KR20140063296A - Method for preparation of extracts of berries and colored potatos comprising anthocyanin-based pigments using pectinex ultra sp-l - Google Patents

Abstract

The present invention relates to a method for preparing extracts of berries and colored potatoes with increased anthocyanin-based pigments by pre-treating pectinex enzymes, and more specifically, to a method for preparing extracts of berries and colored potatoes, wherein the method includes a step of pre-treating berries and colored potatoes with pectinex enzymes and a step of extracting materials treated with the pectinex enzymes using water, alcohol with 1-6 carbon atoms, or its mixture solvent.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for preparing an extract of berries or colored potatoes containing anthocyanin-based pigments using PECTINEX ULTRA SP-L,

The present invention relates to a method for preparing an extract of berries or color potatoes having an increased anthocyanin pigment content by pretreatment with a pectinex enzyme, and more particularly, to a method for producing beret or color potato extract comprising: a) treating a berries or a color potato with a Pectinex enzyme; And b) extracting the raw material treated with the Pectinex enzyme with water, an alcohol having 1 to 6 carbon atoms or a mixed solvent thereof, a method for producing an extract of berries or colored potatoes, ≪ / RTI > Or a composition containing the same, and a method for separating anthocyanin from an extract prepared by the method.

Natural pigments are found in natural flora and fauna, or are produced by microorganisms. Unlike artificial pigments, they have high in-vivo stability and high reliability. Since natural pigments have a natural color tone, they can be combined in various ways, and they can be safely used in various fields because they are food ingredients that can be edible. In addition, the coloring effect is excellent, and the production and purification techniques are simpler than the artificial coloring, which also has a manufacturing cost reduction effect. In addition to the above advantages, natural pigments have various physiologically active functions according to raw materials and colors, and thus their functional and nutritional value are highly evaluated.

Anthocyanin is one of the representative natural pigments. It is a pigment that expresses various colors ranging from red to blue in leaves, flowers and fruits of various plants, and is also referred to as a flower. More than 600 species of anthocyanins found in plants are all anthocyanin glycosides, which have a structure in which the basic structure of flavylium (2-phenylbenzopyrilim) is substituted with a hydroxy group or a methoxy group.

The major features of anthocyanin pigments are relatively soluble in water, but they are insoluble in non-hydroxy solvents and possess potential properties very sensitive to pH changes. In particular, anthocyanin pigments have antioxidant efficacy and whitening effect, which are used as raw materials in the cosmetics industry and have a preventive effect against chronic diseases such as cardiovascular diseases and cancerous diseases such as cancer. In addition, it has a strong antioxidant activity in foods, and thus can improve the health function of the food.

Recently, in an age of LOHAS (Life Style of Health and Sustainability), efforts are being made to separate anthocyanin pigments from natural raw materials. The most important raw materials of anthocyanin-based natural pigments are olive, berry-like berries, purple sweet potatoes, and color potatoes. However, most of these crops are crops affected by climate. In particular, the anthocyanin pigments have various kinds and contents depending on the plant, and their content and physiological performance are changed in the processing such as compression, sterilization, and drying after harvest. In addition, color potatoes and audi have higher anthocyanin content than grapes, and thus are highly evaluated for their use as an anthocyanin pigment. However, in Korea, crops capable of separating anthocyanin pigment are rare, and most of them depend on imports.

Accordingly, the present inventors have made intensive efforts to develop a technology capable of separating large amounts of anthocyanin-based pigments from color potatoes (autonomous and red marl) and berries (ody and macromolecule) which are domestic functional crops. As a result, And that the extraction efficiency of the anthocyanin pigment can be remarkably increased when the Pectinex enzyme is extracted and extracted before the extraction. Thus, the present invention has been completed.

It is an object of the present invention to provide a method for preparing an extract of berries or colored potatoes with an increased anthocyanin pigment content, which comprises pre-treating pectinex enzyme to berries or colored potatoes.

Another object of the present invention is to provide an extract of berries or color potato prepared by the above method; And a composition comprising the same.

It is another object of the present invention to provide an anthocyanin preparation method comprising separating anthocyanin from berries or color potato extract containing anthocyanin produced by the above method.

In one aspect of the present invention, there is provided a method for producing an extract of berries or color potato having an increased anthocyanin pigment content, comprising the step of pre-treating pectinex enzyme in berries or colored potatoes.

