KR20170106738A

KR20170106738A - Functional cosmetic composition using microalgae extracts and method for manufacturing the same

Info

Publication number: KR20170106738A
Application number: KR1020160030201A
Authority: KR
Inventors: 손경제
Original assignee: 농업회사법인 미션알지파워 주식회사
Priority date: 2016-03-14
Filing date: 2016-03-14
Publication date: 2017-09-22
Also published as: KR101814523B1

Abstract

The present invention relates to a functional cosmetic composition using a microalgae extract and a method for producing the same. The present invention provides a cosmetic composition which can mass-produce a cosmetic material containing a functional material derived from microalgae in a large amount, and has excellent functional effects such as skin wrinkle improvement and whitening improvement, and a method for producing the same.

Description

TECHNICAL FIELD The present invention relates to a functional cosmetic composition comprising a microalgae extract and a method for preparing the functional cosmetic composition using the microalgae extract,

The present invention relates to a functional cosmetic composition using a microalgae extract and a method for preparing the same, and more particularly, to a cosmetic composition containing a functional material derived from a microalgae and capable of mass- And a method for producing the same.

There are various causes such as skin wrinkles and aging, but it is generally known that wrinkles and aging occur due to an increase in lipid peroxidation, which is harmful to the skin, and pigment deposition. In addition, when the blood circulation of the skin is poor, when the nutrient is inadequate, when the elastic fibers in the dermis are degenerated and contracted, when moisture and skin fat are reduced, and free radicals are generated from the active oxygen, It is known that the elasticity of the skin and the shrinkability of the pores are weakened due to various reasons such as the lack of collagen and the increase of the activity of elastase, which is a decomposition factor of elastic fiber, and the wrinkles and aging of the skin are caused.

With increasing interest in the beauty and health of the skin, functional cosmetics for improving the wrinkles and whitening of the skin have been developed in various ways, and the research is being actively carried out. For example, retinol (a vitamin A), a-hydroxy acid (AHA), and adenosine increase the synthesis of collagen and normalize the epidermal keratinization process, thereby contributing to skin regeneration. Functional substances for skin whitening include arbutin, ascorbyl glucoside, and magnesium ascorbyl phosphate.

However, most of the chemical and synthetic products are not uniformly dispersed in cosmetic formulations, and have side effects such as irritation to eyes, restriction of use of pregnant women, swelling and itching (pruritus). Accordingly, the preference for cosmetic products using natural materials is significantly increased as a functional material for improving wrinkles and whitening of skin, rather than synthetic products.

Recently, a technique of using microalgae as a natural material in a cosmetic composition has been attempted. For example, Korean Patent Publication No. 10-2009-0025431, Korean Patent Publication No. 10-2012-0041404, Korean Patent No. 10-1410632 and Korean Patent No. 10-1502359, etc., A cosmetic composition comprising a functional substance (extract) is disclosed.

In general, algae are classified into macroalgae growing up to tens of meters in size, and microalgae, which are single-celled organisms of several micrometres in size, such as phytoplankton. Microalgae is one of the first creatures to appear in the Earth's oceans about 3.5 billion years ago called phytoplankton.

Microalgae is a generic term for all photosynthetic organisms except for land plants. It is not a taxonomic term but refers to a wide variety of taxa. Among them, monocellular algae that can be observed under a microscope is called microalgae, and most phytoplankton belongs to it. Microalgae is estimated to be responsible for 90% of the total photosynthesis on Earth and is a very important location as a primary producer of the global ecosystem. Microalgae have various characteristics and rapid growth rate. It is estimated that there are more than 40,000 species known, and more than 300,000 species are included if they are unregistered. Most of them absorb oxygen through photosynthesis in the sea and produce oxygen.

In addition, microalgae can grow (cultivate) unlimitedly by supplying water, sunlight and CO ₂ . Microalgae are mainly classified into plant groups that grow and propagate through photosynthesis or, in a broad sense, some bacteria, and there are many and many species and numbers. In general, microalgae grow much faster than ground plants and have high viable cell productivity. They are easy to grow in the natural environment where they can obtain light energy as well as fresh water or seawater. In addition, It can be said that there is a possibility that it is an important bio-industrial material because it can produce a substance having a specific physiological function at a high concentration as well as an industrially interesting biomolecule material such as a pigment. Accordingly, industrial application fields using microalgae are widely varied, such as wastewater treatment, air pollution purification (CO ₂ immobilization), aquaculture feed, health food material, bioactive material production, processing material and pharmaceutical material.

In addition, the microalgae, which are photosynthetic microorganisms, have the characteristics that light is an essential energy source as in the case of the ground plants, and that organic materials are synthesized using CO ₂ as a substrate, and its organic productivity is 4 to 20 times higher than that of plants . Organic materials produced by microalgae contain various useful substances (eg, unsaturated fatty acids, proteins, vitamins, polysaccharides, etc.) and can be used for biological drugs, physiologically active substances, dietary supplements, It can be used in the process of producing value-added products.

Accordingly, microalgae are being utilized not only for functional cosmetics but also for health food, alternative energy source, feed for concentrated aquaculture, production of pharmaceutical raw materials and production of biochemical materials. Full-scale studies on the industrial use of microalgae began in Germany during the Second World War for the production of vegetable fats and oils through the mass culture of diatoms. Research on the production of fats and proteins from the green algae chlorella and scenedesmus has been extensively followed.

As the potential of microalgae is enormous, it is expected that utilization will be expanded mainly in the fields of energy, chemistry and environment. In recent years, representative microalgae, which are increasingly utilized industrially, are as follows.

(1) Chlorella

It is the most widely studied microalgae of single-celled colony-type algae. Asexually grown cells divide into 4, 8, or rarely 16 cells and grow in both sea water and fresh water and are easily separated from each other, and several different species are introduced. This is not only in appearance, but also in a variety of species depending on the growth-requiring substance or growth characteristics (organic substance need, coloring culture, etc.), salt (1 to 5% NaCl) tolerance known as an important factor for forming carotenoid pigment Chlorella sp. Which is an important classification characteristic of

Chlorella sp . The content of chlorophyll pigment and the amount of chlorophyll pigment vary greatly depending on culture condition. The content of protein is 50 ~ 60%, saccharide 15 ~ 25% and lipid 2 ~ 65% The content of constituents is known to be closely related to chlorella sp . Depending on the species. In addition, Chlorella sp . Is widely used as a health food material because it contains a large amount of protein and is also widely used as a food for fish culture in Korea. Some materials (CGF: chlorella growth factor) .

(2) Spirulina

Spirulina sp . Is a multicellular, filamentous cyanobacterial spiral that slides along the axis of the fiber. Spirals vary in shape by species. Spirulina sp . Is characterized by rapid growth in warm, shallow, highly concentrated brine lakes and is commonly found in African alkaline saline reservoirs, but several species of Spirulina sp . Have been found in a variety of environments.

Spirulina sp . It is rich in proteins, lipid (γ-linolenic acid), vitamins (B-12, B-2), minerals and β-carotene and protein pigments. Diet, wound healing, hormones, anticancer, aging control and enzyme activity.

(3) Dunaliella

Dunaliella sp . Is a single cell, which has two cilia motility and, like other microalgae, is affected by temperature, light intensity, and carbon dioxide in growth. Dunaliella sp . Provided an important clue to the understanding of the osmotic regulating metabolic machinery, and in this connection, it was confirmed that the content of glycerol was an important osmolality regulator. Dunaliella sp . One of the important materials produced by β-carotene is β-carotene, which is produced and sold as a food industry or functional material.

(4) Porphyridium

Porphyridium sp . Is grown in a variety of habitats and is known to grow on fresh water, salt water, seawater as well as on moist soil surfaces. Porphyridium sp. Is characterized in that it mass-produces an acidic heteropolymer composed of a sulfate and particularly forms a very large molecular weight by bonding with a divalent metal ion and produces a polysaccharide in a large amount during a growth stopping period. Using these characteristics, industrial porphyran polysaccharides are produced, and the cells are also used as biomaterials for producing fatty acids. In recent years, studies on the production of new physiological functional materials by modifying porphyran polysaccharide are underway.

In general, microalgae are cultured in open air or closed culture (photobioreactor, etc.) under appropriate culture conditions. For example, Korean Patent Laid-Open Publication No. 10-2012-0095826 discloses a method of culturing a microalgae in a closed biochemical bioreactor and harvesting (recovering) the microalgae through agglutination.

In order to utilize microalgae having various usability industrially, mass culture technology is important. That is, in order to commercially and economically apply microalgae to fields such as cosmetics (cosmetics, etc.), biodiesel, and food supplements, it is important to cultivate microalgae capable of mass-producing microalgae in high density. In addition, mass production as well as lipid content in cells must be considered in culturing microalgae. That is, the most effective components contained in microalgae, especially functional materials useful for physiological activity, are contained in lipids of microalgae. In order to apply such microalgae to cosmetic compositions at low cost, for example, the lipid content should be increased.

However, the conventional culture method is difficult to mass-produce. That is, the conventional culture method has a low cell growth rate and biomass productivity of microalgae. Above all, the conventional culture method has a low lipid content in cultured microalgae cells. As a result, the extraction rate of the functional material derived from the microalgae is low, which makes it difficult to mass-produce large quantities of cosmetics (cosmetics) using microalgae and raises the price of the cosmetics product itself. In addition, in the case of microalgae extract according to the prior art, there is a problem that the effect of improving the function such as skin wrinkle improvement and whitening improvement is low.

