JP2006061091A - Fermentation product obtained from leaf of perilla frutescens crispa - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a fermentation product having high physiological activities, and to obtain an oral composition and a percutaneous composition each using the fermentation product. <P>SOLUTION: A microorganism (especially lactobacillus) was made to act on the leaves of Perilla frutescens crispa to obtain the fermentation product having high physiological activities. Concretely, the fermentation product having excellent physiological activities such as a tyrosinase-inhibiting activity, an α-glucosidase-inhibiting activity, and an elastase-inhibiting activity was obtained. Especially, a fermentation with lactobacillus is suitable, and the fermentation product can be used as a raw material for many foods, cosmetics, medicines, and quasi-medicines. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a fermented product obtained by allowing microorganisms to act on perilla leaves, and particularly to a fermented product having various physiological effects by fermenting perilla leaves.

  Fermentation using microorganisms is one of the processing techniques that have been used for a long time. For example, by causing microorganisms to act on raw materials derived from animals and plants, the palatability of raw materials can be improved, and certain substances and new materials can be used. It has been used to produce useful substances.

In recent years, fermenting not only improves the production and palatability of useful substances, but also acquires new physiological activities, such as having an effect of enhancing the physiological activity over the raw material before fermentation. Studies on fermented products with high physiological activity have been made. (For example, Patent Documents 1 to 3)
Patent No. 3060965 No. 06-011216 Japanese Patent No. 3490938

  However, depending on the raw material used, the raw material processing method, the microorganism used, etc., there is a problem that the fermented product does not always have physiological activity, and may not even be fermented. In other words, even if fermentation is performed to obtain a fermented product with high physiological activity, if the method and material in the fermentation are wrongly selected, only a fermented product with low or no physiological activity can be obtained. There is a point.

  The present invention has been made in view of the above problems, and its object is to provide a fermented product having high physiological activity, and an oral composition and a transdermal composition using the fermented product. It is in.

  The present inventor has conducted intensive studies on fermented products obtained by allowing microorganisms to act on animal and plant raw materials, and found that fermented products having excellent physiological activity can be obtained by fermenting perilla leaves with microorganisms. The present invention has been reached.

  That is, the fermented product of the present invention is a fermented product obtained by allowing microorganisms to act on perilla leaves and has enzyme inhibitory activity.

  Preferably, the microorganism is a lactic acid bacterium.

  More preferably, the fermented product has at least one of tyrosinase inhibitory activity, glucosidase inhibitory activity, and elastase inhibitory activity.

  Moreover, the composition for oral use of this invention is characterized by containing the said fermented material.

  Furthermore, the transdermal composition of the present invention is characterized by containing the fermented product.

  According to the present invention, when a microorganism is allowed to act on perilla leaves under predetermined conditions, a fermented product having physiological activities such as tyrosinase inhibitory activity, glucosidase inhibitory activity (for example, α-glucosidase inhibitory activity), and elastase inhibitory activity is obtained. Can do. For example, if a lactic acid bacterium is used as a microorganism, a fermented product with high physiological activity can be obtained. Furthermore, if the fermented product is used, an oral composition and a transdermal composition having a cosmetic effect can be obtained.

  Hereinafter, the fermented product of the present invention will be described. In addition, the form demonstrated below does not limit this invention. That is, the present invention can be variously modified within the scope of the gist of the present invention.

(Perilla leaves)
The perilla leaf used in the present invention is a leaf of a plant belonging to the family Lamiaceae, and includes, for example, a leaf used for food as perilla. In the present invention, red-purple leaves or green leaves may be used as perilla leaves.

  Perilla leaves are rich in α-linolenic acid and vitamin B. Furthermore, perilla has been shown to have an effect of reducing allergic symptoms such as hay fever, and has attracted attention in recent years.

  When obtaining the fermented product of the present invention, the perilla leaves that have been washed or the perilla leaves that have been crushed by a method commonly used by those skilled in the art such as cutting and grinding after washing are used. When the crushed perilla leaves are used, the size of the crushed material is not particularly limited, but a crushed material having a particle size of 5000 μm or less, more preferably about 20 μm to 5000 μm is preferably used.

  In the present invention, a solution (squeezed juice) obtained by solid-liquid separation after crushing can also be used as a crushed product. Furthermore, after extracting the perilla leaf or its crushed material, it may be used after solid-liquid separation. In terms of the ability to ferment all the components of perilla leaves, ie, insoluble components and water-soluble components, it is preferable to use perilla leaves or their crushed material without solid-liquid separation.

  The perilla leaf and its crushed material may be subjected to heat treatment or may be stored frozen for the purpose of preventing the growth of germs. In particular, frozen storage is preferable from the viewpoint of maintaining components and flavor contained in perilla leaves, and is employed when the next step is not performed immediately.

