KR20190023974A - A Suspension comprising Microalgae-Derived Pigment - Google Patents

Abstract

The present invention relates to a suspension comprising c-phycocyanin and chlorophyll a. Since the suspension is effective for preventing the photo-oxidation of the chlorophyll a, the suspension can be usefully used as pigment or the like capable of maintaining blue-green of chlorophyll a for a long time.

Description

A Suspension-containing Microalgae-Derived Pigment containing microalgae-

The present invention relates to a suspension comprising c-phycocyanin and chlorophyll a.

In general, a coloring matter is a substance to be added for coloring a product, and conventionally, an artificial coloring matter such as a tar coloring matter has been mainly used, but its use amount has been strictly defined in the presence of harmful components. Therefore, in recent years, the use frequency of natural colorants such as chlorophyll has been increased rather than the use of synthetic colorants in various foods and cosmetics in the world, but chlorophyll may be discolored or discolored during processing or preservation, It is difficult to carry out the present invention.

As a method for extracting chlorophyll-a from a natural raw material, a method of using an organic solvent such as ethanol or acetone has been widely used for the convenience of the process. However, chlorophyll-a extract containing an organic solvent can not be directly used as a health food, There is a problem that the chlorophyll is destroyed at a high rate on the extract. Further, in order to extract chlorophyll and use it as food, chlorophyll is extracted and then the organic solvent is removed by a method such as concentration under reduced pressure. In this process, a considerable amount of chlorophyll is lost.

In order to solve this problem, there has been developed a method in which chlorophyll-a separated and purified is subjected to a glycation reaction to apply it to health food and food materials, or gradually to replace the organic solvent with a large amount of aqueous solution, Methods are difficult to add only a single component of chlorophyll in a powdered form of health supplement and functional food, and it is difficult to maintain the stability of chlorophyll-a.

Accordingly, the inventors of the present invention have made extensive efforts to discover a composition for stabilizing chlorophyll-a, and as a result, confirmed that chlorophyll-a on the suspension is stabilized by phycocyanin, thereby completing the present invention.

Korean Patent Registration No. 10-1281858

One object of the present invention is to provide a suspension comprising c-phycocyanin and chlorophyll a.

Another object of the present invention is to provide a cosmetic composition for improving skin comprising a suspension containing c-phycocyanin and chlorophyll a.

It is another object of the present invention to provide a natural pigment comprising a suspension comprising c-phycocyanin and chlorophyll a.

One aspect of the present invention provides a suspension comprising c-phycocyanin and chlorophyll a.

The term "suspension " as used in the present invention means that particles of minute solid particles are dispersed and floating in a liquid.

The c-ficocyanin may be commercially available or may be derived from an extract of Spirulina without limitation, and the form may be powder or liquid.

Chlorophyll-a can be isolated from microalgae or plants by conventional methods used in the art. For example, it can be extracted by low temperature dark condition extraction, room temperature extraction, ultrasonic extraction, and reflux extraction. In order to obtain a large amount of chlorophyll a, reflux extraction is preferable, and ultrasonic extraction is also possible for convenience of work.

According to one embodiment of the present invention, the c-phycocyanin may be derived from a spirulina extract.

The spirulina extract of the present invention can be prepared by extracting with a conventional extraction solvent, and can be prepared by (a) reacting an anhydrous or a lower alcohol having 1 to 4 carbon atoms (e.g. methanol, ethanol, propanol, butanol, n-propanol, Butanol or the like), (b) a mixed solvent of the lower alcohol and water, (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, , (h) diethyl ether, (i) butyl acetate or (j) water. The extract may be impregnated or warmed at room temperature, and may be subjected to filtration and / or concentration during the extraction process, but may be prepared as an extract crude without purification.

According to one embodiment of the present invention, the extract may be obtained by extracting spirulina with an aqueous solution of calcium chloride (CaCl ₂ ).

According to one embodiment of the present invention, the suspension may include any one solvent selected from the group consisting of water, an organic solvent, and a mixed solvent thereof.

The organic solvent is not particularly limited as long as it is a solvent commonly used in the production of a suspension, and a mixed solvent of an organic solvent and water can be used, but it is preferable to prepare a suspension using water as a solvent.

