Malva sylvestris fermentation product and its use

Abstract

Provided is a Malva sylvestris-derived material that has been improved in order to increase its activity.　This fermentation product is provided, using Malva sylvestris or a processed material therefrom as starting material, by one or two or more microorganisms selected from the group consisting of microorganisms belonging to genus Lactobacillus, microorganisms belonging to genus Lactococcus, and microorganisms belonging to genus Pediococcus. The fermentation product is useful, for example, as an ingredient for incorporation in a topical agent or cosmetic.

Description

[Technical field]

[0001] The present invention relates to a plant fermentation product useful as an external skin preparation or the like, and more specifically, to a mallow fermentation product and an external skin preparation containing the same. [Background technology]

[0002] Malva sylvestris (sometimes called mallow) is a wild perennial plant native to Europe, but is widely cultivated as an herb or herbal medicine. Conventionally, plant extracts and fermented products have been used in cosmetics, but the use of malva sylvestris as an external skin preparation has been reported. That is, for example, Patent Document 1 describes that malva sylvestris extract suppresses the increase in expression of bFGFmRNA, the expression of which increases with ultraviolet light irradiation. It is described that this makes it possible to prevent, treat, or improve age spots, freckles, skin pigmentation, and the like. In addition, for example, Patent Document 2 describes that malva sylvestris extract (malva sylvestris extract) suppresses the expression level of kinesin in human epidermis-derived pigment cells. It is also described that it has the effect of improving age spots, freckles, and pigmentation. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2011-1328 A [Patent Document 2] JP 2012-219091 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, conventional methods using extracts have not been able to fully bring out the activity of mallow.

[0005] Therefore, an object of the present invention is to provide an improved mallow-derived material for improving activity, and thereby to provide a novel external skin preparation. [Means for solving the problem]

[0006] In order to achieve the above object, the present inventors have conducted extensive research and have completed the present invention.

[0007] The first aspect of the present invention provides a fermentation product using mallow or a processed product thereof as a raw material, with one or more microorganisms selected from the group consisting of microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Lactococcus, and microorganisms belonging to the genus Pediococcus.

[0008] In the fermented product of the present invention, the microorganism is preferably one or more microorganisms selected from the group consisting of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus zeae, Lactobacillus mali, Lactobacillus fabifermentans, Lactobacillus hordei, Lactococcus lactis, Pediococcus acidilactici, and Pediococcus pentosaceus.

[0009] A second aspect of the present invention provides an external skin preparation containing the above-mentioned fermented product.

[0010] The topical skin preparation according to the present invention is preferably used to bring about a whitening effect.

[0011] In the external skin preparation according to the present invention, the fermented product preferably has an inhibitory effect on melanin production.

[0012] In the skin external preparation according to the present invention, the fermented product preferably has a tyrosinase inhibitory effect. Effect of the Invention

[0013] According to the present invention, the fermented product is obtained by treating mallow or a processed product thereof with a specific microorganism, and therefore has excellent whitening effects such as melanin production suppression activity and tyrosinase inhibition activity, etc. Thus, a novel skin external preparation can be provided. [Brief description of the drawings]

[0014] [Figure 1] 13 is a graph showing the results of investigating the melanin production inhibitory activity in Test Example 4, in which (A) is a graph showing the results of investigating the change in intracellular melanin production amount for each final concentration of mallow fermentation product added to melanin-producing cells (the results are shown as a percentage relative to the intracellular melanin amount when unfermented mallow extract was added at the same final concentration), and (B) is a graph showing the results of a similar test performed on hibiscus or black mallow. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In this specification, "malva" has the same meaning as a plant generally understood by those skilled in the art, and specifically includes malva sylvestris (scientific name: Malva sylvestris, also known as blue mallow) of the genus Malva in the family Malvaceae, and its variant, Malva mauritiana (scientific name: Malva mauritiana, also known as common mallow). Malva is a plant native to Europe, widely cultivated as an herb or herbal medicine, and easily available.

