pressure homogenizer (LAB40), about 200-1000kgf / cm ^2, preferably about 300-800kgf / cm ² condition, or Sanwa It can be carried out under conditions of 150 kg / cm ² or more using a machine industry homogenizer (product number: HA × 4571, H20-A2, etc.). By this homogenization, a beverage product having an excellent texture, particularly smoothness, in particular a fermented soymilk beverage product can be obtained. In this homogenization, it is necessary to dilute appropriately as necessary, add organic acids for pH adjustment, and produce beverages such as sugars, fruit juices, thickeners, surfactants, and fragrances. Various additives that are usually used can be added as appropriate. Specific examples of preferable additives and the amount thereof added (% by weight based on the weight of the carded fermented product) include, for example, glucose 8% (% by weight, the same applies hereinafter), sugar 8%, dextrin 8%, citric acid 0.1%, glycerin Mention may be made of 0.2% fatty acid esters and 0.1% fragrances.

  The lactic acid bacteria beverage of the present invention such as a fermented soymilk beverage thus obtained can be aseptically filled into a suitable container according to a conventional method to obtain a product. The product has a smooth throat texture and flavor.

The amount of administration (ingestion) is appropriately determined according to the age, sex, body weight, degree of disease, etc. of the living body ingesting it, and is not particularly limited. In general, a beverage product prepared with a microbial content of 10 ⁸ -10 ⁹ cells / mL may be taken at about 100-300 mL per day.

  Other specific examples of the composition of the present invention in the form of food include those in the form of foamed preparations. This product comprises 0.01-50% (weight%, hereinafter the same) of the microorganism of the present invention (cell lyophilized product), sodium carbonate and / or sodium bicarbonate 10-35%, and neutralizer 20-70%. It can prepare by mix | blending as a foaming component. The neutralizing agent used here is an acidic compound capable of generating carbon dioxide gas by neutralizing the sodium carbonate and sodium hydrogen carbonate. Examples of the compound typically include organic acids such as L-tartaric acid, citric acid, fumaric acid, and ascorbic acid.

  The blending ratio of the foaming component in the foamed preparation of the present invention is such that when the obtained present preparation is dissolved in water, the solution exhibits acidity, particularly about pH of about 3.5-4.6. It is good. More specifically, the proportion is selected from the range of sodium carbonate and / or sodium bicarbonate 10-35% and neutralizer 20-70%. In particular, sodium carbonate should be selected from the range of 11-31%, preferably 22-26%, and sodium hydrogen carbonate of 10-35%, preferably 20-30%. Among them, it is most preferable to use sodium hydrogen carbonate alone in the range of 20-25%. The neutralizing agent is selected from the range of 20-70%, preferably 30-40%, and most preferably L-tartaric acid is used in the range of 20-25% and ascorbic acid is used in the range of 8-15%. .

  The foamed preparation comprises the microorganism of the present invention and a foaming component as essential components, and various other additive components that are generally known, such as excipients, binders, disintegrants, lubricants, thickeners, surface activity. Agents, osmotic pressure regulators, electrolytes, sweeteners, fragrances, pigments, pH regulators and the like may be appropriately added and blended as necessary. Examples of the additive include starches such as wheat starch, potato starch, corn starch and dextrin; sugars such as sucrose, glucose, fructose, maltose, xylose and lactose; sugar alcohols such as sorbitol, mannitol, maltitol and xylitol Glucose rearranged glycosides such as coupling sugar and palatinose; excipients such as calcium phosphate and calcium sulfate; starch, saccharides, gelatin, gum arabic, dextrin, methylthycellulose, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropylcellulose, xanthan gum , Pectin, tragacanth gum, casein, alginic acid binder or thickener; leucine, isoleucine, L-valine, sugar ester, hydrogenated oil, stearic acid, magnesium stearate, talc, ma Lubricants such as Logol; disintegrating agents such as crystalline cellulose ("Avicel" manufactured by Asahi Kasei Corporation), carboxymethylcellulose (CMC), sodium carboxymethylcellulose (CMC-Na), potassium carboxymethylcellulose (CMC-Ca); polyoxyethylene sorbitan Examples thereof include surfactants such as fatty acid esters (polysorbate) and lecithin; dipeptides such as aspartame and alitame; and other sweeteners such as stevia and saccharin. These can be used by appropriately selecting appropriate amounts in consideration of the relationship with essential components, the properties of the preparation, the production method, and the like.