According to one embodiment of the present invention, the suspension may contain c-phycocyanin and chlorophyll-a in a weight ratio of 3 to 4: 1 to 2, preferably c-phycocyanin and chlorophyll-a comprise 7 : 3. ≪ / RTI >

Another aspect of the present invention provides a cosmetic composition for skin improvement comprising a suspension comprising c-phycocyanin and chlorophyll a.

The cosmetic composition of the present invention may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizing agents, foaming agents, fragrances, surfactants, Or other cosmetically acceptable adjuvants, such as ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, And may further include adjuvants conventionally used in the same cosmetics field.

The cosmetic composition of the present invention may contain various bases and / or additives which are necessary and suitable for the formulation of the formulations, and may contain nonionic surfactants, silicone polymers, extender pigments, flavorings, preservatives, But are not limited to, disinfectants, oxidative stabilizers, organic solvents, ionic or nonionic thickeners, plasticizers, antioxidants, free radical scavengers, opacifiers, stabilizers, emollients, silicones, It may further comprise known compounds such as vitamins, insect repellents, perfumes, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, basic or acidifying agents, or coloring agents.

The cosmetic composition of the present invention may be in the form of a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in a water phase, a suspension, a microemulsion, a microcapsule, a microgranule, an ionic (liposome) And may be provided in the form of a fecal dispersant, a cream, a skin, a lotion, a powder, an ointment, a spray, or a conical stick. It can also be prepared in the form of a foam or an aerosol composition further comprising a compressed propellant.

Another aspect of the present invention provides a natural pigment comprising a suspension comprising c-phycocyanin and chlorophyll a.

The term "natural pigment" as used in the present invention means a coloring agent to be added for imparting color to a product, which is not artificially chemically synthesized.

The natural coloring matter of the present invention may contain various kinds of bases and / or additives necessary for formulation of the formulation, and may further include known functional ingredients within the range not to lose the function of the coloring matter.

The natural coloring matter of the present invention may further comprise a known anti-fading material.

The natural coloring matter of the present invention is a natural coloring matter selected from the group consisting of paprika pigment, anato pigment, cochineal pigment, carrot pigment, algae pigment, beryllium pigment, hibiscus pigment, seiso pigment, grape juice pigment, grape skin pigment, red cabbage pigment, May be further provided with a known natural food coloring matter such as a coloring matter, safflower yellow, cacao color, ukon coloring, ragoo coloring, chlorophyll coloring, red coloring pigment, Is not particularly limited.

The natural coloring matter of the present invention can be used as a beverage additive, a food additive, a textile dye or a cosmetic additive which is commonly used in the art, but is not limited thereto.

Since the suspension containing c-phycocyanin and chlorophyll a of the present invention is effective in preventing the photo-oxidation of chlorophyll-a, it can be usefully used as a coloring matter capable of maintaining the cyan color of chlorophyll-a for a long period of time have.

