JP2010138147A - Melanogenesis inhibitor, antioxidant, anti-inflammatory agent, external preparation for skin, and food and beverage - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an external preparation for the skin utilizing a newly found fermentation product of lactobacillus which has both excellent melanogenesis inhibiting effect and anti-oxidation effect and exhibits high whitening effect and anti-aging effect for the skin. <P>SOLUTION: The external preparation for the skin includes, as an effective ingredient, a fermentation product or its extract obtained by fermenting pineapple juice with a lactobacillus selected from the group consisting of Lactobacillus casei and Lactobacillus paracasei. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  Ultraviolet rays are said to damage the collagen fibers that form the skin, accelerate skin aging, and affect DNA damage and skin cancer risk. As a defense against ultraviolet radiation, the human body secretes a brown pigment, melanin, and attempts to lower the average level of ultraviolet radiation by tanning. This dye blocks UV penetration and reduces damage to deeper skin tissue. This melanin pigmentation is excreted by turnover, but if it is exposed to a large amount of ultraviolet light for a long period of time, skin cells are damaged, causing abnormal melanin pigmentation due to abnormal activation of melanocytes.

  In addition, it is known that ultraviolet rays activate melanocytes, while suppressing the activity of fibroblasts that maintain skin elasticity. Furthermore, it is known that active oxygen generated by ultraviolet rays or the like is greatly related to skin aging such as inflammation. For example, active oxygen decomposes, denatures or cross-links biological tissues such as collagen, or generates lipid peroxides that oxidize fats and oils and damage cells, thereby forming skin wrinkles and reducing skin elasticity. It is thought to cause skin aging and inflammation. Much research has been conducted to protect against such pigment deposition and skin aging, and to date skin external preparations having several effects such as melanin production inhibitory action and antioxidant action have been developed.

  For example, as a natural substance, plants such as an extract of Duabanga (Patent Document 1), a leaf of a genus Bamboo and / or an extract thereof (Patent Document 2), a white flower serpentine plant or an extract thereof (Patent Document 3) A topical skin preparation using an extract as an active ingredient has been reported.

  On the other hand, utilization of lactic acid bacteria and fermented products thereof as active ingredients has also been studied. For example, it is known that fermented lactic acid bacteria of various fruit juices have tyrosinase inhibitory activity and antioxidant activity (Patent Document 4). . However, the fermented lactic acid bacteria disclosed in this document cannot be said to have sufficient tyrosinase inhibitory activity or antioxidant activity.

JP 2008-74728 A JP 2008-56587 A Japanese Patent No. 2007-269552 Japanese Patent No. 2006-8666

  Therefore, the present invention finds a fermented lactic acid bacterium that has an excellent melanin production inhibitory effect and antioxidant effect, and exhibits a high whitening effect and skin aging prevention effect, and provides a skin external preparation and food using the same. It is to be an issue.

  As a result of diligent research to solve the above problems, the present inventors have found that a fermented product obtained by fermenting pineapple juice with a specific lactic acid bacterium of the genus Lactobacillus has an extremely excellent melanin production inhibitory action. The inventors have found that it has an antioxidant action and have completed the present invention.

  That is, the present invention contains, as an active ingredient, a fermented product obtained by fermenting pineapple juice with a lactic acid bacterium selected from the group consisting of Lactobacillus casei and Lactobacillus paracasei, or an extract thereof. A melanin production inhibitor, an antioxidant, and an anti-inflammatory agent.

  Moreover, this invention is a skin external preparation or foodstuff which contains the fermented material obtained by fermenting with the said lactic acid bacteria, or its extract as an active ingredient.

  The melanin production inhibitor of this invention shows the outstanding melanin production inhibitory effect based on high tyrosinase inhibitory activity etc. Further, the antioxidant and anti-inflammatory agent of the present invention have high DPPH radical scavenging ability and macrophage-producing active oxygen scavenging ability, and have excellent antioxidant effect and anti-inflammatory effect. Furthermore, the external preparation for skin and foods and beverages of the present invention can be applied to or ingested with the skin to obtain an excellent whitening effect, anti-inflammatory effect and prevention / improvement effect against skin aging such as wrinkles and sagging. is there.

