KR20210046412A - Compositions containing defatted microalgae extract - Google Patents

Abstract

The present invention relates to a cosmetic composition containing defatted microalgae enzyme decomposition product as an active ingredient. The cosmetic composition is effective in skin whitening and skin regeneration by containing defatted microalgae formed by defatting microalgae as an active ingredient.

Description

Compositions containing defatted microalgae extract as an active ingredient

The present invention relates to a cosmetic composition and a health functional food comprising an enzyme decomposition product of skim microalgae as an active ingredient.

In recent years, due to the destruction of the ozone layer, skin damage due to an increase in ultraviolet rays reaching the surface has been continuously increasing. When the skin is stimulated with ultraviolet rays, hormones and nitrogen monoxide are secreted from keratinocytes, increasing skin pigmentation, promoting skin pigmentation and aging.

Skin pigmentation is caused by melanin pigment and is known to protect the skin from ultraviolet rays, but excessive melanin production is known to cause diseases such as skin cancer in addition to pigmentation. Melanin is converted to DOPA quinone through 3,4-dihydroxy-phenylalanine (DOPA) by tyrosine as a substrate using tyrosine as a substrate, and is produced through DOPA chrome through automatic oxidation and enzymatic reactions. Tyrosinase is known as a major enzyme in the process of melanin production, and recently, studies on inhibition of tyrosinase activity have been actively conducted in order to reduce or eliminate the production of melanin pigments.

Currently, the most widely used skin whitening agents are arbutin and kojic acid, which are chemically synthesized substances. Arbutin is known as an inhibitor that acts competitively with tyrosine, and kojic acid inactivates the active site of tyrosine to produce DOPA. It is known to inhibit the production of melanin pigment by interfering with. However, use of kojic acid is limited due to stability problems such as skin discoloration and skin cancer potential, and arbutin is known to produce hydroquinone, which is suspected as a carcinogen, by light or enzymes, and has a risk for long-term use.

Accordingly, there is a growing need for the development of natural-derived whitening materials in order to overcome the shortcomings of existing chemically synthesized whitening agents. Most of the whitening or anti-aging materials derived from nature occupy a high proportion of land plants, such as chrysanthemum, serrata, cockscomb extract, or medicinal herbs, but marine organisms have a variety of species and produce physiologically active substances with excellent performance due to the peculiarity of the marine environment. It is reported that it is becoming a high value-added material.

Among marine plants, seaweeds are attracting a lot of attention as a material for the production of physiologically active substances because they contain various proteins and polysaccharides and contain abundant minerals and vitamins. According to Manou et al., seaweeds contain various physiologically active substances including antioxidants, pharmacological activities, anti-tumor, anti-inflammatory and immunomodulatory, which are superior to land plants, so they can be used as whitening and antioxidant functional cosmetic materials.

It is reported that the physiologically active function of these algae is due to the low-molecular form of polysaccharides and proteins. Peptides produced through protein decomposition present in natural products are small in size and can be easily absorbed into living tissues, and have the properties of improving wrinkles and increasing collagen synthesis, so various researches are ongoing in the cosmetics and medicine fields as a next-generation material. . In particular, peptides composed of 18 or less amino acids are known to be effective in inhibiting free radicals, regenerating skin cells, promoting cell division, and improving wrinkles, and are widely used in cosmetics, functional foods, and pharmaceuticals.

However, in protein decomposition, chemical decomposition using acids or bases is restricted due to safety issues and increased consumer rejection in the use of food and cosmetics, and enzymatic decomposition using commercial enzymes is a problem with permission for food and cosmetic additives. It has the disadvantage of increasing product cost due to the expensive enzyme price. In order to overcome the shortcomings of the production of peptides by chemical decomposition and the production of peptides using commercial enzymes, the need for production of peptides using more safe and low-cost enzymes is increasing.

Meanwhile, as the need for new and renewable energy increases, microalgae, a type of seaweed, is a third generation biomass that produces biodiesel through lipid extraction, and its utilization is increasing. However, despite the high protein content in skim microalgae discarded after lipid production, it is used or discarded as a low-grade feed, so research on recycling is insufficient.

Therefore, there is a need for a composition comprising an enzymatic digestion product obtained by hydrolyzing defatted microalgae using a new naturally-derived enzyme instead of a conventional chemical digestion or enzymatic digestion using a commercial enzyme.

Republic of Korea Patent Publication No. 10-2019-0008509 Republic of Korea Patent Publication No. 10-2019-0094326

It is an object of the present invention to provide a cosmetic composition comprising an enzyme decomposition product of skim microalgae as an active ingredient.

In addition, another object of the present invention is to provide a health functional food comprising an enzyme decomposition product of skim microalgae as an active ingredient.

In addition, another object of the present invention is to provide a new strain isolated from traditional paste.

