Marjoram ferment and its use

Abstract

Provided is a marjoram-derived material having been improved to enhance the activity thereof.　This material is a product obtained by fermenting a starting material, which is optionally treated marjoram, with the use of one or more microorganisms selected from among microorganisms belonging to the genus Lactobacillus. This fermentation product is useful as a material to be blended in skin external agents and cosmetics, etc.

Description

Technical Field

[0001] The present invention relates to a plant ferment useful as a topical skin preparation or the like, and more particularly to a marjoram ferment and a topical skin preparation containing the same.

Background Art

[0002] Marjoram is a perennial plant native to the Mediterranean region and is a wild species, but it is widely cultivated for use as a herb or crude drug. Conventionally, plant extracts and ferments have been used in cosmetics and the like, but the use of marjoram as a topical skin preparation has been reported. That is, for example, Patent Document 1 describes that an ethanol extract of marjoram promotes the expression of heat shock proteins and suppresses melanin production. It is also described that it may be provided as a topical skin preparation.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, with the conventional method using extracts, the activity of marjoram could not be sufficiently elicited.

[0005] Therefore, an object of the present invention is to provide a marjoram-derived material improved for enhancing activity. Further, thereby, to provide a novel topical skin preparation.

Means for Solving the Problems

[0006] To achieve the above object, the present inventors have conducted intensive studies and completed the present invention.

[0007] The first aspect of the present invention provides a fermented product by one or more microorganisms selected from microorganisms belonging to the genus Lactobacillus, using marjoram or a processed product thereof as a raw material.

[0008] In the fermented product according to the present invention, the microorganism is preferably one or more microorganisms selected from the group consisting of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus mali, Lactobacillus fabifermentans, and Lactobacillus hordei.

[0009] The second aspect of the present invention provides a topical skin preparation containing the above fermented product.

[0010] In the topical skin preparation according to the present invention, the topical skin preparation is preferably used to provide a whitening effect.

[0011] In the topical skin preparation according to the present invention, the fermented product preferably has an action of suppressing melanin production.

[0012] In the topical skin preparation according to the present invention, the fermented product preferably has a tyrosinase inhibitory action.

Advantages of the Invention

[0013] According to the present invention, since it is a fermented product using marjoram or a processed product thereof as a raw material and treating it with specific microorganisms, it is excellent in activities that bring about whitening effects such as melanin production inhibitory activity and tyrosinase inhibitory activity. Therefore, a novel topical skin preparation can be provided thereby.

Brief Description of the Drawings

[0014]

Figure 1

Mode for Carrying Out the Invention

[0015] In this specification, "marjoram" is synonymous with the plant commonly understood by those skilled in the art, and specifically, it means including marjoram of the genus Origanum in the family Lamiaceae (scientific name: Origanum majorana, also called mayolana in Japanese). Marjoram is a plant native to the Mediterranean region and is widely cultivated for use as herbs and crude drugs and is easily available.

[0016] In the present invention, microorganisms are allowed to act on marjoram or a processed product thereof to form a fermented product by the microorganisms. Examples of the part of the marjoram plant body on which the microorganisms act include leaf parts, flower parts, stem parts, above-ground parts, root parts, whole plants, or mixtures of these parts, etc., but preferably leaf parts, above-ground parts, or mixtures of these parts, etc., and more preferably leaf parts. Examples of the shape of the plant body on which the microorganisms act include crushed products of plant protoplasts or their dried products, squeezed juices, extracts, or mixtures of these, etc., and are not particularly limited. Preferably, they are squeezed juices, extracts, or mixtures of these, etc., and more preferably extracts.

