JP2009124943A - New lactobacillus, lactobacillus composition and plant extract as well as method for producing plant extract and low molecular weight polyphenol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new and safely ingestible Lactobacillus that has strong tannase activity and no gallic acid decarboxylase activity, and can suitably colonize in the intestine, and to provide a Lactobacillus composition containing the Lactobacillus, as well as a method for producing plant extract and a low molecular weight polyphenol using the Lactobacillus. <P>SOLUTION: Disclosed is a Lactobacillus that has tannase activity and no gallic acid decarboxylase activity, wherein the Lactobacillus has inulin assimilation. Any one of Lactobacillus plantarum 22A-1 (FERM AP-21409), Lactobacillus plantarum 22A-3 (FERM AP-21411) and Lactobacillus plantarum 22B-2 (FERM AP-21410) is desirable as the Lactobacillus. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention is a novel lactic acid bacterium having strong tannase activity, having no gallic acid decarboxylase activity, being able to be suitably established in the intestine, and being able to be safely ingested, a lactic acid bacterium composition and plant extract containing this lactic acid bacterium, and The present invention relates to a plant extract using this lactic acid bacterium and a method for producing a low molecular weight polyphenol.

  Tannins are plant-derived polyphenols that are a group of compounds that react with proteins, basic substances, metals, and the like to form precipitates that are sparingly soluble in water. As its action, various physiological actions such as an astringent action, a skin tanning action, an antitumor action and an antioxidant action are known. Tannins are roughly classified into hydrolyzable tannins in which gallic acid or ellagic acid and sugar are ester-bonded, and condensed tannins in which flavonoid compounds are polymerized.

Catechins are a kind of condensed tannin, and various physiological actions such as antitumor action, antioxidant action, anti-aging action, and blood pressure lowering action are known. Catechins are roughly classified into gallate-type catechins having a galloyl group and non-gallate-type catechins having no galloyl group because of their structures. Examples of the gallate catechin include epigallocatechin gallate (EGCg), epicatechin gallate (ECg), catechin gallate (Cg), gallocatechin gallate (GCg) and the like. Non-gallate catechins include epigallocatechin (EGC), epicatechin (EC), catechin (C), gallocatechin (GC) and the like. Catechins are known to be abundant in green tea.
According to a report from the Vegetable Tea Leaf Research Institute, Functional Analysis Department, and Tea Quality Chemistry Laboratory in 2005, gallate-type catechins show high reactivity with pectin and proteins. Moreover, as a result of measuring AUC (area under the blood concentration-time curve) of catechins after ingesting tea, it has been reported that EGCg has lower AUC than EGC (for example, non- Patent Document 1). These facts suggest that gallate-type catechins have a reduced absorption efficiency from the intestinal tract by forming a complex with pectin and protein.

  Tannase (tannin acylhydrolase, EC 3.1.1.20) is an enzyme that catalyzes a reaction of hydrolyzing the ester bond of hydrolyzed tannin. Tannase also catalyzes a reaction that hydrolyzes the ester bond of gallate catechin. It has been reported that tannase is contained in Aspergillus oryzae, Aspergillus niger and the like, which are filamentous fungi.

  Conventionally, lactic acid bacteria were thought to have no tannase activity, but some lactic acid bacteria were reported to have tannase activity (see, for example, Patent Document 1). Therefore, the lactic acid bacterium having tannase activity is used for hydrolysis of gallate catechin and hydrolyzable tannin during the production of food or in the intestine for the purpose of improving the absorption efficiency of gallate catechin and hydrolyzable tannin. It is expected that.

  However, the lactic acid bacteria described in Patent Document 1 have tannase activity, but also have gallic acid decarboxylase activity. Gallic acid decarboxylase is an enzyme that decarboxylates gallic acid to produce pyrogallol. It is known that low molecular weight polyphenols such as gallic acid and ellagic acid that are liberated after tannase treatment of hydrolyzable tannin or gallate-type catechin have an antioxidant effect. In addition, when gallic acid and pyrogallol are compared, it has been reported that pyrogallol has a higher DNA damage ability and is likely to cause colorectal cancer (see, for example, Patent Document 2). Accordingly, there is a need for a lactic acid bacterium that can produce low molecular weight polyphenols such as gallic acid and ellagic acid and that does not convert the produced gallic acid into pyrogallol.

In recent years, a strain that has tannase activity and does not have gallic acid decarboxylase activity has been reported in Lactobacillus plantarum, which is a lactic acid bacterium (see, for example, Non-Patent Documents 2 and 3). According to the said nonpatent literatures 2 and 3, the lactic acid strain which can improve said problem can be provided.
However, the Lactobacillus plantarum strain described in Non-Patent Documents 2 and 3 that has tannase activity and does not have gallic acid decarboxylase activity does not necessarily have strong tannase activity.

Inulin, on the other hand, is a structure in which fructose is mainly polymerized. It reaches the intestine without being digested in the stomach and duodenum, and is preferably used for the growth of intestinal bacteria. Attention has been paid. Inulin is abundantly contained in chicory, garlic, burdock and the like, and in recent years, it is often added to health foods. Therefore, a bacterium having inulin assimilation ability can assimilate inulin in the intestine, so that it is easy to grow and settle. Such properties are strongly sought for bacteria that are beneficial in the intestine, such as lactic acid bacteria.
However, it is generally known that Lactobacillus plantarum has low inulin assimilation properties (see, for example, Non-Patent Documents 2 and 4). None of the Lactobacillus plantarum strains described in Non-Patent Documents 2 and 3 that have tannase activity and do not have gallic acid decarboxylase activity have inulin assimilation properties.

