KR20220012564A - Method for manufacturing triple misses with antioxidant and whitening activity - Google Patents

Abstract

The present invention relates to a method for manufacturing triple misses having antioxidant and whitening activities. More specifically, related is a method for manufacturing triple misses having antioxidant and whitening activities, which contains elder flowers, chamomile flowers, and calendula flowers as active ingredients, and conducts fermentation through inoculation of galactomyces to ensure antioxidant activity and whitening activity.

Description

Method for manufacturing triple misses with antioxidant and whitening activity

The present invention relates to a method for preparing Triple Mrs. having antioxidant and whitening activity. More particularly, it relates to a method for preparing triple Mrs. comprising elder flower, chamomile flower, and calendula flower as an active ingredient, and fermenting it through Galactomyces inoculation to have antioxidant activity and whitening activity.

In modern society, clean and white skin is becoming a measure of beauty. As the standard of living has also improved and the average lifespan has been extended, interest in skin beauty has increased, and interest in skin beauty and whitening has grown even more due to an increase in UV exposure and environmental pollution. Therefore, research to find a whitening material that is stable and less irritating to the skin is currently being actively conducted. Currently, studies on whitening include inhibiting tyrosinase activity, inhibiting oxidation of 3,4-dihydroxy-phenylalanine (DOPA), and blocking UV rays.

In order to verify the whitening effect, it is most important to suppress the formation of melanin, because the pigmentation of the skin and even the color of the skin are determined according to the amount of melanin in the keratinocytes. Melanin protects essential vitamins such as folic acid, riboflavin, and vitamin E of the vitamin B group, which are easily destroyed by ultraviolet rays, and also plays a positive role in promoting vitamin D synthesis and inhibiting infection and growth of pathogens. In addition, melanin (melanin) has a function against ultraviolet rays, exists in the skin of the human body, plays a role in removing toxins in the human body, and finally protects the skin. However, when it is overproduced, spots and freckles are formed on the skin, which further accelerates skin aging and is known to cause skin cancer called melanoma.

There are three major approaches to the development of skin whitening agents to control excessive melanin synthesis. First, it inhibits the expression of tyrosinase, TRP-1, and TRP-2, which are enzymes directly involved in melanin synthesis, secondly regulates factors affecting melanin synthesis such as ROS, and finally melanin-forming cells There is a method of inhibiting the movement of melanin from keratinocytes to keratinocytes, and the main whitening raw materials that control melanin synthesis are kojic acid, ascorbic acid, arbutin, and the like. However, kojic acid is carcinogenic or genotoxic, ascorbic acid is decomposed in water-soluble, and arbutin is a cosmetic whitening ingredient due to cell permeability problems. For that reason, the search for natural whitening materials with fewer side effects is continuously being pursued.

Among those with antioxidant and whitening properties, elder, calendula, and chamomile and galactomyces used in the present invention are attracting attention, and these will be briefly described.

First, Galactomyces is a yeast of the genus Saccharomyces and has been used to make fermented food since ancient times, and it is a natural moisturizing factor (NMF) precursor, profilagrin. It is reported to help convert into moisturizing factors and maintain skin moisture.

In addition, amino acids, vitamins, minerals, and polysaccharides contained in the fermented galactomyces filtrate prevent moisture from evaporating from the skin surface, so that the skin does not become dry and excellent skin moisturizing effect can be obtained. According to the research results, it has effects such as moisturizing the skin, improving the skin barrier, improving skin tone, removing dead skin cells, whitening, and improving skin troubles.

In addition, the elder (Sambucus nigra) tree grows in shady and humid areas of Europe, Asia, North Africa and North America and is a deciduous tree with blue-black elderberry and cream-white elderflower, used as a diuretic to reduce inflammation and symptoms of diabetes in herbal medicine, as a diuretic for colds and flu It has been used for a long time in treatment, and studies have shown that it has various drug effects such as antioxidant, anti-inflammatory, antiviral, anti-allergic, angioprotective and anti-cancer.

