KR102260080B1 - Compositions containing defatted microalgae extract - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising degreasing microalgae enzyme decomposition product as an active ingredient. By including defatted microalgae degreasing microalgae as an active ingredient, it is effective in skin whitening and skin regeneration.

Description

Composition containing defatted microalgae enzyme decomposition product as an active ingredient {Compositions containing defatted microalgae extract}

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

Recently, due to the destruction of the ozone layer, the damage to the skin due to the increase in ultraviolet rays reaching the earth's surface is continuously increasing. When the skin is stimulated by UV rays, hormones and nitric oxide are secreted from keratinocytes, which increases skin pigmentation and accelerates skin pigmentation and aging.

Skin pigmentation is caused by melanin pigment and is known to protect the skin from UV 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 tyrosinase with tyrosine as a substrate, and is produced through DOPA chromium through autoxidation 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 to reduce or remove the production of melanin pigment.

Currently, the most widely used skin whitening agents are chemical synthetic substances arbutin and kojic acid. Arbutin is known as an inhibitor that competes with tyrosine, and kojic acid inactivates the tyrosinase active site to produce DOPA. It is known to interfere with the production of melanin pigment. However, the use of kojic acid is limited due to stability problems such as skin discoloration and the possibility of skin cancer, and arbutin is known to generate hydroquinone, which is suspected to be a carcinogen by light or enzymes, and there is 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 disadvantages of existing chemically synthesized whitening agents. Most of the natural-derived whitening or anti-aging materials are terrestrial plants such as chrysanthemum, chrysanthemum, cockscomb extract, or herbal medicine, but marine organisms are diverse and bioactive substances with excellent performance are produced due to the specificity of the marine environment. It is reported that it is in the spotlight as a raw material for high value added materials.

Among marine plants, seaweeds are receiving a lot of attention as a material for the production of physiologically active substances, as they contain various proteins and polysaccharides and contain abundant minerals and vitamins. According to Manou et al., seaweed contains various physiologically active substances including antioxidant, pharmacological activity, antitumor, anti-inflammatory and immune regulation superior to land plants, so it can be used as a material for whitening and antioxidant functional cosmetics.

It is reported that these physiologically active functions of seaweed originate from polysaccharides and peptides, which are low molecular weight forms of proteins. Peptides produced through proteolysis existing in natural products are small in size, so they can be easily absorbed into living tissues, and have the characteristics of improving wrinkles and increasing collagen synthesis, so various studies are continuing in the cosmetic and pharmaceutical fields as a next-generation material. . In particular, peptides composed of 18 or less amino acids are known to be effective in inhibiting active oxygen, regenerating skin cells, promoting cell division, and improving wrinkles, and thus their use is expanding as cosmetics, functional foods, and pharmaceuticals.

However, chemical degradation using acids or bases for protein degradation is limited due to safety issues and increased consumer rejection in the use of food and cosmetics, and enzymatic degradation using commercial enzymes is associated with licensing issues as food and cosmetic additives. It has the disadvantage of increasing product cost due to high enzyme price. In order to overcome the disadvantages of the production of peptides by chemical degradation and the production of peptides using commercial enzymes, the need for more safe and low-cost peptide production using enzymes is increasing.

Meanwhile, as the need for new and renewable energy increases, microalgae, a type of seaweed, is increasingly being used as a third-generation biomass that produces biodiesel through lipid extraction. However, despite the high protein content in defatted microalgae that are discarded after lipid production, research on recycling is insufficient because they are used or discarded as low-grade feed.

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

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

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

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

In addition, another object of the present invention is to provide a novel strain isolated from traditional soy sauce.

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

The defatted microalgae may be defatted by tetracelmis, spirulina, chlorella or phytoplankton.

The protease may be a supernatant (coenzyme solution) in which Bacillus tequilensis (B. tequilensis ) SM18 [Accession No.: BP1429751] is cultured.

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

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

In addition, the health functional food of the present invention for achieving the above-mentioned other object may include an enzyme decomposition product of defatted microalgae hydrolyzed by a proteolytic enzyme as an active ingredient.

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

The composition comprising the degreasing microalgae enzyme decomposition product of the present invention uses the enzyme B. tequilensis SM18 ; Accession No. BP1429751) separated from traditional soybeans instead of conventional chemical degradation or enzymatic degradation using commercial enzymes. It is non-toxic by decomposing algae and is effective for skin whitening and skin regeneration.