  Therefore, a new lactic acid strain that has strong tannase activity, does not have gallic acid decarboxylase activity, can be well established in the intestine, and can be safely ingested has not yet been provided, and its prompt provision is strong. The current situation is what is required.

JP-T-2006-521817 Japanese Patent No. 3822813 Henning SM, Niu Y, Lee NH, Thomas GD, Minutti RR, Wang H, Go VL, Heber D. Bioavailability and antioxidant activity of tea flavanols after consumption of green tea, black tea, or a green tea extract supplement. Am J Clin Nutr. 2004; 80: 1558-64. Osawa, R.A. K. Kuroiso, S .; Goto, and A.A. Shimizu (2000). Isolation of tannin-degrading lactobacilli in humans and fermenter foods. Applied and Environmental Microbiology. 66 (7): 3093-3097. Nishitani, Y. et al. , Sasaki, E .; , Fujisawa, T .; , Osawa, R (2004). Genotypic Analyzes of Lactobacilli with a Range of Tannase Activities Isolated from Human Faces and Fermented Foods. System. Appl. Microbiol. 27: 109-117. Kandler, O .; And Weiss, N.M. 1986. Genus Lactobacillus. In: Sneath, P.M. H. A. Mair, N .; S. Sharpe, M .; E. and Holt, J .; G. , Editors, 1986. Bergey's Manual of Systematic Bacteriology vol. 2, Williams & Wilkins, Baltimore, pp. 1209-1234.

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention is a novel lactic acid bacterium having strong tannase activity, having no gallic acid decarboxylase activity, being able to be suitably established in the intestine, and being able to be taken safely, a lactic acid bacterium composition and a plant extract containing the lactic acid bacterium, And it aims at providing the manufacturing method of the plant extract and low molecular weight polyphenol which use this lactic acid bacteria.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, when the properties of a novel lactic acid bacterium isolated from pickles were analyzed, the lactic acid bacterium had a strong tannase activity, no gallic acid decarboxylase activity, and further an inulin assimilation property. The lactic acid bacteria can effectively hydrolyze hydrolysable tannin and gallate catechins, and are excellent in colonization in the intestines. This is a finding that it can be suitably used as a lactic acid bacterium used in the production of molecular polyphenols.

  As described above, lactic acid bacteria having tannase activity and gallic acid decarboxylase activity have already been reported. However, it has not been known at all that there are lactic acid bacteria having strong tannase activity compared to conventional lactic acid bacteria, no gallic acid decarboxylase activity, and even inulin assimilation ability. These are new findings.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A lactic acid bacterium having tannase activity and not having gallic acid decarboxylase activity, wherein the lactic acid bacterium has an inulin assimilation property.
<2> The lactic acid bacterium according to <1>, which is Lactobacillus plantarum.
<3> Lactobacillus plantarum 22A-1 (FERM AP-21409), Lactobacillus plantarum 22A-3 (FERM AP-21411), or Lactobacillus plantarum 22B-2 (FERM AP-21410) The lactic acid bacterium according to <2>.
<4> A lactic acid bacterium composition comprising the lactic acid bacterium according to any one of <1> to <3>.
<5> A plant extract comprising the lactic acid bacterium according to any one of <1> to <3> and an extract from a plant.
<6> A method for producing a plant extract, comprising a step of hydrolyzing a gallate catechin contained in an extract from a plant using the lactic acid bacterium according to any one of <1> to <3>.
<7> A method for producing a plant extract comprising a step of hydrolyzing hydrolyzable tannin contained in an extract from a plant using the lactic acid bacterium according to any one of claims 1 to 3.
<8> Production of a low molecular weight polyphenol comprising a step of hydrolyzing at least one of hydrolyzed tannin, gallate catechin and methyl gallate using the lactic acid bacterium according to any one of <1> to <3> Method.

  According to the present invention, various conventional problems can be solved, a novel lactic acid bacterium having a strong tannase activity, no gallic acid decarboxylase activity, suitable for colonization in the intestine, and safe ingestion, It is possible to provide a lactic acid bacteria composition and a plant extract containing lactic acid bacteria, and a method for producing a plant extract and a low molecular weight polyphenol using the lactic acid bacteria.

(Lactic acid bacteria)
The lactic acid bacterium of the present invention is a lactic acid bacterium having tannase activity and not having gallic acid decarboxylase activity, characterized by having inulin assimilation properties, and further having other properties as required. .

-Tannase activity-
The tannase activity is an activity that catalyzes a reaction of hydrolyzing the ester bond of hydrolyzed tannin. When the ester bond is hydrolyzed in the hydrolyzed tannin, low molecular weight polyphenols such as gallic acid and ellagic acid are usually released.
The tannase activity is also an activity that catalyzes a reaction of hydrolyzing the ester bond of gallate catechin. When an ester bond is hydrolyzed in gallate catechin, gallate catechin is usually converted into non-gallate catechin and gallic acid is released.

  A method for examining whether or not the lactic acid bacterium has tannase activity is not particularly limited, but a method of detecting gallic acid as a reaction product by reacting methyl gallate as a substrate is common. The method for detecting gallic acid is not particularly limited. For example, it can be detected using HPLC (High Performance Liquid Chromatography) or a mass spectrometer, or can be colored from green to brown derived from liberated gallic acid. Can be detected using a spectrophotometer or visually.