  Further, vitamins, particularly cyanocobalamin and ascorbic acid (vitamin C) can be added and blended in the foamed preparation of the present invention. The blending ratio is not particularly limited, but for vitamin C, it is usually selected from an amount of up to 30%, preferably about 5-25%.

  The production method of the foamed preparation of the present invention can be basically the same as the production method of this ordinary foamed tablet. That is, the preparation of the present invention in the form of an effervescent tablet can be prepared according to a direct powder compression method, a dry type or a wet granule compression method, etc. by weighing and mixing predetermined amounts of each component.

  The preparation of the present invention thus obtained becomes a beverage form suitable for oral administration by simply adding it to water, and this is orally administered.

  The amount of administration (ingestion) is appropriately determined according to the age, sex, body weight, degree of disease, etc. of the living body to which it is applied, and is not particularly limited, but generally about 1.5-6.0 g per tablet. The effervescent tablet of the present invention prepared in 1-2 may be dissolved in 100-300 mL of water at a time and taken.

The particularly preferred mixing ratio of daidzein or daidzein-containing substance as a substrate in the composition of the present invention, a specific lactic acid bacterium, and other components added and blended as necessary is 100 dag of daidzein or daidzein. It is desirable that the substance-containing substance is in the range of about 10-50 mg in terms of daidzein, 10 ⁹ to ¹⁰ 10 lactic acid bacteria (as the number of viable bacteria), and oligosaccharides in the range of about 1-5 g.

  As described above, the equol-producing lactic acid bacterium-containing composition of the present invention contains microorganisms (mainly viable bacteria), and therefore, when commercializing the composition, it is necessary to adopt conditions such as heating and pressurization. Not very good. Therefore, in preparing the composition of the present invention into a product form such as a bar, granule, powder, tablet, etc., the microorganism is directly formulated as a freeze-dried cell, or the freeze-dried cell is processed with an appropriate coating agent. It is preferable to use it.

  However, the equol-producing lactic acid bacterium-containing composition of the present invention does not necessarily contain live bacteria. In the case where the composition of the present invention in which live bacteria and daidzein that can be assimilated thereof are blended has already produced the desired equol, the composition is heat sterilized according to a conventional method, and the composition You may kill the microbe which exists in a thing. According to the adoption of such heat sterilization treatment, in the case of the composition of the present invention in which viable bacteria and daidzein which can be assimilated are blended, the viable bacteria present in the composition are stored in the composition. In some cases, it is possible to avoid the risk of degrading the taste, flavor, etc. due to excessive fermentation in the middle or distribution process.

(3-5) Pharmaceutical Form The equol-producing lactic acid bacterium-containing composition of the present invention is generally prepared in a pharmaceutical preparation form containing the specific lactic acid bacterium as an essential component together with an appropriate pharmaceutically acceptable preparation carrier. .

  As a pharmaceutical carrier, diluents or excipients such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, lubricants and the like that are usually known in this field Can be illustrated. These are appropriately selected and used depending on the dosage unit form of the preparation to be obtained.

  Various forms can be selected as the dosage unit form of the pharmaceutical preparation. Typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories and the like.

  In the case of forming into a tablet form, the above-mentioned preparation carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc .; water, ethanol , Propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone and other binders; sodium carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried starch, alginic acid Disintegrating agents such as sodium, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate; polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate Detergents such as sucrose, stearin, cocoa butter, hydrogenated oil; absorption promoters such as quaternary ammonium base and sodium lauryl sulfate; humectants such as glycerin and starch; Adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; lubricants such as purified talc, stearate, boric acid powder and polyethylene glycol can be used.

  Further, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

  When forming into a pill form, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, etc. as binders; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol; Disintegrants such as laminaran and agar can be used.

  In forming into a suppository, for example, polyethylene glycol, cacao butter, higher alcohol, higher alcohol esters, gelatin, semi-synthetic glyceride and the like can be used as a pharmaceutical carrier. Capsules are usually prepared by mixing the microorganism of the present invention with the various preparation carriers exemplified above and filling them into hard capsules, soft capsules and the like according to conventional methods.

  Furthermore, in the preparation of the present invention, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other medicines may be contained as necessary.