  In the present invention, a fermented product obtained by fermenting pineapple juice with lactic acid bacteria belonging to the genus Lactobacillus is used as an active ingredient. The pineapple fruit juice is a liquid obtained by crushing or squeezing pineapple fruit by a conventional method, and may contain a solid content, or may be a solid-liquid separated according to a conventional method to remove the solid content. . Commercially available pineapple juice can also be used.

  Lactic acid bacteria selected from the group consisting of Lactobacillus casei and Lactobacillus paracasei are used as the lactic acid bacteria that act on the pineapple juice. As long as it belongs to these species, it can be used without particular limitation, and either a commercially available strain or a strain isolated from the environment by a conventional method may be used. Arbitrary hybrids can also be used, and these can be used alone or in combination of two or more.

  In fermenting the pineapple juice with the lactic acid bacteria, the lactic acid bacteria are usually 0.0001 to 5% by mass (hereinafter simply referred to as “%”) with respect to the pineapple juice, preferably 0.1 to 3%. %Added. This range is preferable because a fermented product having high tyrosinase inhibitory activity and high antioxidant activity can be obtained.

  Moreover, nutrient components, such as culture media, such as sugar, a yeast extract, and a peptone, a vitamin, and a mineral, can be added to a pineapple fruit juice as needed. Although the addition amount is not particularly limited, it is preferably about 0.01 to 1% with respect to pineapple juice. Further, the pineapple juice may be diluted by adding water such as sterilized distilled water as necessary. Note that the pineapple fruit juice may be heat-treated.

  Fermentation conditions are not particularly limited, and the temperature is usually in the range of 20 ° C to 45 ° C, preferably 25 to 40 ° C, and the time is usually 8 hours to 10 days, preferably 3 to 7 days. Such a range is preferable because a fermented product having high tyrosinase inhibitory activity and antioxidant activity can be obtained. Moreover, the range of preferable pH is 5-8.

  The fermented product obtained in this manner can be used as it is, but may be further subjected to treatment such as concentration / drying as necessary, and such concentrate, dried product, etc. are also included in the present invention. Included in the fermented product used. Further, the fermented product may be further extracted. Examples of such an extract include water, physiological saline, phosphate buffer, polar organic solvent, supercritical fluid and subcritical fluid, or a mixture thereof. An extract using an extraction solvent such as Although extraction conditions can be set suitably, what is necessary is just to carry out about several minutes-overnight at 10-50 degreeC in the extraction solvent about 50-1000 times with respect to a concentrate (dry weight).

  Among these, an extract with a polar organic solvent such as alcohol is preferable, and an extract with ethanol for food addition is particularly preferable. More specifically, after removing moisture from the obtained fermented product by a conventional drying means such as freeze-drying, about 100 to 200 ml of ethanol is added to 10 g of the fermented product at 30 ° C. overnight. By performing a degree of extraction, an ethanol extract of the fermented product is obtained. This ethanol extract can be used as it is, but if necessary, ethanol may be distilled off and used.

  Fermented product of Lactobacillus casei and / or Lactobacillus paracasei of the pineapple juice obtained as described above (hereinafter sometimes referred to as “pineapple lactic acid bacteria fermented product”) or its extract is high. It has melanin production inhibitory action based on tyrosinase inhibitory activity etc. and antioxidant action based on DPPH radical and active oxygen scavenging activity etc., and this pineapple lactic acid bacteria fermented product or extract thereof as an active ingredient, It can be set as the outstanding melanin production inhibitor, antioxidant, and anti-inflammatory agent by mixing suitably and formulating.

  As the carrier for formulation, for example, starch, lactose, sucrose, mannitol and the like can be used, and additives such as binders, disintegrants, surfactants and lubricants can be further blended as necessary. . Moreover, the form is not specifically limited, It can be set as a powder, a powder, a granule, a tablet, a capsule, a liquid agent, an ointment, a cream agent, etc.