  As a method for quantifying tannase activity using the coloration from green to brown derived from gallic acid, for example, a paper by Nishitani et al. (Nishitani, Y., Osawa, R. (2003) A novel colorimetric method to quantify) tannase activity of viable bacteria.Journal of Microbiological Methods 54 (2): 281-284.).

  The strength of tannase activity possessed by the lactic acid bacteria is not particularly limited and can be appropriately selected according to the purpose. However, when measured by the tannase activity quantification method described in the Nishitani et al. 0.0 mU / mL or more is preferable, and 6.0 mU / mL or more is particularly preferable. It is preferable that the strength of the tannase activity is 2.0 mU / mL or more from the viewpoint that the tannase activity is stronger than the known lactic acid bacteria having tannase activity and gallic acid decarboxylase activity. It is preferable that the tannase activity is 6.0 mU / mL or more in terms of strong tannase activity as compared with known lactic acid bacteria having tannase activity.

-Gallic acid decarboxylase activity-
The gallic acid decarboxylase activity means an activity of decarboxylating gallic acid to generate pyrogallol.
The method for examining whether or not the lactic acid bacterium has gallic acid decarboxylase activity is not particularly limited, but a general method is to react with gallic acid as a substrate and detect pyrogallol as a reaction product. . The method for detecting pyrogallol is not particularly limited. For example, pyrogallol generated using HPLC or a mass spectrometer is detected, or coloration to orange derived from the generated pyrogallol is performed using a spectrophotometer. Or can be detected visually.

-Inulin utilization-
The inulin assimilation means that it can be grown using inulin as a carbon source.
A method for examining whether or not the lactic acid bacterium has inulin assimilation is not particularly limited, but there is a method of inoculating the lactic acid bacterium on an agar medium containing only inulin as a carbon source and observing its growth. Can be mentioned. In addition, the presence or absence of the inulin assimilation can be easily examined using, for example, a commercially available bacterial identification kit.

-Lactic acid bacteria-
The lactic acid bacterium is not particularly limited and is a bacterium that has tannase activity and does not have gallic acid decarboxylase activity and has inulin assimilation ability among bacteria that can produce lactic acid by fermentation. Can be appropriately selected according to the purpose. Examples of the lactic acid bacteria include, for example, Lactobacillus, Pediococcus, Bifidobacterium, Lactococcus, Leuconostoc, and Enterococcus cc. And bacteria belonging to

  The seeds of the lactic acid bacteria, e.g., as Lactobacillus, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivalius), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus casei (Lactobacillus casei), Rakutobachiru Kitasatonisu (Lactobacillus kitasatonisi), Rakutobachiru Reuteri (Lactobacillus reuteri), Lactobacillus johnsonii (Lactobacillus jhonsonii), Lactobacillus acidophilus (Lactobacillus acidophilus) ), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Fal'cie Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus); as Pediococcus sp Pediococcus damnosus, Pediococcus halophyllus, Pedio Pseudococcus parvulus, Pediococcus pentosaceus; Bifidobacterium as Bifidobacterium adolescentes, Bifidobium fid Lactococcus cremoris, Lactococcus lactis; as the genus Leuconostoc, Leuconostoc oenos, Leuconostoc lactis (Leuconostost) lactis), Leuconostoc mesenteroides (Leuconostoc mesenteroides); as Enterococcus, and the like Enterococcus faecalis (Enterococcus faecalis). Among them, Lactobacillus plantarum is particularly preferable because it proliferates efficiently and the activity of the obtained fermented product is strong.

  There is no restriction | limiting in particular as said Lactobacillus plantarum, Although it can select suitably according to the objective, Lactobacillus plantarum 22A-1 (FERM AP-21409), Lactobacillus plantarum 22A-3 (FERM AP- 21411) and Lactobacillus plantarum 22B-2 (FERM AP-21410) are particularly preferred.

-22A-1 strain, 22A-3 strain and 22B-2 strain-
Three strains of Lactobacillus plantarum 22A-1 (FERM AP-21409), Lactobacillus plantarum 22A-3 (FERM AP-21411), and Lactobacillus plantarum 22B-2 (FERM AP-21410) are the present inventors. Is a strain isolated and identified from pickles and deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

The Lactobacillus plantarum 22A-1 has tannase activity, does not have gallic acid decarboxylase activity, and has inulin utilization. The tannase activity of Lactobacillus plantarum 22A-1 was 6.64 mU / mL as a result of measurement by the tannase quantification method described in the above-mentioned article of Nishitani et al.
The Lactobacillus plantarum 22A-3 has tannase activity, does not have gallic acid decarboxylase activity, and has inulin utilization. The tannase activity of the Lactobacillus plantarum 22A-3 was 7.18 mU / mL as a result of measurement by the tannase quantification method described in the above-mentioned article by Nishitani et al.
The Lactobacillus plantarum 22B-2 has tannase activity, does not have gallic acid decarboxylase activity, and has inulin utilization. The tannase activity of the Lactobacillus plantarum 22B-2 was 6.95 mU / mL as a result of measurement by the tannase quantification method described in the above-mentioned article by Nishitani et al.