In addition, Calendula (Calendula officinalis) is a dicotyledonous plant belonging to the Asteraceae and its origin is in the Mediterranean region. It has been reported that calendula flowers contain phenolic compounds and flavonoids and have anti-inflammatory, antibacterial and anticancer activities. Calendula is used as a dye or fragrance in the food field, and in the medical field for inflammatory diseases of the skin, can also be used as an insecticide, has antimicrobial, antiseptic, wound and ulcer treatment, blood pressure lowering agent It can be utilized as hypotensive properties and has been used as a herbal remedy in the West for its function of inhibiting auto-oxidation and protecting against cell damage induced by oxidation.

Chamomile (Matricaria chamomilla L.) is native to Europe, North Africa, and North Asia, has a sweet and refreshing apple scent, and has skin sterilization and body beauty effects, so it is used as a bathing agent for beauty. It is good for antiseptic, anthelmintic, and convulsions, and it is mainly used as a tea to treat inflammatory diseases, fever, diarrhea, and digestion of the small intestine and liver because it is known for its effects such as preventing colds, relieving insomnia, and sedation due to gastric and sweating action. do. The flowers of chamomile contain components such as terpenes, flavonoids, coumarin, calicacid, and minerals. It has been reported that 120 kinds of compounds including ingredients, 36 kinds of flavonoids (apogenin, etc.) and 52 kinds of organic acids are contained. It is effective in treatment, and chamazulene has an anti-inflammatory lipid peroxidation inhibitory effect, so it is effective in alleviating inflammation.

Lastly, Galactomyces, a yeast of the genus Saccharomyces, has been used to make fermented foods since ancient times, It is known to help maintain skin moisture by helping According to the research results, it is reported that there are effects such as skin moisturizing, skin barrier improvement, skin tone improvement, dead skin removal, whitening, and skin trouble improvement.

Although the raw materials as described above have various effects, the raw material extraction, culture, fermentation technology, and extraction technology at a ratio beneficial to whitening and antioxidant activity are weak, and thus the above effects cannot be actively implemented.

Prior art document: KR Registered Patent Publication No. 198032 (published on Jul. 8, 2019)

The present invention has been devised to solve the above problems. In particular, it contains elder, calendula, and chamomile as active ingredients, and is fermented through Galactomyces inoculation to have antioxidant and whitening activity. An object of the present invention is to provide a method for manufacturing triple Mrs.

The triple Mrs. manufacturing method having antioxidant and whitening activity according to the present invention, which was devised to achieve the above object, comprises putting dried elder flowers, chamomile flowers, and calendula flowers in a grinder, and pulverizing them. mixing and pulverizing step; A medium mixing and sterilization step of sterilizing the elder flower, chamomile flower, and calendula flower pulverized by the mixing grinding step by mixing it with a microbial culture medium; Galactomyces inoculation step of inoculating Galactomyces for fermentation of the mixture subjected to medium mixing and sterilization; A culturing step of culturing the mixture subjected to the Galactomyces inoculation step in an incubator; A filter aid mixing step of mixing a filter aid containing silicon dioxide with the culture solution subjected to the culture step to perform adsorption, decolorization, and deodorization of lipids or heavy metals; A filtration step of filtration under reduced pressure using a filter press on the culture solution subjected to the filter aid mixing step to obtain only the pure extract excluding the residue; A sterilization step of heating the pure extract subjected to the filtration step to inhibit bacterial activity and further fermentation; And it may include a formulation step of adding a preservative including 1,2-hexanediol and butylene glycol to the pure extract subjected to the sterilization step.

In addition, in the Galactomyces inoculation step, after performing the medium mixing and sterilization step, when the temperature of the extraction medium is 40°C or higher and 50°C or lower, citric acid is added to adjust the pH to 3 to 4, and the temperature of the extraction medium is 30°C or higher When it enters below 36°C, it may include inoculating Galactomyces at a rate of 1% or more and 5% or less of the medium amount.

In addition, the culturing step may include culturing the mixture subjected to the Galactomyces inoculation step in an incubator at 20° C. or more and 40° C. or less for 24 hours or more and 48 hours or less.

According to the present invention, it is effective to prepare a complex extract having antioxidant activity and whitening activity by fermenting elder, calendula, and chamomile as active ingredients through Galactomyces inoculation.

In addition, according to the present invention, it is effective in inhibiting infection of bacteria and microorganisms by controlling temperature and time and controlling acidity in the Galactomyces inoculation step, preventing color change and minimizing off-flavor.

