KR102112949B1 - A method of manufacturing cosmetics materials that extracts cosmetics materials by using insoluble protein with compound yeast fermentation - Google Patents

Abstract

The present invention relates to a cosmetic raw material producing method of extracting a cosmetic raw material by solubilizing an insoluble protein through composite yeast fermentation. The method comprises the steps of: preparing an egg yolk which is an insoluble protein; extracting a raw material protein by partially removing lipids, which are impurities in the egg yolk, by using a solvent; fermenting the raw material protein on the basis of composite yeast composed of Galactomyces candidus and Saccharomyces cerevisiae; and extracting cosmetic raw materials by sterilizing an obtained fermented liquid and filtering remaining strains. According to the present invention, advantages are secured in terms of costs, and production efficiency is enhanced.

Description

A method of manufacturing cosmetics materials that extracts cosmetics materials by using insoluble protein with compound yeast fermentation}

The present invention relates to a method for producing a cosmetic raw material for extracting a cosmetic raw material by solubilizing an insoluble protein by complex yeast fermentation, and more specifically, forming a medium composed of starch, germinated brown rice, germinated barley, raw sugar, and purified water and proteolytic strain The present invention relates to a method for producing a cosmetic material for extracting a cosmetic material containing free amino acids and AHA components by fermenting a raw protein based on a complex yeast composed of Galactomyces candidus and Saccharomyces cerevisiae.

As is known, most of the various peptides used as cosmetic raw materials are produced synthetically or through fermentation. In particular, expensive peptides are known to cost more than 300,000 won per gram. Among them, EGF is a representative peptide used as a medicine. These peptides are known to help skin rejuvenation by pharmacological action, but they are also raw materials that have problems such as less than 1% being added due to the usage limit of the Ministry of Food and Drug Safety.

On the other hand, dipeptide diaminobutyroyl benzylamide diacetate, also known as snake venom peptide, is a structure in which functional groups are attached to proline and alanine, and is known to play a role in inducing relaxation and improving wrinkles by inhibiting Na movement of cells in the human body. Most of these peptides are water-soluble and have the advantage of being easily absorbed by the skin.

In addition, Hydrolyzed Silk Protein, also known as silk amino acid, is produced by hydrolyzing the external silk component of silkworm cocoon and is used in skin protection and hair elasticity recovery.

These peptide components are widely used and applied industrially as cosmetic ingredients with a variety of products, from high-priced products with a specific structure to mid-low-priced products through hydrolysis.

Recently, the release of cosmetics containing AHA and BHA components among cosmetic ingredients has become popular. In particular, AHA ingredient is a raw material that is easily used without a limited amount, and the majority of ingredients made through chemical synthesis.

The AHA component is short for α-hydroxy acid, and the -OH group is collectively attached to C next to the COOH group. In particular, it is commonly used as an organic acid, and one well-known component is Lactic acid. The main effect is known as wrinkle improvement and exfoliation.

For this reason, the present invention, which will be described later, intends to propose a solubilization method soluble in water from an insoluble protein component and a process for recovering AHA components produced through fermentation.

Existing hydrolysis methods include acid hydrolysis with inorganic acids, base hydrolysis with basic substances, enzymatic hydrolysis, and hydrolysis through fermentation. Among them, in the case of hydrolysis with the addition of inorganic acids, certain amino acids (tryptophan, Try) are mostly decomposed, which makes it difficult to commercialize them industrially. In addition, in the case of alkaline hydrolysis, it was used industrially in the past, but recently, in the course of neutralization, the problem of heavy metal contamination of the neutralizing agent emerged, and the yield due to the formation of salt during neutralization was lowered, resulting in high cost of filtration process to remove salt. have.

In recent years, protein hydrolysis is processed by enzymatic treatment, and known enzymes include protamax, flavourzyme, and the like, and are widely used in industry. However, enzyme activity is different depending on the pH and temperature, and it is difficult to use, such as endo type and exo type, which are worth using as foods. In addition, commercially available enzymes are expensive and highly import-dependent, so the production price of protein hydrolysates is high.