Korea Patent Publication No. 10-2009-0025431 Korean Patent Publication No. 10-2012-0041404 Korean Patent No. 10-1410632 Korean Patent No. 10-1502359 Korean Patent Publication No. 10-2012-0095826

Accordingly, it is an object of the present invention to provide an improved method for culturing microalgae, and mass production of a cosmetic (cosmetic) material containing a functional material derived from microalgae through the method, And to provide a cosmetic composition and a process for producing the same.

In order to achieve the above object, the present invention provides a method for culturing microalgae in a microalgae culture reactor, wherein microalgae are cultured in the presence of carbon dioxide (CO ₂ ) at a pH of 7.5-13.

Also, the present invention provides a method for culturing microalgae, comprising: a first culture step of culturing microalgae in a culture medium of a microalgae culture reactor, wherein the microalgae is cultured in the presence of carbon dioxide (CO ₂ ) and an organic carbon source at a pH of 7.5 to 13; And a second culturing step of culturing in the presence of carbon dioxide (CO ₂ ) and an organic carbon source when the nitrogen is depleted by the first culture, wherein the second culture is a second culture in a nitrogen deficiency culture broth.

At this time, the initial concentration of microalgae contained in the culture solution in the 1 culture step may be 0.01 to 5.0 g / L and the initial concentration of organic carbon source may be 0.5 to 2 g / L. If the organic carbon source is depleted by the first culture, the organic carbon source is injected at a concentration of 0.5 to 2 g / L, and the organic carbon source is selected from the group consisting of glycerol and methanol It can be more than one. In addition, the microalgae that have undergone the second culturing step may have a lipid content of 30 wt% or more.

The present invention also provides a method for culturing microalgae, comprising: culturing a microalgae; An extraction step of extracting microalgae cultured in the culturing step to obtain a microalgae extract; And a mixing step of mixing the microalgae extract obtained in the extraction step with the cosmetic. At this time, the culturing process is according to the culturing method of the present invention.

In addition, the present invention includes a microalgae extract obtained by extracting microalgae having a lipid content of 30 wt% or more in a cell by culturing, and the microalgae extract is an effective ingredient for at least one selected from wrinkle improvement and whitening improvement Functional cosmetic composition.

According to the present invention, it is possible to mass-cultivate microalgae and has an excellent effect on the cell growth rate and biomass productivity of microalgae. In addition, it has high cell growth rate and high lipid content in cells. Thus, according to the present invention, it is possible to mass-produce a cosmetic (cosmetics, etc.) containing a functional material derived from microalgae in a large amount, and has an excellent effect such as improvement of skin wrinkles and whitening by a high amount of lipids .

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a configuration diagram showing a microalgae culture apparatus according to an exemplary embodiment of the present invention. Fig.
FIG. 2 shows the results of measurement of cell growth and biomass production of microalgae according to an embodiment of the present invention.
FIG. 3 shows the results of measurement of the amount of nitrogen remaining in the microalgae culture according to the embodiment of the present invention.
FIG. 4 shows the results of measurement of the pH of the microalgae culture solution according to the embodiment of the present invention.
FIG. 5 shows the results of measurement of biochemical composition of microalgae cultured according to the embodiment of the present invention.
FIG. 6 is a result of measuring the optical optical density (OD) of the microalgae cultured according to the embodiment of the present invention.
FIG. 7 shows the results of measurement of the nitrogen concentration in the microalgae culture liquid according to the embodiment of the present invention.
FIG. 8 shows the results of measurement of the pH of the microalgae culture according to the example of the present invention.
FIG. 9 shows the results of measurement of cell dry weight (DCW) of microalgae cultured according to an embodiment of the present invention.
FIG. 10 shows the result of measurement of biomass biomass of microalgae cultured according to an embodiment of the present invention and nitrogen concentration of the culture. FIG.
FIG. 11 shows the result of measurement of biochemical composition of microalgae cultured according to the embodiment of the present invention.
FIG. 12 shows the results of measurement of biochemical composition of microalgae cultured with phosphorus deficiency (P) according to an embodiment of the present invention.
FIG. 13 shows the results of measurement of cell optical density (OD) alc lipid content of microalgae cultured according to an embodiment of the present invention.
FIG. 14 shows the results of measurement of cell dry weight (DCW) of microalgae cultured according to an embodiment of the present invention.
15 is a result of measuring the optical optical density (OD) of the microalgae cultured according to the embodiment of the present invention.
16 is a result of measuring the organic / inorganic carbon source of the culture liquid according to the embodiment of the present invention.
FIG. 17 shows the results of measurement of cell dry weight (DCW), lipid content and organic / inorganic carbon source of microalgae and culture according to an embodiment of the present invention.
FIG. 18 is a systematic diagram showing a systematic fraction extraction process of microalgae extract according to an embodiment of the present invention. FIG.
Fig. 19 shows the result of skin cell stability test.
Fig. 20 is a test result of skin cell collagen synthesis promoting ability.
Fig. 21 shows the result of inhibition of tyrosinase activity.
22 shows the measurement results of the skin moisture content of the cosmetic composition according to the embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

As used herein, the term "and / or" is used to include at least one of the preceding and following elements. The term "one or more" as used in the present invention means one or more than two.

The present invention provides a method for culturing microalgae capable of at least mass culture at least according to the first aspect. The present invention provides a cosmetic composition comprising a microalgae extract having improved functionality according to the second aspect. According to a third aspect of the present invention, there is provided a method for producing a cosmetic composition capable of mass-producing cosmetics using microalgae in large quantities.

First, a method for culturing microalgae according to the present invention (hereinafter referred to as "culture method") will be described with reference to Fig.

1 is a view showing a microalgae culture apparatus. The culture method according to the present invention can be implemented through the culture apparatus shown in FIG. The culture method according to the present invention includes a culturing step of culturing the microalgae in the culture medium of the microalgae culture reactor (10). At this time, the pH is maintained at 7.5 to 13 during the culturing of microalgae. That is, the culture solution contained in the culture reactor 10 is maintained in the range of pH 7.5 to 13.

According to the present invention, when culturing is carried out while maintaining the pH of the culture solution within the above-mentioned range, high cell growth rate of microalgae can be shown and mass culture can be performed. It also shows a constant growth rate regardless of the species of microalgae. According to a more specific embodiment, the culture medium in the culture reactor 10 can be maintained in the range of pH 8 to 12. In addition, cultured in the presence of carbon dioxide (CO ₂₎ from a culture of microalgae. Referring to FIG. 1, the carbon dioxide (CO ₂ ) may be supplied into the culture reactor 10 in the carbon dioxide feed tank 40.

In the present invention, the kind of microalgae is not particularly limited. Microalgae should be classified as microalgae species. Microalgae may include, for example, the four species mentioned above. Specifically, the microalgae can be selected from Chlorella, Spirulina, Dunaliella and / or Porphyridium and the like. Examples of the microalgae include, but are not limited to, the above species and other examples, such as Scenedesmus, Botryococcus and / or Micractinium.

Other conditions for culturing the microalgae are not limited. The microalgae can be cultured in one or more nutrient conditions selected from, for example, photoautotrophic, mixotrophic and heterotrophic. Preferably in photoautotrophic and / or mixotrophic conditions.

Referring to FIG. 1, microalgae and a culture medium may be initially supplied to the culture reactor 10. At this time, the culture liquid can be injected and supplied into the culture reactor 10 through the culture liquid feed tank 20. The culture reactor 10 may be connected to the air supply tank 30 and the carbon dioxide supply tank 40. At least the air supply tank 30 and the carbon dioxide supply tank 40 are connected to the lower end of the culture reactor 10 so that air and carbon dioxide CO ₂ can be supplied upwardly into the culture reactor 10. have. In this case, circulation flow is generated in the culture reactor 10 by blowing, and growth of microalgae can be promoted.

The culture solution is not limited. As the culture solution, those conventionally used may be used. The culture medium may contain an inorganic nutrient source, an organic carbon source, and / or an inorganic carbon source necessary for the growth of microalgae. At this time, the inorganic nutrient source includes nitrogen (N) and phosphorus (P) salts, but may include other inorganic salts. Such a culture medium may be, for example, a nutrient medium used in the art, such as BG-11.

In addition, as another example of the culture liquid, a liquid fertilizer may be used. In one example, the culture solution may be a solution having a total nitrogen (N) content of 60 mg / L or more and a total phosphorus (P) content of 15 mg / L or more based on 1 liter (liters) of the culture solution. For example, an aqueous solution having a total nitrogen (N) content of 80 to 250 mg / L and a total phosphorus (P) content of 20 to 120 mg / L can be used as a culture solution. At this time, the aqueous solution includes seawater and / or fresh water as well as distilled water.

According to a specific embodiment, the initial concentration of the microalgae contained in the culture liquid may be 0.01 to 5.0 g / L, for example, 0.5 to 2.0 g / L, based on 1 L of the culture liquid. The culture solution preferably contains an organic carbon source for promoting growth of microalgae, and the organic carbon source is not particularly limited as long as it promotes the growth of microalgae. At this time, the organic carbon source may be contained in an amount of 0.1 to 8 g / L based on 1 L of culture. That is, in order to promote the growth of microalgae, the initial concentration of the organic carbon source contained in the culture liquid may be 0.1 to 8 g / L. Preferably, when the initial concentration of the microalgae is 0.01 to 5.0 g / L, the initial concentration of the organic carbon source is preferably 0.5 to 2 g / L.

