Althea fermented product and its utilization

Abstract

The subject of this invention is to provide a modified medicinal marshmallow material for enhancing its activity. The fermented product of this invention uses marshmallow or its processed form as raw material and is formed by one or more microorganisms selected from the genera *Lactobacillus*, *Lactococcus*, *Leuconostoc*, and *Pediococcus*. This fermented product can be used as a formulation material in topical skin agents and cosmetics.

Description

[Technical Field]

[0001] The present invention relates to plant ferments for use as skin topical agents, and more specifically, to hibiscus ferments and skin topical agents containing the same. [Previous Technology]

[0002] Hibiscus is a perennial herb native to Europe and naturally grows along the Mediterranean coast. It is widely cultivated as a medicinal herb and crude drug. In the past, plant extracts and ferments have been used in cosmetics, and it has been reported that hibiscus has also been used as a topical skin agent. For example, Patent Document 1 describes that the 1,3-butanediol extract of hibiscus has tyrosinase inhibitory activity, and it is used as a formulation material for whitening cosmetics.

[0002] [Previous Technical Documents]

[0002] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 60-104005

[0004] However, previous methods have failed to fully utilize the activity caused by hibiscus.

[0005] Therefore, the object of the present invention is to provide a modified hibiscus-derived material for enhancing its activity. Furthermore, this provides a novel topical skin preparation.

[0006] In order to achieve the above objectives, the inventors have conducted careful research and thus completed the present invention.

[0007] The first aspect of the present invention is to provide a fermented product, which is made from hibiscus or its processed form as raw material and is formed by one or more microorganisms selected from the genera of lactobacilli, lactococcus, Leuconostoc, and Pediococcus.

[0008] In the fermentation product according to the present invention, when the aforementioned microorganism is a microorganism belonging to lactobacilli, the microorganism is preferably selected from one or more microorganisms of the genera *Lactiplantibacillus*, *Lacticaseibacillus*, and *Liquorilactobacillus*.

[0009] In the fermentation product according to the present invention, the aforementioned microorganisms are preferably selected from one or more microorganisms selected from the group consisting of Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Lactobacillus zeae, Liquorilactobacillus mali, Lactobacillus paracasei, Lactiplantibacillus fabifermentans, Liquorilactobacillus hordei, Lactococcus lactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Pediococcus acidilactici, and Pediococcus pentosaceus.

[0010] The second aspect of the present invention provides a skin topical agent containing the above-mentioned fermented product.

[0011] In the topical skin agent according to the present invention, the topical skin agent is preferably used to provide a whitening effect.

[0012] In the topical skin preparation of the present invention, the aforementioned fermentation product is preferably one that has an inhibitory effect on melanin production.

[0013] In the topical skin preparation of the present invention, the aforementioned fermentation product is preferably one that has tyrosinase inhibitory activity.

[0014] Since the present invention uses hibiscus or its processed form as raw material, and obtains a fermentation product by treating it with specific microorganisms, it exhibits excellent whitening effects due to its melanin production inhibition activity, tyrosinase inhibition activity, and other similar activities. Accordingly, a novel topical skin agent can be provided.

Implementation Method

[0017] As for microorganisms that act on hibiscus or its treated products, examples include microorganisms belonging to the genus *Lactobacilli*, the genus *Lactococcus*, the genus *Leuconostoc*, and the genus *Pediococcus*. More specifically, these include *Lactiplantibacillus plantarum*, *Lactiplantibacillus pentosus*, *Lactobacillus zeae*, *Liquorilactobacillus mali*, *Lactobacillus paracasei*, *Lactobacillus fabifermentans*, *Liquorilactobacillus hordei*, *Lactococcus lactis*, *Leuconostoc pseudomesenteroides*, *Leuconostoc mesenteroides*, *Pediococcus acidilactici*, and *Pediococcus pentosaceus*. Microorganisms belonging to this category can effectively promote the fermentation of hibiscus and provide excellent whitening effects. However, this does not mean that the microorganisms used in this invention are limited to such species. One type of microorganism may be used alone in the treatment of the above-mentioned raw materials, or two or more types of microorganisms may be used in combination in the treatment of the above-mentioned raw materials. That is, treatment may be carried out sequentially by two or more different types of microorganisms, or treatment may be carried out simultaneously by two or more different types of microorganisms, and such treatments may be combined.