Due to these problems, recently, as active research and development is progressing in the development of process processes to which protein hydrolysis is applied through fermentation, microorganisms are attracting attention. Industrial uses of proteolytic strains include Bacillus subtilis, Aspergillus oryzae, and induce fermentation of intestines and alcohol from these strains.

However, these strains are small-sized strains that require more time in the filtration process after fermentation and tend to have higher production costs. In particular, Bacillus subtilis generates spores and has problems such as re-expression if it is not completely sterilized. It is difficult to utilize. Currently, industrial sterilization is processed at 100 ℃, but complete sterilization such as 20 minutes sterilization at 121 ℃ is not used industrially.

Aspergillus oryzae is known as yeast mold and is used to decompose proteins and starches, and is a strain frequently used in the food industry due to its rapid decomposition action. However, since the mycelium is formed with spores and has a property of spreading in a dusty form, there is a problem in management such as the rapid contamination of the surrounding fungi.

1. Patent Application No. 10-2003-0011470, Name of invention: Cosmetic composition using natural herbal fermentation 2. Patent Application No. 10-2012-0018188, Name of the invention: Method for manufacturing fermented cosmetics containing fermented products of germinated grains as a main component 3. Patent Application No. 10-2013-0132897, Name of invention: Cosmetic composition and manufacturing method using sweet persimmon fermentation

Accordingly, in the present invention to be described later, the hydrolysis of protein is induced due to fermentation, and it is intended to utilize a yeast strain known as a strain that is relatively free from contamination after fermentation and has a relatively large cell size. For example, the present invention is based on the use of Galactomyces candidus and Saccharomyces cerevisiae among yeast strains, and has the advantages of differentiating from the existing methods in terms of cost, such as production cost and neutralization reaction, and increasing production efficiency. I would like to suggest.

Galactomyces candidus and Saccharomyces cerevisiae are a type of yeast strain. Galactomyces candidus is characterized by growing by fermentation on the top surface, and Saccharomyces cerevisiae is grown by fermentation by the bottom surface. This is because there is a characteristic of not receiving.

In addition, the present invention aims to develop cosmetic raw materials such as inducing hydrolysis by protein solubilization using Geotrichum candidum, and adding AHA components through fermentation of Saccharomyces cerevisiae to the skin to give value as cosmetic ingredients. do.

That is, the present invention is to propose a method for extracting a cosmetic material containing free amino acids and AHA components by fermenting a raw protein from which a predetermined lipid is removed from an insoluble protein such as egg yolk with a predetermined strain.

In addition, the present invention seeks to devise a method for providing cosmetic raw materials with cost-efficiency by promoting differentiation from existing cosmetic raw material manufacturing methods and reducing material costs.

A method of manufacturing a cosmetic raw material for extracting a cosmetic raw material by solubilizing an insoluble protein through complex yeast fermentation, which is a technical idea proposed in the present invention,

Preparing an insoluble protein yolk;

Impurities in the yolk using a solvent composed of 1 to 5% by weight of dichloromethane, 1 to 5% by weight of n-hexane, 1 to 15% by weight of ethyl ether, 10 to 60% by weight of alcohol, and 10 to 80% by weight of distilled water Removing the phospholipids to extract raw proteins;

Fermentation to form a medium consisting of starch, germinated brown rice, germinated barley, raw sugar, and purified water, and fermenting the raw material protein based on a complex yeast composed of Galactomyces candidus and Saccharomyces cerevisiae having a weight ratio of 1: 1 as a proteolytic strain. Processing steps;

It characterized in that it consists of a step of sterilizing the fermentation broth (culture solution) obtained after the fermentation treatment step and filtering the remaining strain to extract cosmetic raw materials.

In the present invention, the medium is 5 to 20% by weight of the starch, 1 to 10% by weight of the germinated brown rice, 1 to 10% by weight of the germinated barley, 5 to 20% by weight of the raw sugar, and the purified water 10 It consists of ~ 50%,

The fermentation time using the complex yeast is 24 to 48 hours, and the temperature at the time of fermentation is 20 to 28 ° C.