  Bacteriological properties of the three strains of Lactobacillus plantarum 22A-1 (FERM AP-21409), Lactobacillus plantarum 22A-3 (FERM AP-21411), and Lactobacillus plantarum 22B-2 (FERM AP-21410) Are shown in Table 1 and Table 2 below. In Table 2, the sugar degradability of known Lactobacillus plantarum ATCC 14917 is also shown as a comparison target of the three strains.

（＋：陽性、−：陰性）

(+: Positive,-: negative)

（＋：陽性、−：陰性）

(+: Positive,-: negative)

(Plant extract)
The plant extract of this invention contains the said lactic acid bacteria and the extract from a plant, and also contains another component as needed.

-Lactic acid bacteria-
There is no restriction | limiting in particular as said lactic acid bacteria in the said plant extract, It can select suitably from the lactic acid bacteria described in the item of the lactic acid bacteria of the above-mentioned this invention. The said lactic acid bacteria may be used individually by 1 type, and may use 2 or more types together. There is no restriction | limiting in particular as content of the said lactic acid bacteria in the said lactic acid bacteria composition, According to the objective, it can select suitably.

-Extracts from plants-
There is no restriction | limiting in particular as said plant, Although it can select suitably according to the objective, It is preferable to contain a gallate type catechin and hydrolyzable tannin, and it is especially preferable to contain a gallate type catechin.
There is no restriction | limiting in particular as said gallate type catechin, For example, epigallocatechin gallate (EGCg), epicatechin gallate (ECg), catechin gallate (Cg), gallocatechin gallate (GCg) etc. are mentioned.

  Examples of the plant include a thistle, an acha, an opium, an aloe bear, a ginkgo, a fennel, a turmeric, a common beetle, a larva, an age, an elephant, a life-prolonging grass, a yellow spirit, an oat, an ogon, an oak, a barley, a hypericum grass, a cocoon, a chamomile, and a licorice. , Kidachi Aloe, Gymnema, Cabbage, Jade Bamboo, Kiraya, Gold and Silver Flower, Chrysanthemum, Chrysanthemum, Red Ginseng, Ginseng, Kumagusu, Mulberry, Laurel Leaves, Akiko, Gentiana, Wheat, Rice, Burdock, Sesame, Salvia, Sanza, Shiso, Sanshishi, Sanche, Yam, Yamayaku, Shiitake mushrooms, Purple candy, Glaze, Car forage, Ten ginseng, White ginger, Horsetail, Stevia, Cyprus, Senna, Assembly, Buckwheat, Radish, Taiso, Soy, Tamarind, Cod, Chimpi, Green tea , Tenninka, Tokachi, Tochu, Cordyceps, Corn, Sashimi, Ginseng, Shinobyu, Parsley, Beach windbreak, Mamellisu, himematsu mushroom, bilberry, loquat, bukuro, grape, blueberry, loofah, hematin, linden tree, peony, hops, pine needles, peaches, melissa, yucca, yokoinin, mugwort, rye, lakanka, green tea, apple, rooibos, lucas, Examples include ganoderma, reindeer grass, rose hips, rosemary, and bitumen. Among these, green tea is preferable because it contains abundant gallate catechins.

  The plant extract can be easily obtained by a method generally used for plant extraction. The extract from the plant includes a plant extract, a dried product obtained by drying a diluted solution of the extract, or a crude product or a purified product thereof.

  The plant extraction raw material is not particularly limited and may be appropriately selected depending on the purpose. For example, a plant leaf part, a stem part, a flower (bud) part, a seed (these are referred to as above-ground parts), A root or the like can be used.

  The plant that is the raw material for extraction can be obtained by drying or pulverizing it as it is or using a crusher and subjecting it to solvent extraction. Drying may be performed in the sun or using a commonly used dryer. The plant may be used as a raw material for extraction after pretreatment such as degreasing with a nonpolar solvent such as hexane or benzene. In addition, the extraction process by the polar solvent of a plant can be performed efficiently by performing pretreatments, such as degreasing.

As the solvent used for the extraction, it is preferable to use water, a hydrophilic organic solvent, or a mixed solvent thereof at a temperature from room temperature to the boiling point of the solvent.
Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. The water that can be used as the extraction solvent includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol, propylene glycol, Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
In addition, when using the mixed solvent of the said water and a hydrophilic organic solvent, in the case of a lower alcohol, 1 mass part-90 mass parts with respect to 10 mass parts of water, and in the case of a lower aliphatic ketone, 10 masses of water. It is preferable to add 1 to 40 parts by mass with respect to parts. In the case of polyhydric alcohol, it is preferable to add 1 part by mass to 90 parts by mass with respect to 10 parts by mass of water.

  In the present invention, when extracting an extract from a plant as an extraction raw material, it is not necessary to adopt a special extraction method, and the extraction can be performed using an arbitrary extraction device at room temperature or under reflux heating.

  Specifically, in a treatment tank filled with the extraction solvent, the plant as an extraction raw material is added, and further, with occasional stirring as necessary, after standing for 30 minutes to 2 hours to elute soluble components, The solid matter is removed by filtration, and the extraction solvent is distilled off from the obtained extract, and the extract is obtained by drying. The amount of the extraction solvent is usually 5 to 15 times (mass ratio) of the extraction raw material. The extraction conditions are usually about 1 to 4 hours at 50 to 95 ° C. when water is used as the extraction solvent. Moreover, when the mixed solvent of water and ethanol is used as an extraction solvent, it is normally 30 minutes-about 4 hours at 40 to 80 degreeC. In addition, the extraction liquid obtained by extracting with a solvent can be used as it is as the plant extract of the present invention after adding the lactic acid bacteria, as long as the extraction solvent is highly safe.