A) treating the vertebrate or colored potato with a Pectinex enzyme at a temperature of 20 ° C to 60 ° C for 2 to 8 hours; And b) extracting the raw material treated with the Pectinex enzyme with water, an alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof.

The step a) is a step of treating the pectinex enzyme in order to improve the extraction yield of anthocyanin pigment in the production of berries or color potato extract. The treatment of the pectinex enzyme can be performed by adding Pectinex Ultra SP-L to a suspension of berries or color potatoes adjusted to pH 4, but is not limited thereto.

The term "berries" in the present invention means small fruits such as shrubs of berries. For the purpose of the present invention, the berries include anthocyanins and can be used for extracting anthocyanins. Examples of the berries include strawberry, blackberry, mulberry, blueberry, raspberry, currant, Cranberry, cherry, gooseberry, acerola, elderberry, and the like, preferably brambles or audi, but are not limited thereto. The brambler is a generic name of a wild raspberry belonging to the rose family. It contains a large amount of polyphenol in the fruit and is known to have an anticancer effect, inhibition of aging, and prevention of arteriosclerosis. For the purpose of the present invention, the bokbunja refers to a fruit capable of increasing the extraction of anthocyanin pigment upon extraction through pretreatment of pectinex enzyme. The above-mentioned Odi is a fruit of Morus alba L., a deciduous arboreous tree belonging to the mulberry family, and is a fruit of flavonoids, stilbenes, prenylflavonoids, coumarin, It is known to contain various physiologically active substances such as deoxynoijirimycin and rutin. In particular, unlike ordinary fruits, audi contains pigment in pulp as well as in pericarp.

In the present invention, the term "color potato" is a potato having the same color as that of purple color. Examples thereof include, but are not limited to, The self-portrait is a type of color potato, which means potatoes developed by the Agricultural Research Center of Higher Education in 2007 Korea Rural Development Administration. It is purple in both the inside and outside, and contains a large amount of anthocyanin, which is not present in the white potatoes, and is excellent for prostate cancer, aging, gout and hypertension. The above-mentioned Hongyoung is a kind of color potato, and it means a potato which was developed by the Agricultural Research Center for Senior Citizens in the Republic of Korea, Rural Development Administration in 2007 and contains a large amount of anthocyanin component which is not present in the existing white potatoes. Hongyoung is known to have anti-cancer activity and whitening effect in both the inside and the inside.

Colored potatoes such as berries such as bokbunja or audi, and self-colored or red-colored potatoes, including anthocyanin-based pigments, can be used as raw materials for the extraction of anthocyanin-based pigments. In the method of the present invention, the content of the anthocyanin-based pigment is increased through pretreatment with a pectinex enzyme to a color potato such as berries such as bokbunja or audi, or self-smear or red tincture before extraction.

In the present invention, the term "anthocyanin pigment" is a pigment which expresses various colors ranging from red to blue in leaves, flowers and fruits of various plants. In the basic structure of flavorium (2-phenylbenzopyrilim) The compounds having a structure in which the substituents are substituted are present in more than 600 kinds in plants. Examples of the anthocyanin dye include Pelargonidin, Cyanidin, Delphinidin, Peonidin, Petunidin, Malyidin, and the like. It does not. The anthocyanin pigment has antioxidative effect and whitening effect and can be used as a raw material for the cosmetics industry and has a preventive and therapeutic effect on chronic diseases such as cardiovascular diseases and cancer. In addition, many anthocyanin pigments have strong antioxidant activity and are known to improve the health function of foods. Despite this demand, a method of extracting the anthocyanin pigment in a high yield was required because the content of the anthocyanin pigment was low in the plant. The method of the present invention is characterized by dramatically improving the content and extraction yield of anthocyanin pigment.

The term "Pectinex enzyme" in the present invention refers to an enzyme preparation comprising polygalacturonase, a species of pectinase derived from Aspergillus aculeatus , Is Novozyme's Pectinex Ultra SP-L, Novozyme enzyme. In one embodiment of the present invention, optimized treatment conditions of the Pectinex Ultra SP-L were determined in order to extract a high content of anthocyanin-based pigments from bokbunja, oudy, selfish or red mullet.