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

1 is a photograph confirming strains forming a clear zone by inoculating various types of strains isolated from 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 changes in protein molecular weight according to chemical hydrolysis according to Comparative Examples 5 to 8;
4 is a comparison of protein molecular weights by using SDS-PAGE to confirm changes in protein molecular weight according 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 changes in protein molecular weight according to enzymatic hydrolysis when different enzymes are used according to Example 1 and Comparative Example 1.

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

Hereinafter, the present invention will be described in detail.

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

Microalgae are phytoplankton that inhabit the sea, and plankton such as Coclodinium, which often causes red tides, also belong to microalgae. Microalgae that marine bioenergy research pays attention to are microalgae species rich in lipids, that is, oil components. Its size is about 10 μm (micron, 1 millionth of 1 m), about one-tenth the thickness of a human hair.

The defatted microalgae (LEM) of the present invention refers to a solid by-product remaining after extracting lipids after culturing the microalgae, and generally accounts for 30-60% of the dry weight of the microalgae. Defatted microalgae contain a large amount of protein in addition to residual lipids. Compared to other grains, defatted microalgae contain a large amount of protein (22-25%), and the amino acid composition of the protein is diverse, so essential amino acids are also abundantly included.

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

As a result of identifying the strains isolated from the traditional soybeans, it belongs to Bacillus tequilensis ( B. tequilensis ), and it is named Bacillus tequilensis SM18, and as of March 04, 2019, the Center for Biological Resources (KCTC: Korean Collection for Type Cultures) and was given an accession number as BP1429751.

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

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

As used herein, the term 'contained as an active ingredient' means including an amount sufficient to achieve the efficacy or activity of the degreasing microalgal enzyme decomposition product. As an example, the degreasing microalgal enzyme decomposition product is used at a concentration of 20 to 400 unit/ml, preferably 100 to 200 unit/ml. Since the degreasing microalgal enzymatic decomposition product is a natural product and has no side effects even when used in excess, the upper limit of the quantity of degreasing microalgal enzymatic decomposition product included in the composition of the present invention can be selected and carried out by those 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 commonly formulated in cosmetics may be blended as necessary. Examples of such blending ingredients include oil and fat ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, Organic powder, UV absorber, preservative, disinfectant, antioxidant, pH adjuster, alcohol, colorant, fragrance, blood circulation promoter, cooling agent, limiting agent, purified water, water-soluble vitamin, fat-soluble vitamin, polymer peptide, polymer polysaccharide, sphingolipid and seaweed an extract, etc. are mentioned.

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

In addition, the components included in the cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the above components as active ingredients, for example, conventional adjuvants such as stabilizers, pigments and natural fragrances; 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, nourishing lotion, massage cream, nourishing cream, sunscreen cream , moisture cream, hand cream, foundation, essence, nourishing essence, mask pack, press powder, loose powder, eye shadow, etc., and 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, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a 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, additional chlorofluorohydrocarbon, propane , butane or propellants such as dimethyl ether.

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

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

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

Health functional food is a food prepared by adding defatted microalgae enzyme decomposition product to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspension, etc. It means to bring it, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time because it uses food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be ingested on a daily basis. The amount of defatted microalgae enzyme decomposition product in such health functional food cannot be uniformly defined because it varies depending on the type of health functional food, but it can be added within a range that does not impair the original taste of the food, and is added to the target 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 a health functional food in the form of pills, granules, tablets or capsules, it is usually added in an amount 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, preferred examples are presented to help the understanding of the present invention, but the following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It goes without saying that such variations and modifications fall within the scope of the appended claims.