[0016] In the present invention, a microorganism is allowed to act on Malva sylvestris to produce a fermented product by the microorganism. The part of the Malva sylvestris plant body that is allowed to act on the microorganism may be the flower part, the leaf part, the stem, the above-ground part, the root part, the whole plant, or a mixture of these parts, etc., but is preferably the flower part, the leaf part, or a mixture of these parts, etc., and is more preferably the flower part. The form of the plant body that is allowed to act on the microorganism is not particularly limited, and may be a crushed product of the plant raw material or its dried product, a squeezed juice, an extract, or a mixture of these, etc., but is preferably the squeezed juice, an extract, or a mixture of these, etc., and is more preferably the extract.

[0017] Examples of microorganisms that are allowed to act on mallow include microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Lactococcus, microorganisms belonging to the genus Leuconostoc, and microorganisms belonging to the genus Pediococcus. More specifically, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus zeae, Lactobacillus mali, Lactobacillus fabifermentans, Lactobacillus hordei, Lactococcus lactis, Pediococcus acidilactici, Pediococcus pentosaceus, etc. These microorganisms can promote the fermentation of mallow and have excellent whitening activity. However, this does not mean that the microorganisms that can be used in the present invention are limited to these species. A single type of microorganism may be used to treat the raw material, or two or more types may be used in combination to treat the raw material. That is, treatment with two or more different types of microorganisms may be carried out sequentially, treatment with two or more different types of microorganisms may be carried out simultaneously, or these treatments may be combined.

[0018] The conditions under which the microorganisms are allowed to act on mallow are not particularly limited as long as they are conditions under which the components of mallow are changed in some way by the microorganisms, but for example, the growth conditions are preferably such that the microorganisms allowed to act on mallow grow to 2 to 10,000 times, typically 5 to 1,000 times, and more typically 10 to 1,000 times the initial number of bacteria. If the growth is poor, fermentation does not proceed well.

[0019] When the above-mentioned microorganisms are allowed to act on mallow, materials that aid in the growth of the microorganisms may be used, such as sugars such as glucose, fructose, sucrose, and oligosaccharides; amino acids such as alanine, arginine, tryptophan, and cysteine; peptides such as casein hydrolysates and protein hydrolysates; extracts such as yeast extract, meat extract, and soybean extract; surfactants having fatty acids in their side chains, such as polyoxyethylene sorbitan oleate; or standard medium compositions for lactic acid bacteria, such as Lactobacillus MRS Broth (Difco).

[0020] However, if other components remain in addition to the components derived from mallow, the quality of the fermented product obtained may be affected in terms of preservative properties, usability, etc., so it is not desirable to mix more components than necessary other than the components derived from mallow during fermentation. Therefore, when using the above-mentioned auxiliary material, for example, the composition ratio of the other components is preferably 0.001 to 5.0 parts by mass, and more preferably 0.01 to 0.5 parts by mass, per 100 parts by mass of the material derived from mallow. On the other hand, the material may be subjected to treatment with the above-mentioned microorganisms without adding any raw materials not derived from mallow.

[0021] Hereinafter, any non-limiting embodiment for obtaining the fermented product according to the present invention will be described in more detail.

[0022] As a raw material to be acted on by the above microorganisms, for example, an extract of mallow 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-exchanged water, tap water, well water, distilled water, ultrapure water, etc. may be used) is added to the dry powder of the plant body and heated to obtain a hot water extract, which is used as the raw material as it is in the form of an extract suspension, or the water is optionally evaporated and concentrated to use 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 to use as the raw material, and the above microorganisms are inoculated at an appropriate initial concentration on such a raw material and acted on.

[0023] The treatment with the above-mentioned microorganisms can be carried out by a method similar to ordinary aeration and static culture. In this case, the initial bacterial concentration is preferably 1×10 4 CFU / mL or more 5×10 7 CFU / mL or less, more preferably 1×10 5 CFU / mL or more 1×10 7 CFU / mL or less. The temperature conditions are 20°C to 40°C, more preferably 25°C to 37°C. The treatment period is 12 hours to 10 days, more preferably 1 to 3 days. By such static culture, a fermented product can be obtained in which the microorganisms are grown to, for example, 2 to 10,000 times, typically 5 to 1,000 times, more typically 10 to 1,000 times the initial number of bacteria. After the treatment with the microorganism, the fermented product according to the present invention may be obtained by still containing the microorganism used, but from the viewpoint of the above-mentioned antiseptic properties, usability, etc., it is preferable to remove the cells of the microorganism used in the fermentation by solid-liquid separation means such as filter filtration and centrifugation, and to obtain the supernatant as the fermented product. In addition, after the treatment with the microorganism, various solvents can be added to the treated product to prepare an extract or dilution, which can be used as the fermented product of the present invention. The solvent used here includes, but is not limited to, 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, and may be used alone or in combination of two or more kinds.