The amount of the microorganism of the present invention to be contained in the preparation of the present invention is not particularly limited and is appropriately selected from a wide range. Usually, it is good to contain about 10 ⁸ -10 ¹⁰ pieces / g in a pharmaceutical preparation.

  The administration method of the pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient age, sex and other conditions, the degree of disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally, and suppositories are administered rectally.

  The dosage of the above pharmaceutical preparation is appropriately selected depending on its usage, patient age, gender and other conditions, disease severity, etc., but the amount of the microorganism of the present invention, which is usually an active ingredient, is about 0.5-kg / kg body weight per day. The dosage is preferably about 20 mg, and the preparation can be administered in 1 to 4 divided doses per day.

  In addition, the composition of the present invention can reach the lower gastrointestinal tract alive or settle as a resident bacterium by ingestion (administration), and thus has the desired effect. A particularly preferred preparation is in the form of an enteric-coated tablet, whereby microorganisms can reach the intestine without being invaded by gastric acid.

  The equol-producing lactic acid bacteria-containing composition of the present invention thus obtained is useful for the prevention and treatment of symptoms such as osteoporosis and climacteric disorder associated with indefinite complaints or menopause in middle-aged and elderly women. Such prevention and treatment is carried out by administering or ingesting an effective amount of the above-mentioned composition of the present invention to a middle-aged woman who is required to do so. The effective amount is not particularly limited as long as administration of the composition of the present invention can prevent and treat symptoms such as osteoporosis and menopause associated with indefinite complaints or menopause in middle-aged women. In general, the amount of urinary equol excretion in a human who has taken the composition of the present invention is 5 μmole (about 1.2 mg) / day or more.

Hereinafter, preparation examples of the equol-producing lactic acid bacteria-containing composition of the present invention will be described as examples in order to describe the present invention in more detail, but the present invention is not limited to these examples.
Example 1
(1) Preparation of Fermented Soymilk Beverage Each component of the following formulation was weighed and mixed to prepare the composition of the present invention in the form of a fermented soymilk beverage.

100mL water-soluble soy protein fermentation culture
Vitamins and minerals Appropriate amount Fragrance Appropriate amount Water Appropriate amount Total amount 150mL

The above fermented culture of water-soluble soy protein was prepared by adding 10 ⁸ -10 ⁹ lactococcus 20-92 (FERM BP-10036) to a solution of 13 g of water-soluble soy protein in 100 mL of water, and -Fermented for 48 hours. The water-soluble soy protein used contains about 1-2 mg of daidzein in terms of daidzein in 1 g.

(2) Preparation of fermented milk Each component of the following formulation was weighed and mixed to prepare the composition of the present invention in the form of fermented milk.

Lactococcus 20-92 Fermented milk 100mL
Vitamins and minerals Appropriate amount Fragrance Appropriate amount Water Appropriate amount Total amount 150mL

Lactococcus 20-92 fermented milk contains 10 ⁸ -10 ⁹ lactococcus 20-92 (FERM BP-10036) to 1 liter of milk (non-fat milk solid content 8.5% or more, milk fat content 3.8% or more). In addition, it was fermented at 37 ° C for 24-48 hours.

(3) Fermented soymilk lyophilized powder preparation Lactococcus 20-92 (FERM BP-10036) to about 10 ⁹ using soy milk (soy solids content of 10%, daidzein content: 10-15 mg as daidzein equivalent amount) 100 g Was lactic acid fermented at 37 ° C. for 72-96 hours to produce equol. This was freeze-dried to obtain a powder. The equol content in the powder was 0.1-0.3% by weight as a result of HPLC measurement.

  Using the powder, each component of the following formulation was weighed and mixed to prepare the composition of the present invention (food form and pharmaceutical form) in powder form.

Fermented soymilk freeze-dried powder 2.2g
(Containing equol 0.005g)
Excipient (corn starch) 17g
Vitamins and minerals Appropriate amount Fragrance Appropriate amount Total amount 20g

(4) Preparation of powder The components of the following formulation were weighed and mixed to prepare the composition of the present invention in powder form (food form and pharmaceutical form).

Lactococcus 20-92 freeze-dried powder 4.1g
Excipient (lactose) 1.0g
Vitamins and minerals Appropriate amount Fragrance Appropriate amount Total amount 20g

Lactococcus 20-92 freeze-dried powder was collected after culturing Lactococcus 20-92 (FERM BP-10036) in an appropriate liquid medium (MRS) capable of growing (37 ° C, 24-48 hours). Was suspended in 10% skim milk and then freeze-dried, and its cell content was 10 ⁹ -10 ¹⁰ cells / g.