Preparation Example 1. Proteolytic enzyme

Isolation of fermented food-derived proteolytic enzyme strains

In order to isolate strains with excellent proteolytic ability, cheonggukjang prepared in 2017 was purchased from traditional food manufacturers in 18 regions across the country, and strains with excellent proteolytic enzyme activity were isolated and used through a culture process. 1 g of the purchased Cheonggukjang sample was suspended in 20 ml of sterile distilled water, stirred at 200 rpm for 20 minutes at room temperature, the suspension was smeared on MRS solid medium, and cultured at 35 ° C. for 48 hours to obtain a single cell body. A single cell was inoculated into MRS broth and cultured at 37 °C for 24 hours to be used as a seed for enzymatic digestion. Thereafter, dried defatted microalgae were added to the LB medium at a ratio of 1:20 (w/v), the seed cells were inoculated with 1% (v/v) compared to the LB medium, and enzymatic digestion was performed at 50° C. for 72 hours. To measure protease activity at 72 hours of enzymatic digestion, 1 ml of the supernatant was taken and centrifuged to remove the solids by centrifugation (10,000 g, 20 minutes, 4 ℃), and the enzyme activity was determined by separating the crude enzyme solution. 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 MRS solid medium, and the universal primers 27F (S'-AGAGTTTGATCATGGCTCAG-T) and 1492R (5'-GGATACCTTGTTACGACTT-3') primers used for 16S rDNA sequencing were used. was used for PCR amplification. After the PCR product was electrophoresed on a 1% agarose gel (5 μl, X-gal 30 mg/ml), the band was checked, and the PCR product whose amplification was confirmed was requested to Cosmogenetech Co., Ltd. and identified.

Specifically, cheonggukjang prepared in a traditional fermentation method was purchased, and a total of 18 types were isolated according to the colony type of the strain, and each strain was inoculated on a skim milk plate to first separate the bacteria forming a clear zone. Among 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 strain was measured, the proteolytic activity was observed in 12 strains, and the highest activity of 95.9 unit/ml was obtained in strain 18, obtaining the same result as the first selection result (FIG. 2). As a result of analyzing the 16S rDNA base sequence of the selected strain No. 18, it was found to be Bacillus sp ., and as a result of investigating the homology with the standard Bacillus species, it was confirmed as a strain with high homology with Bacillus tequilensis. Bacillus tequilensis ( B. tequilensis ) It was named SM18 (SEQ ID NO: 1) and deposited at the Center for Biological Resources (KCTC).

For the production of a crude enzyme solution containing proteolytic enzymes using a seed strain, LB medium, which is a production medium optimized for protein production, was used. The components of the optimized production medium include 3% soybean extract, 2.1% fructose, and 2.25% beef extract added to LB medium. 0.5 ml of the seed culture was inoculated into 50 ml of production medium and cultured at 37 ° C. for 72 hours at 200 rpm. After that, the supernatant was recovered by centrifugation (10,000Xg, 20 minutes, 4 ℃) and used as a crude enzyme solution for degreasing microalgal protein.

Example 1. Use of cheonggukjang-derived proteolytic enzyme

defatted microalgae

The defatted microalgae were provided with Tetraselmis KCTC 12236BP, which was cultured in an indoor incubator and finished with a degreasing process from the Inha University Marine Bio Research Center, and the sample was dried in a hot air dryer at 60 ° C for 24 hours and then stored in a refrigerator at -18 ° C.

enzymatic hydrolysis

For enzymatic decomposition of defatted microalgae protein, the supernatant (crude enzyme solution) obtained by culturing defatted microalgae and the protease (B. tequilensis SM18 ) deposited in Preparation Example 1 was mixed with a solid-liquid ratio of 1:20 (w/v) After mixing, enzymatic decomposition was performed in a shaking incubator 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. Using Alcalase protease

The same procedure as in Example 1, except that Alcalase (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 a defatted microalgae enzyme lysate did.

Comparative Example 2. Use of Protamax protease

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

Comparative Example 3. Use of Neutrase protease

It was carried out in the same manner as in Example 1, except that neutrase (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 solution of defatted microalgae. .

Comparative Example 4. Flavorzyme (Flavourzyme) using proteolytic enzyme

It was carried out in the same manner as in Example 1, but instead of Bacillus tequilensis SM18 as a proteolytic enzyme, flavorzyme (Novozyme A/S) was provided and diluted 200 times compared to the buffer solution and used as an enzyme solution to obtain an enzyme degreasing product of microalgae. .

comparative example 5. hot water decomposition

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

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

Proteolysis using strong acid was performed _{by mixing defatted microalgae and 1 MH 2} SO ₄ at a solid-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 to obtain a degreasing microalgae chemical decomposition product.

Comparative Example 7. 6 M HCl hydrolysis using

It was carried out in the same manner as in Comparative Example 6, except that _{6 M HCl was used instead of 1 MH 2} SO ₄ to obtain a degreasing microalgae chemically decomposed product.

Comparative Example 8. Hydrolysis using 1 M NaOH

For protein degradation using a base, degreasing microalgae and 1 M NaOH were mixed at a solid-liquid ratio of 1:20 (w/v), and then reacted 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 x g, 20 minutes) to obtain a degreasing microalgae chemical decomposition product.