  In order to perform fermentation efficiently, the perilla leaf or its crushed material may be added with water as necessary. There is no restriction | limiting in particular in the amount of water. For example, the lower limit of the amount of water to be added is 0.1 parts by mass or more, preferably 0.5 parts by mass or more, and the upper limit of the amount of water to be added is 1 part by mass of perilla leaves or its crushed material It may be 30 parts by mass or less, preferably 10 parts by mass or less.

(Enzyme treatment process and neutralization process)
Next, a process for promoting fermentation of perilla leaves or crushed materials thereof will be described. Examples of the process for promoting such fermentation include a cell wall decomposing process using an enzyme and a neutralizing process. In addition, this process may use only 1 type of the said cell wall decomposition | disassembly process process and a neutralization process process, and may use both.

  Examples of the enzyme that can be used in the cell wall degradation treatment step with an enzyme include amylase, pectinase, endoarabanase, cellulase, hemicellulase, endo β-glucanase, exo β-glucanase, xylase, β-glucosidase and the like. . Of these, pectinase is preferably used. Of course, you may mix and use the said enzyme. Particularly preferred is a mixed enzyme of pectinase and at least one enzyme selected from the group consisting of amylase, endoarabanase, endo β-glucanase, exo β-glucanase, xylanase, and β-glucosidase. The amount of addition varies depending on the type of enzyme, but is usually added so that the enzyme concentration is 0.0001 to 0.5 mass / volume%, preferably 0.0005 to 0.3 mass / volume%. The

  The conditions for enzyme treatment may be appropriately set according to the shape of perilla leaves to be treated, the type of enzyme, and the enzyme concentration. For example, the lower limit of the temperature in the enzyme treatment may be 30 ° C or higher, preferably 35 ° C or higher, and the upper limit may be 75 ° C or lower, preferably 70 ° C or lower. The time for enzyme treatment (enzyme reaction time) may be, for example, a lower limit of 30 minutes or longer, preferably 1 hour or longer, and an upper limit of 72 hours or shorter, preferably 48 hours or shorter.

  When the enzyme treatment is performed, for example, a fermentation inhibiting factor (for example, a polysaccharide constituting a cell wall such as pectin) can be decomposed, and as a result, the fermentation can be completed in a short time. Thus, if fermentation can be completed in a short time, for example, when there is a component (such as ascorbic acid) that is not desired to be lost by fermentation, the loss of such component can be minimized. . Specifically, fermented perilla leaves can be obtained with less loss due to fermentation (for example, loss of ascorbic acid).

  Furthermore, for example, when a polysaccharide is decomposed by an enzyme treatment, a degradation product (monosaccharide, oligosaccharide, etc.) of the polysaccharide is generated. These become new components in the fermented fern leaves obtained. For example, oligosaccharides have various effects such as intestinal regulation and promoting solubilization of fermented products. Monosaccharides are thought to further become nutrients necessary for the growth of microorganisms and promote the production of useful substances of microorganisms.

  The neutralization treatment is performed for fermentation at an optimum pH. For example, the pH of perilla leaves or crushed material thereof is adjusted to 4.0 to 7.5. Adjustment of pH is performed using the usual method used by those skilled in the art. For example, pH adjusters such as sodium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium citrate, etc., electrolyzed water, buffer solution (eg citrate buffer solution, acetate buffer solution, phosphate buffer solution) etc. are added. PH can be adjusted.

  Both the enzyme treatment and the neutralization treatment described above are useful in that the loss of effective components contained in perilla leaves can be reduced by accelerating fermentation and fermenting in a relatively short time. is there. If fermented in the following fermentation process by combining enzyme-treated or neutralized perilla leaves or a crushed product thereof with a microorganism having a high fermentation ability (for example, a specific lactic acid bacterium described later), it is relatively short. Fermentation can be completed in time (eg, 24 hours to 72 hours).

(Fermentation process)
Next, the fermentation process will be described. The fermented product of the present invention can be obtained by fermenting the perilla leaf or its crushed material, or fermenting perilla treated with at least one of the enzyme treatment and neutralization treatment.

  Examples of fermentation include lactic acid fermentation, citric acid fermentation, alcohol fermentation, and acetic acid fermentation. Among these, lactic acid fermentation is preferable, and these fermentations may be combined. The fermentation time is preferably finished, for example, in 24 hours to 72 hours. Moreover, lactic acid bacteria, yeasts, acetic acid bacteria, etc. are used according to the kind of fermentation. Yeast and acetic acid bacteria are particularly preferably used because of their strong acid resistance.

(Lactic acid fermentation)
Lactic acid fermentation is performed by contacting the perilla leaves or crushed materials thereof with lactic acid bacteria. Lactic acid fermentation is preferable in that lactic acid is contained in the obtained fermented product. Regarding the amount of lactic acid contained, per 100 g of dry mass of the fermented product, lactic acid is 0.1 mg (0.0001 mass%) or more, preferably 0.5 mg (0.0005 mass%) or more, in terms of dry mass. Preferably it is 1 mg (0.001 mass%) or more.