1 is a flowchart of a method for preparing triple Mrs. having antioxidant and whitening activity according to a preferred embodiment of the present invention;
2 to 6 are graphs showing the experimental results using the triple Mrs. manufacturing method having antioxidant and whitening activity according to a preferred embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. First of all, it should be noted that in adding reference numerals to the components of each drawing, the same components are given the same reference numerals as much as possible even though they are indicated on different drawings. In addition, in describing the present invention, if it is determined that a detailed description of a related known configuration or function may obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, preferred embodiments of the present invention will be described below, but the technical spirit of the present invention is not limited thereto and may be variously implemented by those skilled in the art without being limited thereto.

1 is a flowchart of a method (S100) for preparing Triple Mrs. having antioxidant and whitening activity according to a preferred embodiment of the present invention, and FIGS. 2 to 6 are Triple Mrs. Experimental results using the manufacturing method (S100) are shown in a graph.

The present invention relates to a method (S100) for preparing triple Mrs. having antioxidant and whitening activity. More specifically, it relates to a manufacturing method (S100) of triple Mrs. containing elder flower, chamomile flower, and calendula flower as an active ingredient, and fermented through Galactomyces inoculation to have antioxidant activity and whitening activity will be.

1, the present invention is a mixing and grinding step (S10), medium mixing and sterilization step (S20), galactomyces inoculation step (S30), culture step (S40), filter aid mixing step (S50), filtration step It consists of (S60), a sterilization step (S70), and a formulation step (S80), and will be described in detail from the mixing and grinding step (S10).

In the mixing and grinding step (S10), the dried elder flowers, chamomile flowers, and calendula flowers are put into a grinder, and then pulverized. This is to increase the extraction yield by increasing the surface area of the raw material.

In the medium mixing and sterilization step (S20), the elder flower, chamomile flower, and calendula flower crushed in the mixing and pulverizing step (S10) are mixed with a microbial culture medium at a temperature of 90 ℃ or more and 120 ℃ or less, 1 kgf / cm ² or more Sterilize for 15 minutes or more and 30 minutes or less at a pressure of 2kgf/cm ² or less.

This is, when the pulverized raw material and the microbial culture medium are sterilized at less than 90 ° C, it is not possible to sterilize the bacteria surviving at a high temperature, and when it exceeds 120 ° C, the cooling time is exceeded in the subsequent step, which increases the process time and has a significant effect Since there is no sterilization, it is preferable to perform sterilization in the above temperature range.

In addition, during sterilization, if the pressure is less than 1kgf/cm ² , even the spores of microorganisms cannot be sterilized ^. It is preferable to perform sterilization in the range.

In addition, if the sterilization reaction time is less than 15 minutes, it is impossible to sterilize even the spores of microorganisms, and if it exceeds 30 minutes, the entire process time including cooling time and pressure release time becomes longer and there is no significant effect, so sterilization during the time It is preferable to proceed with

In addition, the composition of the Galactomyces culture medium in the medium mixing and sterilization step (S20) is based on 1 L of purified water, 1 g or more of peptones and 5 g or less, 1 g or more of yeast extract and 5 g or less, and 1 g or more of malt extract 5 g or less, it is preferable to comprise 10 g or more and 20 g or less of dextroxse.

If it is out of the above range, the growth rate of Galactomyces becomes slow or fast, making handling difficult, so it is preferable to create a culture medium within the above range.

In the Galactomyces inoculation step (S30), Galactomyces is inoculated for fermentation of the mixture that has been subjected to medium mixing and sterilization step (S20).

In addition, in the Galactomyces inoculation step (S30), after performing the medium mixing and sterilization step (S20), when the temperature of the extraction medium is 40° C. or higher and 50° C. or lower, citric acid is added to adjust the pH to 3 to 4, and the extraction medium Inoculate Galactomyces at a weight ratio of 1% or more and 5% or less of the total weight of the medium when the temperature of the

In addition, if the temperature of the extraction medium before pH adjustment in the Galactomyces inoculation step (S30) is less than 40° C., infection of microorganisms may occur when the pH is titrated, and when the pH is higher than 50° C., at room temperature when the pH is titrated. Since it changes differently from the pH of the extraction medium, the temperature of the extraction medium before pH adjustment is preferably set at 40°C or higher and 50°C or lower.