In the present invention, the cosmetic raw material is characterized in that it contains a free amino acid and AHA components.

When using the method of manufacturing a cosmetic raw material to extract the cosmetic raw material by solubilizing the insoluble protein by the complex yeast fermentation proposed in the present invention, there is an advantage that can reduce the production time and input cost of conventional cosmetic raw materials.

1 is a flow chart showing a conceptual diagram of a cosmetic material manufacturing method (process) proposed in the present invention.

Hereinafter, a method of manufacturing a cosmetic raw material for extracting a cosmetic raw material by solubilizing an insoluble protein through complex yeast fermentation, which is a technical idea proposed in the present invention, will be described with reference to the drawings.

1 is a flow chart showing a conceptual diagram of a cosmetic material manufacturing method (process) proposed in the present invention.

As shown, the method of manufacturing a cosmetic material of the present invention comprises the steps of preparing an insoluble protein raw material; Removing impurities (eg lipids) from the insoluble protein; Extracting the raw material protein through a filtration process; It consists of the step of extracting a predetermined cosmetic raw material by culturing the raw protein and fermenting the recovered fermentation broth, heating, sterilizing and filtering the raw protein.

First, there may be various types of insoluble protein, but industrially used include silk, egg yolk, isolated soy protein, whey protein, and skim milk powder. Among them, egg yolk, isolated soy protein, skim milk powder, etc. have the highest industrial utilization, but isolated soy protein lacks essential amino acids methionine and tryptophan, which are known to be incomplete proteins.

In the case of whey protein, the protein content is about 70%, but the carbohydrate content that is rapidly absorbed is also high, so it has the disadvantage of quick decomposition and high production cost, so it is used as a powder product with a moisture content less than 10% in terms of industrial use, price, etc. Due to the problem, the scope of use is limited.

In addition, in the case of skim milk powder, fat and oil are removed from milk, which is known as a complete food containing all essential amino acids, and it is a raw material with a protein content of 35%. .

Therefore, among the above listed raw materials, the most insoluble protein suitable for the present invention is egg yolk. Yolk, which is derived from egg, a complete food, refers to the yolk in the egg and is used most industrially. It extracts oil from the yolk and consumes it as lecithin and yolk oil, and reprocesses the sludge (the yolk left after extracting the fat). There are advantages such as being able to obtain an insoluble protein with 80% protein.

In the present invention, after preparing a yolk with an insoluble protein, a process of removing impurities in the yolk is performed.

Insoluble proteins, such as egg yolk, are known as structural proteins and are characterized by retaining their shape as they do not dissolve in purified water (collectively, tap water or distilled water). This component can be absorbed only after being converted to a component that is soluble in water through hydrolysis, and has a disadvantage that can be utilized as a cosmetic raw material.

As described above, the egg yolk suitable for industrial use contains a lipid content of about 45%, so an impurity removal process such as removing lipids is essential.

The process of removing impurities from the insoluble protein refers to a process of removing components other than proteins (eg, lipids, carbohydrates, etc.).

Removal of impurities is carried out using a solvent.

In general, purified water, alcohol, n-hexane, acetone, dichloromethane, etc. are also used. However, it is limited to purified water and alcohol because it is limited to a solvent that can be used in food.

The mixed solvent is dichloromethane (1 ~ 5%), n-hexane (1 ~ 5%), ethyl ether (1 ~ 15%), alcohol (10 ~ 60%), distilled water (10 ~ 80%), respectively. It is a step to remove other ingredients by mixing. Among them, dichloromethane and n-hexane may not be used depending on the situation.

The lipid content in the yolk according to the temperature and the solvent was measured and indicated as residual lipid, and the examples are as follows.

[Table 1]

The result of fixing the temperature through the results obtained in Examples 1-8 and confirming the content of lipids in the yolk over time is the same as in Examples 9-16 below.