  Examples of lactic acid bacteria that can be used in the present invention include lactic acid bacteria classified into the genus Leuconostoc, Lactobacillus, Streptococcus, Bifidobacterium, Pediococcus, Enterococcus, and Pediococcus. Can be mentioned. Specific examples of lactic acid bacteria that can be used in the present invention include Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus brevis, Lactobacillus delvis. Lactobacillus delbrueckii (more specifically, Lactobacillus delbrueckii subsp. Bulgaricus), Lactobacillus gasseri (Lactobacillus gassili), Lactobacillus acidophilus acillus casei), Streptococcus thermophilus, Streptococcus faecalis (Bifidobacterium longum), Bifidobacterium longum (Bifidobacterium longum) lactis), Bacillus mesentericus, and the like.

  In the present invention, since perilla leaves are used as a material to be fermented, lactic acid bacteria that do not require milk components, such as lactic acid bacteria found in pickles, may be used. Specific examples of such lactic acid bacteria include Lactobacillus plantarum, Leuconostoc mesenteroides, Enterococcus faecalis, Enterococcus faecalis, Enterococcus faecalis, (Lactobacillus brevis), Pediococcus acidilactici, Pediococcus pentoaceaceus, Pediococcus halophyllus (Pediococcus halophilus) lus).

  In the present invention, the lactic acid bacteria may be used alone or in combination with different types of lactic acid bacteria.

  In the present invention, when fermentation is performed using a microorganism (particularly lactic acid bacteria), the obtained fermented product has at least one physiological activity of tyrosinase inhibitory activity, glucosidase inhibitory activity (for example, α-glucosidase inhibitory activity), and elastase inhibitory activity. Excellent physiological activity. Therefore, it is useful to perform fermentation using microorganisms (particularly lactic acid bacteria).

  In performing the fermentation, the microorganism (particularly lactic acid bacteria) is 0.005 to 10 parts by mass, preferably 0.01 to 5.0 parts by mass with respect to 100 parts by mass of perilla leaves or crushed material thereof. It is good to add.

  Moreover, you may add the additive for growth for the preferential growth of lactic acid bacteria. Examples of the growth additive herein include glutamic acid or a salt thereof, yeast extract, peptone, and the like. The amount of the additive for growth is not particularly limited, but it is about 0.05 to 1% by mass, preferably about 0.2% by mass, based on perilla leaves or crushed materials thereof.

  Moreover, in order to promote fermentation of lactic acid bacteria, lactic acid bacteria metabolic sugar may be added. This addition of sugar is useful when fermenting a plant having a low sugar content (sugar content of less than 1% by mass). Of course, sugar may be added for the purpose of promoting fermentation and adding sweetness to the fermented product. When sugar is added, the type is not particularly limited, but it is preferable to add sugar that can be used for growth or fermentation of lactic acid bacteria. For example, sucrose, glucose, fructose, maltose and the like are preferably added. Of course, sugars other than the sugars exemplified here may be added. About the quantity of the sugar to add, it is preferable to add so that sugar content may be about 1-6 mass% in total with the sugar content of a perilla leaf.

  In lactic acid fermentation, in order to create an environment in which lactic acid bacteria can preferentially grow, it is preferable to previously adjust the pH of perilla leaves or crushed materials thereof by neutralization treatment. For example, in the case of using Lactobacillus plantarum, if the fermentation is started after adjusting the pH to about 4.0, the fermentation can be completed in a short period of time.

  Lactic acid fermentation is preferably performed under anaerobic conditions from the viewpoint of suppressing the decomposition of ascorbic acid. Anaerobic conditions are, for example, by putting perilla leaves or their crushed material into a fermenter and then degassing, or sealing the fermenter, filling with a gas such as nitrogen or carbon dioxide, or reducing the pressure. Or by combining them. Moreover, the flavor of the fermented material obtained by performing fermentation under anaerobic conditions also becomes good.

  There are no particular restrictions on the conditions for lactic acid fermentation. Fermentation temperature can be normally performed at 4 to 50 degreeC. What is necessary is just to set fermentation time suitably according to fermentation temperature, and when performing fermentation at 20 to 50 degreeC, they are 12 hours-72 hours, Preferably they are 24 hours-72 hours. Furthermore, when performing low-temperature fermentation at 4 ° C. to 10 ° C. for the purpose of enhancing the flavor, it is preferably 5 to 14 days in consideration of loss of components contained in perilla leaves such as ascorbic acid.

  Lactic acid fermentation can be stopped by adding sugar. Examples of such sugars include sugar alcohols (for example, sorbitol), oligosaccharides (for example, maltooligosaccharide, chitooligosaccharide, fructooligosaccharide) and the like. Such oligosaccharides are effective for intestinal regulation, prevention of dental caries, and the like, and can impart functionality to the obtained fermented product.