  The obtained plant extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crudely purified product or purified product thereof. Processing may be performed.

Since the plant extract contains an extract from the plant, it can exert various physiological actions that the extract from the plant has.
When the extract from the plant contains at least one of tannin and catechins, the plant extract can exert a physiological action possessed by at least one of tannin and catechins. The physiological action of at least one of the tannins and catechins is not particularly limited, and examples thereof include an antitumor action, an antioxidant action, an anti-aging action, and a blood pressure lowering action.
Furthermore, since the plant extract contains the lactic acid bacteria having strong tannase activity, the tannin excellent in intestinal absorption obtained by hydrolysis of hydrolyzable tannin and the intestinal absorption obtained by hydrolysis of gallate catechin are excellent. Abundantly contains at least one of the catechins. Therefore, the said plant extract can exhibit more effectively the physiological effect which tannin and catechin have.

In addition, since the plant extract contains the lactic acid bacterium having strong tannase activity, low molecular polyphenols such as gallic acid and ellagic acid produced when at least one of hydrolyzed tannin and gallate catechin is hydrolyzed. Contains abundantly. Therefore, the plant extract can exert the physiological action of the low molecular weight polyphenol. There is no restriction | limiting in particular as a physiological effect which the said low molecular polyphenol has, For example, an antioxidant effect etc. are mentioned. Note that the lactic acid bacteria contained in the plant extract does not have gallic acid decarboxylase activity and does not convert gallic acid into pyrogallol. As one.
Moreover, since the lactic acid bacteria contained in the plant extract have inulin assimilation properties, the lactic acid bacteria grow and settle in the intestine after the plant extract is taken orally, thereby improving the intestinal bacterial flora. be able to.

  The method for using the plant extract is not particularly limited, and can be widely used regardless of the field of food, medicine, compound production, etc. For example, as an additive to be taken orally or added to a ready-made food It can be used as a raw material for purifying low molecular weight polyphenols such as gallic acid and ellagic acid.

  There is no restriction | limiting in particular as said foodstuff, Although it can select suitably according to the objective, For example, drinks, such as a tea drink, a soft drink, a carbonated drink, a nutritive drink, a fruit drink, a lactic acid drink; Ice cream, ice sherbet , Shaved ice and other frozen desserts; buckwheat noodles such as noodles, udon noodles, harsame, gyoza zest, sushi mai, noodles such as Chinese noodles and instant noodles; rice cakes, candy, gum, chocolate, tablet confectionery, snacks, biscuits, jelly, jam, cream, Sweets such as baked goods and bread; crab, salmon, clams, tuna, sardines, shrimp, skipjack, mackerel, whale, oyster, saury, squid, red sea bream, scallop, abalone, sea urchin, sea bream, tocobushi, etc .; Processed fishery and livestock products such as ham and sausage; Dairy products such as processed milk and fermented milk; salad oil, tempura oil, margarine, mayonnaise, Fats and processed foods such as yotoning, whipped cream, dressing, etc .; seasonings such as sauces, sauces; curry, stew, oyakodon, rice cake, miscellaneous cooking, Chinese rice cakes, tempura, eel rice cake, hayashi rice, oden, marbodorf, Retort pouch foods such as beef bowl, meat sauce, egg soup, omelet rice, dumplings, shumai, hamburger and meatballs; various forms of health foods and dietary supplements.

(Lactic acid bacteria composition)
The lactic acid bacteria composition of this invention contains the said lactic acid bacteria, and also contains another component as needed.
Here, the lactic acid bacteria composition is one that is less likely to harm human health and is taken by oral or gastrointestinal administration in normal social life. It is not limited to categories such as quasi-drugs, and includes, for example, a wide range of functional foods, quasi-drugs, and pharmaceuticals that are taken orally.

-Lactic acid bacteria-
There is no restriction | limiting in particular as said lactic acid bacteria in the said lactic acid bacteria composition, It can select suitably from the lactic acid bacteria described by the item of the lactic acid bacteria of the above-mentioned this invention. The said lactic acid bacteria may be used individually by 1 type, and may use 2 or more types together. There is no restriction | limiting in particular as content of the said lactic acid bacteria in the said lactic acid bacteria composition, According to the objective, it can select suitably. The lactic acid bacterium composition may be the lactic acid bacterium itself.

-Other ingredients-
There is no restriction | limiting in particular as said other component, Although it can select suitably according to the utilization form of the said lactic acid bacteria composition, For example, the support | carrier accept | permitted pharmacologically is mentioned.
The carrier is not particularly limited as long as the effects of the present invention are not impaired. For example, when the active ingredient is used in various dosage forms, it can be appropriately selected depending on the dosage form. The dosage form is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the administration method. For example, oral solids (tablets, coated tablets, granules, powders, capsules) , Lozenges, etc.), oral solutions (internal solutions, syrups, elixirs, etc.).

As the oral solid preparation, for example, an excipient, and if necessary, additives such as a binder, a disintegrant, a lubricant, a coloring agent, a flavoring / flavoring agent, etc. are added to the lactic acid bacterium, and manufactured by a conventional method. Can do.
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid.