[0018] Furthermore, as described in the academic journal published in April 2020 listed below, the genus of lactic acid bacteria that previously belonged to the genus Lactobacillus has been reclassified, and the genus name has been changed in one part of the species.

[0019] (Reclassification of Lactic Acid Bacteria)

[0019] ‧Zheng et al., "A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae." Int. J. Syst. Evol. Microbiol. 2020 Apr; 70(4):2782-2858 DOI 10.1099 / ijsem.0.004107

[0020] Hereinafter, this specification shows the notation of the new classification after reclassification. Furthermore, microorganisms belonging to the genus Lactobacillus in the old classification and which are now represented in the new classification are collectively referred to as "microorganisms belonging to Lactobacilli" and are included in the microorganisms that can be used in this invention.

[0021] Furthermore, as stated in the academic journals published in 2000 listed below, due to historical classification and nomenclature issues of lactic acid bacteria, many lactic acid bacteria that were called Lactobacillus casei were sometimes called Lactobacillus paracasei.

[0022] (Synonyms for the former name "Lactobacillus paracasei")

[0022] ‧Kenichiro Suzuki, “The current status and problems of the classification of lactic acid bacteria, especially regarding nomenclature” Japanese Journal of Lactic Acid Bacteria 2000 Vol.11, No. 2, pp49-59.

[0023] In this specification, the lactic acid bacteria previously known as Lactobacillus paracasei include those previously known as Lactobacillus casei as its synonym.

[0024] For example, the aforementioned *Lactiplantibacillus plantarum* was formerly known as *Lactobacillus plantarum*, the aforementioned *Lactiplantibacillus pentosus* was formerly known as *Lactobacillus pentosus*, the aforementioned *Lactobacillus zeae* was formerly known as *Lactobacillus zeae*, the aforementioned *Liquorilactobacillus mali* was formerly known as *Lactobacillus mali*, the aforementioned *Lactobacillus paracasei* was formerly known as *Lactobacillus paracasei*, and the aforementioned *Lactiplantibacillus fabifermentans* was formerly known as *Lactobacillus fabifermentans*. The aforementioned *Liquorilactobacillus hordei*, formerly known as *Lactobacillus hordei*, is a microorganism belonging to the *Lactobacillus* family and is included in the microorganisms used in this invention.

[0025] Regarding the conditions under which the aforementioned microorganisms act on hibiscus or its treated product, there are no particular limitations as long as the components of hibiscus are altered in some way by the aforementioned microorganisms. However, for example, it is preferable to allow the microorganisms acting on hibiscus or its treated product to multiply to 2 to 10,000 times the initial bacterial count, typically 5 to 1,000 times, and more typically 10 to 1,000 times. Poor growth will prevent proper fermentation.

[0026] When the above-mentioned microorganisms act on hibiscus or its treated products, as growth aids for the microorganisms, sugars such as glucose, fructose, sucrose, oligosaccharides, amino acids such as alanine, arginine, tryptophan, cysteine, peptides such as casein decomposed products and protein decomposed products, extracts such as yeast extract, meat extract, soybean extract, surfactants with fatty acid side chains such as polyoxyethylene sorbitan oleate, or, for example, Lactobacillus MRS culture medium (Difco) which is composed of standard lactic acid bacteria culture media.

[0027] However, when other components besides those derived from hibiscus remain, from the viewpoints of preservative properties and user experience, the quality of the obtained fermented product may be affected. Therefore, it is undesirable to add more components than needed from hibiscus during fermentation. Accordingly, when using the aforementioned adjuvant, for example, relative to 100 parts by weight of hibiscus or its processed product used as fermentation raw material, it is preferable to set the composition ratio of the aforementioned adjuvant to be 0.001 parts by weight or 5.0 parts by weight, and more preferably 0.01 parts by weight or 0.5 parts by weight or less. On the other hand, no raw materials not derived from hibiscus may be added, and the product may be processed by the aforementioned microorganisms.