Specifically, in the case of pretreatment of pectinex enzyme to bokbunja, the treatment temperature and the treatment time have a greater effect on the anthocyanin content than the concentration considering the enzyme activity. Therefore, the treatment of the Pectinex Ultra SP-L enzyme can be preferably performed at a temperature of 20 to 60 캜 for 2 to 8 hours, more preferably at a temperature of 30 to 50 캜 for 2 to 3 hours Can be performed.

In an embodiment of the present invention, when the enzyme was treated with 1.5 mL / 100 L of enzyme, the enzyme treatment temperature was 40 ° C and the enzyme treatment time was 2 hours, the anthocyanin content of the Pectinex Ultra SP-L enzyme was increased 8.7 times or more (Experimental Examples 2-3 and Table 9). It was also confirmed that when the enzyme concentration of Pectinex Ultra SP-L was performed at an enzyme concentration of 1.0 mL / 100 L, an enzyme treatment temperature of 40 DEG C, and an enzyme treatment time of 5 hours, the content of polyphenol could be enhanced compared with that of the control Examples 2-6 and Table 12). In the case of flavonoid, the highest flavonoid content was obtained at the enzyme concentration of 1.0 mL / 100 L, the enzyme treatment temperature of 20 ° C and the enzyme treatment time of 8 hours. In addition, the pretreatment of Pectinex Ultra SP-L enzyme before bacteriological extraction showed an increase in DPPH radical scavenging activity and SOD-like activity, suggesting that it may be useful for preparing antioxidant-active extracts Reference).

In addition, in the case of pre-extraction treatment of the ODiE pectinex enzyme, the treatment temperature has a greater effect on the anthocyanin content than the concentration considering the enzyme activity and the treatment time. Thus, the treatment of the pectinex enzyme can be carried out preferably at a temperature of 20 ° C to 60 ° C, more preferably at a temperature of 20 ° C to 40 ° C, but is not limited thereto. In addition, the treatment of the pectinex enzyme can be carried out preferably at a temperature of 20 ° C to 60 ° C and for 2 hours to 8 hours, more preferably at a temperature of 20 ° C to 40 ° C and for 2 hours to 5 hours, Do not.

In an embodiment of the present invention, when Pectinex Ultra SP-L enzyme was treated with an enzyme concentration of 1.0 mL / 100 L, an enzyme treatment temperature of 20 ° C and an enzyme treatment time of 2 hours, the anthocyanin content was about 7 times higher than that of the control group (Experimental Examples 3-3 and Table 16). When Pectinex Ultra SP-L enzyme was treated at an enzyme concentration of 1.0 mL / 100 L, an enzyme treatment temperature of 20 ° C, and an enzyme treatment time of 2 hours, it was confirmed that the content of polyphenol could be increased compared with the control. In the case of flavonoid, the highest flavonoid content was shown at an enzyme concentration of 1.0 mL / 100 L, an enzyme treatment temperature of 40 ° C and an enzyme treatment time of 5 hours (Experimental Examples 3-6 and 20). In addition, pretreatment of Pectinex Ultra SP-L enzyme before ODI extraction showed an increase in DPPH radical scavenging activity and SOD-like activity, suggesting that it may be useful for preparing antioxidant-active extracts Reference).

In addition, in the case of pretreatment of pectinex enzyme for self-administration, the treatment temperature and the treatment time have a greater effect on the anthocyanin content than the concentration considering the enzyme activity. Thus, the treatment of the Pectinex Ultra SP-L enzyme can be preferably performed at a temperature of 20 to 60 캜 for 2 to 8 hours, more preferably at a temperature of 20 to 40 캜 for 2 to 3 hours Can be performed.

In an embodiment of the present invention, when the Pectinex Ultra SP-L enzyme was treated at an enzyme concentration of 0.5 mL / 100 L, an enzyme treatment temperature of 20 ° C and an enzyme treatment time of 5 hours, the anthocyanin content of the enzyme was 3.1 times higher than that of the control (Experimental Example 4-3 and Table 24). In addition, it was confirmed that when the enzyme concentration of Pectinex Ultra SP-L was 0.5 mL / 100 L, the enzyme treatment temperature was 40 ° C, and the enzyme treatment time was 2 hours, the content of polyphenol could be increased compared to the control. In the case of flavonoid, the highest flavonoid content was exhibited at an enzyme concentration of 1.5 mL / 100 L, an enzyme treatment temperature of 60 ° C and an enzyme treatment time of 5 hours (Experimental Examples 4-6 and Table 28). In addition, pretreatment of Pectinex Ultra SP-L enzyme before self-extraction showed an increase in SOD-like activity, suggesting that it may be useful for preparing antioxidant-active extracts (see Table 29).

