TW202029956A - Fermented cereal hydrolysate and use thereof for preparing skin care cosmetics - Google Patents

Abstract

The present invention provides a fermented cereal hydrolysate having a skin caring effect, wherein the fermented cereal comprises a rice, a sputum bacteria, a yeast, and a fermentation product, which are subjected to protein quantification followed by a hydrolyzation, an acidification, a cold-stabilization, and a plate and frame filter pressure filtration. Further, the present invention provides a use of the fermented cereal hydrolysate for the manufacture of a skin caring cosmetic, and a cosmetic or pharmaceutical composition comprising the fermented rice hydrolysate.

Description

Fermented rice hydrolysate and its use for preparing skin care cosmetics

The present invention relates to a fermented rice hydrolysate and its use for preparing skin care cosmetics.

Skin aging includes the appearance of wrinkles, skin dullness, atrophy, sagging, and dryness. The demand for skin care has never been reduced, but has increased day by day. Therefore, researchers in related fields continue to seek and develop safe skin care products.

There are many patents for using novel yeast strains to ferment and cause skin care cosmetics. For example, US Patent No. 4,503,041 discloses a novel strain, its culture product or culture solution, and its use for preparing skin treatments. In addition, US Patent No. 4,554,161 discloses skin treatments composed of the specific strain culture solution and cosmetics containing the specific strain culture solution. However, the two patents disclosed above are yeast fermentation filtrate, which is mainly composed of culture broth and components naturally released by yeast growth, and contains only a small amount of protein.

National Taiwan University Food Science and Technology Research Institute Lai Manning’s 014 master's thesis "Using rice aspergillus and yeast fermentation to develop products with anti-aging effects" reveals the use of rice and soybean meal as a fermentation substrate. The first part uses rice aspergillus ( Aspergillus oryzae BCRC30428) for the preparation of rice koji and soybean koji; the second part uses two different yeasts ( Saccharomyces cerevisiae K901 and YCL1087) to inoculate the rice koji and soybean koji to complete the preparation of the fermented product. Some samples were extracted with water and alcohol to explore the anti-aging effects of fermented extracts on human skin fibroblasts. The results show that in the test of the ability to remove active oxygen substances, it is found that the wine extract group of the product fermented by Saccharomyces cerevisiae YCL1087 has a good effect of removing active oxygen substances. The results of promoting the proliferation of type 1 procollagen showed that the samples fermented by Saccharomyces cerevisiae YCL1087, at low concentrations, the Koji mold unfermented group, the Koji mold fermented for 36 hours, the rice wine extraction group, and the Koji mold was not fermented but yeast Compared with the control group, the soybean wine extract group that has been fermented for 72 hours can still maintain the content of type 1 procollagen. Therefore, it does not have the fermented product that inhibits the synthesis of type 1 procollagen, and can remove the active oxygen substance content in the cell, which is beneficial to prevent the oxidative stress caused by the skin cells caused by ultraviolet rays, and it still has anti-aging effects. The potential of the product. However, after the fermented product is extracted with water and alcohol, the type of the functional ingredients in the filtrate obtained is not clear, and the concentration of the functional ingredients is also low due to dilution or extraction efficiency, which prevents subsequent development into commercial products.

On the other hand, peptides are often used in skin care products or cosmetics in recent years because of their easy absorption and good effects. At present, most of the peptide raw materials used in cosmetics on the market are artificially synthesized, ranging in length from 2 to 9 peptides, each with its skin care effect, but whether long-term use will cause physiological interference problems is still inconclusive and has potential risks.

Natural fermentation products or protein hydrolysates, which contain nutrients such as protein, peptides, and amino acids, are prone to browning due to processing methods such as sterilization, or precipitation during storage. It produces negative effects on product appearance, color, smell, stability and applicability.

Therefore, there is still room for the development of novel skin care cosmetics using bacterial fermentation products or protein hydrolysates.

In view of the problems of the prior art: (1) Most of the peptide raw materials of commercially available cosmetics are artificially synthesized, and long-term use has potential risks; and (2) The fermentation products or protein hydrolysates of bacterial strains are easy to process due to industrial mass production. Causes poor stability. In order to solve the above-mentioned problems, the present invention provides a fermented rice hydrolysate and its use for preparing skin care cosmetics.