[0028] Hereinafter, in order to obtain the fermentation product according to the present invention, a more specific description will be given for any undefined state sample.

[0029] As a raw material for enabling the above-mentioned microorganisms to function, for example, an extract of hibiscus can be obtained and used as a raw material. At this time, for example, 10 to 200 times the amount of water or hot water (e.g., reverse osmosis membrane treated water, ion-exchange water, tap water, well water, distilled water, ultrapure water, etc.) can be added relative to the dried powder of the plant and heated to obtain a hot water extract. This can be used directly as a raw material in the form of an extract suspension, or the water can be evaporated and concentrated as needed to use it as a raw material. Alternatively, the supernatant obtained by removing the solids through solid-liquid separation means such as filtration or centrifugation can be used as a raw material. The above-mentioned microorganisms can be inoculated at an appropriate initial concentration relative to such a raw material to enable them to function.

[0030] The treatment using the aforementioned microorganisms can be carried out using conventional aeration and static culture methods. The initial bacterial count concentration is preferably 1×10⁴ CFU / mL to 5×10⁷ CFU / mL, more preferably 1×10⁵ CFU / mL to 1×10⁷ CFU / mL. The temperature conditions are 20°C to 40°C, preferably 25°C to 37°C. The treatment period is 12 hours to 10 days, more preferably 1 to 3 days. Through such treatment, for example, the microorganisms can be multiplied to 2 to 10,000 times the initial bacterial count, typically 5 to 1,000 times, and more typically 10 to 1,000 times, thereby obtaining a ferment. After treatment by microorganisms, the fermentation product containing the used microorganisms can be used directly as the fermentation product of this invention. However, from the viewpoints of preservation and user experience, it is preferable to remove the cell bodies of the microorganisms used in the fermentation by solid-liquid separation methods such as filtration or centrifugation, and use the obtained supernatant as the fermentation product. Furthermore, after treatment by microorganisms, various solvents can be added to the treated product to prepare extracts or diluents for use as the fermentation product of this invention. Examples of solvents used include water, lower alcohols such as ethanol and propanol, higher alcohols such as cetyl alcohol and stearyl alcohol, and polyols such as 1,3-butanediol, 1,3-propanediol, and glycerin, commonly used in cosmetics. However, these are not limited to these, and two or more solvents can be used alone or in combination.

[0031] The fermentation product according to the present invention can be used directly as a topical skin agent, or it can be formulated and used during the manufacturing process of a topical skin agent. Specifically, for example, it can be suitably used as a cosmetic ingredient or raw material in the form of lotion, cream, cleanser, massage cream, sunscreen, foundation, cream foundation, etc. Furthermore, such cosmetic ingredients refer to pharmaceuticals, quasi-pharmaceuticals, and cosmetics as defined in laws related to ensuring the quality, efficacy, and safety of pharmaceuticals, medical devices, etc.

[0032] Furthermore, in terms of the form of the topical skin preparation, it can be a pack, a mask, a gel, etc., in which the skin-acting ingredient is held in a suitable substrate portion. That is, it can be a skin-acting ingredient suitable for use in such a topical skin preparation.

[0033] Furthermore, in any form not limited to the present invention, the above-mentioned topical skin agent may be a product that is labeled as having functions such as bringing about whitening effects, inhibiting melanin production, and inhibiting tyrosinase.

[0033] [Example]

[0034] The following examples are provided to illustrate the present invention in detail, but these examples are not intended to limit the scope of the present invention.

[0035] 〔1. Preparation of Hibiscus extract〕

[0035] 1-1. Plants

[0035] The dried leaves or roots of commercially available hibiscus (scientific name: Althaea officinalis) were used in the experiment.