  Content of the pineapple lactic acid bacteria fermented product or its extract in the melanin production inhibitor of this invention is 0.00001-100 mass% normally in dry matter conversion, and 0.001-50 mass% is preferable. This melanin production inhibitor improves pigmentation symptoms such as spots and freckles and exhibits an excellent whitening effect due to high tyrosinase inhibitory activity and the like.

  The content of the fermented pineapple lactic acid bacterium or the extract thereof in the antioxidant and anti-inflammatory agent of the present invention is the same as that of the melanin production inhibitor. The antioxidant and anti-inflammatory agent of the present invention exhibit excellent antioxidant and anti-inflammatory effects based on high DPPH radical scavenging action and scavenging activity of reactive oxygen species produced by macrophages.

  Also, by blending pineapple lactic acid bacteria fermented product or its extract into a skin external preparation, based on its excellent melanin production inhibitory action and antioxidant action, it is used for skin whitening, anti-inflammatory use, wrinkle, tarmi and other skin aging prevention It can be used as a skin external preparation.

  The content of the pineapple lactic acid bacteria fermented product or the extract thereof in the external preparation for skin of the present invention is usually 0.00001 to 50% in terms of dry matter, and 0.001 to 20% from the standpoint of effects and stability. Is more preferable, and 0.1 to 10% is more preferable.

  The external preparation for skin of the present invention includes cosmetics, quasi-drugs, and pharmaceuticals. In addition to the lactic acid fermented product, it is usually blended into cosmetics, quasi-drugs, and pharmaceuticals as necessary. , Oil components, surfactants, moisturizers, pigments, powders, dyes, emulsifiers, solubilizers, cleaning agents, UV absorbers, thickeners, drugs, fragrances, resins, antibacterial and antifungal agents, alcohols, etc. Can be appropriately blended. Moreover, in the range which does not impair the effect of this invention, combined use with another melanin production inhibitor, an antioxidant, or an anti-inflammatory agent is also possible.

  The dosage form of the external preparation for skin of the present invention is arbitrary, and can be provided, for example, as a solubilizing system such as lotion, an emulsifying system such as cream or milky lotion, or a dispersing system such as calamine lotion. Furthermore, it can be set as various dosage forms, such as aerosol and ointment filled with the propellant.

  In addition, when it is used as a cosmetic, the type is not particularly limited. For example, basic cosmetics such as lotion, milky lotion, cosmetic liquid, moisturizing cream; sunscreen cream, sunscreen lotion, suntan oil, carmine lotion, etc. Sun care products: makeup cosmetics such as foundation, eyeliner, mascara, eye color, teak color, lipstick, etc .; cleansing agents such as face wash, body shampoo, hair shampoo; rinse, treatment, hair cream, hair oil, hair conditioner, etc. Hair cosmetics; perfume or deodorant antiperspirant.

  Furthermore, by mixing the above lactic acid bacteria fermented product with an appropriate food and drink material to make a food and drink, the whitening, anti-inflammatory, based on the excellent melanin production inhibitory action and antioxidant action possessed by the pineapple lactic acid bacteria fermented product or its extract And foods for preventing skin aging such as wrinkles and sagging. Specific examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks; confectionery such as chewing gum, candy, gum, chocolate, and tablet confectionery; health in the form of tablets, capsules, drinks, etc. -Nutritional supplements can be exemplified. The content of the fermented pineapple lactic acid bacteria or the extract thereof in the food or drink can be appropriately set according to the type of food or drink, etc., but the content that the daily intake per adult is about 1 to 10 g in terms of dry matter It can be an amount.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not restrict | limited to these Examples at all.