[0024] The fermented product according to the present invention may be used as an external skin preparation as it is, or may be incorporated in the manufacturing process of the external skin preparation. Specifically, it may be suitably used as a cosmetic preparation or a raw material thereof in the form of, for example, a milky lotion, cream, cleansing, massage, sunscreen, makeup base, cream foundation, etc. Note that the cosmetic preparation referred to here includes drugs, quasi-drugs, and cosmetics as defined in the Law Concerning the Assurance of Quality, Effectiveness, and Safety of Drugs, Medical Devices, etc.

[0025] The form of the topical skin preparation may be a pack, mask, gel, etc., in which the component to be applied to the skin is supported on a suitable base material. In other words, the topical skin preparation can be suitably used as the component to be applied to the skin in such topical skin preparations.

[0026] Furthermore, in any non-limiting aspect of the present invention, the above-mentioned topical skin preparation may be a product that displays functionality such as being used to provide a whitening effect, having a melanin production inhibitory effect, or having a tyrosinase inhibitory effect. EXAMPLES

[0027] The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0028] 1. Preparation of Malva Sylvestris Extract 1-1.Plants The dried flowers of commercially available mallow (scientific name: Malva sylvestris) were used in the test.

[0029] 1-2. Extraction Malva sylvestris extract for preculture was prepared as follows: Water (water treated with reverse osmosis membrane, hereafter referred to as "RO water") was added to give a plant powder:water ratio of 1:20 (mass ratio), glucose was added to a final concentration of 0.2 w / v%, and yeast extract (Difco) was added to a final concentration of 0.1 w / v%, and the mixture was stirred thoroughly to prepare a suspension. The suspension was dispensed into test tubes (3 mL each), aluminum capped, and autoclaved at 121°C for 15 minutes to obtain a hot water extract.

[0030] Two types of mallow extracts for main culture were prepared: (1) no additives, and (2) 0.2 w / v% glucose and 0.1 w / v% yeast extract. That is, RO water was added to make the plant powder:water ratio 1:20 (mass ratio), and suspensions were prepared by adding no additives for (1) above, or adding glucose to a final concentration of 0.2 w / v% and yeast extract (Difco) to a final concentration of 0.1 w / v% for (2) above, and stirring thoroughly. The suspensions were dispensed into test tubes in 10 mL portions, sealed with silicone 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 inoculated into Lactobacilli MRS Broth (Difco) using strains stored in DMSO at -80°C, cultured for 20 hours at the optimal culture temperature, and then subcultured for one more generation under the same conditions. This was used as a bacterial liquid for pre-culture of the mallow extract. All 11 types of lactic acid bacteria (except for No. 5 and No. 11) were type strains representative of the genus and species of each lactic acid bacteria. The 13 types of strains used are available from the depositories listed in Table 1.

[0032] [Table 1]

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

[0034] For the main culture, 10 mL of the Malva sieboldii extract was inoculated with the culture after the preculture at 1 v / v% (initial bacterial concentration: approximately 0.5 × 10 5 ~5×10 6 CFU / mL) and cultured at the optimum temperature under aerobic conditions for 72 hours.

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

[0036] [Table 2]

[0037] As a result, no viable cells were detected in lactic acid bacteria No. 13 (Streptococcus thermophilus) after pre-culture, and the species or strain was not suitable for preparing a fermented product using mallow as a raw material.

[0038] In the other 12 strains of lactic acid bacteria, the viable cell count increased by at least 10 times compared to the initial cell count when cultured in mallow extract. The effects of adding glucose and yeast extract to mallow extract varied depending on the type of lactic acid bacteria, with some showing an increase in the viable cell count (e.g., Lactobacillus No. 7 (Lactobacillus hordei)), others showing an almost equal increase (e.g., Lactococcus No. 8 (Lactococcus lactis subsp. lactis)), and others showing a suppression (e.g., Lactobacillus No. 3 (Lactobacillus zeae)). No consistent tendency was observed. The degree of effect on bacterial growth was not particularly significant in any case.