[0017] Examples of microorganisms that act on marjoram or processed products thereof include microorganisms belonging to the genus Lactobacillus. More specifically, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus mali, Lactobacillus fabifermentans, Lactobacillus hordei, etc. These microorganisms can promote the fermentation of marjoram well and are excellent in the activity that brings about a whitening effect. However, it does not mean that the microorganisms that can be used in the present invention are limited to these bacterial species. The microorganisms may be used alone for the treatment with the above raw materials, or two or more kinds may be used in combination for the treatment with the above raw materials. That is, the treatment with two or more different kinds of microorganisms may be carried out sequentially, or two or more different kinds of microorganisms may be used in combination at the same time for the treatment, or these treatments may be combined.

[0018] Regarding the conditions for allowing the above microorganisms to act on marjoram or processed products thereof, any conditions may be used as long as the components of marjoram are changed by the above microorganisms in some way, and there is no particular limitation. For example, it is more preferable that the conditions are such that the microorganisms acting on marjoram or processed products thereof grow to 2 to 10,000 times the initial cell count, typically 5 to 1,000 times, and more typically 10 to 1,000 times. If the growth is poor, the fermentation will not proceed well.

[0019] When allowing the above microorganism to act on marjoram or a processed product thereof, as an auxiliary material for the growth of the microorganism, for example, saccharides such as glucose, fructose, sucrose, oligosaccharides, amino acids such as alanine, arginine, tryptophan, cysteine, peptides such as casein degradation products and protein degradation products, extracts such as yeast extract, meat extract, soybean extract, surfactants having fatty acids such as polyoxyethylene sorbitan oleate in the side chain, or, for example, Lactobacillus MRS broth (Difco) etc. which is a standard medium composition for lactic acid bacteria may be used.

[0020] However, if other components remain in addition to the components derived from marjoram, it may affect the quality of the resulting fermented product from the viewpoints of preservability, usability, etc. Therefore, when fermenting, it is not desirable to blend components other than the components derived from marjoram more than necessary. Thus, when using the above auxiliary material, for example, it is preferable that the composition ratio of the others is 0.001 parts by mass or more and 5.0 parts by mass or less, and more preferably 0.01 parts by mass or more and 0.5 parts by mass or less with respect to 100 parts by mass of the marjoram-derived material. On the other hand, it may be used for the treatment by the above microorganism without adding any raw materials not derived from marjoram.

[0021] Hereinafter, any arbitrary embodiments (not limited) for obtaining the fermented product according to the present invention will be described more specifically.

[0022] As the raw material to be allowed to act on the above microorganism, for example, an extract of marjoram can be obtained and used as the raw material. In this case, for example, 10 to 200 times the amount of water or hot water (for example, reverse osmosis membrane treated water, ion exchange water, tap water, well water, distilled water, ultrapure water, etc. may be used) is added to the dry powder of the plant body and heat-treated to obtain a hot water extract, which can be used as the raw material in the state of the extraction suspension as it is, or optionally concentrated by evaporating water to be used as the raw material, or the solid content is removed by solid-liquid separation means such as filter filtration or centrifugation to obtain a supernatant and used as the raw material, and the above microorganism is inoculated at an appropriate initial concentration to act on such a raw material.

[0023] The treatment with the above-mentioned microorganism can be carried out by a method according to normal aeration and static culture. In this case, as the initial bacterial count concentration, preferably 1×10 4 CFU / mL or more and 5×10 7 CFU / mL or less, more preferably 1×10 5 CFU / mL or more and 1×10 7 CFU / mL or less. As the temperature condition, it is 20°C to 40°C, more preferably 25°C to 37°C. As the treatment period, it is 12 hours to 10 days, more preferably 1 to 3 days. By such static culture, for example, a fermented product can be obtained in which the microorganism is grown to 2 to 10,000 times the amount of the initial bacterial count, typically 5 to 1,000 times the amount, more typically 10 to 1,000 times the amount. After the treatment with the microorganism, it may be used as the fermented product according to the present invention while still containing the used microorganism. However, from the viewpoints of the above-mentioned preservativeness, usability, etc., the cells of the microorganism used in the fermentation are preferably removed by solid-liquid separation means such as filter filtration and centrifugation, and the obtained supernatant is used as the fermented product. Further, after the treatment with the microorganism, various solvents can be added to the treated product to prepare an extract or a dilution, which can also be used as the fermented product of the present invention. The solvents used here include solvents commonly used in cosmetics such as water, lower alcohols such as ethanol and propanol, higher alcohols such as cetyl alcohol and stearyl alcohol, and polyhydric alcohols such as 1,3-butylene glycol, 1,3-propanediol, and glycerin. However, they are not limited to these and can be used alone or in a mixture of two or more.