The cosmetic composition of the present invention for achieving the above object may include, as an active ingredient, an enzyme decomposition product of a skim microalgae obtained by hydrolyzing a lipid-extracted microalgae with a proteolytic enzyme.

The defatted microalgae may be one obtained by defatting tetracelmis, spirulina, chlorella, or vegetable plakton.

The protease may be a supernatant (coenzyme solution) obtained by culturing Bacillus tequillensis (B. tequilensis) SM18 [Accession No. BP1429751].

The skim microalgae and proteolytic enzyme may be mixed in a high-liquid ratio of 1:2-20.

The cosmetic composition may have skin whitening and skin regeneration effects.

In addition, the health functional food of the present invention for achieving the above-described other object may contain as an active ingredient an enzyme decomposition product of defatted microalgae obtained by hydrolyzing a lipid-extracted microalgae with a proteolytic enzyme.

In addition, the novel strain of the present invention for achieving the another object described above may be Bacillus tequillensis isolated from traditional paste (B. tequilensis SM18 ; accession number BP1429751).

The composition comprising the enzyme decomposition product of skim microalgae of the present invention is finely degreased using the enzyme B. tequilensis SM18 ; Accession No. BP1429751) isolated from traditional soybeans instead of conventional chemical degradation or enzymatic degradation using commercial enzymes. It is not toxic by decomposing algae, and is effective for skin whitening and skin regeneration.

The novel Bacillus Bacillus tequillensis (B. tequilensis SM18 ; Accession No. BP1429751) enzyme isolated from the traditional paste is safe and inexpensive, as well as excellent protease activity.

1 is a photograph confirming the strain forming a clear zone by inoculating various strains isolated in Cheonggukjang.
2 is a graph measuring the proteolytic activity of 18 strains isolated from Cheonggukjang.
3 is a comparison of protein molecular weights by using SDS-PAGE to confirm the change in protein molecular weight due to chemical hydrolysis according to Comparative Examples 5 to 8.
4 is a comparison of protein molecular weights by using SDS-PAGE in order to confirm the change in protein molecular weight due to enzymatic hydrolysis using commercially available enzymes according to Comparative Examples 1 to 4.
5 is a comparison of protein molecular weights by using SDS-PAGE to confirm the change in protein molecular weight due to enzymatic hydrolysis when using different enzymes according to Example 1 and Comparative Example 1. FIG.

The present invention relates to a cosmetic composition and a health functional food comprising an enzyme decomposition product of skim microalgae as an active ingredient.

Hereinafter, the present invention will be described in detail.

The cosmetic composition of the present invention contains an enzyme decomposition product of skim microalgae as an active ingredient.

Microalgae are phytoplankton living in the sea, and plankton such as coclodinium, which often causes red tides, are also included in microalgae. The microalgae that marine bioenergy research focuses on are particularly lipid-rich microalgae species. The size is about 10 μm (micron, 1 millionth of 1 m), about a tenth of the thickness of a hair.

The defatted microalgae (LEM) of the present invention refers to a solid by-product remaining after extracting lipids after cultivation of microalgae, and generally accounts for 30-60% of the dry weight of microalgae. In addition to residual lipids, skim microalgae contains a large amount of protein. Compared to other cereals, skim microalgae contain a large amount of protein (22-25%), and the protein amino acid composition is varied, so essential amino acids are also abundant.

The proteolytic enzyme for hydrolyzing skim microalgae of the present invention is a supernatant obtained by centrifugation after culturing a strain isolated from traditional paste, and the traditional paste may include cheonggukjang, soybean paste, or soy sauce, preferably cheonggukjang Can be mentioned. The traditional paste may be a food whose stability has been verified.

As a result of identifying each strain isolated from the traditional paste, it belongs to Bacillus tequillensis (B. tequilensis ), which was named as Bacillus tequillensis (B. tequilensis) SM18, and as of March 04, 2019, the Center for Biological Resources (KCTC: Korean Collection for Type Cultures), and the deposit number was assigned as BP1429751.

The skim microalgae and the proteolytic enzyme are mixed in a high-liquid ratio of 1:2-20, preferably in a high-liquid ratio of 1:5-20. If the content of the proteolytic enzyme based on the content of skim microalgae is less than the lower limit, the hydrolysis of the protein may not be performed, and if it exceeds the upper limit, skin trouble may occur, and thus it may not be used as a cosmetic composition.

The enzyme decomposition product of skim microalgae of the present invention can be used in health functional foods in addition to cosmetic compositions.

In the present specification, the term "contained as an active ingredient" means including an amount sufficient to achieve the efficacy or activity of the enzyme decomposition product of defatted microalgae. For example, the enzyme decomposition product of skim microalgae is used at a concentration of 20 to 400 unit/ml, preferably 100 to 200 unit/ml. Since the degreasing microalgal enzyme decomposition product is a natural product and does not have side effects on the human body even if it is used in an excessive amount, the upper limit of the quantity of the defatted microalgal enzyme decomposition product contained in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.