  In addition, the above-mentioned powder can be made into a daidzein-containing powder by further blending 4.1 g of roughly purified soybean isoflavone powder.

  According to the intake of daidzein-containing powder thus obtained, it is confirmed that urinary equol excretion of about 5 μmole (about 1.2 mg) per day can be observed, and therefore equol corresponding to the excretion amount can be produced in the body. The

(5) Preparation of granules The components of the following formulation were weighed and mixed to prepare the composition of the present invention in the form of granules (food form and pharmaceutical form).

Crude soy isoflavone powder 4.1g
Lactococcus 20-92 freeze-dried powder 1.0g
Sucrose ester appropriate amount Vitamin / mineral appropriate amount Fragrance appropriate amount Total amount 20g

  As the Lactococcus 20-92 freeze-dried powder, the same as the above (1) was used.

  By taking this product, daidzein and equol-producing bacteria can reach the large intestine together, thus producing equol in the large intestine.

  Examples of tests conducted on the equol-producing lactic acid bacteria of the present invention will be given below.

Test example 1
Proliferation and equol production (activity) and yield test
(1) Test method After culturing Lactococcus 20-92 (10 ⁷ -10 ⁹ / g) in 5 ml of BHI broth (liquid medium for growth (basal medium)) at 37 ° C for 24 hours, Dilutions were prepared by diluting to 10 ² and 10 ⁴ with basal medium.

  Mix 0.2 mL each of the culture solution after completion of the culture and each dilution thereof with 5 mL each of daidzein-containing basal medium (BHI broth added with daidzein in an amount of 10 μg / mL), milk and soy milk, The cells were cultured at 37 ° C under anaerobic conditions. The culture time was 8 hours, 24 hours, 48 hours, 72 hours, and 96 hours for basal medium containing 10 μg / mL daidzein and soy milk, and 8 hours, 24 hours, and 48 hours for milk.

  Before the start of culture and at the end of each culture, 0.1 mL and 0.2 mL of the culture solution were sampled and used for the measurement of the number of bacteria and the measurement of equol production ability (activity), respectively. Further, for 10 μg / mL daidzein-containing basal medium and soymilk, 0.5 mL of the culture solution was sampled before the start of culture and at the end of each culture, and the amount of equol produced in the solution was measured.

The number of bacteria was measured as follows. That is, dilute 0.1 mL of each sample with PBS (-) solution (Nissui) to make 10 ⁴ , 10 ⁵ , 10 ⁶ and 10 ⁷ dilutions, and 0.1 mL of each dilution to GAM agar. After coating, the cells were cultured under aerobic conditions at 37 ° C. for 24 hours, and the number of colonies growing on the medium was counted to obtain the number of bacteria.

The equol production ability (activity) was measured as follows. That is, 0.2 mL of each sample was mixed with 5 mL of daidzein-containing basal medium (3 each) and cultured at 37 ° C. for 96 hours under anaerobic conditions. After completion of the culture, 0.5 mL of each culture was sampled and mixed with ethyl acetate. After extraction twice with 5 mL, daidzein, dihydrodaidzein (intermediate) and equol in the extract were measured by HPLC, and the proportion of equol was calculated from the total amount. The obtained results were scored in the following 5 levels, and the average score of 3 samples was used as an index of equol production ability (activity).
4: Over 90% equol,
3: Equol production, daidzein decreased to less than 50% (with intermediate),
2: Equol production, daidzein 50% or more remains (with intermediate),
1: Intermediate formation, no equol formation
0: No intermediate and equol formation, daidzein reduction.

The equol production was measured as follows. That is, 0.5 mL of each sample was extracted twice with 5 mL of ethyl acetate, and daidzein, dihydrodaidzein (intermediate) and equol in the extract were measured by HPLC. Each concentration was calculated as equol production.
(2) Test results
(2-1) The results of examining the number of bacteria (proliferation) are shown in FIG.
In the figure, (1) is the result when using daidzein-containing basal medium, (2) is the result when using soy milk, and (3) is the result when using milk. In each figure, the horizontal axis represents the culture time (hr), and the vertical axis represents the viable cell count (Log cfu / ml).