Example 1
Tyrosinase inhibitory activity test (1):
(Acquisition of lactic acid bacteria)
Lactic acid bacteria were isolated from various environmental samples. MRS agar medium (source: Nippon Becton Decktonson Co., Ltd.) was used to isolate microorganisms that formed halos (circles in which calcium carbonate was dissolved) around the colonies using a separation medium containing 0.5% calcium carbonate. The lactic acid bacteria were isolated. The isolated lactic acid bacteria extracted DNA, and 16S rRNA PCR products were obtained using universal primers (11F and 1451R) corresponding to 16S rRNA. The base sequence of 16S rRNA was identified with a DNA sequencer, and the sequence was searched for species with high homology by Blast search (Japan DNA Data Bank). Alignment of these strains with 16S rRNA sequences was performed to generate a phylogenetic tree and identified at the species level. The separated lactic acid bacteria were cultured in an MRS liquid medium at 28 ° C. for 3 days. After culturing, the culture supernatants were collected.
(Measurement of tyrosinase inhibitory activity)
The collected culture supernatant was evaluated for tyrosinase inhibitory activity by the following method. L-DOPA (final concentration 2.5 mM) and mushroom tyrosinase (maximum final concentration 2.5 U) were dissolved in 10 mM phosphate buffer (pH 6.5). A sample in which 40 μL of phosphate buffer, 25 μL of L-DOPA, and DMSO were dissolved was dispensed in the order of 10 μL to a 96-well plate, and the reaction was started by adding a tyrosinase solution. The reaction was incubated for 30 minutes at 25 ° C. and DOPA chromium formation was measured with a plate reader. The amount of DOPA chromium was estimated from the absorbance at 475 nm (ε = 3700 M ⁻¹ cm ⁻¹ ). Tyrosinase inhibitory activity was calculated according to the following formula. The results are shown in Table 1.
Tyrosinase inhibitory activity (%) = sample A475 nm / no added A475 nm × 100

  As shown in Table 1, when the culture supernatant of lactic acid bacteria was used, high tyrosinase inhibitory activity was observed in the genus Lactobacillus and Enterococcus among lactic acid bacteria. This revealed that Lactobacillus and Enterococcus produced more tyrosinase inhibitory active substances than other lactic acid bacteria.

Example 2
Tyrosinase inhibitory activity test (2):
The tyrosinase inhibitory activity of various species belonging to the genus Lactobacillus and Enterococcus was evaluated. Using strains obtained from microorganism preservation organizations (National Institute of Technology and Evaluation) and strains isolated from Okinawa's special fruits (pineapples, etc.), bark, flowers and marine environment in the same manner as in Example 1, The tyrosinase inhibitory activity of was evaluated in the same manner as in Example 1. Table 2 shows the results.

  As shown in Table 2, tyrosinase inhibitory activity was confirmed to be strong in Lactobacillus casei, Lactobacillus paracasei, and some Enterococcus sulfurous. However, stronger tyrosinase inhibitory activity was observed in common with Lactobacillus genus strains, especially Lactobacillus casei and Lactobacillus paracasei than with Enterococcus strains. It can be seen that the tyrosinase inhibitory activity is a physiological activity common to strains of the genus Lactobacillus and Enterococcus. Among them, particularly strong tyrosinase inhibitory activity was observed in Lactobacillus casei and Lactobacillus paracasei.

Example 3
Selection of fruit juice as culture medium:
Fermented lactic acid bacteria were prepared using various fruit juices or vegetable juices as culture media, and tyrosinase inhibitory activity was evaluated in the same manner as in Example 1. As the fruit juice or vegetable juice, a commercially available 100% reduction-concentration type was used. Lactobacillus paracasei isolated from the pineapple fruit obtained in Example 2 was used as the lactic acid bacterium. Culturing was carried out under the conditions of 72 hours at 28 ° C. with 2% by mass of lactic acid bacteria added to the culture medium. Moreover, the tyrosinase inhibitory activity of each fruit juice or vegetable juice itself was similarly evaluated (unfermented). Table 3 shows the results.

  As shown in Table 3, when pineapple juice was used as a fermentation group, an extremely high tyrosinase inhibitory activity was observed compared to other fruit juices or vegetable juices. In addition, this fermented product had a markedly increased activity compared to the tyrosinase inhibitory activity of unfermented pineapple juice itself. On the other hand, the lactic acid bacteria fermented product of known fruit juice (Patent Document 4) is only slightly increased in activity as compared with unfermented fruit juice. Pineapple fruit juice and Lactobacillus casei or Lactobacillus paracasei It was shown that the tyrosinase inhibitory activity is particularly enhanced by the combination. The unfermented and fermented products are diluted with sterilized water so that the activity can be evaluated at the same dilution rate.