Figure 1 is a photograph showing the color change of chlorophyll-a for 10 days under normal light conditions.
2 is a photograph showing the color change of chlorophyll-a for 10 days under strong light conditions,
3 is a graph showing the residual amount of chlorophyll-a for 10 days under ordinary light conditions.
4 is a graph showing the residual amount of chlorophyll-a for 24 hours under strong light conditions.
Figure 5 shows the spectra of chlorophyll-a for 7 days under normal light and EtOH conditions.
Figure 6 is a graph showing the spectrum of chlorophyll-a for 7 days under normal light and c-picosane conditions.
Figure 7 shows the spectra of chlorophyll-a for 24 hours under strong light and EtOH conditions.
8 is a graph showing the spectrum of chlorophyll-a for 48 hours under strong light and c-picosane conditions.
Figure 9 is an HPLC chromatogram of chlorophyll a before light irradiation under c-phycocyanin conditions.
10 is an HPLC chromatogram of chlorophyll-a after intense light irradiation for 6 hours under c-phycocyanin condition.
FIG. 11 is an HPLC chromatogram of chlorophyll a before irradiation with light under EtOH conditions. FIG.
12 is an HPLC chromatogram of chlorophyll-a after irradiating strong light for 6 hours under EtOH condition.
FIG. 13 is an HPLC chromatogram of chlorophyll a before light irradiation in the third distilled water condition. FIG.
14 is an HPLC chromatogram of chlorophyll-a after intense light irradiation for 6 hours under the condition of the third distilled water.
FIG. 15 is a view showing Orbitrap MS spectra of chlorophyll a before irradiation of light under EtOH condition or c-picosian condition.
FIG. 16 shows a mass chromatogram showing no detection of chlorophyll-a after irradiation of light for 4 hours under EtOH condition. FIG.
FIG. 17 is a diagram showing a mass chromatogram of chlorophyll-a detected after 4 hours of light irradiation under c-picosyne condition.
18 is a graph comparing residual amounts of chlorophyll-a in ascorbic acid or c-ficosianne aqueous solution.
19 is a graph showing changes in MDA content due to photo-oxidation prevention of chlorophyll-a in EtOH, tertiary distilled water, or c-ficocyanin aqueous solution.
20 is a photograph showing the color change according to the ratio of each of phycocyanin and chlorophyll.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  1. Analysis of chlorophyll-a stabilization effect of c-ficocyanin

≪ 1-1 > Preparation method of chlorophyll-a suspension

1-1-1: Extraction method of chlorophyll-a

The extraction of chlorophyll-a was carried out by a modification of the method of Henriques et al. (Communicating Current Research and Educational Topics and Trends in Applied Microbiology Formatex (2007): 586-593). The microalgae, Chlorella vulgaris, was removed by centrifugation or filtration to remove the microalgae culture, and then vigorously vortexed for more than 20 seconds by adding more than 97% ethanol to the chlorella pellets. Chlorophyll-a was extracted at 4 ° C for 24 hours, and centrifuged to collect only the extract.

1-1-2: Extraction method of c-ficocyanin

The microalgae, Spirulina sp., Was centrifuged or filtrated to remove the microalgae culture, and then the c-phycocyanin was extracted with 20 to 30 mM CaCl ₂ at 4 ° C for 24 hours.

1-1-3: By each concentration  Preparation of chlorophyll-a suspension

The extract crude extract (extract of CaCl ₂ phase) extracted in Example 1-1-1 and the unpurified Example 1-1-2 was diluted with water at 0, 10, 20, 30, 40, 50, 60, 70, 90 and 100% (v / v), respectively, to prepare a chlorophyll-a suspension of each concentration.

<1-3> Measurement of color change of chlorophyll-a

A sample was prepared by mixing c-phycocyanin at the same concentration of chlorophyll-a at concentrations of 250, 500, 600, 1500 and 2000 ppm. Each sample was stirred on a stirrer (compact rocker CR300, FinePCR). Three white LEDs (Stech LED, Korea) were placed at the top of 20 cm from the sample and irradiated with light. Under the normal light (3 μmol · m ^-2 · s ^-1 ) condition, the color change of the suspension was observed at the same time every day for 10 days. In the condition of strong light (100 μmol · m ^-2 · s ^-1 ) Color change was confirmed.

<1-4> Residual amount analysis of chlorophyll-a

The residual amount of chlorophyll-a on the suspension over time under normal light and intense light conditions was determined using an absorbance meter (Genesis 10S UV / Vis, Thermo Scientific, USA). 630, 645, 663 and 750 nm, and the content of chlorophyll-a was analyzed using the Kaczmar equation of the following formula (1).

<1-5> Spectral analysis of chlorophyll-a

The spectra of chlorophyll-a on the suspension over time under normal light and strong light conditions were confirmed using an absorbance meter (Genesis 10S UV / Vis, Thermo Scientific, USA).

<1-6> HPLC  Chromatogram analysis

Chlorophyll-a was analyzed using HPLC (Agilent 1100 series). As control, chlorophyll-a (from spinach) purchased from Sigma-Aldrich, USA was used. The chlorophyll extract was injected with 20 μl of sample at a flow rate of 1 ml / min and a wavelength of 443 nm on an Eclipse XDB C18 column (150 × 4.6 mm, 5 μm particle sizes, Agilent Technologies, Palo Alto, USA) EA (75: 25) at room temperature.