From the results shown in each figure, the growth of this strain was good, and any of daidzein-containing basal medium, soy milk, and milk reached a steady state in 8 hours of culture regardless of the inoculation amount. Number of bacteria, maintaining a 10 ^9.1-9.4 cells / mL daidzein-containing basal medium, 10 ^8.5-8.7 cells / mL in soy milk, the milk was found to be maintained 10 ^8.0-8.4 cells / mL.

(2-2) The results of determining equol production ability (activity) are shown in FIG.
In FIG. 2, (1) is the result when using daidzein-containing basal medium, (2) is the result when using soy milk, and (3) is the result when using milk. In each figure, the horizontal axis indicates the culture time (hr), and the vertical axis indicates the score.

  From the results shown in the figure, it was confirmed that the equol production ability (activity) tended to increase with time in any of the basic medium containing daidzein, soy milk and milk. Moreover, it was confirmed that the equol production ability (activity) of this strain was maintained even when milk and soy milk were used.

(2-3) Measurement result of equol production The results of measurement of the amount of equol produced in the basic medium containing daidzein and soy milk (approximately 80 μg / mL in terms of daidzein equivalent) are as shown in FIG.

  In FIG. 3, (1) shows the results when using daidzein-containing basal medium, and (2) shows the results when using soymilk. In each figure, the horizontal axis indicates the culture time (hr), and the vertical axis indicates the equol concentration (μg / ml).

  In both media, equol production was observed from 48 hours after the start of culture. When soymilk was used, a difference in the amount of equol produced due to changes in the inoculum was observed, and in particular, a significant amount of equol produced at 57.0 μg / mL was observed in 96 hours of culture when inoculated with 4.00%.

  In soy milk, 90% or more of daidzein, which is a substrate for equol, exists as a glycoside (in a state where glucose is bound), but the peak of the glycoside disappears on the measured chromatogram. From this, it is considered that this strain metabolizes the daidzein to equol after degrading the glycoside (β-glucosidase activity) to produce daidzein.

Test example 2
Equol production pathway by Lactococcus 20-92
(1) Test method After culturing Lactococcus 20-92 strain (10 ⁷ / g) in 5 ml of BHI broth (liquid medium for growth, basal medium) under anaerobic condition at 37 ° C for 24 hours, 0.2 ml of culture solution Was mixed with 5 mL of daidzein-containing basal medium and cultured at 37 ° C. under anaerobic conditions. The culture time was 8 hours, 24 hours, 30 hours, 36 hours, 48 hours, 51 hours, 54 hours, 60 hours, 84 hours and 96 hours.

  0.5 mL was sampled before the start of culture and at the end of each culture, and the concentrations of daidzein, dihydrodaidzein (intermediate) and equol in the sample were measured.

(2) Results The results obtained are shown in FIG.

  FIG. 4 shows changes in concentrations of daidzein (left), dihydrodaidzein (middle) and equol (right) over time. In the figure, the horizontal axis indicates the culture time (hr), and the vertical axis indicates the concentration (μg / ml) of each substance.

  The results shown in FIG. 4 confirm that the daidzein concentration begins to decrease at 48 hours after culturing, the intermediate dihydrodaidzein appears at 48-60 hours, and equol is produced at 48 hours. . It can also be seen that the metabolism of daidzein to equol is almost completed at 60 hours.

  It was confirmed that the metabolism of daidzein to equol occurred via dihydrodaidzein as an intermediate, but it was speculated that dihydrodaidzein formation and metabolism to equol occurred in parallel.

Test example 3
Low temperature stability of fermented milk containing Lactococcus 20-92
(1) Test method Lactococcus 20-92 strain was cultured for 24 hours at 37 ° C under anaerobic conditions in 5 mL of growth medium (basic medium), and then 1L and 2L of milk and commercially available skim milk (10% solids) 1 L was inoculated with 4%, and statically cultured at 37 ° C. for 48 hours under aerobic conditions. After incubation, it was stored at 4 ° C.

  For milk, equol production ability (activity) was measured weekly at the end of the culture and up to 4 weeks after storage at 4 ° C. Furthermore, only two of them were stored until the 42nd and 51st days, and the activity was measured.

  For skim milk, the activity was measured at the end of the culture and at 1 week and 34 days after low-temperature storage at 4 ° C.