In the cosmetic composition of the present invention, in addition to the cosmetic composition described above, other ingredients that are usually blended in the cosmetic may be blended as needed, and such blending ingredients include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, Organic powders, UV absorbers, preservatives, disinfectants, antioxidants, pH adjusters, alcohols, pigments, fragrances, blood circulation accelerators, cooling agents, restrictors, purified water, water-soluble vitamins, fat-soluble vitamins, polymer peptides, polymer polysaccharides, sphingo lipids and seaweed Extract, etc. are mentioned.

The cosmetic composition of the present invention may be prepared in the form of an emulsified formulation and a solubilized formulation commonly used in the art.

In addition, the ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the above ingredients as an active ingredient. For example, conventional auxiliary agents such as stabilizers, pigments and natural perfumes, and It may further include a carrier.

Products to which the composition of the present invention can be added include, for example, mist, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, sunscreen cream , Moisture Cream, Hand Cream, Foundation, Essence, Nutrition Essence, Mask Pack, Press Powder, Loose Powder, Cosmetics such as Eye Shadow, Soap, Cleansing Foam, Cleansing Lotion, Cleansing Cream, Body Lotion and Body Cleanser.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane , Butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solvating agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

In addition, the present invention provides a health functional food composition containing an enzyme decomposition product of skim microalgae as an active ingredient.

Health functional foods are foods prepared by adding enzyme decomposition products of skim microalgae to food materials such as beverages, teas, spices, gums, confectionery, or encapsulated, powdered, or suspended. It means to bring it, but unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time by using food as raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of enzyme decomposition products of defatted microalgae in such health functional foods cannot be uniformly regulated depending on the type of health functional food to be targeted, but can be added within the range that does not damage the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

Hereinafter, a preferred embodiment is presented to aid in the understanding of the present invention, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the present invention and the scope of the technical idea, but the following examples are only illustrative of the present invention, It is natural that such modifications and modifications fall within the scope of the appended claims.

Preparation Example 1. Proteolytic enzyme

Isolation of proteolytic enzyme strains derived from fermented foods

In order to isolate strains with excellent proteolytic ability, Cheonggukjang, manufactured in 2017, was purchased from traditional food manufacturers in 18 regions nationwide, and strains with excellent protease activity were isolated and used through a cultivation process. 1 g of the purchased Cheonggukjang sample was suspended in 20 ml of sterile distilled water and stirred at 200 rpm for 20 minutes at room temperature. The suspension was spread on MRS solid medium and cultured at 35° C. for 48 hours to obtain single cells obtained. Single cells were inoculated into MRS liquid medium and cultured at 37° C. for 24 hours to be used as a seed for enzymatic digestion. Thereafter, dry skim microalgae was added at a ratio of 1:20 (w/v) to the LB medium, and the seed was inoculated with 1% (v/v) compared to the LB medium, followed by enzymatic digestion at 50° C. for 72 hours. At 72 hours of enzymatic digestion, 1 ml of supernatant is taken to measure the protease activity, and the solid content is removed through centrifugation (10,000 xg, 20 minutes, 4 ℃), and the enzyme activity is measured by separating the crude enzyme solution. It was measured. The proteolytic activity of the crude enzyme solution was measured by the modified Kunitz method, and the strain with the highest proteolytic activity was selected.

Identification of protease-producing strains

To identify the strain isolated above, a single cell was recovered from the MRS solid medium, and the universal primers 27F (S'-AGAGTTTGATCATGGCTCAG-T) and 1492R (5'-GGATACCTTGTTACGACTT-3') primers used for 16S rDNA sequencing were used. PCR amplification was performed using. The PCR product was electrophoresed on a 1% agarose gel (5 µl, X-gal 30 mg/ml), the band was checked, and then the PCR product with amplification was confirmed by requesting Cosmo Genetech Co., Ltd. to identify it.

Specifically, Cheonggukjang prepared by the traditional fermentation method was purchased, and a total of 18 species were separated according to the colony type of the strain. Each strain was inoculated into a skim milk plate to first isolate the bacteria forming a clear zone. Of the strains isolated from Cheonggukjang, the clear zone was most clearly identified in strain 18 (FIG. 1). When the proteolytic enzyme activity of the isolated strains was measured, proteolytic activity was observed in 12 strains, and the highest activity was 95.9 unit/ml in strain 18, resulting in the same results as the first selection result (FIG. 2). As a result of analyzing the 16S rDNA sequence of the selected strain No. 18, it was found as Bacillus sp . As a result of examining homology with the standard Bacillus species, it was identified as a strain having high homology with B. tequilensis. Bacillus tequillensis (B. tequilensis ) It was named SM18 (SEQ ID NO: 1) and deposited with the Center for Biological Resources (KCTC).