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

[0040] [Test Example 2] In Test Example 1, the number of live bacteria after cultivation was 1 x 10 6 Of the 12 types of lactic acid bacteria (No. 1 to 12) that showed CFU / mL or more, No. 1, 2, 4, 6, 9, and 10 lactic acid bacteria were selected, and the melanin production inhibitory activity of the cultures of each lactic acid bacteria was examined.

[0041] Specifically, after the main culture using the additive-free mallow extract was completed, the culture was centrifuged at 3000 rpm (1600×g) for 10 minutes, and the supernatant was sterile filtered through a 0.22 μm filter to obtain a fermentation supernatant. The fermentation supernatant was stored at 4°C in the dark and then frozen at -20°C until measurement. Table 3 shows the pH measurement results and the evaporation residue concentration (mg / mL) of the fermentation supernatant or unfermented mallow extract used, which was measured by heating at 105°C for 3 hours and then cooling in a desiccator.

[0042] [Table 3]

[0043] The melanin production inhibitory activity was measured by the test method shown below. 1. Test Method (1) Cell The cells used were B16 mouse melanoma cells (B16-F1). For the test, cells that were passaged 5 to 10 times from the time of purchase were used. The medium used was DMEM containing 5 or 10% FBS.

[0044] (2) Melanin production inhibition test Place 500 μL of DMEM medium containing 5% FBS in a 24-well plate and incubate with 1.4 × 10 B16 melanoma cells. 4 cells / well (7.5×10 4 cells / cm 2 After seeding, the cells were cultured at 5% CO2 and 37°C for 24 hours. The medium was then replaced with 1 mL of medium containing 0.5 mM theophylline and the test sample, and the cells were cultured for an additional 3 days.

[0045] As test samples, the fermentation supernatant of each lactic acid bacterium or unfermented mallow extract was added to the medium at a mixing ratio (volume ratio) of 0.5%, 1%, 2%, and 5%, with the sample stock solution being 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 mallow extract. In addition, as a positive control, an aqueous solution of arbutin, which is known as a skin whitening ingredient in quasi-drugs, was added to the medium at 1% (final concentration 0.15 to 2.44 mM).

[0046] (3) Intracellular melanin production After the culture was completed, the cells were washed twice with PBS(-) and 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 containing dissolved cells and melanin. After cooling, the entire cell lysate was transferred to a 96-well plate and the absorbance at 405 nm was measured. The amount of intracellular melanin produced per culture well was calculated from a calibration curve using synthetic melanin, and the amount of intracellular melanin produced relative to the control was calculated using the following formula.

[0047]

number

[0048] (4) Intracellular protein amount The 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, and the amount of intracellular protein relative to the control was calculated using the following formula.

[0049]

number

[0050] (5) IC50 value Based on the results of 2 to 5 tests performed for each lactic acid bacteria fermentation supernatant or unfermented mallow extract and a given blend ratio, the 50% inhibitory concentration (IC50 value) was calculated for each intracellular melanin production amount and intracellular protein amount. The IC50 value was calculated as the concentration at 50% or its estimated value, with each value without the addition of the test sample taken as 100%. The concentrations of each fermentation supernatant and unfermented mallow extract were converted into the evaporation residue concentration shown in Table 3 above.

[0051] [2. Results] The results are summarized in Table 4.

[0052] [Table 4]

[0053] As a result, the positive control arbutin had an IC50 value of 340 μg / mL for intracellular melanin production, whereas the unfermented mallow extract had an IC50 value of over 1200 μg / mL, indicating a poor inhibitory effect.

[0054] In addition, for lactic acid bacteria No. 9 (Leuconostoc pseudomesenteroides) and lactic acid bacteria No. 10 (Leuconostoc mesenteroides subsp. mesenteroides), the fermentation supernatants using these lactic acid bacteria showed little inhibitory effect on melanin production, similar to unfermented mallow extract.