<Test Example>

Test Example 1. General component analysis

The content of general components of the sample was analyzed according to the Food Standards Code. Moisture was analyzed by atmospheric heating drying in a dryer at 105 ℃, and ash was analyzed by gravimetric method by direct incineration at 550 ℃ in a kiln. Crude protein was analyzed by Kjeldahl decomposition method, and then it was expressed as percent content (w/w) by multiplying by a nitrogen coefficient of 6.25. . Crude fat was measured by extracting the crude fat contained in the sample with ether using the Soxhlet extraction method. All components, except for moisture, were converted on a dry weight basis through moisture correction, and the component contents before and after degreasing of microalgae were 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 george 15.5±0.37 2.9±0.11 views 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 defatted microalgae had a significantly lower content of crude fat and a higher content of crude protein compared to the microalgae before defatting.

Test Example 2. Electrophoresis Measurement

3 is a comparison of protein molecular weights by using SDS-PAGE to confirm changes in protein molecular weight according to chemical hydrolysis according to Comparative Examples 5 to 8; 4 is a comparison of protein molecular weights by using SDS-PAGE to confirm changes in protein molecular weight according 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 changes in protein molecular weight according to enzymatic hydrolysis when different enzymes are used according to Example 1 and Comparative Example 1.

For electrophoresis, molecular weight was measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli's method. 12% SDS polyacrylamide gel and 15% SDS polyacrylamide gel were used to check the distribution of molecular weights of 1.4 to 26.6 kDa and 6.5 to 200 kDa peptides. A staining buffer of 0.1% coomassie brilliant blue R-250, 45% methanol and 10% acetic acid was used, and a destaining buffer of 10% acetic acid and 45% methanol was used. As a marker for molecular weight measurement, SDS-PAGE molecular weight standards (Biorad, USA) were used. To reconfirm the molecular weight of the peptide, 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 Smartbeam-II laser TYPE.

To compare the effects of various protein degradation methods on the production of defatted microalgal peptides, hydrothermal degradation, chemical degradation, and proteolysis using commercial enzymes and 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, it was confirmed that the water-soluble protein of defatted microalgae had three molecular weights by confirming bands around 3.4, 5.5, and 6.5 kDa.

On the other hand, in the case of base or acid degradation as in Comparative Examples 6 to 8, the band was not distributed between 1.4-200 kDa, so it was confirmed that the defatted microalgal protein was decomposed into peptides of 1.4 kDa or less.

Peptide production by commercial enzymatic digestion

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

As shown in FIG. 4, in the control group, a peptide band degraded between 16 and 26.6 kDa was confirmed, and in Comparative Examples 3 and 4, the same peptide molecular weight as the control was confirmed, so that the degreasing process was performed by neutrase and flavorzyme. It was found that the algal protein was not degraded.

On the other hand, in Comparative Examples 1 and 2, since no band was identified in the control group, it could be predicted that the defatted microalgal protein was degraded to a peptide of 1.4 kDa or less. It showed SDS-PAGA results similar to acid digestion using 6 M HCl, which is generally known to break down proteins into single amino acids, which showed that commercial enzymes Protamax and Alcalase were chemically digested with strong acids and strong bases of defatted microalgal proteins. It shows that it is a very effective enzyme for breaking down into small-molecular peptides.

B. tequilensis Peptide production by SM18 enzymatic digestion

As shown in Figure 5, for the production of functional peptides having whitening and skin regeneration effects through degreasing microalgae protein, degreasing microalgae protein degradation was performed using the crude enzyme solution of B. tequilensis SM18 isolated in Preparation Example 1. It was tried and it was confirmed whether it was degraded using SDS-PAGE.

The hydrolyzate showed bands 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 without the substrate, no bands appeared in all molecular weights of the alkalize, and in Comparative Example 1, which is the hydrolyzate hydrolyzate, the defatted microalgal protein was found to be less than 1.4 kda as in the result of FIG. 4 . Decomposition was confirmed.

In the defatted microalgae enzyme digest of Example 1 using the crude enzyme solution of B. tequilensis SM18, as in Comparative Example 1, three bands in the control group were not confirmed, so it was confirmed that the defatted microalgae protein was decomposed. B. tequilensis derived from Cheonggukjang It was confirmed that the proteolytic enzyme (coenzyme solution) produced by SM18 can replace commercial enzymes.