[Table 2]

Examples for confirming the removal of lipids according to the ratio of the solvent are shown in Table 3 below.

[Table 3]

As a result of confirming with the above Example (1-24), suitable processes are dichloromethane 1-8 wt%, n-hexane 1-8 wt%, alcohol 50-44 wt%, purified water 48-40 wt%, temperature 40-60 (℃), it was confirmed that the time was 20 to 30 minutes.

However, dichloromethane and n-hexane cannot be used for food use, and even as a solvent for cosmetic production, it is a chemical that is likely to cause problems such as heavy metal contamination. %) And distilled water (40 ~ 90%), respectively, to remove other components may be suitable.

In addition, in the step of mixing the mixed solvent and the residual egg yolk together with the above-described embodiment and setting the pressure reduction, temperature, and stirring speed, maintaining the pressure at a level of 1 to 0.15 atm, and maintaining the level at 10,000 to 15,000 RPM using a high-speed homogenizer It may include the steps.

[Table 4]

Next, a process of manufacturing a microbial fermentation broth to prepare a culture medium for fermentation is performed by recovering the egg yolk from which lipids have been removed and adding sub-raw materials (starch, germinated brown rice, germinated barley, yeast, raw sugar).

Addition of sub-materials is intended to induce the cultivation of the strain so that the metabolic reaction can work efficiently and must be included in the fermentation.

The existing culture medium is PDA, and is formed at a high price of 200,000 won per 500 g. As this affects the production cost of the product, it is very important to develop a replacement product. In order to replace the existing medium and to produce more efficiently, a section on medium composition is shown.

The medium composition is as follows. That is, starch (5 to 20%), germinated brown rice (1 to 10%), germinated barley (1 to 10%), raw sugar (5 to 20%), and purified water (10 to 50%).

The purpose of using starch is to make glucose, which is the food of the strain through saccharification, and raw sugar is used to supplement the minerals during fermentation, and germinated brown rice and germinated outer barley are used to supply saccharification and minerals.

There are various types of starch, such as potato, sweet potato, corn, and wheat, but the most suitable is potato and corn starch. Among them, the basic component of PDA medium is potato, so potato starch is used in the composition of this medium.

Since yeast strains do not have starch degrading enzymes, they use the raw materials, easily saccharify and utilize the starch. Especially, they use germinated brown rice and germinated barley as raw materials to help grow yeast strains by supplying minerals and other nitrogen sources. .

The saccharification level of the medium was determined by measuring the content of reducing sugars using several examples and DNS analysis to determine whether saccharification was well related to the medium composition. The DNS analysis method is as follows. Dissolve salicylic acid and 21.4 g of NaOH first in 6.3 g of dinitrogen in 1,000 mL of distilled water, then add 182.0 g of sodium potassium tartrate, 5 g of Na ₂ S ₂ O ₅ and dissolve. Weigh the standard substance to prepare a calibration curve, collect some of the medium, and check the final reducing sugar content by contrasting with the reaction and calibration curve.

[Table 5]. Saccharification reaction according to medium composition

In the yeast fermentation process, proteolytic strains mainly include Bacillus subtilis, Aspergillus oryzae, and industrial enzymes such as protamax and flavozyme, which are proteolytic enzymes, are also used. The problem of the strain is that it is difficult to sterilize and disinfect the bacteria, and the cost of the enzyme is high for the degrading enzyme, which creates a price barrier for industrial use. Therefore, as a method of avoiding the method, it is necessary to produce proteolytic enzymes and to easily sterilize and also to easily disinfect.

Therefore, in the present invention, Galactomyces candidus is proposed as a fermentation strain.

Galactomyces candidus is a class 1 yeast strain that is involved in the fermentation of cheese with a level of biological safety rated by the US Bureau of Disease Control and Prevention. Galactomyces candidus is a strain that produces proteolytic enzymes and is put into a fermentation process for the hydrolysis of raw proteins.