In order to produce a crude enzyme solution containing protease using the seed, LB medium, a production medium optimized for protein production, was used. The components of the optimized production medium were 3% soybean extract, 2.1% fructose, and 2.25% beef extract added to the LB medium. 0.5 ml of the seed was inoculated into 50 ml of the production medium and cultured at 200 rpm for 72 hours at 37°C. After that, the supernatant was collected by centrifugation (10,000Xg, 20 minutes, 4℃) and used as a crude enzyme solution for decomposing defatted microalgae protein.

Example 1. Use of proteolytic enzyme derived from Cheonggukjang

Skim microalgae

Degreasing microalgae was provided with Tetraselmis KCTC 12236BP, which had been cultivated in an indoor incubator and then degreased, from Inha University's Marine Bio Research Center. Samples were dried in a hot air dryer at 60° C. for 24 hours and stored in a refrigerator at -18° C.

Enzyme hydrolysis

The supernatant (coenzyme solution) obtained by culturing the skim microalgae and the proteolytic enzyme (B. tequilensis SM18 ) deposited in Preparation Example 1 for enzymatic decomposition of the skim microalgal protein was mixed with a high-liquid ratio of 1:20 (w/v) After mixing in a shaking incubator, enzymatic digestion was carried out at 50° C. and 200 rpm for 72 hours. To stop the reaction, the enzyme was inactivated by heating in water at 100° C. for 10 minutes, and the supernatant obtained by centrifugation (3000Xg) for 20 minutes was recovered and used in the experiment.

Comparative Example 1. Use of Alcalase Proteolytic Enzyme

In the same manner as in Example 1, but instead of B. tequilensis SM18 as a proteolytic enzyme, an alcalase (Novozyme A/S) was provided, diluted 200 times compared to a buffer solution, and used as an enzyme solution to obtain an enzyme digestion of skim microalgae. I did.

Comparative Example 2. Using Protamax proteolytic enzyme

Conducted in the same manner as in Example 1, except that Protamax (Novozyme A/S) was provided instead of B. tequilensis SM18 as a proteolytic enzyme, diluted 200 times compared to the buffer solution, and used as an enzyme solution to obtain an enzyme digestion of skim microalgae. I did.

Comparative Example 3. Nutrase (Neutrase) using proteolytic enzyme

In the same manner as in Example 1, but instead of B. tequilensis SM18 as a proteolytic enzyme, Nutrase (Novozyme A/S) was provided, diluted 200 times compared to the buffer solution, and used as an enzyme solution, thereby obtaining an enzyme lysate of skim microalgae. .

Comparative Example 4. Flavorzyme Proteolytic Enzyme Use

In the same manner as in Example 1, but instead of Bacillus tequilensis SM18 as a proteolytic enzyme, flavorzyme (Novozyme A/S) was provided, diluted 200 times compared to the buffer, and used as an enzyme solution, thereby obtaining an enzyme digestion of skim microalgae. .

Comparative example 5. Hydrothermal decomposition

20 mL of distilled water was added to 1 g of the defatted microalgae of Example 1, and extraction was performed in a shaking incubator (SIB-05RH, Jeongbio, Korea) at 80° C. and 200 rpm for 16 hours to obtain a hydrolyzed product of the defatted microalgae.

Comparative Example 6. 1 M H ₂ SO ₄ Hydrolysis using

Protein decomposition using strong acid was carried out _{by mixing degreasing microalgae and 1 MH 2} SO ₄ in a high-liquid ratio of 1:20 (w/v), and then reacting at 110° C. for 24 hours using a sand bath (CSB2, Sungwong Science, Korea). Neutralization with CaCO ₃ gave a chemical decomposition product of defatted microalgae.

Comparative Example 7. 6 M HCl Hydrolysis using

In the same manner as in Comparative Example 6, a chemical decomposition product of defatted microalgae was obtained using 6 M HCl instead of _{1 MH 2} SO _4.

Comparative Example 8. Hydrolysis using 1 M NaOH

Protein decomposition using a base was carried out by mixing skim microalgae and 1 M NaOH in a high-liquid ratio of 1:20 (w/v), and then reacting at 110° C. for 24 hours using a sand bath (CSB2, Sungwong Science, Korea), and then at room temperature. After cooling, the supernatant was recovered by centrifugation (3000 xg, 20 minutes) to obtain a chemical decomposition product of defatted microalgae.