The culture apparatus may further include a drain port 12 connected to the lower portion of the culture reactor 10 and a microalgae separator 51 may be connected to the drain port 12. In addition, the microalgae separator 51 is connected to the microalgae collection tank 52, and the microalgae collection tank 52 can be connected to the culture reactor 10.

The microalgae are supplied to the microalgae separator 51 through the discharge port 12 and the microalgae separator 51 separates the microalgae and water. In the microalgae separator 51, microalgae can be separated by, for example, sedimentation, filtration and / or flocculation. The microalgae separated through the microalgae separator (51) are collected (collected) and stored in the microalgae collection tank (52). At this time, the microalgae stored in the microalgae collection tank 52 are recycled to the culture reactor 10 through the air blower 53 as occasion demands. By repeating this recirculation, microalgae can be mass-produced at a high density in a small-scale culture reactor (10).

The culture reactor 10 is not limited as long as it is capable of culturing microalgae, which can be selected from a cylindrical shape and a round shape. The culture reactor 10 may be provided with a light illumination (not shown) for light-independent nutrition or mixed nutrition, and the light illumination may be selected from, for example, an LED and / or a fluorescent lamp. In addition, the incubation reactor 10 can be incubated in an enclosed state for blocking external contaminants.

According to a preferred embodiment, the culturing method according to the present invention comprises a first culturing step of culturing microalgae in a culture medium of a microalgae culture reactor 10, wherein the microalgae are cultured in the presence of carbon dioxide (CO ₂ ) and an organic carbon source, And a second incubation step of culturing in the presence of carbon dioxide (CO ₂ ) and an organic carbon source when the nitrogen is exhausted by the first incubation, wherein the second incubation is performed in a nitrogen deficient culture broth.

At this time, in the first culturing step, the pH of the culture liquid is maintained at 7.5 to 13 and cultured. Also, in the second culturing step, the culture can be cultured while maintaining the pH of the culture at 7.5 to 13. Specifically, in the first culturing and the second culturing, the pH of each culturing liquid may be maintained within a range of 8 to 12.

According to a preferred embodiment of the present invention, when the culture is performed through the two steps as described above, it is possible to cultivate microalgae, which are capable of mass culture of microalgae, and also have high biomass productivity and lipid content. Generally, it is difficult to obtain maximum biomass productivity and maximum lipid content simultaneously due to the metabolism of microalgae.

However, according to a preferred embodiment of the present invention, the maximum biomass productivity and the maximum lipid content can be simultaneously realized when the culture is performed through the two steps. That is, after a large amount of microalgae are cultured at a high concentration under the maximum growth condition through the first culturing step and then cultured under the nitrogen-deficient condition through the second culturing step, microalgae having a high lipid content can be cultured have. This can be confirmed by the following examples.

According to a specific embodiment, the initial concentration of the microalgae contained in the culture solution in the first culture step is 0.01 to 5.0 g / L and the initial concentration of the organic carbon source is 0.5 to 2 g / L. If the organic carbon source is depleted by the first culture in the second culture step, it is preferable to inject organic carbon source at a concentration of 0.5 to 2 g / L. Thus, after the first incubation, re-injection of the organic carbon source promotes the growth of microalgae and improves the productivity of the biomass.

In addition, the organic carbon source used in the first culture and the second culture may be one or more selected from glucose, glycerol, and methanol, for example. At this time, glycerol and methanol are economically advantageous when using by-products generated in the production process of biodiesel.

According to a specific embodiment, when the nitrogen (N) and the organic carbon source are depleted by the first culture in the second culturing step, a nitrogen deficiency culture liquid containing a nutrient source other than nitrogen (N) and an organic carbon source is supplied to a culture reactor 10).

At this time, the nitrogen deficiency culture solution does not contain a nitrogen source such as nitrate, and it may be any one containing an inorganic nutrient source other than nitrogen (P) and an organic carbon source. Specifically, the nitrogen-deficient culture medium contains inorganic salts such as phosphorus (P), and may contain other inorganic salt nutrients. In one example, the nitrogen-deficient culture medium may contain a phosphorus (P) compound such as phosphate as an inorganic nutrient and glucose and / or glycerol as an organic carbon source.

According to a preferred embodiment of the present invention, when culturing is carried out through the maximum growth conditions (first culture) and the maximum lipid content condition (second culture) through nitrogen deficiency as the above two-stage culture conditions, maximum biomass productivity And maximum lipid content can be realized at the same time. The microalgae that have undergone the second culturing step may have a lipid content of 30 wt% or more, for example. The lipid content of the microalgae that has undergone the second culturing step may be 30 wt% to 60 wt%, and more specifically, 30 wt% to 52 wt%, for example.

According to another embodiment, the method of culturing according to the present invention may comprise repeating the first culturing step and the second culturing step one cycle, and repeating this one cycle a plurality of times at least twice.

In addition, the method for producing a cosmetic composition according to the present invention includes the above-described culture method of the present invention. Specifically, the method for producing a cosmetic composition according to the present invention comprises the steps of: culturing a microalgae by the culturing method of the present invention; extracting microalgae by extracting microalgae cultured in the culturing step; And a mixing step of mixing the microalgae extract obtained in the extraction step with the cosmetic.

The extraction step is not particularly limited. The extraction process is in accordance with known extraction methods in the art, and may include, for example, at least solvent extraction. The extraction step may be carried out in the following manner. Specifically, a solvent extraction step using water and / or an organic solvent, a concentration step in which the extract obtained through the solvent extraction is concentrated, a drying step in which the concentrate obtained through the concentration step is dried (vacuum freeze- And / or a systematic fractionation step of purifying / separating the functional active ingredient through the systematic fraction of the extract, concentrate or dried product.

In addition, the cosmetic composition according to the present invention includes a microalgae extract. The cosmetic composition according to the present invention can be produced through the above-described production method of the present invention. Herein, the microalgae extract includes those obtained by extracting microalgae having a lipid content of 30% by weight or more through the culturing method of the present invention. The microalgae extract serves as an active ingredient for at least one selected from the group consisting of wrinkle improvement and whitening improvement in the cosmetic composition. In another example, the microalgae extract may act as an active ingredient for skin moisturizing and / or ultraviolet screening in cosmetic compositions.

In the present invention, the microalgae extract may be obtained by extracting a liquid phase obtained by solvent extraction, a concentrate obtained by concentrating the liquid extract, a solid powder obtained by drying (vacuum freeze drying) the concentrate, and / Concentrate, powder) is purified and separated through a systematic fraction, and the like. The microalgae extract may be contained in the total weight of the cosmetic composition according to the present invention, for example, in an amount of 0.001 to 20% by weight, and specifically 0.01 to 10% by weight, for example, no.

In the present invention, the kind and formulation of the cosmetic composition are not limited. In the present invention, the cosmetic composition is included in a skin or hair of a human body. Specifically, the cosmetic composition according to the present invention can be applied to cosmetics for aesthetic beauty, skin protection and the like, And a skin cleanser for the cosmetic and cleansing of the skin; Hair cleanser (shampoo, conditioner, soap, etc.) for cleaning of hair; Hair cosmetics for the beauty of hair; And a hair loss preventing agent to prevent hair loss.

The cosmetic composition according to the present invention may be a product selected from, for example, cosmetics, soap, body cleanser, shampoo, rinse, hair spray, hair gel, hair curdening agent and the like. The cosmetic composition according to the present invention may be solid, liquid, spray, ointment and / or gel, but is not limited thereto.

The cosmetic composition according to the present invention may contain the microalgae extract, and may include a cosmetic base according to its kind or formulation. The cosmetic base is not particularly limited. The cosmetic base may be variously formed depending on the kind of the product. The cosmetic base may be selected from, for example, base components such as cosmetics, soaps, body cleansers, shampoos and rinses as described above, which may be formulated as usual.

The cosmetic base includes a water-in-oil, an oil-in-water or a water-in-oil mixed formulation. The cosmetic base may be, for example, a skin and / or hair base, which may be at least one composition selected from cosmetic bases, soap bases, body cleanser bases, shampoo bases and rinse bases, for example. In addition, each of the cosmetic bases may be different depending on the product. However, the cosmetic base may be a surfactant (anionic, cationic, nonionic, amphoteric), an emulsifier, an emulsifier, a glycol, an alcohol, a moisturizer, , A viscosity adjusting agent, a chelating agent, an ultraviolet screening agent, a perfume and a pH adjusting agent, and the like. Since each of these cosmetic base components can be prepared as usual, a detailed description thereof will be omitted.

In addition, when the cosmetic base is a skin cosmetic composition, for example, the cosmetic composition includes liquid, cream, paste, foam, gel and solid. Specifically, the cosmetic composition may be a cosmetic composition such as a softening agent, a convergent lotion, a nutritional lotion, a nutritional cream, a massage cream, an eye cream, an eye essence, an essence, a cleansing cream, a cleansing lotion, a cleansing foam, a cleansing water, Lotions, body creams, body essences, body cleansers, ointments, gels, patches, sprays, skin-adhesive types and food creams. In addition, when the cosmetic base is a soap composition, it includes soaps such as liquid or solid.

According to the present invention described above, it is possible to mass-cultivate microalgae and improve the cell growth rate and biomass productivity of microalgae. In addition, it has an excellent cell growth rate and a high lipid content in cells. Accordingly, it is possible to mass-produce a cosmetic product containing a functional substance derived from microalgae, and a high content of lipids improves the function of improving skin wrinkles and whitening.

Hereinafter, embodiments of the present invention will be exemplified. The following examples are provided to illustrate the present invention in order to facilitate understanding of the present invention, and thus the technical scope of the present invention is not limited thereto.