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

Test Example 3. Measurement of the activity of proteolytic enzymes

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

The protease activity measurement experiment 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 crude enzyme solution was added to 0.6% casein, and the reaction was allowed to proceed at 37 ° C. for 10 minutes. Then, 0.44 mol trichloroacetic acid was added to carry out the enzymatic 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 centrifugation of the sample solution in which the reaction was stopped, and the reaction was allowed to proceed for 30 minutes, followed by a spectrometer (UV-1650PC, Shimadzu) at 660 nm. , Kyoto, Japan) to measure the absorbance. Using tyrosine as a standard material, a standard CO line was drawn and the protein activity was converted and displayed.

division Protease activity (unit/mL) B. tequilensis SM18 crude enzyme solution (Example 1) 427.6 Alcalase (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 proteolytic enzyme activity of four commercial enzymes was measured in the order of flavorzyme < neutrase < protamax = alkalase, which was found in the previous SDS-PAGE experiment with alkalase and protamax. The superior peptide degradation compared to flavorzyme and neutrase was attributed to the high enzymatic activity.

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

Test Example 4. Tyrosinase (Tyrosinase) and collagenase (Collagenase) inhibitory activity measurement

4-1. 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, 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 non-added group) X 100

Melanin has a beneficial action to protect the skin by blocking UV rays, but excessive melanin production is known to cause spots, freckles and darkening of the skin and cause skin aging and skin cancer. Tyrosinase is an enzyme that plays an important role in the melanin synthesis step. As tyrosine activity increases, tyrosine is converted to DOPA and melanin production is known to increase. Therefore, inhibition of tyrosinase activity is essential for skin whitening.

4-2. Collagenase inhibitory activity was measured 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. After mixing 0.125 mL of substrate and 0.05 mL of sample, 0.075 mL of collagenase (Clostridium histolyticum, 1 mg/mL) was added and left at room temperature for 20 minutes, and then 0.5 mL of 6% citric acid was added to stop the reaction. Then, 2 mL of ethyl acetate was added, and absorbance was measured at 320 nm with a spectrometer.

[Equation 2]

Collagenase inhibition rate (%) = (1-Sample added group/Sample unadded group)X100

The dermis is a connective tissue that exists between the epidermis and the subcutaneous fat layer and serves to structurally support the epidermis by maintaining the elasticity and tension of the skin. It is known that collagen, which is a major structural protein of the dermal layer, along with elastin, is decomposed by an increase in collagenase activity as aging progresses, leading to a decrease in 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 tissue, strengthening the bond between tissues, and maintaining the firmness of the skin. Recently, research on skin regeneration and hair treatment has been actively conducted through the extraction of collagen materials using proteins from marine resources. is in progress In order to confirm the wrinkle improvement effect, the inhibition rate of collagenase activity is measured.

In Control 1, peptide extraction of defatted microalgae was performed under the same conditions as for enzymatic digestion using distilled water, and in Control 2, peptide extraction was performed under the same conditions as for proteolysis using 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 defatted microalgal enzyme decomposition product prepared according to Example 1 of the present invention had superior tyrosinase inhibitory ability and collagenase inhibitory ability compared to other groups. In 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 Alcalase , which showed that the protein degradation of defatted microalgae using the coenzyme solution of B. This suggests that it is more effective than the existing commercially used enzymes in the production of cosmetic materials for whitening and skin regeneration.

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

When comparing Comparative Example 6 decomposed using 6 M HCl and Comparative Example 1 decomposed using alkalinease, the tyrosinase inhibitory ability of Comparative Example 1 is superior, so it is decomposed into peptides with a low molecular weight of 1.4 kDa or less rather than a single amino acid. It can be seen that the tyrosinase inhibitory activity is excellent when

In addition, the collagenase inhibitory ability for the hydrolyzate of defatted microalgae of Comparative Example 5 was 4.1%, which is considered to have insignificant skin regeneration effect.

In inhibition of tyrosinase and collagenase activity, it was confirmed that the inhibition of tyrosinase and collagenase activity of the peptide produced by alkaline was superior when comparing treatment with alkaline and strong acid (6 mol HCl). This is thought to be because the low molecular weight peptide produced by the enzyme was more effective in inhibiting collagenase activity than the single amino acid produced by the strong acid.