<Test Example>

Test Example 1. General component analysis

The content of general components of the sample was analyzed according to the food code. Moisture was analyzed by atmospheric pressure drying method in a drying machine at 105°C, and ash was directly incinerated in an incinerator at 550°C and analyzed by gravimetric method. Crude protein was analyzed by Kjeldahl decomposition method and then multiplied by a nitrogen coefficient of 6.25 and expressed as percent content (w/w) . Crude fat was measured by extracting the crude fat contained in the sample with ether using the Soxhlet extraction method. All components except moisture were converted on a dry weight basis through moisture correction, and the content of components before and after degreasing of microalgae was compared. Carbohydrates were calculated by the following formula and converted into dry weight. Carbohydrates were analyzed based on the content of cellulose and hemicellulose, and the content of each sugar component was analyzed according to the NREL Laboratory Analytical Procedures of the National Renewable Energy Laboratory (NREL).

division(%) Microalgae before degreasing Microalgae after degreasing Crude protein 23.5±0.72 56.7±1.8 Crude fat 15.5±0.37 2.9±0.11 Minutes 32.7±0.53 22.0±0.41 carbohydrate 13.4±0.54 10.1±0.34

As shown in Table 1 above, it was confirmed that the content of crude fat was significantly lower in the degreasing microalgae compared to the microalgae before degreasing, and the content of crude protein was increased.

Test Example 2. Electrophoresis measurement

3 is a comparison of protein molecular weights by using SDS-PAGE to confirm the change in protein molecular weight due to chemical hydrolysis according to Comparative Examples 5 to 8; 4 is a comparison of protein molecular weights by using SDS-PAGE to confirm the change in protein molecular weight due to enzymatic hydrolysis using commercially available enzymes according to Comparative Examples 1 to 4; 5 is a comparison of protein molecular weights by using SDS-PAGE in order to confirm the change in protein molecular weight due to enzymatic hydrolysis when using different enzymes according to Example 1 and Comparative Example 1. FIG.

Electrophoresis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli's method. A 12% SDS polyacrylamide gel and a 15% SDS polyacrylamide gel were used to check the molecular weight distribution of 1.4 to 26.6 kDa to identify peptides with a molecular weight of 6.5 to 200 kDa. The staining buffer was used by mixing 0.1% coomassie brilliant blue R-250, 45% methanol and 10% acetic acid, and the destaining buffer was used by mixing 10% acetic acid and 45% methanol. SDS-PAGE molecular weight standards (Biorad, USA) were used as a marker for molecular weight measurement. To reconfirm the peptide molecular weight, MALDI-TOF-MS analysis was performed, and the peptide molecular weight was measured in positive linear mode using an Autoflex speed TOF/TOF (Bruker MA, USA) model equipped with a Smartbeam-II laser TYPE.

In order to compare the effects of various proteolytic methods on the production of skim microalgae peptides, hydrolysis, chemical digestion, commercial enzymes, and protein digestion using B. tequilensis SM18 were performed.

Peptide production by chemical degradation

As shown in FIG. 3, when the protein was hydrolyzed as in Comparative Example 5, bands were observed around 3.4, 5.5, and 6.5 kDa to confirm that the water-soluble protein of skim microalgae had three types of molecular weight.

On the other hand, in the case of base or acid degradation as in Comparative Examples 6 to 8, since the band was not distributed between 1.4-200 kDa, it was confirmed that the skim microalgal protein was degraded into a peptide of 1.4 kDa or less.

Peptide production by commercial enzymatic digestion

As a control, a sodium phosphate buffer was used to extract peptides under the same conditions as those for protein degradation.

As shown in Figure 4, the control group was confirmed to be a peptide band decomposed between 16 ~ 26.6 kDa, Comparative Example 3 and Comparative Example 4 also confirmed the same peptide molecular weight as the control group, degreasing microscopically by neutrase and flavorzyme It was found that the algal protein was not degraded.

On the other hand, in Comparative Examples 1 and 2, the band was not identified in the control group, so it could be predicted that the skim microalgal protein was degraded into a peptide of 1.4 kDa or less. In general, SDS-PAGA results similar to acid digestion using 6 M HCl, which is known to degrade proteins into single amino acids, showed that commercial enzymes, Protamax and Alkalase, were used for chemical degradation using strong acids and strong bases of defatted microalgae proteins. Likewise, it shows that it is a very effective enzyme for decomposing into small molecular peptides.

B. tequilensis Peptide production by SM18 enzyme digestion

As shown in Figure 5, for the production of functional peptides having whitening and skin regeneration effects through decomposition of skim microalgae, protein decomposition of skim microalgae was performed using the coenzyme solution of B. tequilensis SM18 isolated in Preparation Example 1 above. Attempts were made to confirm whether they were degraded using SDS-PAGE.

As for the hydrolyzed product, bands were identified around 3.4, 5.5 and 6.5 kDa as in the SDS-PAGE result of FIG. 3. As a result of confirming the protein molecular weight of the enzyme solution containing no substrate, alkarease did not show a band at all molecular weights, and in Comparative Example 1, which is an alkalase hydrolyzate, the defatted microalgal protein was 1.4 kda or less as in the result of FIG. 4. It was confirmed that it was degraded.