[0024] The fermented product according to the present invention may be used as it is as a skin external preparation, or may be used by being formulated in the manufacturing process of a skin external preparation. Specifically, for example, it can be suitably used as a cosmetic in the form of an emulsion, a cream, a cleansing, a massage, a sunscreen, a makeup base, a cream foundation, etc. or as a raw material thereof. Here, the cosmetics mentioned herein mean those including pharmaceuticals, quasi-drugs, and cosmetics as defined by the laws regarding the quality, effectiveness, and safety assurance of pharmaceuticals, medical devices, etc.

[0025] In addition, as the form of the external preparation for skin, it may be a pack, a mask, a gel, etc. in which the component acting on the skin is supported on an appropriate base material part, that is, it can be suitably used as the component acting on the skin of such an external preparation for skin.

[0026] In addition, in any optional aspect of the present invention that is not limited, the above external preparation for skin may be a product that exhibits functions such as being used to bring about a whitening effect, having a melanin production inhibitory action, having a tyrosinase inhibitory action, etc.

Example

[0027] The present invention will be specifically described with the following examples, but these examples do not limit the scope of the present invention.

[0028] 〔1. Preparation of marjoram extract〕 1-1. Plant The dried product of the leaves of commercially available marjoram (scientific name: Origanum majorana) was used for the test.

[0029] 1-2. Extraction The marjoram extract for preculture was prepared as follows. That is, water (reverse osmosis membrane treated water, hereinafter referred to as "RO water") was added so that the ratio of plant powder: water = 1:20 (mass ratio), glucose was added so that the final concentration was 0.2 w / v%, and yeast extract (Difco) was added so that the final concentration was 0.1 w / v%. After stirring well, a suspension was prepared. 3 mL of the suspension was dispensed into test tubes, capped with an aluminum cap, and autoclaved at 121 °C for 15 minutes to obtain a hot water extract.

[0030] As the marjoram extract for this cultivation, two types were prepared: (1) without addition and (2) with the addition of 0.2 w / v% glucose and 0.1 w / v% yeast extract. That is, RO water was added so that the ratio of plant powder to water was 1:20 (mass ratio). For (1), no addition was made, or for (2), glucose was added so that the final concentration was 0.2 w / v%, and yeast extract (Difco) was added so that the final concentration was 0.1 w / v%. After thorough stirring, suspension liquids were prepared respectively. 10 mL of each suspension liquid was dispensed into test tubes, stoppered with silicon stoppers, and autoclaved at 98 °C for 100 minutes to obtain hot water extracts.

[0031] [2. Lactic acid bacteria] The 13 types of lactic acid bacteria used are shown in Table 1. The bacteria were obtained by inoculating the -80 °C DMSO-preserved strain into Lactobacilli MRS Broth (Difco), culturing at the optimal culture temperature for 20 hours, and then subculturing once more under the same conditions. The resulting bacterial solution was used for the preculture of the marjoram extract. Among the 11 types of lactic acid bacteria used (except for No. 5 and No. 10), reference strains representing the genus and species of each lactic acid bacterium were obtained and used. The 13 types of strains used can be obtained from the depositories described in Table 1.

[0032] [Table 1]

[0033] [Test Example 1] For preculture, the bacterial solution was inoculated at 0.5 v / v% into 3 mL of the marjoram extract for preculture (initial bacterial concentration: approximately 0.5×10 6 ~2×10 7 CFU / mL), and statically cultured at the optimal temperature for 48 hours under aerobic conditions.