In one aspect, the present invention provides a method for preparing fermented rice hydrolysate, which comprises the following steps: (1) Mixing and fermenting the white rice, water, koji bacteria, and fermenting yeast at a temperature of 5-20°C to produce a fermented rice mixture (2) Select a certain quality of the fermented rice mixture and quantify its protein content, weigh the calculated fermented rice mixture, and crush it in an emulsifying machine to obtain fine fermented rice grains. Mix the rice grains with hot water and stir evenly, add 0.5~5% of Bacillus licheniformis protease, 0.5~2% subtilisin protease and 0.5~5% Koji mold protease based on total protein content. The value is pH 6.5~7.5 and temperature 50~60℃ to react for 3~5 hours to hydrolyze it; (3) Add food grade citric acid at the end of hydrolysis to adjust the pH value of the overall hydrolysate to pH 4~5, thereby stopping Enzyme reaction and increase storage stability. After the hydrolysate is placed in refrigeration for 1~3 days for cold stabilization, the plate and frame filter press is used for circulating pressure filtration, and then heated in a hot water bath at 70~85°C to obtain the fermentation Millet hydrolysate.

In a specific embodiment, the fermented rice mixture includes rice, koji bacteria, yeast, and fermentation products.

On the other hand, the present invention provides a fermented rice hydrolysate prepared by the above method, which has skin care effects.

In a specific embodiment, the fermented rice hydrolysate of the present invention has the effects of whitening, allergy suppression, anti-aging, etc., without skin irritation.

In another aspect, the present invention provides a use of fermented rice hydrolysate for manufacturing skin care cosmetics, wherein the fermented rice hydrolysate is prepared by the method of the present invention.

In a specific embodiment, the skin care cosmetics made according to the present invention have skin care effects.

In another specific embodiment, the skin care cosmetic prepared according to the present invention has whitening, anti-sensitization, and anti-aging effects.

In yet another aspect, the present invention provides a skin care cosmetic or pharmaceutical composition comprising the fermented rice hydrolysate and/or physiologically or pharmaceutically acceptable salts, and at least one cosmetically or pharmaceutically acceptable carrier .

In a specific embodiment, the skin care cosmetic or medical composition of the present invention has whitening, anti-sensitization, and anti-aging effects.

It should be understood that the foregoing general description and the following detailed description are only illustrative and explanatory, rather than limiting the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. It should be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

As used herein, the singular forms "one" (a, an) and "the" include plural references unless the content is clearly indicated otherwise. Therefore, for example, a reference to "a sample" includes a plurality of these samples and their equivalents known to those familiar with the technology.

As used herein, "fermentation" refers to the decomposition process of organic matter by an organism, where the organism includes microorganisms, plant cells, yeasts, etc., which decompose organic matter to produce energy in an aerobic or anaerobic state.

As used herein, "grain rice" refers to gramineous food crops and their seeds, including rice, wheat, corn, millet and other miscellaneous grains, such as sorghum, wild rice, oats, barley rice, etc. The main nutrients are sugars And protein, etc., are the main food in many regions.

According to the present invention, a method for preparing fermented rice hydrolysate is provided. This method directly uses fermented rice, which is a natural source of nutrients, and contains rice, koji bacteria, yeast, and fermentation products instead of just fermentation broth. The fermented rice hydrolysate is after quantitatively fermenting the protein in the rice, it undergoes enzymatic hydrolysis to increase the content of small molecule proteins, peptides, and amino acids in the product to promote absorption and enhance efficacy. After the fermented rice hydrolysate is hydrolyzed by the enzyme, steps such as acidification, cold stabilization, and plate-and-frame cycle filter press are added, which can effectively improve the darkening of the color, poor odor and precipitation after sterilization, which is beneficial to increase the product Stability and its subsequent applications such as formula preparation.