[Example 1] Culture of microalgae

In order to extract useful substances from microalgae and commercialize them, it is important to mass-cultivate microalgae first, and to select microalgae species suitable for mass culture and optimize culture conditions.

To this end, Chlorella vulgaris AG10032, C hlorella vulgaris AG30007, Scenedesmus accuminatus , Scenedesmus quadricauda , and Botryococcus sp. 5 species were selected. These microalgae were distributed from the BRC Center of Korea Biotechnology Research Institute. Experiments were carried out to compare the 5 kinds of microalgae under photoautotrophic, mixotrophic and heterotrophic conditions. BG-11 and PAL- 1 was used. At this time, all the medium was sterilized by high pressure steam for 30 minutes at 120 ° C in an autoclave to prevent contamination from the outside.

BG-11 (Allen and Stanier, 1968) is a nutrient medium suitable for culturing freshwater algae microalgae under light and autotrophic conditions. It is rich in NaNO _{3, which} is a nitrogen source, and Na ₂ CO ₃ and contains 6 kinds of water-soluble boron and molybdenum as trace elements.

PAL-1 is a liquid fertilizer, which contains N and P nutrients and carbon sources suitable for microalgae growth as shown in [Table 2] below. This was filtered with a 0.45 mu m filter for use as a culture medium (culture medium).

<Composition of BG-11 culture medium> Composition of BG-11 medium Component Final Concentration (M) NaNO ₃ 1.76 x 10 ^-4 K ₂ HPO ₄ 1.75 x 10 ^-4 MgSO ₄ .7H ₂ O 3.04 x 10 ^-4 CaCl ₂ .2H ₂ O 2.45 x 10 ^-4 Na ₂ CO ₃ 1.89 x 10 ^-4 Citric acid 3.12 x 10 ^-4 Ferric ammonium citrate 3.00 x 10 ^-4 Na ₂ EDTA 2.79 x 10 ^-4 H ₃ BO ₃ 4.63 x 10 ^-5 MnCl ₂ .4H ₂ O 19.15 x 10 ^-6 ZnSO ₄ .7H ₂ O 7.65 x 10 ^-7 Na ₂ MoO ₄ .2H ₂ O 1.61 x 10 ^-8 CuSO4 · 5H ₂ O 3.16 x 10 ^-7 Co (NO ₃ ) ₂ .6H ₂ O 1.70 x 10 ^-7

<Composition of PAL-1 culture medium> Component Concentration (mg / L) T-N 85 - 105 NO ₃ -N 55 - 65 NH ₄ -N 6 - 8 PO ₄ -P 30 - 35 COD 200 TOC 231

Each of the five microalgae was subjected to the following culture experiment according to the respective nutritional conditions (light independent nutrition, mixed nutrition, and heterotrophic nutrition), and then the microalgae and the culture solution cultured were analyzed.

1. Experiments on microalgae culture

First, micro-algae were inoculated in a 250 mL Erlenmeyer flask containing 100 mL of BG-11 medium and incubated in a shaking incubator (VS-8480SR, Vision Science Co., Ltd.) for aseptic manipulation on a clean bench for sub- , Light intensity 90 μmol m ^-2 s ^-1 , temperature 25 ° C., stirring rate 160 rpm. [Picture 1] below is a photograph of some flask samples cultivated as above.

After reaching the stationary phase of the growth of the microalgae of the 250 mL flask cultivated in the subculture, a part of the microalgae culture liquid was separated and recovered by a centrifuge in order to secure enough microalgae to be used in the experiment, 2], a conical-ended photobioreactor with a conical tip of 95 mm in outer diameter, 2.5 mm in thickness, and an effective volume of 2 L was carried out. Then, the culture medium was periodically subcultured in a 2 L incubator in the same manner, and the medium used for the culture was replaced with a fresh medium.

Inorganic carbon sources supplied at photoautotrophic and mixedotrophic are CO ₂ gas (1 to 5 v / v%), organic nutrients supplied at mixed nutrition and heterotrophic Experiments were performed to compare the effects of glucose with other organic carbon sources such as glycerol and methanol.

In the heterotrophic culture experiment, 1000 mL flasks were shaded with aluminum foil on a shaker (25 ° C, 250 rpm) as shown in [Picture 3] below. 4], and cultured in a conical photobioreactor (6.5 x 37 cm) having an effective capacity of 1 L. In the lower part of the reactor, CO ₂ mixed with sterilized air through a 0.2 μm PTFE membrane at a rate of 0.1 vvm . At this time, in other cultivation experiments except for heterotrophic culture, light was continuously supplied for 24 hours at a light intensity of 120 to 150 μmol / m ² / s using a white fluorescent lamp as an artificial light source.

2. Analysis method

(1) Cell dry weight (DCW) and cell optical density (OD)

Dry cell weight (DCW) and optical density (OD) were measured to confirm microbial growth rate and biomass productivity in each culture experiment.

In order to measure the cell dry weight (DCW), a portion of the culture broth was taken from a reactor under culture, and 5.0 mL of the culture broth was subjected to solid-liquid separation using a centrifuge, washed with distilled water, drying oven for 12 hours, weighing (W ₀ ), and then filtering with a cellulose nitrate filter (47 mm, 0.45 μm). Filtered, dried in a drying oven under the same conditions, stored in a desiccator, cooled to room temperature, and the weight (W ₁ ) was measured. Dry cell weight (DCW) was estimated by subtracting the W ₀ from _{W 1. (DCW = W 1} W 0)

Cell optical density (OD) is a method for indirectly measuring the density of microalgae. 1.0 mL of the microscopic algae culture solution was diluted 10 times with distilled water and analyzed with a spectrophotometer (DR4000U, Hach, USA) at 680 nm and 750 nm wavelength Respectively.

(2) Nitrogen concentration

Nitrate concentration in microalgae culture is an important factor affecting the growth and biochemical composition of microalgae. Therefore, it is necessary to monitor the change of nitrogen concentration during microalgae culture. Nitrogen detected in the form of nitrate-nitrogen (NO ₃ ^- ) was measured according to the Standard Method (APHA, 1995). That is, the nitrogen concentration was determined by filtering the sample taken from the culture solution with a 0.45 μm syringe filter (Whatman, UK) to remove microalgal cells and suspended solids, diluting 50 times with distilled water Respectively. In this case, 1N HCl (1:50, HCl: sample v / v) was added to the diluent to oxidize the interfering substances at the measurement wavelength, and the mixture was mixed using a voltex mixer. Absorbance at 220 nm and 275 nm, And the concentration was quantified by substituting the previously prepared calibration curve equation.

(3) Biochemical composition and fatty acids

For biochemical composition and fatty acid analysis of microalgae, a certain amount of recovered biomass was subjected to a vacuum oven (vacuum) at 40 ° C in accordance with the preparation method of Moisture-free biomass sample of NREL (Wychen and Laurens, 2013) oven for 24 hours, and finely ground and homogenized using a mortar and mortar. The homogenized microalgae biomass was completely dried and dried in a vacuum drier at 40 ° C for 6 hours to remove moisture and weighed. After drying, the microbial biomass was kept in a desiccator while shutting off the inflow of air. [Picture 5] below is a sample picture obtained through this process.

For lipid content analysis, weigh accurately (50.0 mg) from the above dried biomass and solvent was extracted. For the measurement of carbohydrate and protein content, 2.0 mg was accurately weighed and dissolved in 5.0 mL of distilled water for analysis. 10.0 mg dry biomass was used for fatty acid analysis.

(a) Lipid analysis

Lipid extraction was carried out by using 7.5 mL of a mixed solvent (Chloroform / methanol / water) (1/2 / 0.8, v / v / v) in a 50.0 mg microalgae dry biomass sample according to the solvent extraction method of Bligh & Dyer ), And sonication (cell disruption) was performed for 1 minute using an ultrasonicator (VCS 130, Sonics & Materials IN., CT, USA) for smooth extraction from microalgae cells. 2.0 mL of chloroform and 2.0 mL of distilled water were added thereto, and the mixture was ultrasonically pulverized by the same method for 1 minute, mixed with a vortex mixer for 1 minute, and centrifuged at 4500 rpm for 5 minutes.

After centrifugation, the chloroform layer containing crude lipid in the layered mixture was transferred to a pre-weighed aluminum dish using a pipette, placed in a drying oven at 80 ° C, chloroform) were all evaporated. [Picture 6] below is a chloroform layer transferred to an aluminum plate. Thereafter, the cells were stored in a desiccator, cooled to room temperature, weighed with an electronic balance (GH-200, A & D Company, Japan), and the measured lipid was expressed as weight% of cell dry weight.

(b) Carbohydrate analysis

Carbohydrate content was analyzed according to the phenol-sulfuric acid method (Dubois et al., 1956). Specifically, a 2.0 mg dried moisture-free biomass sample was dissolved in 5 mL of distilled water and mixed well. Take 0.5 mL of the solution and transfer it to a glass vial. A 5 wt% phenol solution and 2.5 mL of sulfuric acid (95 wt% purity) were injected and vigorously mixed for 1 min in a vortex mixer. Thereafter, it was confirmed that the color was orange, and after cooling to room temperature (25 ° C) for about 10 minutes, absorbance was measured at a wavelength of 490 nm, and the absorbance was substituted into the previously prepared calibration curve formula.