In addition, when adjusting the pH in the Galactomyces inoculation step (S30), adjusting the pH to 3 or more to pH 4 or less may cause infection of bacteria and microorganisms during the culture process, and to create an environment to inhibit their growth. it is for

In addition, after adjusting the pH in the Galactomyces inoculation step (S30), when the temperature of the extraction medium is 20° C. or more and 40° C. or less, the growth of Galactomyces stops when the temperature of the extraction medium is less than 20° C., and 40° C. or more Since yeast and protein are inactivated and Galactomyces cannot be cultured, it is preferable to make the composition within the above temperature range.

In addition, when the Galactomyces inoculation weight ratio is less than 1% of the total medium weight, the growth of Galactomyces is delayed and the culture time and process time are lengthened, making it unsuitable for mass production. When it exceeds 5%, the growth rate of Galactomyces is It is preferable to perform the inoculation at the above inoculation weight ratio because it cannot be used as a raw material due to deterioration of physical properties, color, and odor because

In the culturing step (S40), the mixture subjected to the galactomyces inoculation step (S30) is cultured in an incubator.

In addition, in the culturing step (S40), the mixture subjected to the Galactomyces inoculation step (S30) is cultured in an incubator at 20° C. or higher and 40° C. or lower for 24 hours or more and 48 hours or less.

This is because when the temperature in the incubator of the culturing step (S40) is less than 20 ° C, the growth of Galactomyces stops, and when it exceeds 40 ° C, yeast and proteins are inactivated and Galactomyces cannot be cultured within the above temperature range. Incubation is preferred.

In addition, Galactomyces in the culturing step (S40) is preferably cultured (fermented) for 24 hours or more and 48 hours or less. This is because, when fermented for more than 48 hours, the physical properties, color and flavor of the fermentation product are altered and cannot be used as a raw material.

In the filter aid mixing step (S50), 1% or more and 2% or less of the filter aid containing silicon dioxide is mixed with the culture solution subjected to the culture step (S40) with respect to the total weight of the culture solution and reacted for 20 minutes or more and 40 minutes or less. Filtration performs adsorption of fats and heavy metals, decolorization, and deodorization.

This is, if the content of the filter aid mixed in the filter aid mixing step (S50) is less than 1% relative to the total weight of the culture medium, the reaction amount is insufficient, so lipid or heavy metal adsorption does not occur smoothly, and when it exceeds 2%, the reaction amount increases and the filtration rate And the extraction yield is reduced, and when the reaction time is less than 20 minutes, the reaction time is shortened so that adsorption of lipids or heavy metals does not occur smoothly, and when it exceeds 40 minutes, the reaction time is prolonged and extraction due to adsorption with physiologically active substances Since the yield is reduced, it is preferable to proceed with mixing with the filter aid within the above range.

In the filtration step (S60), the culture solution subjected to the filter aid mixing step (S50) is filtered under reduced pressure using a filter press at a pressure of 500 mmHg or more and 600 mmHg or less to obtain only the pure extract excluding the residue.

This is, when the pressure value of the filtration step (S60) is less than 500mmHg, the filtration speed is slowed and the process time is prolonged. It is preferable to proceed to the above pressure range because the risk of

In the sterilization step (S70), the pure extract subjected to the filtration step (S60) is heated for 3 minutes or more and 7 minutes or less to inhibit bacterial activity and further fermentation.

If the heating time in the sterilization step (S70) is less than 3 minutes, the sterilization time is short and the activity of microorganisms is not inhibited. It is preferred to heat during this time.

In the formulation step (S80), a preservative containing 1,2-hexanediol and butylene glycol is added to the pure extract subjected to the sterilization step (S70) to facilitate storage and extend the shelf life.

<Example 1>

In this example, the effect of scavenging free radicals (DPPH) was observed. Add each sample of various concentrations to 0.5 ml of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) methanol solution, add 1 ml of 10% ethanol solution, mix, and then stand at 37°C for 10 minutes and measure absorbance at 517 nm did. As a result, the free radical scavenging action was obtained as shown in the following equation.