[0036] 1-2. Extraction

[0036] The hibiscus extract for pre-culturing was prepared as follows. Specifically, water (reverse osmosis membrane treated water, hereinafter referred to as "RO water") was added at a plant powder:water ratio of 1:20 (mass ratio), glucose was added to achieve a final concentration of 0.2 w / v%, and yeast extract (Difco) was added to achieve a final concentration of 0.1 w / v%. The mixture was stirred thoroughly to prepare a suspension. 3 mL of the suspension was dispensed into each test tube, capped with an aluminum cap, and autoclaved at 121°C for 15 minutes to obtain the hot water extract.

[0037] Regarding the hibiscus extract used as the main culture medium, two types of wood mold extracts were prepared: (1) without additives, and (2) with 0.2 w / v% glucose and 0.1 w / v% yeast extract. That is, RO water was added in a ratio of plant powder to water of 1:20 (mass ratio), and a suspension was prepared by stirring thoroughly without additives as described above (1), or 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 a suspension was prepared by stirring thoroughly as described above (2). 10 mL of the suspension was dispensed into test tubes, capped with silicone stoppers, and autoclaved at 98°C for 100 minutes to obtain a hot water extract.

[0038] 〔2. Lactic acid bacteria〕

[0038] The 13 types of lactic acid bacteria used are shown in Table 1. The strains were inoculated into Lactobacilli MRS Broth (Difco) at -80°C DMSO and cultured at the optimal temperature for 20 hours. The culture was then subcultured once more under the same conditions to obtain the bacterial culture for the pre-culture of hibiscus extract. Furthermore, the 13 types of strains used were available from the publicly disclosed transferring organizations listed in Table 1. In addition, for 11 types of lactic acid bacteria (excluding No. 5 and No. 10) of the 13 types used, standard strains representing the genera and species of each lactic acid bacteria were obtained and used.

[0039] [Table 1]

[0040] 〔Experimental Example 1〕(Use of Hibiscus Leaves)

[0040] The pre-culture was carried out by inoculating 3 mL of hibiscus leaf extract for pre-culture with 0.5 v / v% bacterial suspension (initial bacterial count concentration: about 1.9 × 10⁶ to 2.4 × 10⁷ CFU / mL) and incubating it under aerobic conditions at the optimal temperature for 48 hours.

[0041] The master culture was carried out by inoculating 10 mL of hibiscus leaf extract used for the master culture with 1 v / v% of the culture after pre-culture (initial bacterial count concentration: about 0.4 × 10⁵ to 2.3 × 10⁶ CFU / mL) and incubating it under aerobic conditions at the optimal temperature for 72 hours.

[0042] Viable cell count was confirmed after the master culture. Specifically, 100 μL of the culture after the master culture was seeded onto Lactobacilli MRS agar plates using an EDDY JET2 spiral plate (IUL Instruments) with either the stock solution or a suitably diluted 0.1 w / v% yeast extract. After incubation at the optimal temperature for 3 days, the colonies formed were counted using a ProtoCOL3 colony counter (SYNBIOSIS) to calculate the CFU (colony forming units) / mL value. The initial cell count before the master culture was also confirmed in the same manner.

[0043] The results obtained are shown in Table 2.

[0044] [Table 2]

[0045] As a result, lactic acid bacteria No. 13 (Streptococcus thermophiles) was not suitable for the preparation of fermentation products using hibiscus leaves as raw materials due to the low number of viable cells after pre-culture.

[0046] Of the 12 lactic acid bacteria strains other than those mentioned above, the viable counts after culturing with hibiscus leaf extract were 6×10⁵ CFU / mL or higher, which was at least 6 times higher than the initial count (as an exception, Lactococcus lactis subsp. lactis with "GY added" was about 4.3 times higher; Lactococcus mesenteroides subsp. mesenteroides without "no added" was about 4.6 times higher). The effects of adding glucose and yeast extracts to hibiscus leaf extract varied depending on the type of lactic acid bacteria. Some showed a tendency to increase viable cell counts (e.g., Lactiplantibacillus pentosus), others were roughly the same (e.g., Lactiplantibacillus fabifermentans), and still others showed a tendency to inhibit it (e.g., Lactiplantibacillus pseudomesenteroides). No consistent trend was observed. The degree of influence on bacterial growth was not significant in any of these cases.