(c) Protein analysis

Protein content was determined by measuring the absorbance at 770 nm according to the Lowry method (Lowry et al., 1951). Specifically, the Lowry solution required for protein determination was prepared on the day and Solution A prepared in advance was prepared by adding 20 g (2 wt%) of Na ₂ CO ₃ + 4 g (0.1 N) of NaOH and 1 g (0.5 wt%) of Solution B CuSO ₄ .5H ₂ O, ) + 2 g (1 wt%) of sodium potassium tartrate were mixed at a volume ratio of 50: 1. Prepare the same amount of carbohydrate. Prepare 0.1 mL of the microalgae biomass sample and add 1.0 mL of the prepared Lowry solution. Mix for 30 seconds with a voltex mixer. After 10 minutes of reaction, add 0.1 mL of 1N Folin solution. Minute. The absorbance of the blue-colored compound was measured at a wavelength of 770 nm, and the concentration was calculated by substituting the previously prepared calibration curve.

(d) Fatty acid analysis

In order to analyze fatty acids in microalgae biomass, transesterification reaction was carried out according to NREL (National Renewable Energy Laboratory) Direct-transesterification method (Wychen and Laurens, 2013). The microalgae samples recovered from the culture broth were separated by solid-liquid separation using a centrifuge, the supernatant was discarded, washed once with distilled water, centrifuged again, and dried in a 40 ° C vacuum dryer for about 24 hours.

After finely homogenizing with a mortar and mortar, exactly 10.0 mg was taken in a glass vial and dried in a vacuum drier at 40 ° C for 6 hours to completely remove water. For transesterification, 0.2 mL of a mixture of Chloroform / MeOH (2: 1, v / v) and 0.3 mL of 0.6 M HCl solution (in methanol) were mixed and heated at 85 ℃ for 1 hour After heating, allow to stand for at least 15 minutes and cool to room temperature. Add 1.0 mL of n- Hexane and mix well. Separate the mixture for 1 hour. Add hexane layer to the mixture using a micropipette And transferred to a GC vial to prepare gas chromatography analysis by gas chromatography. [Picture 7] below is a picture of some samples transferred to GC vials.

From the n- hexane layer containing the fatty acid transferred to the GC vial, exactly 1.0 μL was taken using a micro syringe and the fatty acid composition was analyzed by gas chromatography (GC). The analytical instrument was a GC (YL6500 GC, Younglin Instrument, Korea) equipped with a FID detector and capillary column (30 mx 0.32 mm id x 0.50 ㎛ HP-INNOWAX, Agilient 19091N-213)

FID temperature at 140 ° C for 5 min, 4 ° C / min up to 240 ° C and hold for 10 min, 1.0 ℓ injection at 10: 1 split ratio, inlet temperature of 260 ° C, constant flow of 1 ml / min helium of 260 ° C, flow rate of 300 mL / min, zero air, 35 mL / min H ₂ , 20 mL / min helium.

As a standard material for analysis, FAME mix 14 C8-C24 (Sigma Aldrich # 18918-1AMP) was used, and a retention time (RT) and a calibration curve for the concentration of each fatty acid component were measured by software (YL-Clarity, Yougnlin Instrument Co., Korea), and the same sample was analyzed twice or more, and the average value was analyzed.

(e) Other analyzes

(TC), dissolved inorganic carbon (DIC), and carbon monoxide (CO) were monitored to monitor the changes of the carbon source concentration, which were decreased with the injection concentration of organic / inorganic carbon source and the growth rate during the mixedotrophic and heterotrophic cultivation. Dissolved carbon monoxide) and COD (chemical oxygen demand) were measured.

TC and DIC were obtained by filtering the microalgae culture with 0.45 ㎛ syringe filter and diluting 10 times with TOC analyzer (TOC-V _CSH , Shimadzu, Japan) Samples prepared by the same method were analyzed using the COD High Range kit (20 to 1,500 mg / L, Hach) according to the USEPA Standard Method (5220 D) and the absorbance was measured with a spectrophotometer to determine the concentration.

For all experiments except heterotrophic culture, the light intensity of an artificial light source incident on the incubator was measured using a photometer (Light meter, LI-COR, LI-250A, USA) The pH value was monitored with a pH meter to check the condition of the culture liquid. All cultures except for the heterotrophic culture experiment and the 50.0 L mass culture experiment were carried out in an isolated culture space where the temperature was maintained at 25 ° C.

3. Microalgae culture experiment results

(1) Biomass productivity according to nutritional conditions

FIG. 2 is a result of comparing cell growth and biomass production according to microalgae species and nutrient conditions, which is a result of measurement of cell dry weight (DCW) according to cultivation time. As shown in Fig. 2, the five microalgae showed a faster growth rate under the mixed nutrient conditions than the light-independent nutrient or heterotrophic conditions, in order to select the optimal microalgae.

In particular, in the case of C. vulgaris AG30007 in mixed nutrient conditions, 15-day average biomass productivity is 0.24 g L ^-1 d ^-1, maximum productivity (P _max) is 0.40 g L ^-1 d ^- ¹ represented by the highest biomass Productivity. Scenedesmus quadricauda also showed fast cell growth under mixed nutrient conditions, showing the highest biomass productivity (0.23 g L ^-1 d ^-1 ) after C. vulgaris AG30007.

As shown above, biomass productivity (and cell growth) was higher in mixed nutritional conditions than other nutritional conditions. Especially, productivity of C. vulgaris AG30007 was higher than that of other species.

FIG. 3 shows the result of measurement of nitrogen (N.sub.2) remaining in the culture solution in order to confirm the amount of nitrogen consumed while microalgae grow in each nutrient condition. As shown in FIG. 3, it was confirmed that the growth rate of the microalgae was low in the light independent nutrient condition, so that the consumption of nitrogen was reduced to that extent. In the mixed nutrient condition, Nitrogen was exhausted.

In general, heterotrophic microalgae are known to grow at a high concentration using an organic carbon source as an energy source and a carbon source. However, according to this experimental example, C. vulgaris AG10032 and AG30007 showed a high growth rate even though nitrogen was rapidly consumed in the culture medium I can not. Therefore, it was confirmed that the heterotrophic culture was not suitable as the culture condition of C. vulgaris through this experimental example.

FIG. 4 shows the measured values of the pH in the culture medium according to the nutritional conditions. When microalgae are cultured, the pH increases as the growth progresses. When the pH is changed, the concentration of bicarbonate (HCO ₃ ^- ) and carbonic acid (CO ₃ ^2- ) in the culture fluid changes, and when the pH changes sharply Indicating that it inhibited the growth of microalgae. As a result, it was found that the range of pH change between pH 8 and pH 12 did not show any difference or showed rapid change. In other words, it can be seen from this experimental example that when the pH value is maintained between 8 and 12 at the time of culturing under each nutrient condition, the growth rate is constant regardless of the species of the microalgae. The analytical results (measured values) of each item according to the nutritional conditions are shown in the following [Table 3] to [Table 5].

<Analysis results of each item in the light independent nutrition culture> OD _{750 nm} elapsed
time (d) One 2 3 4 5 0 0.0615 0.062 0.2235 0.0545 0.062 2 0.255 0.28 0.18 0.165 0.19 4 0.46 0.53 0.385 0.27 0.4 7 0.81 0.84 0.665 0.4 0.545 9 1.26 1.54 1.11 0.94 0.775 11 1.475 1.68 1.46 1.13 1.03 14 1.64 1.895 1.75 1.29 1.205 DCW (g / L) elapsed
time (d) One 2 3 4 5 0 0.12 0.08 0.18 0.1 0.1 4 0.42 0.26 0.34 0.24 0.42 7 0.7 0.4 0.74 0.36 0.62 11 1.2 0.76 One 0.94 0.88 14 1.26 0.84 1.22 1.18 1.16 Nitrate (NaNO ₃ g / L) elapsed
time (d) One 2 3 4 5 0 1.48 1.47 1.48 1.48 1.48 2 1.44 1.48 1.39 1.50 1.45 4 1.39 1.41 1.37 1.38 1.36 7 1.18 1.22 1.18 1.22 1.20 9 1.08 1.10 1.08 1.13 1.12 11 1.09 1.05 1.08 1.13 1.12 14 1.07 1.03 1.09 1.11 1.13 pH elapsed
time (d) One 2 3 4 5 0 7.13 7.27 7.03 7.13 6.54 2 11.01 10.89 11.17 11.32 11.3 4 11.72 11.29 11.56 11.56 11.72 7 11.1 11.47 11.35 11.76 11.76 9 12.22 11.56 11.65 12.1 12.1 11 12.24 11.3 11.4 12.15 12.13

<Analysis results of each item in mixed nutrition culture> OD _{750 nm} elapsed
time (d) One 2 3 4 5 0 0.32 0.09 0.30 0.32 0.31 2 0.56 1.32 1.15 0.58 0.74 4 0.85 1.98 1.55 0.85 1.13 7 1.56 2.44 1.75 1.70 1.73 9 2.20 2.71 1.89 1.95 1.85 11 3.16 3.26 3.18 2.48 2.98 14 4.88 5.57 4.69 3.80 3.84 16 4.75 5.76 4.87 4.38 4.22 DCW (g / L) elapsed
time (d) One 2 3 4 5 0 0.40 0.08 0.26 0.36 0.36 2 0.78 0.78 1.06 0.80 1.04 7 1.52 0.96 1.04 1.52 1.56 9 1.74 0.94 1.34 1.66 1.68 11 3.02 1.42 3.02 2.92 3.30 14 4.64 2.60 4.08 4.08 3.66 15 4.50 2.65 4.35 4.59 3.96 Nitrate (NaNO ₃ g / L) elapsed
time (d) One 2 3 4 5 0 1.44 1.46 1.45 1.46 1.46 2 1.45 1.17 1.17 1.45 1.37 4 1.35 1.11 1.10 1.36 1.16 7 1.12 1.05 1.03 0.99 0.95 9 0.96 0.98 1.03 0.92 0.91 11 0.82 0.94 0.82 0.87 0.62 14 0.08 0.18 0.32 0.11 0.00 pH elapsed
time (d) One 2 3 4 5 0 9.64 8.79 7.76 9.67 8.68 2 11.25 9.93 10.22 11.39 11.48 4 11.74 10.85 11.37 11.76 11.92 7 11.94 11.37 11.25 11.13 11.89 9 11.34 11.44 11.43 12.23 12.12 11 9.33 10.41 9.80 10.28 9.80 14 9.44 9.25 9.33 9.43 9.40