  The activity before the start of culture and at the time of each storage was inoculated with 4% (0.2 mL) of 3 mL of basal medium containing 10 μg / ml daidzein according to the method described above, at 37 ° C under anaerobic conditions for 96 hours. After culturing, it was judged (scored) from the concentrations of daidzein, dihydrodaidzein (intermediate) and equol in the medium.

(2) Results The results are shown in FIG. In FIG. 5, the horizontal axis indicates the storage period (days), and the vertical axis indicates the score.

  From the results shown in the figure, it is clear that in both 1L and 2L of milk, equol-producing ability (activity) is maintained up to 4 weeks under low-temperature storage at 4 ° C after completion of the culture. It was also confirmed that 2L of milk maintained the activity until the 51st day after examining its storage stability at 4 ° C. It is clear that commercially available skim milk 1 L also maintains equol-producing ability (activity) until the 34th day after examination after examination at 4 ° C. under low temperature storage.

  From the above, fermented milk prepared using Lactococcus 20-92 strain can maintain its activity even under low-temperature storage, so it is considered that it can be handled as a food from the viewpoint of distribution.

  From the results obtained in Test Example 1-3, the relationship between the growth of Lactococcus 20-92 strain, equol production ability (activity) and equol production amount is summarized as shown in FIG.

  That is, although culturing conditions differ depending on the medium to be used, the equol production ability (activity) can be maintained in both the growth phase and the stationary phase. On the other hand, with regard to equol production, it is considered that the enzyme is expressed or activated after a certain amount of time has passed since the steady state to produce equol.

Test example 4
Gastric juice tolerance test of fermented milk prepared using Lactococcus 20-92
(1) Test method After culturing Lactococcus 20-92 strain in anaerobic bacterial growth medium (BHI broth, basal medium) under anaerobic condition at 37 ° C for 24 hours, culture (10 ⁹ cells / g) was inoculated with 4% of 1 L of milk and statically cultured at 37 ° C. for 48 hours under aerobic conditions. After culturing, the one stored at 4 ° C. was used as fermented milk and subjected to the following test.

  As an artificial gastric juice, 50 mM glycine / hydrochloric acid buffer (pH 2.5 and pH 3.0) containing 0.045% pepsin was prepared. As a control, 50 mM glycine / hydrochloric acid buffer (pH 6.0) was prepared.

  1 mL of fermented milk that had been stored at low temperature was added to 9 mL of the prepared artificial gastric juice, and the mixture was incubated (cultured) in a static condition in a 37 ° C. thermostatic bath in a stationary state.

  The culture time is 1 hour, 2 hours, and 3 hours, and 0.1 mL and 0.2 mL of the culture are sampled before the start of culture and at the end of each culture, respectively, and each sample is counted (in the case of 0.1 mL) and equol production ability It was used for (activity) measurement (in the case of 0.2 mL).

To measure the number of bacteria, 0.1 mL of each cultured culture was sampled according to the method described in Test Example 1- (1), and 10 ⁴ , 10 ⁵ , 10 ⁶ were collected using a PBS (−) solution manufactured by Nissui. After dilution to 10 ⁷ times, 0.1 mL of each diluted solution was applied to a GAM agar medium, cultured at 37 ° C. for 24 hours under aerobic conditions, and the number of colonies grown on the GAM agar medium was counted. Asked.

  In accordance with the method described in Test Example 1- (1), equol production ability (activity) is measured by inoculating 0.2 mL (4%) of a sample into 5 mL (three) of daidzein-containing basic medium for 96 hours. After culturing at 37 ° C. under anaerobic conditions, judgment (scoring) was made from the concentrations of daidzein, dihydrodaidzein (intermediate) and equol in the medium.

(2) Results The obtained results are shown in A (viable count results) and B (equol concentration) in FIG.

  In FIG. 7-A, the horizontal axis represents the culture time (hr), and the vertical axis represents the viable cell count (Log cfu / ml in milk).

  In FIG. 7-B, the horizontal axis represents culture time (hr), and the vertical axis represents equol activity (score).

From the results shown in FIGS. 7-A and 7-B, the following can be understood. That is, the number of viable bacteria was maintained at 10 ⁸ cells / mL for 3 hours in a pH 6.0 buffer solution, and in this case, equol production ability (activity) was also maintained. In artificial gastric juice at pH 3.0, the viable cell count can be maintained up to 3 hours of culture, in which case the activity is also maintained. On the other hand, in artificial gastric fluid at pH 2.5, a significant decrease in the number of viable bacteria is observed from the second hour of culture, and the activity disappears.