  Lactic acid fermentation not only uses components in perilla leaves to produce useful components such as organic acids and oligosaccharides, but also maintains the fermented product at a low pH, thus preventing the propagation of other germs It is. In addition, since lactic acid bacteria are added, fermented perilla leaves with high physiological activity such as improved flavor, intestinal regulation and enzyme inhibition can be obtained.

(Citric acid fermentation)
Citric acid fermentation is generally performed by culturing yeast in contact with perilla leaves or a crushed material thereof under aerobic conditions. In citric acid fermentation, it is preferable to add lactic acid bacteria and ferment, because the growth of yeast is easily promoted and the preference of the obtained fermented product is also increased.

  As yeast, sake yeast, wine yeast, beer yeast, baker's yeast and the like are used. For example, yeast belonging to the genus Saccharomyces, Schizosaccharomyces, etc. is used, and preferred examples include Saccharomyces cerevisiae, Saccharomyces pastorianus, Schizosaccharomyces pombe and the like. In particular, Saccharomyces cerevisiae and its isolates are preferred in terms of producing useful substances such as amino acids and vitamins.

  Yeast is added in an amount of 0.001 to 15 parts by mass, preferably 0.01 to 10 parts by mass, based on 100 parts by mass of perilla leaves or crushed material thereof.

  The citric acid fermentation is carried out at 4 ° C. to 40 ° C., preferably 10 ° C. to 35 ° C. for 24 hours to 14 days while agitating and stirring a perilla leaf or its crushed material and yeast. In particular, when perilla leaves are acidic, aroma components are increased by yeast fermentation, so that a fermented product with higher palatability can be obtained.

  Since citric acid fermentation is performed using yeast, amino acids, proteins, vitamins and the like produced by yeast are contained in the fermented product, which is preferable in terms of high nutritional value and excellent palatability.

(Alcohol fermentation and acetic acid fermentation)
Alcohol fermentation is performed by culturing yeast in contact with perilla leaves or crushed material under anaerobic conditions. The kind and amount of yeast used for alcoholic fermentation are the same as in the case of the citric acid fermentation. The fermentation conditions are the same as in the above citric acid fermentation except that the conditions are anaerobic. It is preferable that the fermented perilla leaf thus obtained by alcohol fermentation is further subjected to acetic acid fermentation described below.

  Acetic acid fermentation is performed by adding alcohol to perilla leaves or a crushed material thereof to obtain a predetermined alcohol concentration, and then adding a microorganism (acetic acid bacterium) capable of acetic acid fermentation. In general, acetic acid fermentation is performed by culturing a microorganism (acetic acid bacterium) in contact with perilla leaves or a crushed material thereof under aerobic conditions. Alternatively, acetic acid bacteria may be added to fermented perilla leaves obtained by the above-described alcoholic fermentation to perform two-stage fermentation.

  The alcohol concentration is not particularly limited as long as it is a concentration at which acetic acid bacteria can grow, and may be appropriately adjusted according to fermentation time and the like. Preferably it is 10 mass / volume% or less, More preferably, it is 1-6 mass / volume%.

  Examples of the acetic acid bacteria include microorganisms belonging to the genus Acetobacter, such as Acetobacter aceti, Acetobacter pasteurianus, Acetobacter hanseni, and the like.

  The acetic acid bacteria are preferably precultured in an appropriate medium at 15 to 40 ° C, preferably 25 to 35 ° C for 6 to 48 hours. The pre-cultured acetic acid bacteria can be obtained, for example, as follows. First, acetic acid bacteria were added to 1 L of acetic acid bacteria medium (pH 7.0) containing 200 g of potato, 30 g of crushed yeast, 5 g of liver extract, 5 g of meat extract, 10 g of thioglycolic acid medium, 5 g of glucose, 15 g of glycerol, and 15 g of calcium carbonate. Add and pre-incubate at 15-40 ° C. for 24 hours. Next, the obtained culture is centrifuged, and the collected cells are washed with sterilized water, and centrifuged again to remove the supernatant, thereby obtaining pre-cultured acetic acid bacteria.

  Acetic acid fermentation can be performed by any of stirring culture, shaking culture, and stationary culture. The fermentation temperature is 10 ° C to 40 ° C, preferably 20 ° C to 35 ° C. Fermentation time is suitably set according to the addition amount of acetic acid bacteria, and usually 1 day to 1 week is suitable.

  After the fermentation, it may be further sterilized or sterilized as necessary. For example, methods commonly used by those skilled in the art such as pressure sterilization, heat exchange sterilization, steam sterilization, and ultrafiltration are used. In the case of heat sterilization, for example, it is performed at 60 ° C. to 120 ° C. for 5 seconds to 12 hours.