<Analysis of each item in heterotrophic cultivation> OD _{750 nm} elapsed
time (d) One 2 3 4 5 0 0.29 0.54 0.28 0.35 0.33 One 0.58 1.19 0.67 0.47 0.62 2 0.46 0.84 0.61 0.34 0.62 3 1.07 1.74 0.92 0.65 1.44 5 1.12 1.43 1.05 0.77 1.33 7 1.09 1.63 1.10 0.94 1.42 9 1.64 3.12 1.86 1.17 2.01 11 1.35 2.27 1.25 1.27 2.03 DCW (g / L) elapsed
time (d) One 2 3 4 5 0 0.36 0.34 0.32 0.58 0.44 One 0.66 0.74 0.64 0.62 0.76 2 0.98 0.96 0.94 0.84 1.12 3 1.30 1.12 0.96 0.94 1.60 5 1.02 0.78 0.68 0.80 1.36 7 1.00 0.76 0.74 0.74 1.32 9 1.64 1.40 1.10 1.20 2.12 11 1.18 1.16 0.80 0.58 1.80 Nitrate (NaNO ₃ g / L) elapsed
time (d) One 2 3 4 5 0 1.26 1.22 1.25 1.25 1.25 One 1.56 1.47 1.54 1.56 1.51 2 1.59 1.16 1.32 1.54 1.45 3 1.42 0.04 0.43 1.55 1.40 5 1.30 0.00 0.46 1.40 1.28 7 1.15 0.00 0.44 1.15 1.17 9 1.06 0.00 0.00 0.90 1.08 11 0.93 0.00 0.00 0.35 1.04 pH elapsed
time (d) One 2 3 4 5 0 7.42 6.94 7.14 6.48 7.25 One 5.60 6.11 6.02 5.93 6.05 2 5.94 6.40 6.34 5.49 6.23 3 5.86 6.73 6.25 4.80 6.53 5 5.98 7.43 7.35 6.00 6.92 7 6.83 7.52 7.60 6.37 6.89 9 6.45 6.93 6.91 6.60 6.53 11 6.67 7.14 7.16 7.14 6.77

(2) Biochemical composition according to microalgae species

In order to determine the change in the biochemical composition while cultivating the 5 kinds of microalgae, the contents of lipid, carbohydrate, and protein were measured at intervals of about 5 days during the culture The results are shown in FIG. The method of measuring the biochemical composition is as described above.

As shown in FIG. 5, the biochemical composition of microalgae varied with species and over time. The carbohydrate content of Botryococcus sp. Was higher than that of lipid and protein, and the carbohydrate content was about 40 wt% after 2 weeks of culture. In addition, the initial lipid content of C. vulgaris AG10032 was about 28 wt%, which was slightly higher than that of carbohydrate and protein, but the lipid content decreased and the carbohydrate and protein content increased slightly .

Scenedesmus Accuminatus and Scenedesmus In the case of quadricauda, the lipid content was relatively low (less than 20 wt%) compared to other microalga species, but the carbohydrate content was high (maximum 45 wt%). In the case of C. vulgaris AG30007, which had the highest biomass productivity, the lipid content was maintained within the range of 20 ~ 30 wt% for 2 weeks of culture, which showed a generally higher lipid content than the other species.

Therefore, when the biomass productivity and lipid content are considered together, C. vulgaris AG30007 among the five freshwater species has relatively high biomass productivity and lipid content is higher than other species. Therefore, C. vulgaris AG30007 is considered to be suitable for mass culture and its application in the future, and as the microalgae, were selected vulgaris AG30007 optimally species (species), in the following experiments were all conducted on C. vulgaris AG30007.

(3) Biomass Productivity of C. vulgaris during 50 L Mass Culture

C. vulgaris AG30007 was selected as the optimal species of microalgae and cultured under the mixed nutrient conditions which showed the best result in the nutritional condition of the previous culture experiment. At this time, a culturing apparatus designed as shown in FIG. 1 was used for expanding to a size larger than 2 L on the laboratory scale. At this time, a 50 L oval photobioreactor made of polycarbonate (PC) having a diameter of 72 cm, a height of 107 cm, and a thickness of 8 cm was installed, It is possible to recover the microalgae concentrate by the bottom port after the culture is mixed with the carbon dioxide and air injected through the aeration port, the operation is stopped after the incubation, It is easy to control the operating conditions.

In order to cultivate the microalgae in the elliptical 50 L photobioreactor, C. vulgaris AG30007 cultivated on a small scale in advance was inoculated in an initial concentration range of about 0.05 to 0.08 g L ^-1 and stirred for carbon dioxide supply Aeration was performed at a flow rate of 0.5 vvm using an air pump. The LED illumination was used as an artificial light source, and light was irradiated by controlling the luminous intensity to about 200 μmol / m² / s by a control panel mounted on the light source. BG-11 containing an inorganic carbon source was used as a basic culture medium (culture medium). Glucose was supplied as an organic carbon source in order to maintain the mixed nutritional condition. After culturing for a certain period of time, The glucose was re-injected and cultured.

The cell concentration (DCW, dry weight) was significantly increased to 0.94 g L ^-1 d ^-1 at the initial 0.08 g L ^-1 2 days after the incubation for about 15 days and average biomass productivity the results are shown for holding the ¹ ^- 0.2 g L ^-1 d. And interval maximum biomass production (P _max) is about 0.43 g L ^-1 d after 2 days incubation ^- appeared to ^1. The The evaluation results of each item in the 50 L large-scale culture are shown in FIGS. 6 to 9. FIG. 6 shows the optical optical density (OD), FIG. 7 shows the nitrogen concentration in the culture medium, FIG. 8 shows the pH change of the culture medium, and FIG. 9 shows the cell dry weight (DCW) as the biomass productivity.

Therefore, when microalgae biomass is produced using a 50 L incubator, the optimal pH is maintained under mixed nutrition conditions, and the optical density (OD) and the organic carbon source concentration are measured during the culturing, and the organic carbon source Yeast, glucose, etc.) can be mass - cultured and highly productive.

(4) Improvement of lipid content

In order to increase the lipid content of C. vulgaris AG30007, we could know the change of lipid content of C. vulgaris by adding nitrogen deficiency, phosphorus deficiency, NaCl addition and various organic carbon sources such as glycerol and methanol as follows saw.

(a) nitrogen (N) deficiency

To investigate how the lipid content of C. vulgaris changed when the nitrogen source was completely depleted during the culture, the culture broth was incubated under photoautotrophic and mixedotrophic conditions for about 2 weeks including before and after depletion of the nitrogen source C. vulgaris Lt; / RTI > In each case, biomass biomass (cell dry weight, DCW) and nitrogen concentration were measured, and the results are shown in FIG. The contents of lipids, carbohydrates and proteins were measured for each case, and the results are shown in Fig.

First, as shown in FIG. 10, the nitrogen source in the culture broth was depleted within 4 days of culturing according to the cell growth of microalgae. In the mixed nutrient condition, cell growth was faster than in the light independent condition and nitrogen depletion was also observed within 2 days It can be seen that it is happening quickly. Further, as shown in Fig. 11, from 2 to 4 days after the nitrogen source began to be completely deficient, thereafter, C. vulgaris And the maximum lipid content was increased up to 48 wt% even in the light independent nutrient condition where organic carbon source was not supplied. Also, as shown in Fig. 11, the carbohydrate and protein contents decreased while the lipid content increased.

The above results indicate that microalgae can be used as an alternative to accumulation of carbohydrates and proteins by changing the carbon flux when subjected to a stress environment condition in which nitrogen (N) is deficient in the culture solution regardless of the light independent nutrition or mixed nutrition condition I could see that I accumulated my lipids.

(b) phosphorus (P) deficiency

In addition, we investigated whether the deficiency of nutrients other than nitrogen (N) could increase the lipid content of C. vulgaris . For this purpose, an experiment was conducted to confirm the deficiency condition of phosphorus (P), which is next to nitrogen source. For more accurate experiments, the same nutritional conditions were introduced into two incubators, respectively, and the results are shown in FIG.

As shown in Fig. 12, phosphorus (P) was depleted within 5 days of culture, and inhibition of cell growth due to phosphorus (P) depletion was not significant. In addition, the lipid content decreased rather than increased. Therefore, it was found that the intracellular lipid accumulation of C. vulgaris due to phosphorus (P) deficiency did not occur.

(c) NaCl addition

In addition, in addition to nitrogen (N) and phosphorus (P) supplied for the microalgae cultivation, the injection concentrations of other trace elements are relatively low, and their deficiency does not have a significant effect on the biochemical composition changes of microalgae It was judged whether or not to increase the lipid content by adding certain elements other than the experiment to restrict nutrients.