Manufacturing example 1
Preparation of ethanol extract of lactic acid bacteria fermentation of pineapple juice:
Lactic acid bacteria were cultured at 28 ° C. for 3 days using MRS (Difco) liquid medium. A pre-culture solution of 2% by mass was added to commercially available pineapple juice (Company A), and static culture was performed at 28 ° C. for 3 days. After culturing, the pineapple lactic acid bacteria fermentation product was freeze-dried, and the same amount of ethanol as the culture solution was added to the dried product. After sufficient stirring, the ethanol was removed by an evaporator. This operation was repeated twice to obtain an ethanol extract of fermented pineapple lactic acid bacteria. The lactic acid bacteria used were isolated from NBRC 15883, NBRC 101979 and pineapple fruit of Example 2 as Lactobacillus casei, and from NBRC 3533, NBRC 15889 and Example 2 pineapple fruit as Lactobacillus paracasei 3 strains.

Example 4
Melanin production inhibitory action test:
The extract of pineapple lactic acid bacteria fermentation product by Lactobacillus casei NBRC 101979 and Lactobacillus paracasei NBRC 15889 obtained in Production Example 1 was used as a sample. B16 mouse melanoma (B16F0) cells were seeded in a 90 mm dish at 25000 cells per dish. The seeding medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS). After culturing for 24 hours, the medium was replaced with a sample-containing culture solution adjusted to the concentration of each sample in 5% by mass FBS-added DMEM medium, and further cultured for 5 days. In addition, what used the 5 mass% FBS addition DEME culture medium without a sample instead of the sample was made into negative control. After completion of the culture, the cells were collected by trypsin treatment, transferred to a 1.5 mL microtube, and centrifuged to obtain a cell precipitate. DMSO was added to the resulting precipitate and heated at 60 ° C. for 30 minutes, then cooled to room temperature, and the absorbance at 500 nm was measured with a spectrophotometer. The inhibitory effect on melanin production was evaluated by the absorbance at 500 nm. The results are shown in FIG.

  As shown in FIG. 1, both the Lactobacillus casei NBRC 101979 strain and the Lactobacillus paracasei NBRC 15889 fermented lactic acid bacteria showed a significant decrease in melanin content compared to the additive-free control. From this, it became clear that the fermented material by Lactobacillus casei and Lactobacillus paracasei has a melanin production inhibitory effect and a whitening effect based thereon.

Example 5
DPPH radical scavenging action test:
The ethanol extract of the pineapple lactic acid bacteria fermentation product obtained in Production Example 1 was used as a sample, and ethanol was added again to prepare a sample solution having a concentration of 100 mg / ml. 90 μL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution was added to each 96-well microplate. Further, 10 μL of the sample solution was added thereto. After thorough mixing, the mixture was allowed to stand in the dark at room temperature for 1 hour, and then the absorbance at 517 nm derived from the DPPH radical was measured. When the absorbance when only ethanol was added instead of the sample solution was (A) and the absorbance when the sample solution was added was (B), the value obtained by the following formula was the DPPH radical elimination rate. The DPPH radical scavenging action was evaluated by this DPPH radical scavenging rate. Moreover, it evaluated similarly about pineapple fruit juice (Company A). Table 4 shows the results.
DPPH radical elimination rate (%) = {1- (B) / (A)} × 100 (%)

  From Table 4, it became clear that the ethanol extract of the pineapple fermented product by any strains of Lactobacillus casei and Lactobacillus paracasei has an antioxidant action based on a high DPPH radical scavenging action. It was also shown that radical scavenging activity was significantly increased compared to unfermented pineapple juice. On the other hand, a conventionally known lactic acid bacteria fermented product of fruit juice (Patent Document 4) shows only a slight increase in activity compared to unfermented fruit juice, and pineapple juice and Lactobacillus casei or Lactobacillus paracasei It was clarified that the antioxidant activity was particularly improved by the combination.