In addition, in the case of pretreatment of pectinex enzyme to Hongyoung, the treatment temperature and the treatment time have a greater influence on the anthocyanin content than the concentration considering the enzyme activity. Thus, the treatment of the Pectinex Ultra SP-L enzyme can be preferably performed at a temperature of 20 to 60 캜 for 2 to 8 hours, more preferably at a temperature of 30 to 50 캜 for 6 to 8 hours Can be performed.

In an embodiment of the present invention, when the Pectinex Ultra SP-L enzyme was treated at 0.5 mL / 100 L of enzyme concentration, 40 DEG C of the enzyme treatment time and 8 hours of the enzyme treatment time, anthocyanin content of about 3.7 times higher than that of the control group (Experimental Examples 5-3 and Table 32). In addition, it was confirmed that when the enzyme concentration of Pectinex Ultra SP-L was performed at an enzyme concentration of 1.5 mL / 100 L, an enzyme treatment temperature of 60 ° C, and an enzyme treatment time of 5 hours, the content of flavonoid could be increased as compared with the control group 5-6 and Table 36). In addition, pretreatment of Pectinex Ultra SP-L enzyme before red-leaf extraction showed an increase in SOD-like activity, suggesting that it may be useful for preparing antioxidant-active extracts (see Table 37).

The step b) comprises extracting berries or color potatoes having an increased anthocyanin pigment content by extracting berries or color potatoes pretreated with pectinex enzyme with water, an alcohol having 1 to 6 carbon atoms or a mixed solvent thereof, to be. When the Pectinex enzyme is extracted in the step b), the extraction yield of the anthocyanin pigment is enhanced compared to the raw material without the pretreatment.

The extract of berries or colored potatoes is an extract obtained by extracting berries or colored potatoes, wherein water is added to the pulverized product of berries or colored potatoes; A mixed solvent having an alcohol polar solvent having 1 to 6 carbon atoms, such as methanol, ethanol, and butanol, preferably a lower alcohol having 1 to 4 carbon atoms, or a mixing ratio of 1: 0.1 to 1:10, However, it is preferable that the concentration of ethanol as ethanol is 80% to 90%.

The ethanol extraction process of the berries or the color potatoes is not particularly limited, but may be performed at a sample to solvent ratio of 10% (w / v) to 20% (w / v) at a temperature of 30 ° C to 70 ° C for 1 hour to 5 hours .

In particular, ethanol extraction of the brambles can preferably be performed at a sample to solvent ratio of 10% (w / v) to 15% (w / v) at a temperature of 30 ° C to 50 ° C for 3 hours to 5 hours. In one embodiment of the present invention, it was confirmed that the largest amount of anthocyanin was exhibited under the conditions of the sample to solvent ratio of 15% (w / v), the extraction temperature of 30 ° C and the extraction time of 1 hour.

In addition, ethanol extraction of Hongyoung can preferably be carried out at a sample to solvent ratio of 15% (w / v) to 20% (w / v) at a temperature of 40 ° C to 60 ° C for 3 hours to 5 hours. In one embodiment of the present invention, it was confirmed that the maximum anthocyanin content was exhibited under the conditions of the sample to solvent ratio of 20% (w / v), the extraction temperature of 50 ° C and the extraction time of 5 hours.

In another aspect, the present invention relates to a berries or colored potato extract prepared by the above method; And a composition comprising the same.

The extract prepared by the method of the present invention contains high anthocyanin pigment and antioxidant activity, and thus can be used not only for the separation process of the anthocyanin pigment, but also for various medicines, health foods and cosmetics fields requiring anthocyanin pigment. The formulations of the above medicines, health foods, cosmetics and the like can be produced through conventional methods in the art.

In another aspect, the present invention provides a method for producing anthocyanins, comprising separating anthocyanin from berries or colored potato extracts comprising anthocyanin produced by the method.

The method for separating anthocyanin from berries or color potato extract prepared by the method of the present invention can be carried out through a conventional separation and purification method in the art. The method for producing an extract of the present invention is a method for producing an extract having an increased anthocyanin content, and thus an anthocyanin can be obtained with high yield.