[0034] For main culture, the culture after preculture was inoculated at 1 v / v% into 10 mL of the marjoram extract for main culture (initial bacterial concentration: approximately 0.2×10 5 ~3×10 6 CFU / mL), and statically cultured at the optimal temperature for 72 hours under aerobic conditions.

[0035] After this culturing, the viable cell count was confirmed. Specifically, the culture after this culturing or a solution appropriately diluted with the stock solution or 0.1 w / v% yeast extract was seeded at 100 μL on a Lactobacilli MRS agar plate medium using a spiral plater EDDY JET2 (IUL Instruments). After culturing for 3 days at the optimum temperature, the formed colonies were counted using a colony counter ProtoCOL3 (SYNBIOSIS), and the value of CFU (colony forming unit) / mL was calculated. Regarding the initial viable cell count before this culturing, it was also confirmed in the same manner.

[0036]

Table 2

[0037] As a result, for Lactobacillus No. 13 (Streptococcus thermophilus), viable bacteria were not detected after preculturing, and it was a bacterial species or strain not suitable for the preparation of a fermented product using marjoram as a raw material.

[0038] For the 12 other Lactobacillus strains, the viable cell count increased by at least 10 times or more compared to the initial viable cell count through culturing with the marjoram extract. The effects when glucose and yeast extract were added to the marjoram extract varied depending on the type of Lactobacillus, showing a tendency to increase (e.g., Lactobacillus No. 11 (Pediococcus acidilactici)), being almost the same (e.g., Lactobacillus No. 5 (Lactobacillus casei), Lactobacillus No. 12 (Pediococcus pentosaceus)), or a tendency to be suppressed (e.g., Lactobacillus No. 3 (Lactobacillus zeae)), and no uniform tendency was observed. The degree of the effect on the growth of the bacteria was not very significant in any case.

[0039] From the above, it has been clarified that the above 12 strains of lactic acid bacteria, namely lactic acid bacterium No. 1 (Lactobacillus plantarum subsp. plantarum), lactic acid bacterium No. 2 (Lactobacillus pentosus), lactic acid bacterium No. 3 (Lactobacillus zeae), lactic acid bacterium No. 4 (Lactobacillus mali), lactic acid bacterium No. 5 (Lactobacillus casei), lactic acid bacterium No. 6 (Lactobacillus fabifermentans), lactic acid bacterium No. 7 (Lactobacillus hordei), lactic acid bacterium No. 8 (Lactococcus lactis subsp. lactis), lactic acid bacterium No. 9 (Leuconostoc pseudomesenteroides), lactic acid bacterium No. 10 (Leuconostoc mesenteroides subsp. mesenteroides), lactic acid bacterium No. 11 (Pediococcus acidilactici), and lactic acid bacterium No. 12 (Pediococcus pentosaceus) are suitable for the preparation of fermented products using marjoram as a raw material.

[0040] [Test Example 2] In Test Example 1, among the 12 types of lactic acid bacteria (No. 1 to No. 12) that showed viable cell counts of 1×10 6 CFU / mL or more after culturing, lactic acid bacteria No. 1, 2, 4, 6, and 7 were selected, and the melanin production inhibitory activity was examined for the cultures by each lactic acid bacterium.

[0041] Specifically, after the completion of the main culture with marjoram extract without added glucose and yeast extract, the culture was centrifuged at 3000 rpm (1600×g) for 10 minutes, and the supernatant was aseptically filtered through a 0.22 μm filter to obtain a fermentation supernatant. The fermentation supernatant was stored in the dark at 4°C and then stored frozen at -20°C until measurement. Table 3 shows the pH measurement results and the evaporation residue concentration (mg / mL) measured by allowing it to cool in a desiccator after heating at 105°C for 3 hours for the used fermentation supernatant or unfermented marjoram extract.