(Fermented product)
The fermented product obtained by fermenting perilla leaves in this manner (fermented perilla leaf) may be used as it is, or, if necessary, used in various modes depending on the processing methods usually used by those skilled in the art. May be. Examples of such an embodiment include a fermented extract obtained by solid-liquid separation of a fermented product as described above and collecting the supernatant, a fermented paste obtained by concentrating the fermented product or the fermented extract, and the fermentation Or a fermented powder or fermented extract powder obtained by drying / pulverizing the product or the fermented extract. These are all aspects of fermented perilla leaves.

  One embodiment of the fermented fern leaf is a fermented powder or fermented extract powder. Specifically, these fermented powder or fermented extract powder can be obtained by drying and pulverizing the fermented product or fermented extract. Drying may be performed by various methods commonly used by those skilled in the art, but freeze drying and spray drying are preferably used. Spray drying is performed by adding excipients such as dextrin, cyclodextrin, starch, and maltose as necessary. When the fermented product is dried, the mixing ratio of the fermented product or fermented extract and the excipient is preferably 1: 5 to 10: 1 by mass ratio, and when the fermented extract is dried, the mass ratio is preferably 1:10 to 10: 1. 5: 1. Browning of the fermented powder or fermented extract powder obtained by drying at the above mixing ratio can be prevented.

  The fermented product of the present invention has a good fragrance and flavor and is excellent in palatability. And by fermentation, the novel function (activity) which a perilla leaf has originally cannot be given, or the physiological activity which a perilla leaf has originally can be strengthened. That is, a fermented product having higher physiological activity can be produced after fermentation than before fermentation. Examples of the novel function (activity) and physiological activity here include enzyme inhibition activity.

  Specific examples of novel functions (activities) that perilla leaves originally do not include elastase inhibitory activity. Specific examples of physiological activities that perilla leaves naturally have and are enhanced include: Examples include tyrosinase inhibitory activity and glucosidase inhibitory activity (α-glucosidase inhibitory activity). Fermented perilla leaves having such activity can be used as various functional foods and pharmaceutical raw materials.

  Since the fermented perilla leaf of the present invention has various physiological activities such as tyrosinase inhibitory activity, α-glucosidase inhibitory activity, and elastase inhibitory activity as described above, various actions and effects can be expected. For example, according to the tyrosinase inhibitory action, effects such as skin whitening effect and improvement of the skin can be expected. According to the α-glucosidase inhibitory action, it can be expected to have an effect of suppressing digestion and absorption of sugar, an effect of suppressing an increase in blood sugar level, an effect of preventing diabetes and diabetic complications, and an anti-obesity effect. Furthermore, according to the elastase inhibitory action, an effect of improving skin elasticity and firmness, an anti-inflammatory effect, and the like can be expected.

(Oral composition and transdermal composition)
The fermented perilla leaf of the present invention has the above activity. Therefore, it is extremely useful to use the perilla leaf fermented product of the present invention as a transdermal composition or oral composition.

  The transdermal composition and oral composition of the present invention contain the fermented product. Of course, other components can be contained as required. The oral composition mentioned here includes foods, pharmaceuticals intended for oral administration, quasi drugs and the like. Further, the transdermal composition referred to here includes cosmetics, pharmaceuticals intended for transdermal administration, quasi-drugs, toiletries and the like.

  In the composition of the present invention, the content of the fermented product is not particularly limited. However, in consideration of obtaining the physiological effect of fermented perilla leaves, the following amounts are preferred. For example, when it is set as an oral composition (when aiming at oral administration, such as food), the lower limit of content of fermented material is 0.001 mass% or more, Preferably it is 0.01 mass% or more. The upper limit of the content of the fermented product is 100% or less, preferably 70% or less. In the case of a transdermal composition (for the purpose of transdermal administration such as cosmetics), the lower limit of the fermented product content is 0.00001% by mass or more, preferably 0.0001% by mass or more. The upper limit of the content of the fermented product is 50% by mass or less, preferably 30% by mass or less.

  As described above, the composition of the present invention may contain various components as necessary. What is necessary is just to determine content of a various component arbitrarily in consideration of a use etc. Examples of the various components mentioned here include components that can be added as ordinary foods, specifically excipients, extenders, binders, thickeners (for example, agar), emulsifiers, lubricants, and wets. Ingredients (base materials, animal and plant extracts, etc.) that can be added as agents, suspensions, colorants (pigments), food additives, seasonings, etc., or quasi-drugs, cosmetics, and toiletries. Of course, these various components may be contained alone or in combination.