[0047] As can be seen from the above, the above-mentioned 12 strains of lactic acid bacteria are, namely, Lactiplantibacillus plantarum subsp. plantarum, Lactiplantibacillus pentosus, Lactiplantibacillus zeae, Lactiplantibacillus mali, Lactiplantibacillus paracasei, Lactiplantibacillus fabifermentans, Lactiplantibacillus hordei, Lactiplantibacillus lactis subsp. lactis, Lactiplantibacillus paracasei, Lactiplantibacillus fabifermentans, Lactiplantibacillus hordei, Lactiplantibacillus lactis subsp. lactis, Lactiplantibacillus pseudomesenteroides, Lactiplantibacillus mesenteroides subsp. mesenteroides, and Lactiplantibacillus petiolus. Lactic acid bacteria (pediococcus pentosaceus) and lactic acid bacteria No. 12 are suitable for the preparation of ferments using hibiscus leaves as raw materials.

[0048] 〔Experimental Example 2〕(Use of Hibiscus Roots)

[0048] The pre-culture was carried out by inoculating 3 mL of hibiscus root extract for pre-culture with 0.5 v / v% bacterial suspension (initial bacterial count concentration: about 3.1 × 106 to 2.8 × 107 CFU / mL) and incubating it under aerobic conditions at the optimal temperature for 48 hours.

[0049] The master culture was carried out by inoculating 10 mL of hibiscus root extract used for the master culture with a 1 v / v% pre-cultured culture (initial bacterial count concentration: about 0.7 × 10⁵ to 3.6 × 10⁶ CFU / mL) and incubating it under aerobic conditions at the optimal temperature for 72 hours.

[0050] The viable count was confirmed after the main culture in the same manner as in Experimental Example 1.

[0051] The results obtained are shown in Table 3.

[0052] [Table 3]

[0053] As a result, lactic acid bacteria No.13 (Streptococcus thermophilus) was unsuitable for the preparation of fermentation products using hibiscus roots as raw materials due to the low number of viable bacteria after pre-culture.

[0054] Of the 12 lactic acid bacteria strains not mentioned above, those cultured with hibiscus root extract showed viable cell counts of 1×10⁶ CFU / mL or higher, representing an increase of at least 10-fold compared to the initial count. The effects of adding glucose and yeast extracts to the hibiscus root extract varied depending on the type of lactic acid bacteria, showing a tendency to increase viable cell counts (e.g., Lactobacillus mali), roughly the same (e.g., Lactobacillus paracasei), and a tendency to inhibit (e.g., Lactobacillus fabifermentans)). No consistent trend was observed. The degree of influence on bacterial growth was not significant in any of these cases.

[0055] As can be seen from the above, the above-mentioned 12 strains of lactic acid bacteria are, namely, Lactiplantibacillus plantarum subsp. plantarum, Lactiplantibacillus pentosus, Lactiplantibacillus zeae, Lactiplantibacillus mali, Lactiplantibacillus paracasei, Lactiplantibacillus fabifermentans, Lactiplantibacillus hordei, Lactiplantibacillus lactis subsp. lactis, Lactiplantibacillus paracasei, Lactiplantibacillus fabifermentans, Lactiplantibacillus hordei, Lactiplantibacillus lactis subsp. lactis, Lactiplantibacillus pseudomesenteroides, Lactiplantibacillus mesenteroides subsp. mesenteroides, and Lactiplantibacillus petiolus. The lactic acid bacteria No. 12 (Pediococcus pentosaceus) are suitable for preparing ferments using hibiscus roots as raw materials.

[0056] 〔Experimental Example 3〕

[0056] In Experiment 1 (use of hibiscus leaves), among the 12 types of lactic acid bacteria (No. 1 to 12) with a viable count of 5×10⁵ CFU / mL or higher after culturing, lactic acid bacteria No. 1, 2, 4, 5, 6, 7, 10, and 11 were selected, and the melanin production inhibition activity was examined for the cultures of each lactic acid bacteria.