[0042]

Table 3

[0043] The measurement of melanin production inhibitory activity was carried out by the test method shown below.

[0044] [1. Test method] (1) Cells B16 mouse melanoma cells (B16-F1) were used as the cells. For the test, cells at passage numbers 5 to 10 counted from the time of purchase were used.

[0045] (2) Melanin production inhibition test 500 μL of DMEM medium containing 5% FBS was placed in a 24-well plate, and B16 melanoma cells were seeded at 1.4×10 4 cells / well (7.5×10 4 cells / cm 2 ). After seeding, it was cultured at 5% CO 2 , 37 °C for 24 hours. Then, it was replaced with 1 mL of 0.5 mM theophylline-containing medium containing the test sample and cultured for another 3 days.

[0046] As the test samples, the fermentation supernatant of each lactic acid bacterium or the unfermented marjoram extract was added to the medium at a mixing ratio (volume ratio) of 0.5%, 1%, 2%, and 5% so that the sample stock solution was 100%. As a control, RO water was added to the medium at the same mixing ratio instead of the fermentation supernatant of each lactic acid bacterium or the unfermented marjoram extract. Also, as a positive control, an aqueous solution of arbutin, which is known as a whitening raw material for quasi-drugs, was added to the medium at 1% (final concentration 0.15 to 2.44 mM).

[0047] (3) Intracellular melanin production amount After the completion of the culture, the cells were washed twice with PBS(-), and then fixed with 99.5% ethanol. After removing the ethanol by air drying, 1N NaOH was added to the wells and heated at 80 °C for 30 minutes to obtain a cell lysate that dissolved the cells and melanin. After cooling, the total amount of the cell lysate was transferred to a 96-well plate, and the absorbance at 405 nm was measured. The amount of intracellular melanin production per culture well was determined from a calibration curve using synthetic melanin, and the amount of intracellular melanin production relative to the control was determined by the following formula.

[0048]

Number

[0049] (4) Amount of intracellular protein The above cell lysate was diluted 10-fold with ultrapure water (Milli-Q water) and quantified by the BCA method (Pierce BCA Protein assay kit) using BSA as the standard protein. The amount of intracellular protein relative to the control was determined by the following formula.

[0050]

Number

[0051] (5) IC50 value Based on the results of 2 - 5 tests for the fermentation supernatant of each lactic acid bacterium or the unfermented marjoram extract and a predetermined blending ratio, the 50% inhibitory concentration (IC50 value) was determined for each of the intracellular melanin production amount and the intracellular protein amount. The IC50 value was calculated by setting each value without adding the test sample as 100% and taking the concentration at which it becomes 50% or its estimated value. The concentrations of each fermentation supernatant and the unfermented marjoram extract were converted based on the evaporation residue concentration shown in Table 3 above.

[0052] [2. Results] The results are summarized in Table 4.

[0053]

Table 4

[0054] As a result, for arbutin, which is a positive control, the IC50 value of the intracellular melanin production amount was 340 μg / mL, while for the unfermented marjoram extract, the IC50 value was 430 mg / mL. On the other hand, for Lactobacillus No.1 (Lactobacillus plantarum subsp. plantarum), Lactobacillus No.2 (Lactobacillus pentosus), Lactobacillus No.4 (Lactobacillus mali), Lactobacillus No.6 (Lactobacillus fabifermentans), and Lactobacillus No.7 (Lactobacillus hordei), in the fermentation supernatants of each lactic acid bacterium, all showed a tendency that the IC50 value was lower than that of the unfermented one. Also, as shown in Fig. 1, there was not much difference in the change in the intracellular protein amount (percentage ratio to the intracellular protein amount when the test sample was not added) compared to the unfermented one, but for intracellular melanin, the decrease in the intracellular melanin production amount (percentage ratio to the intracellular melanin amount when the test sample was not added) was significant compared to the unfermented one.