  Specific examples of the various components include royal jelly, propolis, vitamins (A, B group, C, D, E, K, folic acid, pantothenic acid, biotin, derivatives thereof, etc.) minerals (iron, magnesium, calcium, Zinc, etc.), selenium, chitin / chitosan, lecithin, polyphenol (condensed tannins such as catechins, anthocyanins, proanthocyanidins, hydrolyzed tannins such as gallotannins, flavonoids, derivatives thereof, etc.), carotenoids (lycopene, astaxanthin) Zeaxanthin, lutein, etc.), saponins (isoflavones, ginsaneoids, glycyrrhizic acid, etc.), xanthine derivatives (caffeine, etc.), fatty acids, amino acids, proteins (collagen, elastin, etc.), mucopolysaccharides (hyaluronic acid, chondroitin, derma) , Heparan, heparin, ketalan, and their salts), amino sugars (glucosamine, acetylglucosamine, galactosamine, acetylgalactosamine, neuraminic acid, acetylneuraminic acid, hexosamine, their salts, etc.), dietary fiber (indigestible dextrin) , Alginic acid, guar gum, pectin, glucomannan, etc.), oligosaccharides (isomalto-oligosaccharides, cyclic oligosaccharides, etc.), phospholipids and derivatives thereof (phosphatidylcholine, sphingomyelin, ceramide, etc.), sulfur-containing compounds (allyin, sepaene, Taurine, glutathione, methylsulfonylmethane, etc.), sugar alcohols, quinones (coenzyme Q10, etc.), lignans (sesamin, etc.), animal and plant extracts containing these, root vegetables (turmeric, ginger, etc.), wheat leaves, etc. Green leaves of grass family, ke Such as the green leaves of cruciferous plants such as Le and the like. Furthermore, beverages containing these food additives, for example, plant fermented juice, vegetable juice (for example, carrot juice), plant extract, fruit juice, etc. may be used as the above various components, and by containing these, Beverages with high functionality or nutritional value can be obtained.

  Of course, you may add a seasoning as said various components. Specific examples of the seasoning herein include sweeteners such as granulated sugar, honey and sorbit, acidulants such as alcohol, citric acid, malic acid and tartaric acid, and flavors.

  The composition of the present invention can be prepared and used in various forms according to the purpose. For example, when used as an oral composition for foods, etc., those skilled in the art usually use capsules such as hard capsules and soft capsules, tablets, pills, powders (powder), granules, tea bags, liquids (beverages), pastes, etc. It can be set as the form used. Furthermore, the liquid etc. can be processed into jelly, sorbet, frozen yogurt or ice cream. These may be taken as they are, depending on the shape or preference, or can be taken by dissolving in water, hot water, milk, etc. or leaching the ingredients. In the case of beverages, if the pH is low, it can be sterilized under sterilization conditions of 100 ° C. or lower without complete sterilization at 120 ° C. for 4 minutes. For example, when the pH is 4.0 or less, it can be sufficiently sterilized under sterilization conditions corresponding to 65 ° C. and 10 minutes. When used as a transdermal composition (quasi-drugs, cosmetics, toiletries, etc.), for example, lotion, cosmetic cream, emulsion, pack, hair tonic, shampoo, hair rinse, treatment, body shampoo, facial preparation , Soap, foundation, lipstick, hair restorer, ointment, bathing agent, dentifrice, mouthwash, ship, gel and the like.

  Hereinafter, the present invention will be described with reference to examples. In addition, this invention is not restrict | limited by the following Example.

(Example 1: Preparation of fermented product)
First, edible perilla leaves (green) were crushed with a food processor. Next, by adding ion-exchanged water (2 kg) to the crushed product (1 kg) and stirring, a perilla leaf dilution was prepared.

  Next, lactic acid bacteria and the like were added to the perilla leaf dilution. Specifically, lactic acid bacteria (80 mg) and glucose (5% by mass) were added to perilla leaf dilution (80 g) that was not solid-liquid separated. The Erlenmeyer flask (200 mL) was then sealed with a silicon stopper, and fermented at 35 ° C. for 24 hours with light shaking under anaerobic conditions. Fermentation was carried out by applying a silicon stopper to an Erlenmeyer flask (200 mL) and shaking lightly under anaerobic conditions. After fermentation, the fermented fern leaf obtained was filtered to obtain a fermentation broth.

  The lactic acid bacteria used for the fermentation were (1) Lactobacillus plantarum Viniflora strain (Christian Hansen), (2) Lactobacillus casei (Christian Hansen), (3) Bifidobacterium longum ( (4) Bacillus mecentericus (manufactured by Toa Pharmaceutical Co., Ltd.).

  In addition, when performing fermentation, the acidity before and behind fermentation was measured by the neutralization titration method using 1N sodium hydroxide (NaOH). In the measurement of the acidity, the acidity after fermentation was measured using the fermentation broth. The acidity before fermentation was measured by immediately filtering a product obtained by adding lactic acid bacteria to a perilla leaf dilution and using the filtrate obtained by the filtration. The results are shown in Table 1.

  From Table 1, the acidity was increased 24 hours after adding lactic acid bacteria such as Lactobacillus plantarum Viniflora strain, Lactobacillus casei, Bifidobacterium longum, and Bacillus mecentericus to perilla leaves. . This indicates that any of the four types of lactic acid bacteria can be used to ferment perilla leaves.