  In general, probiotics (microorganisms that live in the intestine and have physiological effects) on the market, when examined in the same test system, the viable cell count does not change at pH 3.0, but pH 2. In 5 it is reported that it drops significantly. Therefore, fermented milk prepared using the Lactococcus 20-92 strain reaches the intestine and is active in the lower intestine or the large intestine because it can be passed through if it has gastric juice resistance of pH 3.0. Can be maintained.

Test Example 5
Bile tolerance test of Lactococcus 20-92 Bile tolerance test was performed using Vitech GPI card (Nihon Biomeryu Co., Ltd.) as an indicator of the viability of this strain in 10% and 40% bile.

(1) Test method Lactococcus 20-92 strain was smeared (10 ^8-9 pieces) on 5% sheep blood-added trypticase soy agar medium and aerobically cultured at 37 ° C for 24 hours. Colonies that grew on the cultured medium were picked and a homogeneous suspension was prepared using 0.5% sterile saline. This was put into a Vitec GPI card, and after culturing at 35 ° C. for 15 hours, the viability of this strain in the presence of bile was determined using a dye (pH indicator). The bile was poured into the card after dissolving a predetermined amount of bile powder in sterile distilled water.

(2) Results As a result of the above test, the Lactococcus 20-92 strain showed viability in 10% and 40% bile. From this, the tolerance to 40% bile was confirmed.

Test Example 6
Hemolytic test of Lactococcus 20-92 strain
(1) Test method Lactococcus 20-92 strain was smeared on 5% sheep blood-added Trypsease soy agar medium (10 ^8-9 pieces), and anaerobic conditions (N ₂ : CO ₂ for 24 to 48 hours at 37 ° C) : H ₂ = 8: 1: 1). The periphery of the colonies grown on the culture medium after the culture was observed, and hemolysis was determined by decomposing (decolorizing or discoloring) blood components.

(2) Results As a result of the above test, no decolorization of blood components (appearance of a transparent and colorless zone) was observed around the grown colonies. Therefore, Lactococcus 20-92 was not β-hemolytic and It was determined to be safe.

Test Example 7
When the lactic acid bacteria ingested into the cell invasive enzyme activity test of Lactococcus 20-92 invades into the living body, a host-side factor may be a decrease in intestinal membrane defense function or damage to the membrane itself. A factor on the bacteria side is that it has an enzyme activity (cell invasive enzyme) that binds lipids and proteins to break down proteoglycans constituting the intestinal membrane.

  This test examined whether the Lactococcus 20-92 strain had collagenase (gelatinase), hyaluronidase and sialidase (neuraminidase) activities as cell invasive enzymes, and was performed as follows.

(1) Test method Lactococcus 20-92 strain was smeared on blood agar medium (smear amount: 10 ^8-9 ) and anaerobic condition (N ₂ : CO ₂ : H ₂ ) at 37 ° C for 24-48 hours = 8: 1: 1).

  Colonies that grew on the cultured medium were picked and suspended in sterile distilled water to prepare a homogeneous suspension. About the obtained suspension, the presence or absence of collagenase (gelatinase) activity was determined by using Apikenki (Nihon Biomeryu Co., Ltd.) identification kit with gelatin degradation as an index.

  In addition, whether or not the Lactococcus 20-92 strain has hyaluronidase and sialidase (neuraminidase) activity was tested in a Tris-HCl buffer (pH 7.0) using hyaluronic acid and sialic acid as substrates, respectively. The 92 strains were incubated (37 ° C., aerobic conditions for 15 minutes for sialidase activity and 24 hours for hyaluronidase activity) to determine whether each substrate concentration was reduced.

(2) Results The Lactococcus 20-92 strain did not show any activity of collagenase (gelatinase), hyaluronidase and sialidase (neuraminidase).

  Therefore, since this strain lacks the cell invasive enzyme which is one factor of infectivity, it was confirmed that the strain is highly safe in terms of infectivity.