[0055] On the other hand, for lactic acid bacteria No.1 (Lactobacillus plantarum subsp. plantarum), No.2 (Lactobacillus pentosus), No.4 (Lactobacillus mali), and No.6 (Lactobacillus fabifermentans), the IC50 values ​​of intracellular melanin production in the fermentation supernatants using each lactic acid bacteria were significantly lower than those in the unfermented sample, and tended to approach the value of arbutin, the positive control. On the other hand, the IC50 values ​​of intracellular protein content in these fermentation supernatants did not show a significant difference from those in the unfermented sample.

[0056] From the above, it was revealed that the fermentation product of the mallow extract with the above-mentioned lactic acid bacteria has enhanced melanin production inhibitory activity without causing a decrease in cell number compared to the unfermented extract.

[0057] [Test Example 3] In Test Example 1, the number of live bacteria after cultivation was 1 x 10 6Of the 12 types of lactic acid bacteria (No. 1 to 12) that showed CFU / mL or more, No. 3, 6, 7, 8, 11, and 12 lactic acid bacteria were selected, and fermentation supernatants were prepared in the same manner as in Test Example 2 (No. 6 was the same as in Test Example 2), and tyrosinase inhibitory activity was examined. Table 5 shows the results of pH measurement for the fermentation supernatants and unfermented mallow extracts used, as well as the evaporation residue concentration (mg / mL) measured by heating at 105°C for 3 hours and then cooling in a desiccator.

[0058] [Table 5]

[0059] The tyrosinase inhibitory activity was measured by a modified 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 an unfermented mallow extract was placed in each well of a 96-well microplate (sample stock solution), and 50 μL of 300 mM phosphate buffer (pH 6.8) was added to each well, mixed, and pre-incubated at room temperature for 10 minutes. Next, 25μL of 270U / mL tyrosinase solution (tyrosinase: mushroom-derived, Sigma-Aldrich) and 25μL of 0.06w / v% 3,4-Dihydroxy-L-phenylalanine (L-DOPA, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to each well and mixed, incubated at room temperature for 5 minutes, and then the absorbance at 475nm, which is the maximum absorption wavelength of dopachrome (an intermediate in melanin biosynthesis), was measured. The final volume of the reaction system was 150μL, of which the final mixing ratio of the fermentation supernatant (sample stock solution) was 50μL / 150μL.

[0060] The tyrosinase inhibition rate was calculated from the measured absorbance using the following formula, where "control" refers to a reaction system in which RO water was added instead of the sample, and "blank" refers to a reaction system in which 50 mM potassium phosphate buffer was added instead of tyrosinase.

[0061]

number

[0062] The tyrosinase reaction test was carried out three times for each fermentation supernatant of each lactic acid bacteria or for the unfermented mallow extract, and the average and standard deviation were calculated. The results are shown in Table 6.

[0063] [Table 6]

[0064] As a result, the tyrosinase inhibition rate of unfermented mallow extract was 2% (SD: ±8%), while that of the fermented products of each lactic acid bacteria was 6% (SD: ±1%) for Lactobacillus No.3 (Lactobacillus zeae), 8% (SD: ±3%) for Lactobacillus No.6 (Lactobacillus fabifermentans), 4% (SD: ±4%) for Lactobacillus No.7 (Lactobacillus hordei), 24% (SD: ±1%) for Lactococcus No.8 (Lactococcus lactis subsp. lactis), 21% (SD: ±1%) for Lactobacillus No.11 (Pediococcus acidilactici), and 17% (SD: ±2%) for Lactobacillus No.12 (Pediococcus pentosaceus).

[0065] From the above, it was revealed that the fermented extract of Malva sylvestris with the above lactic acid bacteria has a higher tyrosinase inhibitory activity than the unfermented extract.

[0066] [Test Example 4] We investigated other species of Malvaceae plants to see whether treatment with lactic acid bacteria enhanced their functionality in the same way as Malva sylvestris.

[0067] [4-1. Plants] In the test, dried calyx and bracts of hibiscus and dried flowers of black mallow were used in addition to the dried flowers of mallow used in Test Examples 1 to 3. Table 7 shows the genus name, scientific name, Japanese name, and common English name of the Malvaceae plants used.