Since the method of preparing the extract of Berry or Color Potato by pretreatment of the Pectinex enzyme of the present invention results in a significant increase in the content of anthocyanin-based natural pigment in the preparation of the extract, , Food or cosmetic field.

FIG. 1 shows the appearance of bokbunja, audi, and color potato (selfishness, Hongyoung). The color of the audi was darker than that of the bokbunja, and the color potato showed more pigmentation in the skin than meat quality. It was a purple line.
FIG. 2 shows the appearance of a 60% ethanol crude extract of bokbunja, audi and color potato (selfish, red), and the appearance of crude extract using 60% ethanol showed the darkest color, and bokbunja, In order.
FIG. 3 is a graph showing the reaction surface contour of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L using brambles.
FIG. 4 is a graph showing reaction surface contours of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L using an audi.
FIG. 5 is a graph showing reaction surface contours of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L using self-administration. FIG.
Fig. 6 shows reaction surface contours of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L using red pepper.
7 is a graph showing reaction surface contours of total anthocyanin content after extraction of ethanol from brambles.
8 is a graph showing reaction surface contours of total anthocyanin content after ethanol extraction of Hongyoung.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  1: Selection of raw materials for anthocyanin-based natural pigments

Example  1-1: Preparation of materials and reagents

The color potatoes used in this experiment were purchased from Auction, and Odin was used in Yongkwang, Jeollanam and bokbunja was harvested in Wonju, Jeonbuk. Reagents were used.

Example  1-2: Crude extract  Produce

The crude extracts were prepared by lyophilization of color potatoes (red, green, blue), olive oil, and bokbunja. The extracts were extracted with 200 ml of 60% ethanol as a solvent on a 1 g (dry basis) After extracting for 1 hour at 40 ° C, it was filtered with Wattman No 2, and the obtained filtrate was concentrated at 40 ° C in a vacuum rotary condenser to prepare a crude extract.

Example  1-3: Determination of total anthocyanin content

The total anthocyanin content was measured as follows using a molecular extinction coefficient of 65.1 according to the Lambert-Beer law.

The resulting mixture was subjected to extraction with an ultrasonic mill for 1 hour after addition of 1.5 ml of 1.5 N HCl-95% EtOH (15: 85, v / v) to 2 g of lyophilized berry, brambles and colored potatoes The solution was diluted with 100 mL of the same solvent, and then allowed to stand at room temperature for 2 hours in a dark place. The supernatant was diluted 20-fold and absorbance was measured at 535 nm.

Example  1-4: Total polyphenol content measurement

The total polyphenol content was measured by slightly modifying the Folin-Denis method. In other words, 2 ml of 2% Na ₂ CO ₃ solution was added to 2 ml of the extract, followed by reaction at room temperature for 3 minutes. Then, 0.4 ml of 50% Folin-ciocalteu solution was added, and the mixture was allowed to stand at room temperature for 30 minutes and absorbance was measured at 725 nm. At this time, the content was determined from a standard calibration curve prepared using tannic acid as a standard substance for obtaining the total phenol content.

Example  1-5: Determination of total flavonoid content

To the total flavonoid content, 1 ml of 10% Al (NO ₃ ) ₃ solution and 0.1 ml of 1 M potassium acetate solution were added to 0.5 ml of the extract, and furthermore, 4.3 ml of distilled water was further added. Then, the sample solution was reacted at room temperature for 40 minutes, and the absorbance was measured at 415 nm. The flavonoid content was measured from the standard calibration curves prepared using quercetin, which is a standard substance for the total flavonoid content.

Example  1-6: DPPH Radical Scatters  Measure

1 ml of the sample was placed in a test tube and 4.0 ml of 95% (v / v) ethyl alcohol was added to prepare an experimental solution. 1.0 ml of a 0.2 mM DPPH solution was added to the solution, and the reaction was carried out at room temperature for 30 minutes. Absorbance was measured at 517 nm using a visible spectrophotometer (UV-1601, Shimadzu, Tokyo, Japan).

In the case of the control group, the same procedure as in the experimental group was performed using distilled water instead of the sample, and the scavenging ability (%) of the free radical DPPH radical was calculated using the following equation.