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

Formulation example 1. Preparation of granules

1,000 mg of the enzyme lysate powder obtained in Example 1

appropriate amount of vitamin mixture

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

1.7 mg of nicotinic acid amide

50 μg folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

potassium phosphate monobasic 15 mg

Dicalcium Phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

Although the composition ratio of the vitamin and mineral mixture is relatively suitable for granules in a preferred embodiment, the mixing ratio may be arbitrarily modified, and after mixing the above ingredients according to a conventional granule manufacturing method, the granules It can be prepared and used in the production of a health functional food composition according to a conventional method.

Formulation Example 2. Preparation of functional beverage

1,000 mg of the enzyme lysate powder obtained in Example 1

1,000 mg citric acid

100 g of oligosaccharides

2 g of plum concentrate

1 g taurine

Add purified water to total 900 mL

After mixing the above ingredients according to the usual health drink manufacturing method, after 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, then refrigerated. It is used to prepare the functional beverage composition of the present invention.

Although the composition ratio is prepared by mixing ingredients suitable for relatively favorite beverages in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and national preferences such as demand class, demand country, and use purpose.

A preparation example of a cosmetic composition suitable for applying the present invention will be presented.

Preparation example 3: lotion

Examples of the preparation of the lotion in the cosmetic containing the degreasing microalgae enzyme decomposition product of Example 1 are shown in Table 4 below.

ingredient content (wt%) Enzyme lysate of Example 1 5.0 glycerin 6.0 1,3-butylene glycol 3.0 PG 1500 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, colors a very small amount Distilled water remaining amount Sum 100

manufacturing example 4: lotion

Preparation examples of lotions in the cosmetic containing the degreasing microalgae enzyme decomposition product of Example 1 are shown in Table 5 below.

ingredient content (wt%) Enzyme lysate 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 Macadamina Nut Oil 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.0 Carboxyvinyl Polymer 1.0 preservatives, fragrances, colors a very small amount Distilled water remaining amount Sum 100

manufacturing example 5: Nourishing Cream

Examples of the preparation of the nutritional cream in the cosmetic containing the degreasing microalgae enzyme decomposition product of Example 1 are shown in Table 6 below.

ingredient content (wt%) Enzyme lysate of Example 1 1.0 lipophilic monostearate glycerin 1.5 cetearyl alcohol 1.5 stearic acid 1.0 Polysorbate 60 1.5 Sorbitan Stearate 0.6 Isostearylisostearate 5.0 squalane 5.0 mineral oil 35.0 dimethicone 0.5 Hydroxyethyl Cellulose 0.12 glycerin 6.0 triethanolamine 0.7 preservatives, fragrances, colors a very small amount Distilled water remaining amount Sum 100

manufacturing example 6: Essence

Preparation examples of the essence in the cosmetic containing the degreasing microalgae enzyme decomposition product of Example 1 are shown in Table 7 below.

ingredient content (wt%) Enzyme lysate 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, colors a very small amount Distilled water remaining amount Sum 100

manufacturing example 7: Mask for pack latex

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

ingredient content (wt%) Enzyme lysate 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, colors a very small amount Distilled water remaining amount Sum 100

Biological Resource Center (Korea) 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)

Contains as an active ingredient a degreasing microalgae enzyme hydrolyzate obtained by hydrolyzing lipid-extracted microalgae with a proteolytic enzyme,
The protease is Bacillus tequilensis ( B. tequilensis ) Cosmetic composition, characterized in that the coenzyme solution of SM18 [Accession No.: BP1429751]. The cosmetic composition according to claim 1, wherein the defatted microalgae are defatted by Tetracelmis, Spirulina, Chlorella or Phytoplankton. delete The cosmetic composition according to claim 1, wherein the defatted microalgae and the proteolytic enzyme are mixed in a solid-liquid ratio of 1: 2-20. The cosmetic composition according to claim 1, wherein the cosmetic composition has skin whitening and skin regenerating effects. Contains as an active ingredient a degreasing microalgae enzyme hydrolyzate obtained by hydrolyzing lipid-extracted microalgae with a proteolytic enzyme,
The proteolytic enzyme is Bacillus tequilensis ( B. tequilensis ) Health functional food, characterized in that the coenzyme solution of SM18 [Accession No.: BP1429751]. Bacillus tequilensis isolated from cheonggukjang ( Bacillus tequilensis ) SM18 [Accession No.: BP1429751].

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