[0068] [Table 7]

[0069] 4-2. Preparation of plant extracts The plant extract for preculture was prepared as follows: Water (water treated with reverse osmosis membrane, hereafter referred to as "RO water") was added so that the ratio of plant powder to water was 1:20 (mass ratio), glucose was added to a final concentration of 0.2 w / v%, and yeast extract (Difco) was added to a final concentration of 0.1 w / v%, and the mixture was thoroughly stirred to prepare a suspension. The suspension was dispensed into test tubes (3 mL each), aluminum capped, and autoclaved at 121°C for 15 minutes to obtain a hot water extract. The plant extracts for the main culture were prepared as follows: In the case of mallow and hibiscus, 12 mL of RO water was added to each so that the plant powder:water ratio was 1:20 (mass ratio), and in the case of black mallow, the plant powder:water ratio was 1:50 (mass ratio), and the mixture was stirred thoroughly to prepare a suspension. The suspension was placed in a test tube, sealed with a silicone stopper, and autoclaved at 98°C for 100 minutes to obtain a hot water extract.

[0070] [4-3. Lactic acid bacteria] Lactic acid bacteria No. 1 (Lactobacillus plantarum subsp. plantarum) was used.

[0071] [4-4. Preculture / main culture] Pre-culture and main culture were carried out using Lactic Acid Bacteria No. 1 in the same manner as in Test Example 1, except that the hot water extract prepared above was used as the culture raw material.

[0072] [4-5. Confirmation of viable cell count, pH of fermentation supernatant, and evaporation residue concentration after main culture] The viable cell count after the main culture was confirmed in the same manner as in Test Example 1. Moreover, in the same manner as in Test Example 2, a fermentation supernatant was prepared from the culture after the main culture was completed, and its pH and evaporation residue concentration were confirmed.

[0073] Table 8 summarizes the results.

[0074] [Table 8]

[0075] [4-6. Melanin production inhibitory activity] The melanin production inhibitory activity of the obtained fermentation supernatant or unfermented plant extract was measured using B16 melanoma cells in the same manner as in Test Example 2. Specifically, the sample stock solution was taken as 100%, and was added to the medium at a mixing ratio (volume ratio) of 0.5%, 1%, 2%, or 5%. As a control, RO water was added to the medium at the same mixing ratio.

[0076] Table 9 shows the results of melanin production inhibition activity for each extract of Malvaceae plant, for the presence or absence of fermentation by lactic acid bacteria, and for the blending ratio (volume ratio) of the sample stock solution added to the medium. The upper part of Table 9 (Table 9-1) shows the relative values ​​with the amount of intracellular melanin when the control (RO water) was added as 100%, and the lower part of Table 9 (Table 9-2) shows the relative values ​​with the amount of intracellular melanin production when the unfermented plant extract was added at the same final concentration as 100%. In addition, in Figure 1, the results of the lower part of Table 9 (Table 9-2) are graphed by expressing the final concentration of the sample added to the medium in terms of evaporation residue on the x-axis.

[0077] [Table 9]

[0078] As a result, in the case of mallow, the fermentation supernatant treated with lactic acid bacteria had a higher melanin production inhibitory activity than the unfermented extract, as shown in Test Example 2. In contrast, in the case of other types of mallow plants, such as hibiscus and black mallow, no such enhancement of functionality was observed by treatment with lactic acid bacteria.

Claims

1. A fermentation product using mallow or a processed product thereof as a raw material, with one or more kinds of microorganisms selected from the group consisting of microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Lactococcus, and microorganisms belonging to the genus Pediococcus.

2. The fermented product according to claim 1, wherein the microorganism is one or more microorganisms selected from the group consisting of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus zeae, Lactobacillus mali, Lactobacillus fabifermentans, Lactobacillus hordei, Lactococcus lactis, Pediococcus acidilactici, and Pediococcus pentosaceus.

3. A skin external preparation comprising the fermented product according to claim 1 or 2.

4. The skin external preparation according to claim 3, which is used to provide a whitening effect.

5. The external skin preparation according to claim 3 or 4, wherein the fermented product has an inhibitory effect on melanin production.

6. The skin external preparation according to any one of claims 3 to 5, wherein the fermented product has a tyrosinase inhibitory effect.

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