 DPPH radical scavenging activity (%) = (1-A / B) X 100

A: Absorbance of sample, B: Absorbance of blank

Example  1-7: Hydroxy Radical ( Hydroxy radical ) Scatters

Each sample dissolved in distilled water was added to a predetermined amount of 10 mM potassium phosphate buffer (pH 7.4) containing 2.8 mM 2-deoxy-D-ribose and 1.4 mM H ₂ O ₂ And EDTA / FeCl ₂ (100 uM EDTA pH 7.0, 20 uM FeCl ₂ ) were added to the mixture to make 2.0 mL of the final reaction solution, followed by reaction at 37 ° C for 4 hours. Then, the reaction was stopped with 10% TCA (trichloroacetic acid), mixed well with 1% TBA (thiobarbituronic acid), reacted at 95 ° C for 20 minutes, and then cooled at room temperature and absorbance was measured at 532 nm.

Example  1-8: Hydroxy peroxide ( Hydroxy Peroxide ) Scatters

100 μl of PBS and 20 μl of the sample dissolved in distilled water were added to a 96-well microplate, and 1 mM H ₂ O ₂ was added to the plate for 5 minutes. Then, 30 μl of 1.25 nM ABTS and 30 μl of 1 U / ml peroxidase Was added and reacted at 37 ° C for 10 minutes, and the absorbance was measured at 405 nm.

Examples 1-9: SOD - like ( superoxide dismutase - like ) Active measurement

3 ml of 7.2 mM pyrogallol and 0.2 ml of Tris-HCl buffer (50 mM Tris (hydroxymethyl) aminomethane + 10 mM EDTA, pH 8.5) and 0.2 ml of pyrogallol were added and reacted at 25 ° C for 10 minutes. 1 ml of HCl was added to stop the reaction.

The amount of oxidized pyrogallol in the reaction solution was measured at 420 nm, and the SOD-like activity was expressed as a percentage of absorbance difference between the addition of the sample solution and the addition of no sample. SOD-like activity was calculated using the following equation.

SOD-like activity (%) = (1- (absorbance of sample added / absorbance of non-additive) X 100

Example  1-10: Elastase ( Elastase ) Inhibitory activity

The sample was diluted with a 0.2 M Tris-Cl buffer (pH 8.0) according to the method of James et al. (10-1000 μg / ml) according to the dilution drainage and 0.2 M Tris-Cl buffer (pH 8.0) Was added and then 20 μl of 0.8 mM N-succinyl- (all) 3-p-nitroanilide (N-succinyl- (Ala) 3-p-nitroanilide) was added. The reaction mixture was further incubated at 25 ° C for 10 minutes and then added with 20 μl of 1.0 μg / ml of PPE (porcine pancreatic elastase).

The reaction mixture was further cultured at 25 ° C for 20 minutes, and then the reaction was stopped with ice cold solution and absorbance was measured at 405 nm. As a control, the enzyme activity was measured by adding distilled water instead of the sample. Elastase inhibitory activity was calculated using the following equation.

Elastase inhibitory activity (%) = [1- (B-C) / (A-D)] X 100

B: Absorbance of the sample after reaction by adding enzyme, C: Absorbance of the sample after reaction by adding distilled water instead of enzyme, D: Absorbance of the sample after reaction by adding distilled water instead of enzyme, Instead of the enzyme, the absorbance after the addition of distilled water, respectively,

Example  2: Development of extraction pretreatment technology using enzyme

Example  2-1: Experimental design of enzyme pretreatment process

In the present invention, Pectinex Ultra SP-L from Novozyme (Bagsvaerd, Denmark) was used as a pretreatment process before extracting anthocyanin using ethanol to improve the yield during anthocyanin extraction. Specifically, Pectinex Ultra SP-L is an enzyme preparation for polygalacturonase, a pectinase from Novozyme's Aspergillus aculeatus .

The enzyme pretreatment of Pectinex Ultra SP-L was carried out by adding 8 times water to lyophilized brambles, olive oil, autogenous spots, and red sea bream and adjusting the pH to 4. The Pectinex Ultra SP-L was added at a concentration of 250 rpm (Si-900R, Jeio Tech, Korea) at 20-60 ° C for treatment temperature (X ₂ ) and 2-8 hours for treatment time (X ₃ ). After enzymatic treatment, enzymes were removed by centrifugation at 4000 rpm for 10 minutes with principle separator (UNION 55R, Hanil, Korea).