Free radical scavenging activity (%) = 100 - [(absorbance of experimental group / absorbance of control group) × 100]

2, it shows the free radical scavenging rate according to the concentration. When the concentration of the sample (fermented complex extract) is 31.25ug/mL, it is 45%, when it is 62.5ug/mL, it is 54%, and when it is 125ug/mL, it is 67%. , showed antioxidant activity of 77% at 250ug/mL and 92% at 100%.

<Example 2>

This example observes free radical (ABTS) scavenging action. Accurately weigh 0.142 g of ABTS reagent, dissolve it in 50 mL of distilled water, and accurately weigh 0.033 g of potassium persulfate and mix with the dissolved ABTS reagent. The well-mixed reagent was reacted by blocking light with foil and placing it at room temperature. After 12 hours, the absorbance of the reagent was measured to confirm that the absorbance value was 1, and then the experiment was carried out. 100uL of each sample was dispensed into 96 wells, and 100uL of ABTS reagent was dispensed into 96 wells where samples were dispensed. After dispensing, the mixture was well mixed, and then the 96 well was wrapped with foil to minimize the effect of light, and the reaction was carried out at room temperature for 30 minutes. The measurement was performed using micro-ELISA, and the measurement wavelength was fixed at 740 nm. BHA was used as a positive control to compare the activity of the sample and to check the error of the experiment, and distilled water was used instead of the sample for the blank. As a result, the free radical scavenging action was obtained as shown in the following equation.

Free radical scavenging activity (%) = 100 - [(absorbance of experimental group / absorbance of control group) × 100]

Referring to FIG. 3, the free radical scavenging rate according to the concentration is shown. When the concentration of Triple Myces is 31.25ug/mL, 50%, 62.5ug/mL, 68%, 125ug/mL, 78%, 250ug/mL. The antioxidant activity was 94% at the time of , and 98% at the time of 100%.

<Example 3>

This example is to measure the tyrosinase (tyrosinase) inhibitory activity. For the experimental group, 250 U/mL mushroom tyrosinase (Sigma-Aldrich Co, St. Louis, MO, USA) 0.1 mL and 250 U/mL mushroom tyrosinase (Sigma-Aldrich Co, St. Louis, MO, USA) were mixed with 2.3 mL of 0.1M sodium phosphate buffer (pH 6.8) and 0.4 mL of 1.5 mM L-tyrosine solution. Add 0.2 mL of a sample with a concentration of 50 to 200 ug/mL phenolic compounds, add 0.2 mL of distilled water instead of the sample to the control group, and react at 37°C for 20 minutes. Then, measure the absorbance at 475 nm to determine the inhibition rate (%) (1- The absorbance of the experimental group/the absorbance of the control group) was calculated as X 100.

Referring to FIG. 4, it shows the tyrosinase inhibition rate according to the concentration. When the concentration of triple myses is 31.25ug/mL, 6%, 62.5ug/mL, 18%, 125ug/mL, 23%, The tyrosinase inhibitory activity was 31% at 250 ug/mL and 40% at 100%.

<Example 4>

The cells used in this experiment were cultured using DMEM medium supplemented with 10% FBS and 1% penicillin/steptomycin (100 U/mL), and subcultured at 37°C, adapted to an incubator with 5% CO ₂ . An inverted microscope (Nikon, Tokyo, Japan) was used to check the cells. B16F10 mouse melanoma cells were pretreated for 1 hour by sample solution concentration and then treated with 1 uM of α-melanocyte stimulating hormone (α-MSH) for 48 hours.

Cytotoxicity was measured according to the method of Carmichael et al. (1987). 450uL of B16F10 mouse melanoma cells were aliquoted to 1x10 ⁴ cells/well in a 48 well plate, and after 24 hours, samples were prepared by concentration, added to 50uL, and treated at 37°C, 5% CO ₂ in an incubator for 48 hours. The control group was cultured under the same conditions by adding the same amount of DMEM (-) medium as the sample. To this, 50 uL of MTT solution prepared at a concentration of 5 mg/mL was added, incubated for 4 hours, the culture medium was removed, and 300 uL of dimethyl sulfoxide (DMSO) was added to each well and reacted for 10 minutes at room temperature, followed by enzyme-linked immunosorbent assay (ELISA). ) The absorbance was measured at 540 nm with a reader. The cytotoxicity measurement was expressed as the absorbance reduction rate of the group with and without the addition of the sample solution.