Meanwhile, in the present invention, Saccharomyces cerevisiae is additionally proposed as a fermentation strain.

Saccharomyces cerevisiae is a yeast widely used in the food industry, and is known to prevent the growth of strains other than yeast by producing alcohol during anaerobic fermentation and to lower the pH by generating organic acids to prevent contamination of other strains.

In the present invention, a complex yeast composed of Galactomyces candidus and Saccharomyces cerevisiae in a 1: 1 weight ratio was generated, and then complex fermentation was performed using the same.

Galactomyces candidus was inoculated into the prepared medium, and proteolytic activity was measured by adjusting temperature (15-40 ° C) and incubation time (6-48H) to determine optimal growth conditions.

The proteolytic enzyme activity was measured in the following manner.

That is, the quantitative analysis value of protease in the sample is expressed as a unit, and 1 unit is the amount of protease required to decompose 1 uM of L-tyrosine from casein for 1 minute under conditions for preparing the test solution. . The amount of L-tyrosine in the test solution calculated through the calibration curve was converted to the titer (Unit / g) of the protease contained in 1 g of the sample. About 5.0 g of the strain culture medium was precisely weighed, dissolved in distilled water to make 100 mL, and then filtered to perform the experiment. Add 1 mL of 0.6% casein solution to the test tube, warm it in a 37 ° C constant temperature water bath, add exactly 1 mL of strain culture solution, and shake well to mix. Soon it was placed in a constant temperature water bath at 37 ° C, reacted for exactly 10 minutes, and then 2 mL of 0.4 M trichloroacetic acid was added thereto, and then left at 37 ° C for 25 minutes, and then filtered. Take exactly 1 mL of the filtrate, add 5 mL of 0.4 M sodium carbonate solution and 1 mL of polyline reagent (1: 1) and shake well. After standing at 37 ° C for 20 minutes, the colored solution is used as the test solution. Separately, take exactly 1 mL of distilled water, put it in a test tube, leave it at 37 ° C for 10 minutes, mix it with 2 mL of 0.4 M trichloroacetic acid solution, add 1 mL of 0.6% casein solution, leave it at 37 ° C for 25 minutes, and then Perform the same operation to prepare a blank test solution.

Table 6 is an evaluation table for the activity of protease according to the composition of the medium.

[Table 6]

Fermentation process based on Galactomyces candidus and Saccharomyces cerevisiae was performed. The fermentation process was conducted for a total of 24 to 72 hours, and an appropriate fermentation time can be 24 to 48 hours. In the fermentation, the temperature was set to 10 to 35 ° C, and can be appropriately set to 20 to 28 ° C. It was confirmed that the free amino acid component increased with fermentation time.

Analysis of increasing free amino acid content was determined by total amino nitrogen content.

The amino nitrogen analysis was conducted by Formal analysis with reference to the paper based on the Ministry of Food and Drug Safety.

The calculus is as follows. That is, Amino-Na refers to nitrogen in the amino group (-NH₂) of an amino acid and is a method of displaying the total amount of free amino acids. In a neutral aqueous solution, both the -amine and -carboxyl groups of amino acids are released and ionized. When acting formaldehyde, -amine and formaldehyde react and the basicity of -amine disappears, and amino acid remains acidic because only -carboxyl remains, making it an alkali standard. The amount of amino-Na can be determined by titration with a solution. The culture was taken to 10 mL and adjusted to pH 8.4 with 1N NaOH. 20 mL of 36% formaldehyde was added and titrated to 8.4 with 0.1N NaOH. Apply the formula below to find the total free amino acid content. Table 3 measured the total free amino acid content according to the conditions.

Amino-Na (mg / 100g) = {{(A-B) X 1.4 X F} / Sample g} x100

Here, A: the initial dose at the start of titration, B: the dose used after titration, and F: the purity of the titrant reagent (usually 1).

Table 7 shows the change in total free amino acid with time and temperature.