<1-7> Orbitrap  MS spectra analysis

Mass spectrometry of chlorophyll-a was analyzed by Orbitrap Mass Spectrometer (LTQ orbitrap XL, ThermoSciator Scientific) at Sogang University Organic Reaction Research Center.

<1-8> Residual Stability Analysis of Chlorophyll-a

The absorbance of c-phycocyanin and chlorophyll mixture was measured under the condition of strong light (100 μmol · m ^-2 · s ^-1 ) by mixing the aqueous solution of vitamin C, known as antioxidant, with the same concentration of chlorophyll extract as c- Respectively. The maximum absorbance of chlorophyll-a over time was determined. L-Ascorbic acid was purchased from Sigma-Aldrich, USA and diluted to 1, 10, 50, 100, 500, 600 and 700 ppm in the third distilled water.

<1-9> TBA ( thiobarbituric  acid assay

10% (v / v) Linoleic acid was diluted in ethanol and 0.05 M phosphate buffer was prepared. The sample of Example 1-1-3 was mixed with 3 ml of 10% (v / v) linoleic acid, 3 ml of distilled water and 6 ml of 0.05 M phosphate buffer, and the final volume was adjusted to 15 ml Respectively. Each sample under strong light conditions was sampled every two hours.

0.5 ml of 20% TCA, 0.25 ml of TBA and 3 ml of sample were mixed and heated at 98 ° C for 10 minutes. The sample was cooled and centrifuged at 3000 rpm for 10 minutes. The supernatant was analyzed by UV / VIS spectrophotometer Lt; / RTI &gt; at 532 nm.

Example  2. Chlorophyll a The color change of chlorophyll-a side Check color suppression effect

In order to confirm the stabilizing effect of the chlorophyll-a and not when c- Pico on suspension for 10 days normal light ^{(3μmol · m -2 · s -1} ) , or 24 hours strong light conditions (100μmol · m ^-2 · s ^-1 for ), The color change of chlorophyll-a was measured.

As a result, it was confirmed that the change in color was greatest in the suspension containing EtOH over time in both the normal light (FIG. 1) and the strong light (FIG. 2) conditions.

Namely, through the above results, the chlorophyll-a discoloration inhibiting effect of c-phycocyanin was confirmed.

Example  3. Confirmation of chlorophyll-a stabilization effect of c-ficocyanin by residue analysis

In order to confirm the stabilizing effect of the chlorophyll-a and not when c- Pico, 10 in normal light ^{(3μmol · m -2 · s -1} ) or 48 hours strong light conditions ^{(100μmol · m -2 · s -1} ) while the conditions for , The residual amount (%) of chlorophyll-a was analyzed.

As a result, both the normal light (FIG. 3) and the strong light (FIG. 4) conditions showed the greatest reduction in the residual amount of chlorophyll-a in the suspension containing EtOH over time.

That is, through the above results, the stabilizing effect of chlorophyll a of c-phycocyanin against EtOH was confirmed.

Example  4. Spectrum  Determination of chlorophyll-a stabilization effect of c-ficocyanin according to the analysis

In order to confirm the stabilizing effect of the chlorophyll-a and not when c- Pico on the suspension, the embodiment in normal light ^{(3μmol · m -2 · s -1} ) and strong light conditions ^{(100μmol · m -2 · s -1} ) Condition 1-5 were performed to analyze the chlorophyll-a spectrum for 7 days.

As a result, it was confirmed that the spectral change of chlorophyll-a was large in the suspension containing EtOH, compared with the suspension containing c-phycocyanin in both ordinary light (FIGS. 5 and 6) and strong light (FIG. 7 and FIG. 8) conditions.

That is, through the above results, the stabilizing effect of chlorophyll a of c-phycocyanin against EtOH was confirmed.