Cell viability (%) = (1 - absorbance of experimental group / absorbance of control group) ×100

Referring to FIG. 5 , as a result of analyzing the cell viability in B16F10 melanoma cells of Triple Myces, cytotoxicity according to the concentration was not found and the cell growth rate was slightly increased.

<Example 5>

The amount of melanin was used according to the method of Hosoi et al. (1985). B16F10 mouse melanoma cells were dispensed into 6 wells to 5x10 ⁴ cells/well, and after 24 hours, the DMEM(+) medium was removed. Then, DMEM(-) medium was dispensed into each well, and 200uL of α-MSH was subtracted from the normal group. All wells were aliquoted and stimulated. After dispensing 200uL of samples by concentration, they were treated for 48 hours and incubated in an incubator at 37 °C, 5% CO ₂ . After incubation, all the supernatant was removed, washed with PBS, stored in a C eep reezer for one day, 20uL of M-PER was dispensed at room temperature to collect cells, and centrifuged at 13,000 rpm, 4 °C, for 15 minutes to make a cell precipitate. After removing the supernatant, 200uL of 10% DMSO/1N NaOH was dispensed, dissolved in a heating block at 70°C for 1 hour, and the OD value was measured at absorbance at 405 nm.

Melanin content (%) = (1 - absorbance of experimental group / absorbance of control group) ×100

Referring to FIG. 6 , the intracellular melanin content according to the concentration was shown. When the triple myses were treated, the higher the concentration, the more the melanin production amount was decreased in a concentration-dependent manner. At a concentration of 31.25 ug/mL, melanin production was inhibited by about 5%, but at the highest concentration of 500 ug/mL, melanin production was inhibited by about 38%.

The above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the art to which the present invention pertains may make various modifications, changes and substitutions within the scope without departing from the essential characteristics of the present invention. will be. Accordingly, the embodiments disclosed in the present invention and the accompanying drawings are for explaining, not limiting, the technical spirit of the present invention, and the scope of the technical spirit of the present invention is not limited by these embodiments and the accompanying drawings . The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

S10 - mixing and grinding step
S20 - Medium mixing and sterilization step
S30 - Galactomyces inoculation stage
S40 - Incubation stage
S50 - Filter aid mixing step
S60 - Filtration step
S70 - Sterilization stage
S80 - Formulation stage
S100 - Manufacturing method of Triple Mrs. with antioxidant and whitening activity

Claims (3)

Putting dried elder flowers, chamomile flowers, and calendula flowers into a grinder and crushing them mixing and pulverizing step;
A medium mixing and sterilization step of sterilizing the elder flower, chamomile flower, and calendula flower pulverized by the mixing grinding step by mixing it with a microbial culture medium;
Galactomyces inoculation step of inoculating Galactomyces for fermentation of the mixture subjected to medium mixing and sterilization;
A culturing step of culturing the mixture subjected to the Galactomyces inoculation step in an incubator;
A filter aid mixing step of mixing a filter aid containing silicon dioxide with the culture solution subjected to the culture step to perform adsorption, decolorization, and deodorization of lipids or heavy metals;
A filtration step of filtration under reduced pressure using a filter press on the culture solution subjected to the filter aid mixing step to obtain only the pure extract excluding the residue;
A sterilization step of heating the pure extract subjected to the filtration step to inhibit bacterial activity and further fermentation; and
Formulation step of adding a preservative containing 1,2-hexanediol and butylene glycol to the pure extract subjected to the sterilization step
Triple Mrs. manufacturing method with antioxidant and whitening activity comprising a. 2. The method of claim 1
Galactomyces inoculation stage
After performing the medium mixing and sterilization step, when the temperature of the extraction medium is 40℃ or more and 50℃ or less, citric acid is added to adjust the pH to 3 to 4, and when the temperature of the extraction medium enters 30℃ or more and 36℃ or less, the medium amount Inoculation of Galactomyces at a rate of 1% to 5% of
Triple Mrs. manufacturing method with antioxidant and whitening activity comprising a. 2. The method of claim 1
The incubation stage
Incubating the mixture subjected to the Galactomyces inoculation step in an incubator at 20°C or higher and 40°C or lower for 24 hours or more and 48 hours or less
Triple Mrs. manufacturing method with antioxidant and whitening activity comprising a.

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