[Table 7]

The culture solution obtained after the fermentation process is subjected to a sterilization process through a heat treatment at 50 to 90 ° C. The time of heat treatment can be 10 to 30 minutes, and the most efficient treatment can be 60 to 80 ° C and 15 to 25 minutes. A vacuum filter or press filter, centrifugation, etc. may be used to remove the sterilized strain. When filtering, filtration may be performed using bentonite, activated carbon, silicon dioxide, or baking soda as a filter aid, but it is preferable to use silicon dioxide, salt, and baking soda as the most suitable filter aid. The amount of silicon dioxide is preferably used within 0.1 to 10% of the total amount, but as a more effective use range, it can be used within 0.1 to 5% of the total amount. The amount used may be 1 to 10% of the total capacity, as Na ions in baking soda react with the unsolubilized protein and precipitate. However, the most effective range is around 0.5 ~ 5%.

In order to determine whether it is possible as a cosmetic raw material of the filtered culture medium, the degree of natural organic acid generation for the identification of AHA components was analyzed. The acidity measurement method, published by the Ministry of Food and Drug Safety, and the thesis of the Academic Promotion Foundation were used to identify organic acids.

The method is as follows. That is, after dropping 50 uL of the indicator (phenolphthalein solution) into 20 mL of the culture solution, titrate with 0.1N NaOH. If it appears red, it is the end point. Use the following formula for calculation.

Organic acid content (%) = {0.0090xVxF / S} x100

Here, V: amount of reagent for titration, F: purity of titration reagent (generally 1), S: weight of sample.

Table 8 shows the organic acid content results of Examples 11-15.

It was found that the prepared raw material can be used as a cosmetic raw material as a fermentation broth containing free amino acids and natural AHA components.

As described above, the method for preparing a cosmetic raw material for extracting a cosmetic raw material by solubilizing the insoluble protein through the complex yeast fermentation according to the present invention comprises a medium consisting of starch, germinated brown rice, germinated outer barley, raw sugar, and purified water, and a proteolytic strain It is a method for manufacturing cosmetic raw materials for extracting cosmetic raw materials containing free amino acids and AHA components by fermenting raw protein based on a complex yeast composed of Galactomyces candidus and Saccharomyces cerevisiae. This has the advantage of reducing costs.

Claims (3)

As a method for producing cosmetic raw materials for extracting cosmetic raw materials by solubilizing insoluble proteins through fermentation of complex yeast,
Preparing an insoluble protein yolk;
Impurities in the yolk using a solvent composed of 1 to 5% by weight of dichloromethane, 1 to 5% by weight of n-hexane, 1 to 15% by weight of ethyl ether, 10 to 60% by weight of alcohol, and 10 to 80% by weight of distilled water Removing some of the phospholipids to extract the raw protein;
Fermentation to form a medium consisting of starch, germinated brown rice, germinated barley, raw sugar, and purified water, and fermenting the raw material protein based on a complex yeast composed of Galactomyces candidus and Saccharomyces cerevisiae having a weight ratio of 1: 1 as a proteolytic strain. Processing steps;
Cosmetic raw material manufacturing by extracting cosmetic raw material by solubilizing insoluble protein with complex yeast fermentation, characterized in that it consists of sterilizing fermentation broth (culture solution) obtained after the fermentation treatment step and filtering the remaining strain to extract cosmetic raw materials. Way. According to claim 1,
The medium comprises 5 to 20% by weight of the starch, 1 to 10% by weight of the germinated brown rice, 1 to 10% by weight of the germinated barley, 5 to 20% by weight of the raw sugar, and 10 to 50% of the purified water Lose,
Fermentation time using the complex yeast is 24 to 48 hours, the fermentation temperature is 20 to 28 ℃, characterized in that the complex yeast fermentation method of solubilizing insoluble protein to extract the cosmetic material to extract the cosmetic material. According to claim 2,
The cosmetic raw material manufacturing method of extracting a cosmetic raw material by solubilizing insoluble protein by complex yeast fermentation, characterized in that it contains free amino acids and AHA components.

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