Test Example 8
Acquiring resistance (mutation) to vancomycin resistance test bacteria antibiotics has become a problem in recent years. Patients infected with bacteria that have acquired resistance to such antibiotics often die due to the lack of antibacterial effects of antibiotics. In particular, the emergence of resistant bacteria (VRE) against the antibiotic vancomycin is now a serious problem in the medical field. In addition, when the microorganism ingested in the body has a vancomycin resistance gene, when the microorganism reaches the colon in the intestine and comes into contact with a toxic or infectious fungus (pathogenic fungus), the vancomycin resistant gene moves to the pathogenic fungus, Thus, there is a concern that pathogens acquire vancomycin resistance. For this reason, at least viable bacteria that can be used as probiotics need not be vancomycin-resistant bacteria.

  This test examined the sensitivity of Lactococcus 20-92 strain to vancomycin and was performed as follows.

(1) Test method The sensitivity test for vancomycin was performed using Sensi Disc (manufactured by Nippon Becton Dickinson Co., Ltd.). Specifically, the Lactococcus 20-92 strain was smeared on a GAM agar medium (smear amount: 10 ^8-9 ), a disc containing 30 μg of vancomycin was placed on the medium, and aerobically cultured at 37 ° C. for 24 hours. After culturing, the diameter of the inhibition circle formed around the disc was measured and judged from the judgment table.

(2) Results The inhibition circle diameter of Lactococcus 20-92 strain was 11.9 ± 0.2 mm, which was judged as sensitive based on the judgment table (10 mm or more was judged as sensitive). From this result, it was confirmed that this strain is not a vancomycin-resistant bacterium and is therefore highly safe in this respect.

  Hereinafter, an example of producing equol from daidzein using the lactic acid bacteria of the present invention will be described as Example 2.

Example 2
Manufacture of equol Prepare 1 ml of a suspension of 10 ⁷ to 10 ^{9 of} Lactococcus 20-92 strain (FERM BP-10036) in GAM medium for anaerobic bacteria culture. In addition to anaerobic concentration of about 2.2%, the cells were cultured anaerobically at 37 ° C. for 72 to 96 hours, and the amount of equol produced in the culture was measured by HPLC. The daidzein content in the soymilk is 95 μg / mL in terms of daidzein equivalent.

  As a result, production of equol 10.7 ± 6.3 μg / mL (average value of three times ± standard deviation) was confirmed in the obtained soymilk culture.

  From this, it is clear that equol can be efficiently and inexpensively produced from daidzein in food materials by using the microorganism of the present invention.

FIG. 1 is a graph showing the relationship between the culture time determined according to the method shown in Test Example 1 and the number of viable bacteria. FIG. 2 is a graph showing the relationship between the culture time determined according to the method shown in Test Example 1 and equol production ability (score). FIG. 3 is a graph showing the relationship between the culture time determined according to the method shown in Test Example 1 and the amount of equol production. FIG. 4 is a graph showing changes over time of daidzeins and equol in the culture determined according to the method shown in Test Example 2. FIG. 5 is a graph showing the relationship between the storage period determined according to the method shown in Test Example 3 and equol production ability (score). FIG. 6 is a graph showing the state of equol-producing bacteria (proliferation, equol production ability, and equol production amount change) with the culture time obtained from the test shown in Test Example 1-3. FIG. 7 is a graph showing the relationship between the culture time determined according to the method shown in Test Example 4 and the number of viable bacteria.

Claims (9)

Contains Lactococcus garvieae derived from human feces, which has the ability to assimilate at least one daidzein selected from the group consisting of daidzein glycosides, daidzein and dihydrodaidzein to produce equol A composition containing equol-producing lactic acid bacteria. The composition according to claim 1 , further comprising at least one selected from the group consisting of daidzeins and daidzein-containing substances. The composition according to claim 2 , wherein the daidzein-containing substance is soy flour or soy milk. A composition according to claim 2 in the form of a beverage or dairy product. The composition according to any one of claims 2 to 4 , further comprising equol. The composition according to claim 5 , which is in the form of a fermented soymilk. Lactococcus galbier, which has the ability to assimilate at least one daidzein selected from the group consisting of daidzein glycoside, daidzein and dihydrodaidzein to produce equol, and is derived from human feces ( Lactococcus garvieae). At least one selected from the group consisting of daidzeins and daidzein-containing substances has the ability to produce equol by assimilating at least one daidzein selected from the group consisting of daidzein glycoside, daidzein and dihydrodaidzein , A method for producing equol, characterized by allowing Lactococcus garvieae derived from human feces to act The method according to claim 8 , wherein the daidzein-containing substance is soy flour or soy milk.

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