The additive is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the purpose. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include , Purified talc, stearate, borax, polyethylene glycol and the like, and as the colorant, titanium oxide, iron oxide and the like, and as the flavoring and flavoring agent, If For example, white sugar, orange peel, citric acid, and tartaric acid.
Further, inulin may be added to the lactic acid bacteria composition for the purpose of assisting the colonization of the lactic acid bacteria in the intestine.

  As the oral solution, for example, additives such as a taste-masking / flavoring agent, a buffering agent, a stabilizer and the like can be added to the lactic acid bacteria, and the oral solution can be produced by a conventional method. Here, the additive is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid, and the like. Examples of the buffering agent include sodium citrate. Examples of the stabilizer include tragacanth and gum arabic. And gelatin.

The lactic acid bacterium composition can be taken orally on a daily basis, and can exert a strong tannase activity effectively by the action of the contained lactic acid bacterium. Therefore, hydrolyzable tannin and gallate in the intestine At least one of the type catechins can be hydrolyzed to promote absorption from the intestinal tract of at least one of the tannins and catechins.
Furthermore, since the lactic acid bacterium contained in the lactic acid bacterium composition does not have gallic acid decarboxylase activity, gallic acid produced by tannase activity is not converted to pyrogallol. Therefore, an excellent antioxidant action is exhibited by gallic acid produced in the intestine. Furthermore, it is expected to reduce the risk of colorectal cancer caused by pyrogallol.
In addition, the lactic acid bacteria composition can be taken orally on a daily basis, and the contained lactic acid bacteria have an inulin assimilation property, so that they easily grow and settle in the intestine after being taken orally. The action by the tannase activity can be more effectively exhibited in the intestine.

  In addition, although the plant extract and lactic acid bacteria composition of this invention are suitably applied with respect to a human, as long as each effect is show | played, it can also be applied with respect to animals other than a human.

(Manufacturing method of plant extract)
The plant extract of the present invention includes a step of hydrolyzing at least one of hydrolyzed tannin and gallate catechin contained in an extract from a plant using the lactic acid bacterium, and further, if necessary, removing lactic acid bacterium It contains other processes, such as a process and the manufacturing process of the extract from a plant.

The extract from the lactic acid bacterium and the plant is not particularly limited, and can be appropriately selected from the lactic acid bacterium and the extract from the plant described in the item of the lactic acid bacterium described above.
The method for hydrolyzing at least one of the hydrolyzed tannin and gallate catechin is not particularly limited and may be appropriately selected depending on the intended purpose. Usually, the extract from the lactic acid bacteria and the plant is used. Can be added to the same solution. The conditions for the hydrolysis are not particularly limited, and can be appropriately selected in consideration of the growth conditions for the lactic acid bacteria, the optimum conditions for the enzyme activity of the lactic acid bacteria, and the like.

  The lactic acid bacterium used in the step of hydrolyzing at least one of the hydrolyzable tannin and gallate catechin may remain in the plant extract or may be removed, but the lactic acid bacterium itself Is preferably contained in the plant extract in that it can be safely ingested and is beneficial in the intestine, and does not require a lactic acid bacteria removal step when producing the plant extract.

  According to the method for producing a plant extract, since the lactic acid bacterium used for production has a strong tannase activity, at least one of hydrolyzable tannin and gallate catechin contained in the extract from the plant is hydrolyzed. A plant extract that is decomposed and easily absorbed from the intestinal tract can be produced. Furthermore, since the lactic acid bacteria used in the production method do not have gallic acid decarboxylase activity, gallic acid produced by tannase activity is not converted to pyrogallol. Therefore, a plant extract containing abundant low molecular weight polyphenols having an excellent antioxidant action can be produced.

(Production method of low molecular weight polyphenol)
The method for producing gallic acid of the present invention includes a step of hydrolyzing at least one of hydrolyzed tannin, gallate-type catechin and methyl gallate using lactic acid bacteria, and if necessary, a low molecular polyphenol purification step It contains other processes such as.

There is no restriction | limiting in particular as said lactic acid bacteria, It can select suitably from the lactic acid bacteria described by the item of the lactic acid bacteria of this invention mentioned above.
The method for hydrolyzing at least one of the hydrolyzed tannin, gallate catechin and methyl gallate is not particularly limited and may be appropriately selected depending on the intended purpose. It can be performed by adding at least one of hydrolyzed tannin, gallate catechin and methyl gallate in the same solution. The conditions for the hydrolysis are not particularly limited, and can be appropriately selected in consideration of the growth conditions for the lactic acid bacteria, the optimum conditions for the enzyme activity of the lactic acid bacteria, and the like.

  The low molecular weight polyphenol is not particularly limited as long as it is a low molecular weight polyphenol that can be produced by a tannase treatment using at least one of the hydrolyzed tannin, gallate catechin, and methyl gallate as a substrate. , Gallic acid, ellagic acid and the like.

  According to the method for producing the low molecular weight polyphenol, the lactic acid bacterium used for the production has a strong tannase activity and does not have a gallic acid decarboxylase activity. Therefore, the low molecular weight polyphenol can be produced efficiently. In addition, since the lactic acid bacterium is a bacterium isolated from pickles and highly safe, the reaction solution used for the production of the low molecular weight polyphenol can be allowed to flow as waste water as it is or after simple treatment. It is advantageous.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1
-Identification of strains-
Chromosomal DNA was extracted from the 22A-1 strain, 22A-3 strain and 22B-2 strain isolated from pickles by a known method, and Dong et al. (Dong, X., Xin, Y., Jian, W , Liu, X., Ling, D. (2000) .Bifidobacterium thermophilum sp.nov., Isolated from ananalogous digester.Internal Journal of Systemic 50. 27f (5'-AGAGTTTGATCC / ATGGCTCAG-3 ') and 1541R (5'-AAGGAGGTGATCCAGC) PCR was performed using the 3 ') was amplified a region that substantially covers the 16S rDNA.