[0057] Specifically, after the main culture of hibiscus leaf extract without added glucose and yeast extract was completed, 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 the fermentation supernatant. The fermentation supernatant was stored at 4°C in the dark and then frozen at -20°C until the assay was performed.

[0058] Table 4 shows the results of pH measurement for either the fermentation supernatant or the unfermented hibiscus leaf extract used, as well as the concentration of the evaporation residue (mg / mL) determined by heating at 105°C for 3 hours and then cooling in a desiccator.

[0059] [Table 4]

[0060] The determination of melanin production inhibition activity was carried out by the test method shown below.

[0061] 〔Test Method〕

[0061] (1) Cell

[0061] The cell line used was B16 mouse melanoma cells (B16-F1). Cells from passage 5 to 10 from the time of purchase were used in the experiment.

[0062] (2) Melanin production inhibition test

[0062] 500 μL of DMEM medium containing 5% FBS was added to a 24-well plate, and B16 mouse melanoma cells were seeded at a rate of 1.4 × 10⁴ cells / well (7.5 × 10⁴ cells / cm²). The plates were then cultured at 37°C with 5% CO₂ for 24 hours. Subsequently, the medium was replaced with 1 mL of theophylline-containing medium containing the test sample, and the plates were cultured for another 3 days.

[0063] As the test sample, the fermentation supernatant of each lactic acid bacteria or the unfermented hibiscus leaf extract was used as 100% of the original sample solution and added to the culture medium at a mixing ratio of 5% (volume ratio). As a control, RO water was used to replace the fermentation supernatant of each lactic acid bacteria or the unfermented hibiscus leaf extract and added to the culture medium at the same mixing ratio.

[0064] (3) Intracellular melanin production

[0064] After culture, the cells were washed twice with PBS(-) and fixed with 99.5% ethanol. After air-drying to remove the ethanol, 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 volume of the cell lysate was transferred to a 96-well plate, and the absorbance at 405 nm was measured. The amount of intracellular melanin produced in each culture well was determined from the calibration curve using synthetic melanin, while the amount of intracellular melanin produced relative to the control was determined using the following formula.

[0065]

[0066] (4) Intracellular protein content

[0066] The above cell lysate was diluted 10 times with ultrapure water (Milli-Q water) and quantified using the BCA method (Pierce BCA Protein assay kit) with BSA as the standard protein. The amount of intracellular protein relative to the control was determined by the following formula.

[0067]

[0068] The results obtained are summarized in Table 5.

[0069] [Table 5]

[0070] As a result, regarding intracellular protein levels (the percentage of intracellular protein levels relative to the amount of the test sample not added), the fermentation supernatants of each lactic acid bacteria showed no significant tendency compared to the unfermented hibiscus leaf extract. However, regarding intracellular melanin production (the percentage of intracellular melanin levels relative to the amount of the test sample not added), among the fermentation supernatants of each lactic acid bacteria, the following strains showed the following results: Lactiplantibacillus plantarum subsp. plantarum, Lactiplantibacillus pentosus, Lactiplantibacillus mali, Lactiplantibacillus paracasei, Lactiplantibacillus fabifermentans, Lactiplantibacillus hordei, and Lactiplantibacillus mesenteroides. Compared with unfermented bacteria, *Periflora melanin* subsp. mesenteroides and lactic acid bacteria No. 11 (Pediococcus acidilactici) showed a tendency to reduce intracellular melanin production.

[0071] As can be seen from the above, in the fermented product using the above-mentioned lactic acid bacteria hibiscus leaf extract, the cell number does not decrease compared with the unfermented extract, and the melanin production inhibition activity is increased.

[0072] 〔Experimental Example 4〕

[0072] In Test Example 1 (using hibiscus leaves), among the 12 species (No. 1 to 12) with viable bacterial counts of 5×10⁵ CFU / mL or higher after culture, lactic acid bacteria No. 3, 5, 8, 11 and 12 were selected, and fermentation supernatants were prepared in the same manner as in Test Example 3 (No. 5 and 11 were the same as those used in Test Example 3), and tyrosinase inhibitory activity was examined.