As in the result of Comparative Example 1, it was confirmed that the skim microalgal protein was degraded because the three bands of the control group were not identified in the enzyme decomposition product of skim microalgae of Example 1 using the crude enzyme solution of B. tequilensis SM18. It was confirmed that the proteolytic enzyme (coenzyme solution) produced by SM18 could replace commercial enzymes.

B. tequilensis of the present invention To confirm the molecular weight of the skim microalgal protein degraded by the coenzyme of SM18, the distribution of the peptide was confirmed using MALDI-TOF, and as confirmed by SDS-PAGE, it was confirmed that the skim microalgal protein was degraded into a peptide of 1.4 kDa or less. , It was confirmed that the degraded peptides were 655 da, 890 da and 1,003 da, respectively, and it was confirmed that all of the peptides produced by B. tequilensis SM18 consist of a combination of 9 or less amino acids.

Test Example 3. Measurement of proteolytic enzyme activity

Prior to the production of peptides through hydrolysis of skim microalgae using a proteolytic enzyme, enzyme activity was measured to confirm the proteolytic ability of each enzyme.

The experiment for measuring protease activity was performed by modifying the method of Kim et al. 0.6% casein was added to 50 mM sodium phosphate buffer, and 0.25 mL of each enzyme dilution or coenzyme solution was added to 0.6% casein, and the reaction was allowed to proceed at 37°C for 10 minutes, and then 0.44 mol trichloroacetic acid was added to perform the enzyme reaction. Stopped. _{1 mL of Na 2} CO ₃ and 0.2 mL of Folin &Ciocalteu's phenol solution were added to 0.4 mL of the supernatant obtained by centrifuging the sample solution where the reaction was stopped, and the reaction was allowed to proceed for 30 minutes, followed by a spectroscopic analyzer (UV-1650PC, Shimadzu) at 660 nm. , Kyoto, Japan) to measure the absorbance. Using Tyrosine as a standard material, a standard trunk line was prepared and the protein activity was converted and displayed.

division Protease activity (unit/mL) B. tequilensis SM18 coenzyme solution (Example 1) 427.6 Alkalase (Comparative Example 1) 372.9 Protamax (Comparative Example 2) 347.9 Neutrase (Comparative Example 3) 169.5 Flavorzyme (Comparative Example 4) 110.8

As shown in Table 2 above, the protease activity of four commercial enzymes was measured in the order of flavorzyme <neutraase <protamax = alkalase, which was measured in the order of alkaline enzyme and protamax in the previous SDS-PAGE experiment. It was shown that the high enzyme activity was attributed to the superior peptide degradation compared to flavorzyme and neutraase.

When B. tequilensis SM18 was cultured for 72 hours using the production medium, the protease activity was measured to be 427.6 unit/mL, which was confirmed to be higher than that of the alkaline enzyme, 372.9 unit/mL.

Test Example 4. Measurement of tyrosinase and collagenase inhibitory activity

4-1. The measurement of tyrosinase inhibitory activity was measured according to the method of Yagi et al. Mix 0.4 mL of sodium phosphate buffer (67 mM, pH 6.8) and 0.2 mL of 3,4-dihydroxy phenylalanine (10 mM, L-DOPA) solution, and add 0.1 mL of mushroom tyrosinase (125 unit/mL) and 0.2 mL of sample. After reacting at 37° C. for 20 minutes, absorbance was measured at 475 nm with a spectrometer.

[Equation 1]

Tyrosinase activity inhibition rate (%) = (1-sample added group/sample not added group) X100

Melanin has a beneficial effect of protecting the skin by blocking ultraviolet rays, but excessive melanin production causes spots, freckles and skin blackening, and is known to cause skin aging and skin cancer. Tyrosinase is an enzyme that plays an important role in the melanin synthesis step, and it is known that tyrosine is converted to DOPA as tyrosinase activity increases, thereby increasing melanin production. Therefore, inhibition of tyrosinase activity is essential for skin whitening.

4-2. The measurement of collagenase inhibitory activity was performed according to the method of Wunsh and Heindrich. 4 mM CaCl₂ was added to Tris-HCl buffer (0.1 M, pH 7.5), and 4-phenylazobenzyl oxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (0.3 mg/mL) was used as a substrate. 0.125 mL of the substrate and 0.05 mL of the sample were mixed, 0.075 mL of collagenase (Clostridium histolyticum, 1 mg/mL) was added, left at room temperature for 20 minutes, and 0.5 mL of 6% citric acid was added to stop the reaction. Then, 2 mL of ethyl acetate was added and the absorbance was measured at 320 nm with a spectrometer.