  The obtained PCR product of about 1.6 kb was inserted into pGEM-T EASY Vector (Promega), introduced into competent cells DH5α by the heat shock method, and color screening was performed on LB plate medium supplemented with Xgal-Amiciliin. . A plasmid was extracted from the selected white colonies, and the PCR product (5′-TAATTAGACTACTACTATAGGG-3 ′) and T7 (5′-TAATACGACTCACTATATAGGG-3 ′) designed based on the sequence of pGEM-T EASY Vector were used. 16S rDNA) was determined (22A-1 strain: SEQ ID NO: 1, 22A-3 strain: SEQ ID NO: 2, 22B-2 strain: SEQ ID NO: 3).

As a result of accessing the DNA database (DDBJ) and performing a homology search of the base sequence of 16S rDNA using the FASTA program, the 16S rDNA base sequences of the 22A-1 strain, 22A-3 strain and 22B-2 strain were as follows: , All had high homology with the conventionally known Lactobacillus plantarum WCFS1 strain 16S rDNA base sequence (SEQ ID NO: 4). The results are shown in Table 3.

  In addition, the 22A-1 strain, 22A-3 strain and 22B-2 strain are Lactobacillus plantarum and Lactobacillus pentosus which are very similar to Lactobacillus plantarum in 16S rDNA base sequence. In order to confirm that the 16S / 23S rDNA spacer region of Lactobacillus plantarum has been examined, Berthier et al. (Bertier, F., Ehrhich, SD (1998). Rapid specifications) identification with two groups of closed relaxed using PC As a result of species identification using the PCR method described in Primers that target the 16S / 23S rRNA spacer region. FEMS Microbiological Letters 161, 97-106), 22A-1 strain, 22A-3 strain and 22B-2 All strains were confirmed to be Lactobacillus plantarum.

(Example 2)
-Qualitative tannase test-
The 22A-1 strain, 22A-3 strain and 22B-2 strain were subjected to a qualitative tannase test by the following test method. This test method is described in Osawa et al. (Osawa, R., and T.P. Walsh (1993). A visual reading method for detection of bacterial tannase. Applied and Environmental Micro 125. 125). It is based on the method.
A solution adjusted to pH 5.0 containing sodium dihydrogen phosphate (33 mM; Wako) and methyl gallate (20 mM; Aldrich Chemical Company, Inc., Milwaukee, USA) having a tannin-like structure is sterilized. Using a filter (pore size 0.45 μm; Millipore Co., Bedford, USA), 5 ml each was aseptically dispensed into a test tube to obtain a substrate medium. The test bacteria group cultured for 24 hours at 37 ° C. on MRS agar plate medium was scraped with a sterile cotton swab, suspended in the above-mentioned substrate medium, and O.D. D. The absorbance at 660 nm was adjusted to 0.4. This was statically cultured at 37 ° C. for 24 hours. After culturing, 1 ml of the well-suspended culture solution is collected and transferred to a small test tube, and alkalinized by adding an equal amount of a saturated sodium bicarbonate (Wako) solution (pH 8.6) prepared in advance. Left to stand. A solution colored green or brown after 30 minutes was defined as tannase positive.
As a result of the qualitative tannase test, tannase activity was observed in all of the 22A-1 strain, 22A-3 strain and 22B-2 strain.

(Example 3)
-Qualitative gallic acid decarboxylase test-
The 22A-1 strain, 22A-3 strain, and 22B-2 strain were subjected to a qualitative gallic acid decarboxylase test by the following test method. This test method is described in Osawa et al. (Osawa, R., and T.P. Walsh (1995). Detection of bacterial gallate decylation by visual color discriminating. It complies with the described method.
To 5 ml of MRS liquid medium, 100 mM gallic acid (Wako) solution (pH 5.0) prepared in advance using a sterile filter (pore size 0.45 μm) was aseptically added to a final concentration of 10 mM and mixed well. . This solution was inoculated with 1 colony of freshly isolated strain grown on MRS agar medium, inoculated and anaerobically cultured at 37 ° C. for 24 hours. After culturing, 1 ml of the culture solution was collected and transferred to a small test tube, added with an equal volume of saturated sodium hydrogen carbonate solution (pH 8.6) to be alkalized, and allowed to stand at room temperature. When the solution turned bright orange after 30 minutes, GDase was positive, and when the solution turned deep green or brown, it was regarded as GDase negative. If the color changes to orange, gallic acid is decarboxylated, indicating that the metabolite pyrogallol is present.
As a result of the qualitative gallic acid decarboxylase test, gallic acid decarboxylase activity was not observed in any of the 22A-1 strain, 22A-3 strain and 22B-2 strain.

Example 4
-Quantitative tannase test-
The 22A-1 strain, the 22A-3 strain, and the 22B-2 strain were subjected to a quantitative gallic acid decarboxylase test by the following test method. This test method is described in a paper by Nishitani et al. (Nishitani, Y., Osawa, R. (2003). A novel colorimetric method to quantative activity of 28.Mour.our.Jour. It complies with the described method.