[0073] Table 6 shows the results of pH measurement for the fermentation supernatant or unfermented hibiscus leaf extract used (No. 5 and 11 are the users in Test Example 3), and the concentration of evaporation residue (mg / mL) determined by heating at 105°C for 3 hours and then cooling in a desiccator.

[0074] [Table 6]

[0075] The determination of tyrosinase inhibitory activity was performed by modifying a portion of 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 fermentation supernatant of each lactic acid bacteria or unfermented hibiscus leaf extract was added to each well of a 96-well microplate. 50 μL of 300 mM phosphate buffer (pH 6.8) was added to each well, and the mixture was pre-incubated at room temperature for 10 minutes. Next, 25 μL of 270 U / mL tyrosinase solution (tyrosinase: derived from mushrooms, Sigma-Aldrich) and 25 μL of 0.06 w / v% 3,4-dihydroxy-L-phenylalanine (L-DOPA, Fujifilm and Hikari Pure Chemical Industries Co., Ltd.) were added to each well and mixed. After incubation at room temperature for 5 minutes, the absorbance at 475 nm, the wavelength of maximum absorption of dopachrome (an intermediate in melanin synthesis), was measured. The final volume of the reaction system was 150 μL, with the final ratio of fermentation supernatant (sample stock solution) being 50 μL / 150 μL.

[0076] The tyrosinase inhibition rate is calculated from the measured absorbance according to the following formula. In the formula, "control" means the reaction system in which RO water is added to replace the sample, and "blank" means the reaction system in which 50 mM potassium phosphate buffer is added to replace the tyrosinase.

[0077]

[0077] S475nm: Sample

[0077] SB475nm: Blank Sample

[0077] C475nm: Reference

[0077] CB475nm: Blank control

[0078] The experiment involved three tyrosinase reaction tests on the fermentation supernatant or unfermented hibiscus leaf extract of various lactic acid bacteria to determine the mean and standard deviation. The results are shown in Table 7.

[0079]

Table 7

[0080] As a result, the tyrosinase inhibition rate in the unfermented hibiscus leaf extract was 17% (SD: ±2%). In contrast, among the fermentation supernatants of various lactic acid bacteria, Lactic acid bacteria No.3 (Lacticaseibacillus zeae) had an inhibition rate of 28% (SD: ±1%), Lactic acid bacteria No.5 (Lacticaseibacillus paracasei) had an inhibition rate of 27% (SD: ±8%), Lactic acid bacteria No.8 (Lactococcus lactis subsp. lactis) had an inhibition rate of 33% (SD: ±7%), Lactic acid bacteria No.11 (Pediococcus acidilactici) had an inhibition rate of 39% (SD: ±3%), and Lactic acid bacteria No.12 (Pediococcus pentosaceus) had an inhibition rate of 38% (SD: ±3%).

[0081] As can be seen from the above, the fermented product using the above-mentioned lactic acid bacteria hibiscus leaf extract showed increased tyrosinase inhibitory activity compared to the unfermented extract.

[0082] 〔Experimental Example 5〕

[0082] In Experiment 2 (use of Hibiscus root), 12 types of lactic acid bacteria (No. 1 to 12) with a viable count of 1×106 CFU / mL or higher after culture were selected. Fermentation supernatants of each lactic acid bacteria were prepared in the same manner as in Experiment 3, and their melanin production inhibition activity was examined.

[0083] Table 8 shows the results of pH measurement for either the fermentation supernatant or the unfermented hibiscus root extract used, as well as the concentration of the evaporation residue (mg / mL) determined by adding the extract at 105°C for 3 hours and then cooling it in a desiccator.

[0084] [Table 8]

[0085] The results obtained are summarized in Table 9.