  The fermentation broth was sterilized at 95 ° C. for 8 seconds, and then freeze-dried. By this lyophilization, various dry powders (fermented fermented leaf extract) of fermented perilla leaves fermented with each lactic acid bacterium could be obtained.

(Example 2: Tyrosinase inhibitory activity)
Tyrosinase inhibitory activity (inhibition rate) was measured by the following method.

  First, the fermentation broth (100 μL) described in Example 1 and a tyrosinase solution (100 μL, 112 units / mL) were mixed and held at 37 ° C. for 10 minutes. The tyrosinase solution used here is a solution prepared by dissolving tyrosinase (800 units / mg) manufactured by Funakoshi in a phosphate buffer solution (1/15 M, pH 7.0).

  Then, 0.03 mass / volume% DOPA solution (100 μL) was added, and the mixture was further maintained at 37 ° C. for 5 minutes to obtain a test solution. Then, the absorbance (475 nm) of the test solution was measured. The result obtained by this measurement is defined as a measurement value A. In addition, the 0.03 mass / volume% DOPA solution was prepared by dissolving L-DOPA (manufactured by Wako Pure Chemical Industries, Ltd.) in a phosphate buffer solution (1/15 M, pH 7.0).

  As controls, the following measured values B to D were used.

  The measured value B is a value when no fermentation broth is added. Specifically, in the same manner as the method for obtaining the measurement value A except that a phosphate buffer (1/15 M, pH 7.0) was used instead of the fermentation broth described in Example 1. The measured value B was obtained.

  The measurement value C is a blank of the measurement value A. Specifically, the measurement value C is obtained by the same method as the method for obtaining the measurement value A except that a phosphate buffer (1/15 M, pH 7.0) is used instead of the tyrosinase solution. did.

  The measurement value D is a blank of the measurement value B. Specifically, the measurement value D is obtained by the same method as the method for obtaining the measurement value B except that a phosphate buffer (1/15 M, pH 7.0) is used instead of the tyrosinase solution. did.

  The tyrosinase inhibition rate (%) was calculated using the measured values A to D before and after fermentation for each lactic acid bacterium and the following formula (I). The results are shown in Table 2 in Example 5 below. This measurement is based on a triple measurement (n = 3 per group).

  Moreover, about the tyrosinase inhibition rate before fermentation, it replaces with the fermentation liquid as described in the said Example 1, The method similar to the above except having used the solution before fermentation obtained by filtering the crushed material before fermentation. Calculated with

(Example 3: Elastase inhibitory activity)
The elastase inhibitory activity (inhibition rate) was measured by the following method.

  First, the fermentation broth (100 μL) described in Example 1 and an elastase solution (0.1 unit) were mixed and held at 37 ° C. for 15 minutes. The elastase solution used here was prepared by dissolving elastase (manufactured by Sigma) in Tris buffer (0.2 M, pH 8.0).

  Thereafter, a substrate solution (100 μL, 2.5 mM) was added, and further maintained at 37 ° C. for 10 minutes to obtain a test solution. And the light absorbency (400 nm) of the test liquid was measured. The result obtained by this measurement is defined as a measurement value A. The substrate used here was N-succinyl-Ala-Ala-Ala-p-nitroanilide (manufactured by Wako Pure Chemical Industries, Ltd.), and the substrate solution was Tris buffer (0.2 M, pH 8.0). And a substrate.

  As controls, the following measured values B to D were used.

  The measured value B is a value when no fermentation broth is added. Specifically, a method similar to the method for obtaining the measurement value A except that Tris buffer (0.2 M, pH 8.0) was used instead of the fermentation broth (100 μL) described in Example 1. The measurement value B was acquired.

  The measurement value C is a blank of the measurement value A. Specifically, the measurement value C was obtained in the same manner as the method for obtaining the measurement value A except that a tris buffer (0.2 M, pH 8.0) was used instead of the elastase solution. .

  The measurement value D is a blank of the measurement value B. Specifically, the measurement value D was obtained in the same manner as the method for obtaining the measurement value B, except that a tris buffer (0.2 M, pH 8.0) was used instead of the elastase solution. .

  The elastase inhibition rate was calculated using the measured values A to D before and after fermentation for each lactic acid bacterium and the following formula (II). The results are shown in Table 2 in Example 5 below. This measurement is based on a triple measurement (n = 3 per group).

  Moreover, about the elastase inhibition rate before fermentation, the method similar to the above except having used the solution before fermentation obtained by filtering the crushed material before fermentation instead of the fermentation liquid as described in the said Example 1. Calculated with

(Example 4: α-glucosidase inhibitory activity)
Α-glucosidase inhibitory activity (inhibition rate) was measured by the following method.

  First, the fermentation broth (80 μL) described in Example 1 and a substrate aqueous solution (p-nitrophenyl-α-D-glucopyranoside, 0.02 M, 40 μL) were mixed and held at 37 ° C. for 5 minutes. Next, an α-glucosidase solution (0.04 μg / mL, 40 μL) was added, and the mixture was further maintained at 37 ° C. for 15 minutes.