In the examples of the present invention, it is confirmed that the fermented rice hydrolysate prepared by the method of the present invention has skin care effects. More specifically, the fermented rice hydrolysate has whitening, anti-sensitization, and anti-aging effects without skin irritation. Sex.

Therefore, the present invention provides a use of the fermented rice hydrolysate for manufacturing skin care cosmetics, wherein the skin care cosmetics have whitening, anti-sensitization, and anti-aging effects.

The present invention also provides a cosmetic or pharmaceutical composition, which comprises the fermented rice hydrolysate and/or physiologically or pharmaceutically acceptable salts; and at least one cosmetically or pharmaceutically acceptable carrier.

"Physiologically or pharmaceutically acceptable salts" refer to salts that are approved for use in animals (more particularly humans), and include salts used to form base addition salts, which may be inorganic salts, such as Not limited to lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, or organic salts, such as but not limited to ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, Lysine, histidine or piperazine; or acid addition salts, which are organic salts, such as but not limited to acetate, citrate, lactate, malonate, maleate, Tartrate, fumarate, benzoate, aspartate, glutamate, succinate, oleate, trifluoroacetate, oxalate, pamoate or especially It is gluconate, or inorganic salt, such as but not limited to chloride, sulfate, borate or especially carbonate.

According to the present invention, the cosmetic or pharmaceutical composition may further include a widely used pharmaceutically acceptable carrier, such as but not limited to one or more reagents selected from the following: solvent, buffer, emulsifier, suspension Agents, disintegrants, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and other analog.

According to the present invention, the cosmetic or pharmaceutical composition can be further manufactured into an external preparation suitable for topical application on the skin, including but not limited to emulsifiers, gels, creams, creams, patches, liniments, Powders, aerosols, sprays, emulsions, emulsions, pastes, foams, drops, suspensions, ointments and bandages.

Example

Example 1 Preparation of fermented rice hydrolysate

A white rice is provided for fermentation. The white rice, water, koji bacteria and fermentation yeast are mixed and fermented to produce a white rice fermentation product at a temperature of 5-20°C. Select fermented rice of a certain quality and quantify its protein content. Weigh the calculated fermented rice and crush it in an emulsifying fine-cutter. Mix the fermented rice grains with hot water and stir evenly. Add alkaline protease, neutral protease and compound flavor protease to the aforementioned mixture to hydrolyze it into fermented rice hydrolysate with skin care effect. The hydrolysis step includes: pH 6.5~7.5 and temperature 50~ Reaction at 60°C for 3~5 hours, and based on the total protein content, the alkaline protease added is 0.5~5% licheniform bacillus protease, and the neutral protease is 0.5~2% subtilisin. The compound flavor The protease is 0.5~5% aspergillus oryzae protease. Add food grade citric acid at the end of the hydrolysis to adjust the pH of the overall hydrolysate to pH 4~5, thereby stopping the enzyme reaction and increasing storage stability. After the hydrolysate is placed in refrigeration for 1~3 days for cold stabilization, the plate and frame filter press is used for circulating pressure filtration to obtain the clarified fermented rice hydrolysate, which is then heat-killed at 70~85℃ in a hot water bath to reduce Microbial survival rate.

Example 2 Comparison of fermented rice hydrolysate before and after hydrolysis

Kjeldahl method (CNS 5035_ test method for crude protein in food) and amino acid analyzer (CNS 12632_ fruit and vegetable juice test method-determination of free amino acid) were used to determine the total nitrogen in the sample. (Estimated crude protein content) and total free amino acid content.

In order to compare the fermented rice hydrolysate before and after the hydrolysis, and compared with the commercially available yeast fermentation filtrate products and the fermented rice hydrolysate, the crude protein and total free amino acids of the fermented rice hydrolysate increased significantly ,As shown in Table 1. Table 1. Analysis of the crude protein and total free amino acids of commercially available yeast fermentation filtrate products, fermented rice non-hydrolysates, and fermented rice hydrolysates Commercial yeast fermentation filtrate Fermented rice without hydrolysate Fermented rice hydrolysate Crude protein (%) 0 0.61 2.69 Total free amino acid (mg/100ml) 0 302 1311