[0055] From the above, it became clear that in the fermented product of the marjoram extract by the above lactic acid bacteria, the melanin production inhibitory activity was enhanced without causing a decrease in the cell number compared to the unfermented extract.

[0056] [Test Example 3] Among the 12 types (No.1 to 12) that showed viable cell counts of 1×10 6 CFU / mL or more after culturing in Test Example 1, lactic acid bacteria of No.1, 2, 4, 6, and 7 were selected, and fermentation supernatants were prepared in the same manner as in Test Example 2, and the tyrosinase inhibitory activity was examined.

[0057] The measurement of tyrosinase inhibitory activity was carried out with some modifications based on the method of Matsuda et al. (Matsuda H et al.; Studies of cuticle drugs from natural sources. III. Inhibitory effect of Myrica rubra on melanin biosynthesis., Biol. Pharm. Bull., 18(8), 1148-1150 (1995)). Specifically, 50 μL of the fermentation supernatant by each lactic acid bacterium or the unfermented marjoram extract was placed in each well of a 96-well microplate (sample stock solution). After adding 50 μL of 300 mM phosphate buffer (pH 6.8) to each well and mixing, it was pre-incubated at room temperature for 10 minutes. Then, 25 μL of a 270 U / mL tyrosinase solution (tyrosinase: from mushroom, Sigma-Aldrich) and 25 μL of 0.06 w / v% 3,4-Dihydroxy-L-phenylalanine (L-DOPA, FUJIFILM Wako Pure Chemical Corporation) were added to each well and mixed. After incubating at room temperature for 5 minutes, the absorbance at 475 nm, which is the maximum absorption wavelength of dopachrome (an intermediate in melanin biosynthesis), was measured. The final volume of the reaction system was 150 μL, and the final mixing ratio of the fermentation supernatant (sample stock solution) was 50 μL / 150 μL.

[0058] From the measured absorbance, the tyrosinase inhibition rate was calculated by the following formula. In the formula, "control" represents a reaction system in which RO water was added instead of the sample, and "blank" represents a reaction system in which 50 mM potassium phosphate buffer was added instead of tyrosinase.

[0059]

Equation

[0060] For the fermentation supernatant by each lactic acid bacterium or the unfermented marjoram extract, three tyrosinase reaction tests were conducted to obtain the average and standard deviation. The results are shown in Table 5.

Table 5

[0061] As a result, the tyrosinase inhibition rate of the unfermented marjoram extract was 0% (SD: ±3%), whereas among the fermented products by each lactic acid bacterium, the inhibition rate was 24% (SD: ±4%) for Lactic acid bacterium No. 1 (Lactobacillus plantarum subsp. plantarum), 20% (SD: ±3%) for Y Lactic acid bacterium No. 2 (Lactobacillus pentosus), 24% (SD: ±3%) for Lactic acid bacterium No. 4 (Lactobacillus mali), 36% (SD: ±2%) for Lactic acid bacterium No. 6 (Lactobacillus fabifermentans), and 30% (SD: ±3%) for Lactic acid bacterium No. 7 (Lactobacillus hordei).

[0062] From the above, it was revealed that the fermented products of the marjoram extract by the above lactic acid bacteria had enhanced tyrosinase inhibitory activity compared to the unfermented extract.

Claims

1. A topical skin agent containing a fermented product by one or more microorganisms selected from the genus Lactobacillus, using marjoram or its processed product as a raw material, wherein the microorganism is one or more microorganisms selected from the group consisting of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus mali, Lactobacillus fabifermentans, and Lactobacillus hordei; the topical skin agent.

2. The topical skin agent according to Claim 1, which is used to provide a whitening effect.

3. The topical skin agent according to Claim 1 or 2, wherein the fermented product has an inhibitory effect on melanin production.

4. The topical skin agent according to any one of Claims 1 to 3, wherein the fermented product has a tyrosinase inhibitory effect.

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