As a result of reviewing the literature, there has been proposed a technique for applying salinity change as a factor that causes stress accumulation in freshwater microalgae and causes lipid accumulation. To confirm this, a culture of C. vulgaris is cultivated Changes in cell growth rate (OD) and lipid content during culture were confirmed. That is, NaCl was added to the microalgae culture solution grown at a high concentration for a period of about 72 hours, and the optical density (OD) and lipid content of the microalgae were measured. The results are shown in Fig.

As shown in FIG. 13, the cell growth of C. vulgaris was continued under the condition of fresh water (NaCl 0 M) and low concentration of NaCl (0.1 M), but when the NaCl concentration was 0.25 M or more, It was found that the cell concentration was significantly reduced to less than ½ after 72 hours of incubation. In addition, the lipid content tended to increase slightly after 12 hours of incubation at high concentration of NaCl (0.25 ~ 0.5 M), but there was no significant lipid accumulation. Therefore, the increase of lipid content of C. vulgaris by NaCl addition was not significant, but it was found that the increase of NaCl concentration inhibited cell growth.

As a result, it was possible to increase the lipid content under nitrogen (N) deficiency condition, and to change the salinity through phosphorus (P) deficiency or NaCl injection, the effect of increasing the lipid content of C. vulgaris was remarkable It is not.

(5) Utilization of various organic carbon sources

In addition, in order to investigate the influence of other factors besides nitrogen (N) deficiency on the microalgae cultivation, glucose (glucose) instead of the organic carbon source Of an organic carbon source.

First, glycerol, which is a by-product of the biodiesel production process, was used as a relatively low-priced organic carbon source to replace glucose, and cell growth and biomass productivity were confirmed. BG-11 and PAL-1 (Nitrate) and phosphate (Nitrate), which are the major nutrients, were added to the nutrient solution to improve the biomass productivity.

That is, for the comparison of experiments, comparative experiments were carried out using (1) culture medium in sterilized BG-11 medium and (2) culture medium in distilled water containing only N and P at concentrations such as BG-11 medium. Glycerol as an organic carbon source was mixed with general air at an initial concentration of 1 g / L and CO ₂ at 2.5 v%, and each culture was supplied at a flow rate of 0.2 vvm. That is, (1) a culture solution in which glycerol was mixed at a concentration of 1 g / L in sterilized BG-11 medium, (2) glycerol was added to a mixture of distilled water, nitrate and phosphate, to a 1 g / L concentration in the culture solution, using a mixture, culturing the C. vulgaris in the initial cell density of about 0.2g / L mixed nutrient culture conditions and cell growth for each case, the nitrogen concentration, pH change, etc. Respectively. The results are shown in Fig. 14 and Fig.

First, as shown in Fig. 14, both cultures showed similar growth rates for about 2 days after the start of culture, but the difference began to show from the third day, and the BG-11 medium-based mixture The cell growth rate was significantly higher in nutrient culture, and the cell dry weight (DCW) at 11 days after culture was significantly different.

In addition, the measurement of cell optical density (OD) showed a decrease in the absorbance (680 nm) value at 7 to 9 days, but this was not a decrease in cell growth. C. vulgaris cells formed floc, The optical density was evaluated to be low when the absorbance was measured. As a result, by using glycerol, the optical optical density (OD) and cell dry weight (DCW) are continuously increased, and the phenomenon of growing by forming floc increases the light transmittance of the culture liquid, Can be advantageous. In addition, this has the additional advantage that floc-forming cell clusters can be easily recovered even during cell recovery.

In addition, as shown in FIG. 15, the nitrogen concentration in the culture medium decreased with cell growth, and the nitrogen concentration reduction rate in the distilled water-based culture medium was relatively low as expected. The pH increase due to cell growth was not large and the pH range was maintained at about 7-8. The pH increase after one day of incubation was a phenomenon in which the supply of CO ₂ was stopped midway (carbon dioxide gas cylinder depletion) and the pH returned to about 7-8 after the CO ₂ supply was resumed.

In addition, TC (total carbon) and dissolved inorganic carbon (DIC) were measured with a TOC analyzer to determine the amount of organic / inorganic carbon source that the microalgae grew and absorbed and fixed. The COD (Chemical Oxygen Demand) was measured to confirm the decrease in the glycerol concentration. The results are shown in Fig.

As shown in FIG. 16, when 1.0 g / L glycerol was added to both cultures, the initial TC concentration and COD were 640 and 1260 mg / L, respectively, and organic / inorganic carbon sources were used for the growth of microalgae Gradually, TC and COD concentrations decreased. The concentration of COD decreased to about 130 mg / L (BG-11) on the 11th day of culture, and the DIC concentration in the culture solution was gradually increased by decomposition of organic carbon source and dissolution of carbon dioxide.

Thus, the above results show that glycerol can increase microalgae mass culture. In addition, when the initial cell concentration is about 0.2 g / L, the added concentration of glycerol is about 1 g / L, and the carbon source is depleted during the long-term culture, It was found that it is preferable to re-inject after concentration measurement when it is exhausted.

In addition to glycerol, methanol, another organic carbon source, is a by-product of the biodiesel production process like glycerol, which is specifically added to biodiesel when it is converted, Material. Experiments were carried out to determine whether methanol could be used as another organic carbon source of microalgae such as glycerol and to increase the lipid content. However, it is considered that methanol is a toxic substance to the cells, and when it is injected at a high concentration, it will cause a great inhibition of cell growth. Therefore, an aqueous solution of methanol is put into a concentration range of less than 3 wt% Conditions were determined under the same conditions as in the previous experiment to measure changes in cell growth, COD and lipid content of C. vulgaris. The results are shown in Fig.

As shown in FIG. 17, when C. vulgaris was cultured under various concentrations of methanol, the cell growth continued for about 4 days, and the COD concentration was steadily decreased. As a result, As a result. However, there was no significant difference between the addition of methanol and the change of lipid content according to the concentration of feed, so it did not act as an increase factor of lipid content. In addition, the methanol concentration reached about 1.5 g / L at the initial cell concentration of about 0.5 g / L, and the average biomass productivity of 0.25 g / L / d after 4 days of culture. It could be used as an organic carbon source during culturing.

As a result of culturing microalgae ( C. vulgaris ) under various conditions as described above and analyzing cell growth and lipid content and the like, biomass productivity was found to be a mixture of glucose and glycerol And the highest in nutrient culture. That is, biomass productivity was high even when glycerol generated as a byproduct of biodiesel production process was used as an organic carbon source instead of glucose.

It is sufficient to inject glucose and glycerol at a concentration of 0.5 to 2 g / L during the initial injection into the reactor. Then, the carbon concentration is checked by measuring COD or TOC during the culture, It is preferable to re-inject at the same concentration. In addition, methanol, which is a byproduct of the biodiesel production process, can be used as an organic carbon source to improve biomass productivity if it is injected at a low concentration. That is, when the microalgae were cultured under the above conditions, the average biomass productivity could reach 0.2 g / L / d or more.

On the other hand, it is difficult to obtain maximum biomass productivity and maximum lipid content at the same time due to the metabolism of microalgae. However, as can be seen in the above experimental examples, when the microalgae are first cultured in a high concentration at a high concentration in an optimal growth condition and then cultured under the condition of increasing the lipid content, maximum biomass productivity and maximum lipid content can be simultaneously realized .

In other words, the nitrogen (N) deficient, phosphorus (P) deficiency, salinity changes and results of experiments comparing the like supplied from a wide variety of organic carbon through the supply NaCl, microalgae (C. vulgaris), as identified in the above Experimental Example (Nitrate deficient) is the factor that can maximize the lipid content of fish. For example, in the mixed nutritional condition of the above experimental example, after the nitrogen concentration in the culture liquid in the culture reactor is exhausted, the lipid content of the microalgae ( C. vulgaris ) is increased to 30 wt% or more Day). As a result of further cultivation in the absence of nitrogen (N) and subsequent cultivation in the presence of nutrients or organic carbon sources other than nitrogen (N), the lipid content of microalgae ( C. vulgaris ) , On day 14 after culture). Thus, it can be seen that microalgae containing a high amount of lipid can be cultured / harvested when microalgae of this period are harvested (recovered).

[Example 2] Preparation of cosmetic composition

1. Extraction and fractionation of microalgae

On the basis of the results of Example 1, microalgae were cultured under the most favorable culture conditions and sedimented and separated to harvest (recover) high-concentration microalgae containing a high content of lipid.

Specifically, in this experiment, C. vulgaris was selected as a microalgae, and a heterotrophic condition (light irradiation / CO ₂ / air blower supply) and an initial cell concentration of about 0.2 g / L in BG-11 culture medium , Glucose was initially injected at 1 g / L as an organic carbon source for optimal growth conditions and cultured at pH 8-12. Next, the nitrogen concentration and the carbon source of the culture medium in the reactor were depleted after a certain period of time, and a nitrogen-deficient culture solution containing other nutrients except for nitrogen (N) and glycerol Was injected at 0.1 g / L and cultured at pH 8 ~ 12. Then, the cultivated C. vulgaris was sedimented and dehydrated, and the optimum growth and high content of C. vulgaris were harvested (recovered).

Methanol extraction and phylogenetic fractionation were carried out on C. harvested C. vulgaris in the usual manner to obtain three samples. At this time, the methanol extraction was carried out at room temperature using a 70 wt% aqueous MeOH solution and repeatedly extracted twice for 2 days. Thereafter, filtration was carried out, followed by concentration under reduced pressure. Next, the obtained concentrate was mixed with distilled water, and then hexane extraction was carried out. Thereafter, an EtOAc (ethyl acetate) layer, a BuOH (butanol) layer and an aqueous layer were obtained through a systematic fraction extraction on the water layer from which the hexane layer was removed, and a final fraction The extracts were separately separated and purified to obtain three samples (Sample 1, Sample 2 and Sample 3). FIG. 18 is a flowchart of the systematic fraction extraction process.