[0086] [Table 9]

[0087] As a result, regarding intracellular protein content (the percentage of intracellular protein content relative to the amount of the test sample not added), the fermentation supernatant of each lactic acid bacteria showed no significant tendency compared to the unfermented hibiscus root extract. However, regarding intracellular melanin production (the percentage of intracellular melanin production relative to the amount of the test sample not added), among the fermentation supernatants of each lactic acid bacteria, the following strains showed the following results: Lactiplantibacillus plantarum subsp. plantarum, Lactiplantibacillus pentosus, Lactiplantibacillus zeae, Lactiplantibacillus mali, Lactiplantibacillus paracasei, Lactiplantibacillus fabifermentans, Lactiplantibacillus hordei, and Lactiplantibacillus hordei. Compared with unfermented 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 acidilactici), and lactic acid bacteria No. 12 (Pediococcus pentosaceus) showed a tendency to reduce intracellular melanin production.

[0088] As can be seen from the above, the fermented product of the hibiscus root extract of the above-mentioned lactic acid bacteria does not increase cytotoxicity compared with the unfermented extract, but increases the melanin production inhibition activity.

[0089] 〔Experimental Example 6〕

[0089] In Experiment 2 (using hibiscus root), among the 12 types of lactic acid bacteria (No. 1 to 12) with a viable count of 1×106 CFU / mL or higher after culture, lactic acid bacteria No. 1, 3, 6, 11 and 12 were selected, and fermentation supernatant was prepared in the same manner as in Experiment 5 (using the same method used in Experiment 5) to check tyrosinase inhibitory activity.

[0090] Table 10 shows the results of pH measurement for the fermentation supernatant or unfermented hibiscus root extract used (used in Test Example 5), and the concentration of evaporation residue (mg / mL) determined by heating at 105°C for 3 hours and then cooling in a desiccator.

[0091] [Table 10]

[0092] The results obtained are summarized in Table 11.

[0093] [Table 11]

[0094] As a result, the tyrosinase inhibition rate in the unfermented hibiscus root extract was 10% (SD: ±33%). In contrast, in the fermentation supernatants of various lactic acid bacteria, the inhibition rate was 6% (SD: ±5%) for lactic acid bacteria No.1 (Lactiplantibacillus plantarum subsp. plantarum), 15% (SD: ±6%) for lactic acid bacteria No.3 (Lacticaseibacillus zeae), 16% (SD: ±16%) for lactic acid bacteria No.6 (Lactiplantibacillus fabifermentans), 15% (SD: ±3%) for lactic acid bacteria No.11 (Pediococcus acidilactici), and 11% (SD: ±25%) for lactic acid bacteria No.12 (Pediococcus pentosaceus).

[0095] As can be seen from the above, the fermented product of the hibiscus root extract of the above-mentioned lactic acid bacteria showed increased tyrosinase inhibitory activity compared with the unfermented extract.

Claims

1. A fermented product made from marshmallow or its processed form, and formed by one or more microorganisms selected from the genera *Lactobacillus*, *Lactococcus*, *Leuconostoc*, and *Pediococcus*, wherein... The aforementioned processed material is a pulverized, pressed, extracted, or mixture thereof of plant raw materials or their dried form.

2. The fermentation product as described in claim 1, wherein, In the case where the aforementioned microorganism belongs to the lactobacilli genus, the microorganism is selected from one or more of the genera *Lactiplantibacillus*, *Lacticaseibacillus*, and *Liquorilactobacillus*.

3. The fermentation product as described in claim 1, wherein, The aforementioned microorganisms are selected from one or more species selected from the group consisting of Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Lactobacillus zeae, Liquorilactobacillus mali, Lactobacillus paracasei, Lactobacillus fabifermentans, Liquorilactobacillus hordei, Lactococcus lactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Pediococcus acidilactici, and Pediococcus pentosaceus.

4. A topical skin preparation containing a ferment as described in any one of claims 1 to 3.

5. The topical skin agent as described in claim 4, which is intended to provide a whitening effect.

6. The topical skin agent as described in claim 4, wherein, The aforementioned fermentation products have an inhibitory effect on melanin production.

7. The topical skin agent as described in claim 4, wherein, The aforementioned fermentation products have tyrosinase inhibitory effects.