Example 6
Test for elimination of reactive oxygen species produced by macrophages:
Melanin pigments accumulated by ultraviolet rays are excreted together with the stratum corneum at turnover, but melanin partially dropped into the dermis layer is excreted by the function of active macrophages. Macrophages carry a group of enzymes that produce reactive oxygen species called NADPH oxidase. This enzyme works to break down and remove microorganisms and viruses that invade the body, and is one of the important enzymes for life support. On the other hand, if this enzyme works excessively, it is said to cause collagen disease and allergic diseases. Therefore, the ability of pineapple lactic acid fermentation extract to eliminate reactive oxygen species by macrophages overexpressed with phorbol ester was evaluated.

  The ethanol extract of the pineapple lactic acid bacteria fermentation product by Lactobacillus casei NBRC 101979 and Lactobacillus paracasei NBRC 15889 obtained in Production Example 1 was used as a sample. RAW264.9 macrophage cells were seeded in a 90 mm dish so that there were 20000 cells per dish. As the seeding medium, RPMI medium supplemented with 5% by mass of fetal bovine serum (FBS) was used. After culturing for 72 hours, the cells were collected with a cell scraper and washed three times with sterile PBS. Cells were adjusted to 20000 cells per ml in PBS containing 11 mM glucose. A 96-well microplate for luminescence was dispensed with 100 microliters of cell suspension, 10 μL of pineapple lactic acid fermentation ethanol extract and 100 μM MCLA. 100 nM phorbol ester was added to initiate the reaction. The amount of luminescence for 5 minutes from the start of the reaction was integrated. For the amount of chemiluminescence, a luminescence plate reader (manufactured by Thermoscience) was used. Samples were diluted with DMSO. Also, pineapple juice (Company A) was similarly evaluated (unfermented pine extract). In addition, what used DMSO instead of the sample was made into negative control. The results are shown in FIG.

  From FIG. 2, it was revealed that the ethanol extract of the pineapple fermented product by any strain of Lactobacillus casei and Lactobacillus paracasei has an activity of eliminating reactive oxygen species from macrophages.

  The pineapple lactic acid bacteria fermented product or extract thereof used in the present invention has excellent melanin production inhibitory action and antioxidant action, and is highly safe. Therefore, it is useful as a component for whitening for improving pigmentation symptoms such as stains and kusumi, or for external preparations for skin and for foods and beverages for improving skin aging symptoms such as wrinkles and tarmi.

It is a figure which shows the melanin production inhibitory effect by the ethanol extract of the pineapple fermented material of Lactobacillus casei and Lactobacillus paracasei in Example 3. It is a figure which shows the macrophage production active oxygen scavenging effect by the ethanol extract of the pineapple fermented material of Lactobacillus casei and Lactobacillus paracasei in Example 6.

Claims (10)

  It contains a fermented product obtained by fermenting pineapple juice with lactic acid bacteria selected from the group consisting of Lactobacillus casei and Lactobacillus paracasei or an extract thereof as an active ingredient. Melanin production inhibitor.   The melanin production inhibitor according to claim 1, wherein the fermented product extract is an ethanol extract.   It contains a fermented product obtained by fermenting pineapple juice with lactic acid bacteria selected from the group consisting of Lactobacillus casei and Lactobacillus paracasei or an extract thereof as an active ingredient. Antioxidants.   The antioxidant according to claim 3, wherein the fermented product extract is an ethanol extract.   It contains a fermented product obtained by fermenting pineapple juice with lactic acid bacteria selected from the group consisting of Lactobacillus casei and Lactobacillus paracasei or an extract thereof as an active ingredient. Anti-inflammatory agent.   6. The antioxidant according to claim 5, wherein the fermented product extract is an ethanol extract.   It contains a fermented product obtained by fermenting pineapple juice with lactic acid bacteria selected from the group consisting of Lactobacillus casei and Lactobacillus paracasei or an extract thereof as an active ingredient. Skin external preparation.   The external preparation for skin according to claim 7, wherein the extract of the fermented product is an ethanol extract.   A food or drink comprising a fermented product obtained by fermenting pineapple juice with lactic acid bacteria selected from the group consisting of Lactobacillus casei and Lactobacillus paracasei, or an extract thereof. .   The food or drink according to claim 9, wherein the extract of the fermented product is an ethanol extract.

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