  First, a solution adjusted to pH 5.0 containing tannase (Wako) derived from Aspergillus oryzae and sodium dihydrogen phosphate (33 mM; Wako) was used as an enzyme solution to prepare a calibration curve. At this time, the concentration of tannase derived from Aspergillus oryzae (Wako) contained in the enzyme solution was changed to prepare an enzyme solution having a tannase concentration of 0.63 to 1000 mU. 50 μL each from the enzyme solution was prepared and prepared in advance (methyl gallate (5 mM; Aldrich Chemical Company, Inc., Milwaukee, USA), sodium dihydrogen phosphate (33 mM; Wako)) , PH 5.0) was added and mixed. This was left still at 37 ° C. for 24 hours. After allowing to stand, 100 μL was taken out, made equal by adding an equal amount of a saturated sodium hydrogen carbonate (Wako) solution (pH 8.6) prepared in advance, and allowed to stand at 37 ° C. for 2 hours. After standing, the mixture was stirred well and centrifuged at 8,000 × g for 20 seconds. 100 μL of the supernatant was dispensed into a 96-well microplate, and O.D. was obtained using a microplate reader (Model 550, Bio-Rad). D. Absorbance at 450 nm was measured. A calibration curve was prepared from the obtained absorbance.

  Next, a solution adjusted to pH 5.0 containing sodium dihydrogen phosphate (33 mM; Wako) and methyl gallate (5 mM; Aldrich Chemical Company, Inc., Milwaukee, USA) having a tannin-like structure Was aseptically dispensed 1 ml each into a test tube using a sterilizing filter (pore size 0.45 μm; Millipore Co., Bedford, USA) to obtain a substrate medium. The test bacteria group cultured on MRS agar plate medium at 37 ° C. for 24 hours was scraped with a sterile cotton swab and suspended in the above-mentioned substrate medium. D. The absorbance at 660 nm was adjusted to 0.4. This was statically cultured at 37 ° C. for 24 hours. After the culture, 1 ml of the well-suspended culture solution was collected and transferred to an Eppendorf tube, and centrifuged at 8,000 × g for 5 minutes. 100 μL of the supernatant was collected, made equal by adding an equal amount of a saturated sodium bicarbonate (Wako) solution (pH 8.6) prepared in advance, and allowed to stand at 37 ° C. for 2 hours. After standing, the mixture was stirred well and centrifuged at 8,000 × g for 20 seconds. 100 μL of the supernatant was dispensed into a 96-well microplate, and O.D. was obtained using a microplate reader (Model 550, Bio-Rad). D. Absorbance at 450 nm was measured. Based on the obtained calibration curve, tannase activity (mU / mL) was determined from the obtained absorbance.

As a result of the quantitative tannase test, the tannase activities of the 22A-1 strain, the 22A-3 strain, and the 22B-2 strain were 6.64, 7.18, and 6.95 mU / mL, respectively.
That is, the tannase activity of 22A-1 strain, 22A-3 strain, and 22B-2 strain is the same as that of any Lactobacillus plantarum strain described in the aforementioned Nishitani et al. Paper (maximum 5.73 mU / mL (ATCC 14917). It was shown to be higher than that of the stock)).
Further, the tannase activity of 22A-1 strain, 22A-3 strain and 22B-2 strain has tannase activity among the Lactobacillus plantarum strains described in the above-mentioned Nishitani et al. Paper, and has gallic acid decarboxylase activity. Compared with the tannase activity (up to 1.52 mU / mL (KOG11 strain)) of the Lactobacillus plantarum strain having no E. coli, it was shown to be remarkably high.

  The lactic acid bacterium of the present invention has strong tannase activity, does not have gallic acid decarboxylase activity, can be suitably established in the intestine, and can be safely ingested, for example, lactic acid bacterium for the purpose of improving intestinal flora It can be used as a composition. Further, by adding the lactic acid bacterium of the present invention to an extract from a plant, at least one of hydrolyzable tannin and gallate catechin contained in the plant can be hydrolyzed. An excellent plant extract can be provided. The plant extract can be widely used regardless of the field such as food, medicine and compound production.

Claims (8)

  A lactic acid bacterium having tannase activity and not having gallic acid decarboxylase activity, wherein the lactic acid bacterium has an inulin assimilation property.   The lactic acid bacterium according to claim 1, which is Lactobacillus plantarum.   Lactobacillus plantarum 22A-1 (FERM AP-21409), Lactobacillus plantarum 22A-3 (FERM AP-21411), or Lactobacillus plantarum 22B-2 (FERM AP-21410) Lactic acid bacteria described in 2.   A lactic acid bacteria composition comprising the lactic acid bacteria according to any one of claims 1 to 3.   The plant extract containing the lactic acid bacteria in any one of Claim 1 to 3, and the extract from a plant.   The manufacturing method of a plant extract including the process of hydrolyzing the gallate type catechin contained in the extract from a plant using the lactic acid bacteria in any one of Claim 1 to 3.   The manufacturing method of a plant extract including the process of hydrolyzing the hydrolyzable tannin contained in the extract from a plant using the lactic acid bacteria in any one of Claim 1 to 3.   A method for producing a low molecular weight polyphenol, comprising the step of hydrolyzing at least one of hydrolyzable tannin, gallate catechin and methyl gallate using the lactic acid bacterium according to any one of claims 1 to 3.