[Equation 2]

Collagenase inhibition rate (%) = (1-sample added group/sample not added group) X100

The skin dermis is a connective tissue that exists between the epidermis and the subcutaneous fat layer and plays a role of structurally supporting the epidermis by maintaining the elasticity and tension of the skin. It is known that collagen, a major structural protein of the dermal layer along with elastin, accelerates decomposition by increasing the activity of collagenase as aging progresses, reducing the length and distribution of collagen fibers, leading to skin aging. Collagen is widely used as a cosmetic ingredient because it has functions such as strengthening the resistance of connective tissues, strengthening the bonding between tissues, and maintaining the firmness of the skin. It's going on. In order to confirm the effect of improving wrinkles, the rate of inhibition of collagenase activity is measured.

In the control group 1, the peptide was extracted from the skim microalgae under the same conditions as the enzymatic digestion conditions using distilled water, and in the control group 2, the peptide was extracted under the same conditions as the protein digestion conditions using a sodium phosphate buffer.

division Tyrosinase inhibitory ability (%) Collagenase inhibitory ability (%) Control 1 0.0 10.1 Control 2 0.0 22.3 Example 1 32.0 57.5 Comparative Example 1 20.7 48.0 Comparative Example 2 15.1 42.8 Comparative Example 3 13.5 41.4 Comparative Example 4 12.8 37.7 Comparative Example 5 1.3 4.1 Comparative Example 6 7.4 10.7 Comparative Example 7 10.2 54.0 Comparative Example 8 10.5 30.6

As shown in Table 3 above, it was confirmed that the enzyme decomposition product of skim microalgae prepared according to Example 1 of the present invention has superior tyrosinase inhibitory ability and collagenase inhibitory ability compared to other groups. In the inhibition of tyrosinase and collagenase activity, the coenzyme solution derived from B. tequilensis SM18 showed a higher inhibition rate than that of the commercial enzyme, Alkarease . This suggests that it is more effective than conventional enzymes commercially used in the production of cosmetic materials for whitening and skin regeneration.

On the other hand, it was confirmed that the tyrosinase inhibitory ability for the degreasing microalgae hydrolyzate of Comparative Example 5 was very low as 1.3%, and the chemical decomposition products of Comparative Examples 6 to 8 were also low as 7.4 to 10.5%. In the SDS-PAGE results for base and acid degradation products of Comparative Examples 6 to 8, the protein is degraded to 1.4 kDa or less, and it is known that all proteins are decomposed into a single amino acid under the corresponding degradation conditions. It can be expected as an inhibitory effect.

When comparing Comparative Example 6 digested with 6 M HCl with Comparative Example 1 digested with Alkarease, the tyrosinase inhibitory ability of Comparative Example 1 is more excellent, so it is decomposed into a small molecule peptide of 1.4 kDa or less than a single amino acid. When used, it can be seen that the tyrosinase inhibitory activity is excellent.

In addition, the ability to inhibit collagenase against the hydrolyzate of degreasing microalgae in Comparative Example 5 is 4.1%, which is considered to have a slight skin regeneration effect.

In the inhibition of tyrosinase and collagenase activity, it was confirmed that the inhibition of tyrosinase and collagenase activity of the peptide produced by Alkaraz was more excellent when comparing Alkaraz and strong acid (6 mol HCl) treatment. This is thought to be because the low-molecular peptides produced by enzymes were more effective in inhibiting collagenase activity than single amino acids produced by strong acids.

Hereinafter, examples of the formulation of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit it, but is intended to be described in detail.

Formulation example 1. Preparation of granules

1,000 mg of the enzyme digestion product powder obtained in Example 1

The right amount of vitamin mixture

Vitamin A acetate 70 ㎍

1.0 mg of vitamin E

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

Vitamin B12 0.2 ㎍

10 mg of vitamin C

Biotin 10 ㎍

1.7 mg of nicotinic acid amide

Folic acid 50 ㎍

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

90 mg of potassium citrate

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for granules, but it is also possible to arbitrarily modify the mixing ratio. After mixing the above ingredients according to a conventional granule preparation method, granules And can be used to prepare a health functional food composition according to a conventional method.

Formulation Example 2. Preparation of functional beverage

1,000 mg of the enzyme digestion product powder obtained in Example 1

1,000 mg citric acid

100 g oligosaccharides

2 g of plum concentrate

1 g taurine

900 mL total by adding purified water

After mixing the above ingredients according to a normal health drink manufacturing method, stirring and heating at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator. It is used to prepare the functional beverage composition of the invention.

The composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

An example of preparing a cosmetic composition suitable for applying the present invention will be presented.