2. Evaluation of skin improvement ability

The skin-cell stability test (cytotoxicity test) and skin-improving ability were evaluated for the obtained three fractionated extract samples as follows. 18), Sample 2 is the systematic fraction extract (# 11 in FIG. 18) obtained in the BuOH layer, Sample 3 is the systematic fraction obtained from the system obtained in the water layer Fraction extract (# 5 in Fig. 18).

(1) Cell culture

Human fibroblast (Detroit 551) was inoculated on the bottom of the culture dish and cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with penicillin (100 IU / ml), strap tomaisin (100 μg / ml) and 10% FBS ) Medium and incubated in an incubator containing CO ₂ at 37 ° C.

(2) Skin cell stability test (MTT assay)

fibroblast (Detroit 551 cell) was dispensed in 96 well plate at 1 × 10 ⁴ / well and cultured for 24 hours in cell culture conditions. The medium was discarded and washed with PBS. The medium was changed to a new medium, and the sample 1 and the sample 2 were treated to final concentrations of 0.01, 0.03, 0.1 and 0.5 wt%, and cultured for 24 hours. 4 μl of MTT solution (0.5% 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide solution) was added to each well and incubated for 4 hours. After removing the culture medium, 200 μl of dimethyl sulfoxide (DMSO) solution was added and shaken for 10 minutes. 100 μl of the solution was taken in 96 wells and the absorbance was measured at 540 nm by spectrophotometer.

The degree of cytotoxicity was expressed as a percentage based on the absorbance intensity of the control group using pure water according to Equation (1). The results are shown in Fig.

[Equation 1]

Cell viability (%) = (absorbance of test group / absorbance of control group) x 100

(3) skin cell collagen synthesis promoting ability test

Collagen is synthesized by procollagen in the cell, then secreted extracellularly and polymerized into collagen fiber. At this time, the N-terminal and C-terminal propeptides of procollagen were found to be released by endopeptidase. In this experiment, we investigated the ability of collagen synthesis to stimulate the procollagen in the culture medium secreted from the cells using C-terminal peptide (PIP) specific antibody of human procollagen type I.

First, Fibroblast (Detroit 551 cell) was dispensed into a 48-well plate at 5 × 10 ⁴ / well and cultured for 24 hours under cell culture conditions. The medium was discarded, washed with PBS, and then ground with a fresh starvation medium. Sample 1 and sample 2 were treated to final concentrations of 0.01, 0.1 and 0.5 wt%. Forty-eight hours after the sample preparation, 100 μl of peroxidase-labeled antibody solution was added to each well while the antibody-coated microtiter plate was placed on ice. Cells were centrifuged in each well of the plate, and 20 μl of the supernatant was added to each well of the antibody coated microtiter plate and reacted at 37 ° C for 3 hours. The medium was removed and washed four times with 250 μl of ice-cold PBS. 100 μl of substrate solution was added to each well and incubated for 15 minutes at room temperature in the dark. Then, 100 μl stop solution (1N H2SO4) was added and the absorbance at 450 nm was measured. The results are shown in Fig.

As shown in FIG. 19 and FIG. 20, in the case of Sample 1, the amount of PIP was increased to about 13% as compared with the control group at 0.01 wt% treatment. In the case of 0.05 wt% treatment, PIP synthesis was not affected. In case of 0.1 wt% treatment, PIP tendency decreased due to decrease of viability due to cell cytotoxicity. In the case of sample 2, the concentration of 0.01 wt%, which does not affect cell viability, was similar to that of the control. PIP was confirmed by treatment of 0.05 wt% and 0.1 wt% due to decreased viability.

Thus, it was found that 0.01 wt% of sample 1 had an effect on wrinkle improvement by increasing PIP synthesis without affecting cell viability.

(4) Tyrosinase Inhibition Test

The sample 3 was prepared at a concentration of 0.1, 0.5 and 1 wt% in 0.1 M sodium phosphate buffer (pH 6.8) and 50 μg / ml mushroom tyrosinase was prepared in the same buffer. And mixed.

Remarks Sample Blank Positive control group Sodium phosphate
buffer (pH 6.8) 50 μl 60 μl 50 μl Mushroom
tyrosinase 20 μl - 20 μl sample 10 μl - Positive control 50 μl

Then, 20 μl of 1.5 mM L-Tyrosine was added to each well, and the absorbance was measured at 490 nm every 10 minutes at 37 ° C. for 1 hour using a spectrophotometer. The inhibition rate was evaluated according to the following equation (2), and the results are shown in FIG.

&Quot; (2) "

Inhibition rate (%) = 1 (absorbance of sample solution / absorbance of control group) x 100

As shown in FIG. 21, when L-tyrosine was used as a substrate of tyrosinase, Sample 3 inhibited tyrosinase activity by 39% at 0.1 wt%, 65% at 0.5 wt%, and 88% at 1 wt% Effect. As compared with 2% by weight of Arbutin (34%), which is a raw material for whitening function, which was used as a positive control, Sample 3 showed better inhibitory effect on tyrosinase activity as the concentration was increased.

3. Preparation of cosmetic composition

The above-mentioned sample 1 and sample 2 were mixed with a conventional cream base to prepare a cream-form cosmetic composition. Specifically, a cosmetic composition of cream form was prepared with the components and contents as shown in Table 7 below.

&Lt; Component and content of cosmetic composition, unit weight% > Material name / Product name Formulation Example 1 Formulation Example 2 Danisol M 0.15 0.15 Danisol P 0.03 0.03 Propylene Glycol 5.00 5.00 EDTA-2Na 0.02 0.02 Carbopol # 980 0.64 0.64 Perfume 0.10 0.10 KF-96 (100 cc) 0.10 0.10 KHU 8 0.10 0.10 sample 0.10 0.10 Neo Heliopan E1000 4.50 4.50 Parsol MCX 7.50 7.50 KF-995 5.50 5.50 Al-Stearate 0.50 0.50 Base V 8.0 8.0 KSG-16 0.50 0.50 Abil EM 90 2.20 2.20 D.I.Water 5.00 5.00 TEA 1.14 1.14 Sample 1 0.10 - Sample 2 - 0.10 D.I.Water To 100 To 100

Cream Formulation Example 1 and Formulation 2 according to Table 7 were applied to the skin to evaluate changes in skin moisture content. The moisture content of the skin was measured by the static load capacity measurement method using the Cutometer MPA 580 (Japan) as a skin moisture measuring device and the moisture content of the skin surface was measured. At this time, after each of the formulation examples was applied, the initial measurement time after 30 minutes, 60 minutes, 120 minutes, 240 minutes and 360 minutes after application was measured once a day for 4 days, and the results are shown in FIG. In addition, the control (control) did not contain the microalgae extract (fraction), and it used a commercially available functional cream. In Fig. 22, sam-B is the cosmetic sample according to Formulation Example 1 in Table 7, and sam-D is the cosmetic sample according to Formulation Example 2 in Table 7 above.

As shown in Fig. 22, it was found that the cosmetic composition containing the microalgae extract (fraction) according to the present example had a higher moisture content of the skin than the control group in the market. In addition, the water content was evaluated to be highest after 30 minutes of application.

10: microalgae culture reactor 20: culture medium supply tank
30: air supply tank 40: carbon dioxide supply tank
51: microalgae separator 52: microalgae collection tank
53: Airbrush

Claims

Wherein microalgae are cultured in a culture medium of a microalgae culture reactor and cultured in the presence of carbon dioxide (CO ₂ ) at a pH of from 7.5 to 13.

A first culture step in which a microalgae is cultured in a culture medium of a microalgae culture reactor and cultured in the presence of carbon dioxide (CO ₂ ) and an organic carbon source at a pH of 7.5 to 13;
And a second culturing step of culturing in the presence of carbon dioxide (CO ₂ ) and an organic carbon source when the nitrogen is depleted by the first culture, wherein the second cultivation step is a second culture in a nitrogen deficiency culture broth.

3. The method of claim 2,
The initial concentration of the microalgae contained in the culture solution in the first culture step is 0.01 to 5.0 g / L, the initial concentration of the organic carbon source is 0.5 to 2 g / L,
If the organic carbon source is depleted by the first culture in the second culturing step, the organic carbon source is injected at a concentration of 0.5 to 2 g / L,
The organic carbon source injected in the second culture step may be one or more selected from glycerol and methanol,
Wherein the microalgae that have undergone the second culturing step have a lipid content of 30 wt% or more.

The method according to claim 2 or 3,
Wherein when the nitrogen and organic carbon sources are depleted by the first culture in the second culture step, a nitrogen deficiency culture medium containing a nutrient source other than nitrogen and an organic carbon source is injected into the culture reactor and cultured.

A method for cultivating a microalgae by a culture method according to any one of claims 1 to 4;
An extraction step of extracting microalgae cultured in the culturing step to obtain a microalgae extract; And
And a mixing step of mixing the microalgae extract obtained in the extraction step with the cosmetic.

A microalgae extract obtained by extracting microalgae having a lipid content of 30% by weight or more in a cell by culturing,
Wherein the microalgae extract is contained as an active ingredient for at least one selected from wrinkle improvement and whitening improvement.