Example  5. HPLC  Determination of chlorophyll-a stabilization effect of c-ficocyanin by chromatogram analysis

In order to confirm the effect of stabilizing chlorophyll-a of c-picosien on the suspension of c-picosylan aqueous solution, EtOH or tertiary distilled water, after 6 hours of irradiation with strong light (100 μmol · m ^-2 · s ^-1 ) -6 was performed to analyze the HPLC chromatogram of chlorophyll-a.

The chlorophyll-a on the suspension was compared with the control, and it was separated into a single peak at 4.7 minutes in the same time zone. The chlorophyll-a on the suspension was exposed for 6 hours under strong light conditions and the HPLC chromatogram of chlorophyll-a was analyzed to find the highest single peak in the suspension of c-phycocyanin and chlorophyll-a (Figures 9-14) .

Example  6. Confirmation of chlorophyll-a stabilization effect of c-ficocyanin by mass analysis

In order to confirm the effect of stabilizing chlorophyll-a of c-ficocyanin on c-picosylan aqueous solution or EtOH suspension, Example 1-7 was carried out after 4 hours of irradiation with strong light (100 μmol · m ^-2 · s ^-1 ) And analyzed the Orbitrap MS spectra of chlorophyll a.

Chlorophyll-a was detected in both suspensions containing EtOH or c-phycocyanin before light irradiation (Fig. 15). After 4 hours of intense light irradiation and again analyzed, chlorophyll-a was observed in the suspension containing c-phycocyanin (Fig. 16), whereas chlorophyll-a was not detected in the suspension containing EtOH (Fig. 17).

Namely, through the above-mentioned results, the effect of stabilizing chlorophyll-a of c-phycocyanin was confirmed.

Example  7. Confirmation of chlorophyll-a stabilization effect of c-phycocyanin by absorbance measurement

In order to compare the stabilization effect of chlorophyll a with ascorbic acid and c-picosan, the stability of chlorophyll-a over time was analyzed by performing the above Examples 1-8.

As a result, the content of chlorophyll-a was reduced by 40% or more immediately upon mixing chlorophyll-a with ascorbic acid. However, it was confirmed that the residual amount of chlorophyll-a on the c-phycocyanin suspension remained 50% after 4 hours in strong light (FIG. 18).

Example  8. Chlorophyll a of c-ficocyanin according to TBA analysis Mineralization  Confirm prevention effect

In order to confirm the effect of chlorophyll-a photo-oxidation of c-picosien on the suspension of c-ficosianne aqueous solution, EtOH or tertiary distilled water, the change in the concentration of MDA (malondialdehyde) was measured by carrying out Example 1-9.

As a result, the concentration of MDA was the greatest increase in the suspension containing EtOH, while the concentration of MDA was the lowest in the suspension containing c-phycocyanin (Fig. 19).

That is, through the above-mentioned results, the effect of preventing the photo-oxidation of chlorophyll-a of c-phycocyanin was confirmed.

Example  9. Determine the optimum ratio of c-ficocyanin and chlorophyll-a

In order to confirm the optimum ratio of c-ficocyanin and chlorophyll-a capable of stabilizing chlorophyll-a, strong light (100 μmol · m ^-2 · s ^-1 ) was added to the suspension of each concentration of Example 1-1-3, And the color change of chlorophyll-a was measured.

As a result, a suspension of c-phycocyanin and chlorophyll a 7: 3 ratio (Fig. 20) was observed in that the cyan color of the suspension remained the longest when c-phycocyanin and chlorophyll a were mixed in a ratio of 7: The chlorophyll-a was found to be the most stable.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

A suspension comprising c-phycocyanin and chlorophyll a. The suspension according to claim 1, wherein the c-picosienne is derived from a spirulina extract. The suspension according to claim 2, wherein the extract is a spirulina extracted with an aqueous solution of calcium chloride (CaCl ₂ ). The suspension according to claim 1, wherein the suspension comprises any one solvent selected from the group consisting of water, an organic solvent and a mixed solvent thereof. 2. A suspension according to claim 1 wherein said suspension comprises c-picocyanin and chlorophyll-a in a weight ratio of 3 to 4: 1 to 2. A cosmetic composition for skin improvement comprising the suspension of claim 1. A natural colorant comprising the suspension of claim 1.

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