  Next, an aqueous sodium carbonate solution (0.2 M, 160 μL) was added to obtain a test solution. And the light absorbency (400 nm) of the test liquid was measured. The result obtained by this measurement is defined as a measurement value A.

  The α-glucosidase solution (0.04 μg / mL) was prepared as follows. First, bovine serum albumin is dissolved in a phosphate buffer (1/15 M, pH 7.0) to obtain a 0.2% bovine serum albumin solution. Next, a 0.1 mg / mL solution was prepared using α-glucosidase (manufactured by Wako Pure Chemical Industries, Ltd.) and phosphate buffer (1/15 M, pH 7.0), and the solution was used as the bovine serum albumin solution. To obtain an α-glucosidase solution (0.04 μg / mL).

  As controls, the following measured values B to D were used.

  The measured value B is a value when no fermentation broth is added. Specifically, in the same manner as the method for obtaining the measurement value A except that a phosphate buffer (1/15 M, pH 7.0) was used instead of the fermentation broth described in Example 1. The measured value B was obtained.

  The measurement value C is a blank of the measurement value A. Specifically, the fermentation liquid (80 μL) described in Example 1 and a substrate aqueous solution (p-nitrophenyl-α-D-glucopyranoside, 0.02 M, 40 μL) are mixed and held at 37 ° C. for 20 minutes. did. Next, an aqueous sodium carbonate solution (0.2 M, 160 μL) was added, and an α-glucosidase solution (0.04 μg / mL, 40 μL) was further added to obtain a control test solution. Then, the absorbance (400 nm) of the control test solution was measured. The result obtained by this measurement is defined as a measurement value C.

  The measurement value D is a blank of the measurement value B. Specifically, a phosphate buffer (1/15 M, pH 7.0, 80 μL) and a substrate aqueous solution (p-nitrophenyl-α-D-glucopyranoside, 0.02 M, 40 μL) were mixed, and the mixture was mixed at 37 ° C. For 20 minutes. Next, an aqueous sodium carbonate solution (0.2 M, 160 μL) was added, and an α-glucosidase solution (0.04 μg / mL, 40 μL) was further added to obtain a control test solution. Then, the absorbance (400 nm) of the control test solution was measured. The result obtained by this measurement is defined as a measurement value D.

  The α-glucosidase inhibition rate was calculated using the measured values A to D before and after fermentation for each lactic acid bacterium and the following formula (III). The results are shown in Table 2 in Example 5 below. This measurement is based on a triple measurement (n = 3 per group). Moreover, the α-glucosidase inhibition rate was calculated | required by the method similar to the above except having used the solution before fermentation obtained by filtering the crushed material before fermentation instead of the fermentation liquid of the said Example 1. FIG. .

  Moreover, about the alpha-glucosidase inhibition rate before fermentation, it was the same as the above except having used the solution before fermentation obtained by filtering the crushed material before fermentation instead of the fermentation liquid as described in the said Example 1. It was calculated by the method.

(Example 5: Measurement result)
Each inhibition rate calculated in Examples 2 to 4 is shown in Table 2. In addition, the value of Table 2 has shown the average value +/- standard deviation.

  According to the results in Table 2, it can be seen that the numerical values of the tyrosinase inhibition rate, the elastase inhibition rate, and the α-glucosidase inhibition rate after fermentation are larger than those before fermentation. That is, it can be seen that physiological activities such as enzyme inhibitory activity can be increased or imparted by fermentation. In particular, the tyrosinase inhibitory action and the α-glucosidase inhibitory action showed an inhibitory activity more than doubled by fermentation, and among them, the case of fermenting with Bifidobacterium longum was most excellent.

  Moreover, although the elastase inhibitory action was not detectable before fermentation, it came to show inhibitory activity by fermentation. That is, it can be seen that physiological activity (elastase inhibitory activity) could be imparted by fermentation.

The fermented perilla leaves obtained by allowing microorganisms to act on perilla leaves of the present invention are useful because they have excellent tyrosinase inhibitory action, α-glucosidase inhibitory action and elastase inhibitory action. Fermented perilla leaves having such functions are useful because they can be used as raw materials for oral compositions such as foods and pharmaceuticals, or as raw materials for transdermal compositions such as cosmetics.

Claims (5)

  A fermented product obtained by allowing microorganisms to act on perilla leaves and having enzyme inhibitory activity.   The fermented product according to claim 1, wherein the microorganism is a lactic acid bacterium.   The fermented product according to claim 1 or 2, which has an enzyme inhibitory activity of at least one of tyrosinase inhibitory activity, glucosidase inhibitory activity, and elastase inhibitory activity.   An oral composition comprising the fermented product according to any one of claims 1 to 3. A transdermal composition containing the fermented product according to any one of claims 1 to 3.