Example 3 Skin care efficacy test

3.1 Inhibition test

In order to inhibit the degranulation of rat basophils after stimulation, the anti-sensitization effect of the fermented rice hydrolysate was verified. RBL-2H3 cells were planted in a 96-well cell analysis tray, and the number of cells per well was 5× ^{10 4} . After incubating in a 37 ℃/5% CO2 cell incubator for 24 hours, remove the original cell culture solution and add the diluted sample to the analysis tray. Repeat the test for each sample 3, and add 1 µg/ml A23187 to stimulate 2 after half an hour hour. Aspirate the supernatant for β-Hexosaminidase Release analysis, and analyze the activity of the cells with MTT reagent.

When the concentration of the fermented rice hydrolysate is 1% and 10%, the inhibition rate of degranulation after stimulation is 70% and 95% respectively (as shown in Figure 1), indicating that the fermented rice hydrolysate has Good anti-allergy effect.

3.2 Whitening efficacy test

To determine the inhibition rate of tyrosinase activity in a test tube test, each sample solution of different test concentration, Arbutin solution of the positive control group and phosphate buffer of the control group were transferred to a 96-well plate. Three replicates for each sample. Add phosphate buffer, deionized water, and tyrosinase solution (final concentration of 100 units) to each well plate, shake and mix well, and measure the absorbance value (T0) at OD475 nm (the background value before reaction). Add L-tyrosine solution (the final concentration is 0.825 mM) and wait for 3 minutes, then measure the OD475 nm absorbance (T1), calculate the difference between the 475 nm absorbance before and after the reaction to calculate the tyrosinase activity: Tyrosine Enzyme activity (%) = (absorbance value of sample (T1-T0) / absorbance value of control group (T1-T0)) x 100%

When the concentration of the fermented rice hydrolysate is 1%, 5%, and 10%, the inhibition rates of tyrosinase activity are 26%, 60%, and 65% respectively (as shown in Figure 2), indicating that the fermented rice Rice hydrolysate has the ability to inhibit tyrosinase and has whitening effects.

3.3 Anti-aging test

In the cell experiment, the anti-aging effect of the matrix metalloproteinase MMP-1 released by skin fibroblasts stimulated by inflammatory factors was measured. Hs68 cells were planted in a 96-well cell analysis tray, and the number of cells per well was 1×10 ⁴ . After incubating in a 37 ℃/5% CO ₂ cell incubator for 24 hours, remove the original cell culture solution, and add the samples diluted with the culture solution to the 96-well cell analysis dish, repeat the test for each sample 3, and process it for 6 hours Adding 10 ng/ml TNF-α stimulates the production of MMP-1 in cells. After 18 hours of incubation, the supernatant was transferred to an analysis dish pre-coated with MMP-1 antibody for enzyme immunoassay (ELISA), and the cells were analyzed for activity with MTT reagent.

When the concentration of the fermented rice hydrolysate is 1% and 5%, the inhibition rate of MMP-1 released after stimulation is 50% and 65%, respectively (as shown in Figure 3), indicating that the fermented rice hydrolysate has Anti-aging effect.

3.4 Skin irritation test

A three-week RIPT skin open patch test was conducted for 50 people and the test results showed that the hydrolyzed fraction of fermented rice had no skin irritation and no skin irritation (Table 2). Table 2. Results of skin irritation analysis