Preparation Example 3: Lotion

Examples of the preparation of lotion among cosmetics containing the enzyme decomposition product of skim microalgae of Example 1 are shown in Table 4 below.

ingredient Content (% by weight) Enzyme digest of Example 1 5.0 glycerin 6.0 1,3-butylene glycol 3.0 PG1500 1.0 Allantoin 0.1 DL-panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Sodium hyaluronate 5.0 ethanol 10.0 Polysorbate 20 0.2 Preservatives, fragrances, and pigments a very small amount Distilled water Balance Sum 100

Manufacturing example 4: lotion

Examples of the preparation of lotions among cosmetics containing the enzyme decomposition product of skim microalgae of Example 1 are shown in Table 5 below.

ingredient Content (% by weight) Enzyme digest of Example 1 3.0 Propylene glycol 6.0 glycerin 4.0 Triethanolamine 1.2 Tocopheryl acetate 3.0 Liquid paraffin 5.0 Squalane 3.0 Macadamia Nut Oil 2.0 Polysorbate 60 1.5 Sorbitansquioleate 1.0 Carboxyvinyl polymer 1.0 Preservatives, fragrances, and pigments a very small amount Distilled water Balance Sum 100

Manufacturing example 5: nourishing cream

Examples of the preparation of a nutritional cream among cosmetics containing the enzyme decomposition product of skim microalgae of Example 1 are shown in Table 6 below.

ingredient Content (% by weight) Enzyme digest of Example 1 1.0 Lipotype glycerin monostearate 1.5 Cetearyl alcohol 1.5 Stearic acid 1.0 Polysorbate 60 1.5 Sorbitan stearate 0.6 Isostearyl isostearate 5.0 Squalane 5.0 Mineral oil 35.0 Dimethicone 0.5 Hydroxyethyl cellulose 0.12 glycerin 6.0 Triethanolamine 0.7 Preservatives, fragrances, and pigments a very small amount Distilled water Balance Sum 100

Manufacturing example 6: essence

Examples of the preparation of the essence in the cosmetic composition containing the enzyme decomposition product of skim microalgae of Example 1 are shown in Table 7 below.

ingredient Content (% by weight) Enzyme digest of Example 1 1.5 glycerin 10.0 Betaine 5.0 PEG 1500 2.0 Allantoin 0.1 DL-panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Hydroxyethyl cellulose 0.1 Sodium hyaluronate 8.0 Carboxyvinyl polymer 0.2 Triethanolamine 0.18 Octyldodecanol 0.3 Octyldodeces-16 0.4 ethanol 6.0 Preservatives, fragrances, and pigments a very small amount Distilled water Balance Sum 100

Manufacturing example 7: mask For pack latex

Examples of the preparation of the emulsion for a mask pack among cosmetics containing the enzyme decomposition product of degreasing microalgae of Example 1 are shown in Table 8 below.

ingredient Content (% by weight) Enzyme digest of Example 1 1.0 Polyvinyl alcohol 15.0 Cellulose gum 0.15 glycerin 3.0 PEG 1500 2.0 Cyclodextrin 0.15 DL-panthenol 0.4 Allantoin 0.1 Monoammonium glycyrrhizinate 0.3 Nicotinamide 0.5 ethanol 6.0 PEG 40 hydrogenated castor oil 0.3 Preservatives, fragrances, and pigments a very small amount Distilled water Balance Sum 100

Biological Resource Center (domestic) BP1429751 20190304

<110> Industry-Academic Cooperation Foundation, Sun Moon University <120> Compositions containing defatted microalgae extract <130> HPC-8930 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1455 <212> RNA <213> Unknown <220> <223> Bacillus tequilensis <400> 1 aaaatggggg gcgtgctaat acatgcaagt cgagcggaca gatgggagct tgctccctga 60 tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc 120 cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaaa cataaaaggt 180 ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240 ctcaccaagg caacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300 acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360 tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 420 gaagaacaag taccgttcga atagggcggt accttgacgg tacctaacca gaaagccacg 480 gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540 gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600 gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660 tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720 gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780 cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840 acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 900 gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960 caggtcttga catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga 1020 caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080 agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt 1140 gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200 cacacgtgct acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca 1260 caaatctgtt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct 1320 agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380 tcacaccacg agagtttgta acacccgaag tcggtgaggt aacctttagg agccagccgc 1440 cgaaaggggg accca 1455

Claims (7)

A cosmetic composition comprising, as an active ingredient, an enzyme decomposition product of skim microalgae obtained by hydrolyzing a lipid-extracted microalgae with a proteolytic enzyme. The cosmetic composition according to claim 1, wherein the degreasing microalgae is degreased from tetracelmis, spirulina, chlorella, or vegetable plakton. The cosmetic composition according to claim 1, wherein the proteolytic enzyme is a coenzyme solution of Bacillus tequillensis SM18 [Accession No. BP1429751]. The cosmetic composition according to claim 1, wherein the skim microalgae and the proteolytic enzyme are mixed in a high-liquid ratio of 1:2-20. The cosmetic composition of claim 1, wherein the cosmetic composition has skin whitening and skin regeneration effects. A health functional food containing as an active ingredient the enzyme decomposition product of skim microalgae obtained by hydrolyzing lipid-extracted microalgae with a proteolytic enzyme. Bacillus tequillensis isolated from traditional paste (Bacillus tequilensis) SM18 [Accession No.: BP1429751].

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