Example 4 Stability test

After acidification, cold stabilization, and plate-and-frame circulating filter press, sterilization is performed, and the stability and applicability of the fermented rice hydrolysate is improved (as shown in Table 3). Table 3. Stability analysis of fermented rice hydrolysate after acidification, cold stabilization, plate and frame cycle filter press, and sterilization acidification - - - - + + + + Leng Anding - + - + - + - + Plate and frame circulating filter press - - + + - - + + Minimum temperature and time required for sterilization 95°C, 2 hours 95°C, 2 hours 95°C, 2 hours 95°C, 2 hours 63°C, 0.5 hours 63°C, 0.5 hours 63°C, 0.5 hours 63°C, 0.5 hours Exterior color Dark brown turbid liquid Dark brown turbid liquid Dark brown clear liquid Dark brown clear liquid Light yellow turbid liquid Light yellow turbid liquid Light yellow clear liquid Light yellow clear liquid odor Special smell of dregs Special smell of dregs Special smell of dregs Special smell of dregs Liquor meal, special smell Liquor meal, special smell Liquor meal, special smell Liquor meal, special smell Appearance color after six months Dark brown turbid liquid, white precipitate Dark brown turbid liquid, white precipitate Dark brown clear liquid, white precipitate Dark brown clear liquid Light yellow turbid liquid, white precipitate Light yellow turbid liquid, white precipitate Light yellow clear liquid, white precipitate Light yellow clear liquid Total bacterial count after six months not detected not detected not detected not detected not detected not detected not detected not detected

Those skilled in the art will understand that changes can be made to the specific embodiments described above without departing from the broad inventive concept. Therefore, it should be understood that the present invention is not limited to the specific specific embodiments disclosed, but is intended to cover the spirit and modifications within the scope of the present invention defined by the scope of the appended patent application.

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When read in conjunction with the drawings, one will better understand the foregoing content of the invention and the following detailed description of the invention. For the purpose of illustrating the present invention, the presently preferred specific embodiments are shown in the drawings.

In the attached picture:

Figure 1 shows the analysis of degranulation of rat basophils after stimulation, showing that the fermented rice hydrolysate has an anti-sensitivity effect.

Figure 2 shows the tyrosinase enzyme activity test, showing that the fermented rice hydrolysate has whitening effect.

Figure 3 shows the analysis of matrix metalloproteinase MMP-1 released by skin fibroblasts after stimulation, showing the anti-aging effect of the fermented rice hydrolysate.

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Claims (10)

A preparation method of fermented rice hydrolysate, comprising the following steps: (1) Mix and ferment the white rice, water, koji bacteria and fermenting yeast at a temperature of 5-20°C to produce a fermented rice mixture; (2) Select a certain amount Quality of the fermented rice mixture and quantify its protein content, weigh the calculated fermented rice mixture, crush it in an emulsifying and fine-cutting machine to obtain fermented rice grains, and mix the fermented rice grains with hot water Mix and stir evenly, add 0.5~5% Bacillus licheniformis protease, 0.5~2% subtilisin protease and 0.5~5% Aspergillus oryzae protease based on the total protein content, at a pH of 6.5~7.5 And react at 50~60℃ for 3~5 hours to hydrolyze it; (3) Add food grade citric acid at the end of hydrolysis to adjust the pH value of the overall hydrolysate to pH 4~5, thereby stopping the enzyme reaction and increasing storage stability After the hydrolysate is placed in refrigeration for 1 to 3 days for cold stabilization, it is filtered by a plate and frame filter press, and then heated at 70-85°C in a hot water bath to obtain the fermented rice hydrolysate. The preparation method of claim 1, wherein the fermented rice mixture contains rice, koji fungus, yeast, and fermentation products. A fermented rice hydrolysate prepared by the preparation method of claim 1. Such as the fermented rice hydrolysate of claim 3, which has skin care effects. Such as the fermented rice hydrolysate of claim 3, which has whitening, allergy suppression, anti-aging effects, and no skin irritation. A use of fermented rice hydrolysate for the manufacture of skin care cosmetics, wherein the fermented rice hydrolysate is a fermented rice hydrolysate prepared by the preparation method of claim 1 or 2, or claim 3, 4, or 5 The fermented rice hydrolysate. Such as the use of claim 6, wherein the skin care cosmetic has a skin care effect. Such as the use of claim 6, wherein the skin care cosmetic has whitening, sensitization, and anti-aging effects. A skin care cosmetic or pharmaceutical composition comprising the fermented rice hydrolysate obtained by the preparation method of claim 1 or 2, or the fermented rice hydrolysate of claim 3, 4 or 5, and/or physiologically or medically Acceptable salts; and at least one cosmetically or pharmaceutically acceptable carrier. For example, the skin care cosmetic or medical composition of claim 9, which has whitening, sensitization, and anti-aging effects.

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