Specifically, the pretreatment conditions for the enzyme pretreatment of Pectinex Ultra SP-L were 0.5-1.5 (mL / 100L) for the concentration (X ₁ ) and 20-60 ° C for the treatment temperature (X ₂ ) For the time (X ₃ ), 2-8 hours were set as the interest level (Table 1). These independent variables are coded in three levels (-1, 0, 1) and set according to the central synthetic programming method. Total anthocyanin content, total polyphenol content, total flavonoid content, DPPH radical scavenging ability, SOD-like activity, yield after enzyme pretreatment, and freeze-drying yield were determined as the dependent variables affected by these factor variables. The experimental design for the treatment conditions of the enzyme pretreatment process was based on the central composite design and MINITAB statistical software was used for the reaction surface analysis.

Independent variables Symbol Levels -One 0 One Conc. of enzymatic activity (mL / 100 L) X ₁ 0.5 One 1.5 Temperature (° C) X ₂ 20 40 60 Time (hr) X ₃ 2 5 8

200 mL of 60% ethanol (95%) was added to 1 g (dry basis) of each enzyme-pretreated sample, followed by the addition of 200 mL of an aqueous solution containing 40 mL of water using an ultrasonic washing machine (Power Sonic 420, Hwashintech, KOREA) After filtration with Whatman No. 1, the filtrate obtained was concentrated under reduced pressure at 40 ° C using a vacuum rotary evaporator (Buchi R-114, SWISS) and lyophilized (Ilshin Biobase, KOREA). The analytical method was measured in accordance with the method described in Example 1 above.

Experimental Example  1: Screening and content analysis of anthocyanin-based natural pigments

In order to select raw materials for extracting anthocyanin-based natural pigments by the method described in Example 1, bokbunja, audi, selfishness and red sea were analyzed.

As a result, in the case of the external shape, the color was darker than that of the bokbunja. In the case of the self potatoes such as self potatoes and Hongyoung, the pigment appeared darker on the skin than on the flesh, and selfishness showed a purple line and Hongyoung showed a purple line. In addition, when the appearance of 60% ethanol extract of bokbunja, odi and color potato was checked, Odi showed the darkest color and the color was lighter in the order of bokbunja, hongyoung, and selflessness (Fig. 2).

The total anthocyanin content and color of the 60% ethanol extract are shown in Table 3 below. In the following table, a represents redness and b represents yellowness.

Total anthocyanin
(%, dry weight) Hunter value L a b Bokbunja 1.10 ± 0.01 ^a 45.49 + - 0.01 ^c 72.53 + 0.01 ^a 39.81 ± 0.09 ^a Audi 0.92 ± 0.00 ^b 42.40 ± 0.08 ^d 68.03 0.02 ^b 23.04 + - 0.10 ^c Self-help 0.56 0.04 ^c 56.55 + - 0.21 ^b 65.47 + - 0.21 ^c -14.70 ± 0.17 ^d Hongyoung 0.61 + 0.02 ^d 67.03 0.09 ^a 50.73 ± 0.32 ^d 31.78 ± 0.53 ^b

As a result, as shown in Table 2, the total anthocyanin and red color values were in the order of brambly, audi, red, and green, and in the case of yellowness, the purple selfishness was -14.70

The results for a) total polyphenol and flavonoid content of 60% ethanol extract, b) DPPH radical scavenging activity and SOD-like activity, and c) hydroxy radical, hydrogen peroxide scavenging activity and elastase inhibitory activity are shown in Tables 3 to 5 Respectively.

Total polyphenol contents (mg / g) Total flavonoid contents (mg / ml) Bokbunja 8.25 + 0.04 ^a 4.42 ± 0.01 ^a Audi 5.64 ± 1.40 ^b 3.08 ± 0.11 ^b Self-help 3.74 ± 0.05 ^b 2.26 ± 0.00 ^d Hongyoung 3.83 ± 0.05 ^b 2.47 ± 0.04 ^c

DPPH radical scavenging activity (%) SOD-like activity (%) Bokbunja 11.76 ± 0.34 ^d 1.10 ± 0.93 ^NS Audi 34.17 + - 2.59 ^c 0.88 ± 0.00 ^NS Self-help 50.55 + - 0.01 ^b 0.66 ± 0.31 ^NS Hongyoung 57.90 + - 0.40 ^a 0.88 ± 0.62 ^NS

Hydroxyl radical scavenging activity (%) Hydrogen peroxide scavenging activity (%) Elastase inhibitory activity (%) Bokbunja 80.52 + 0.73 ^a 88.40 ± 0.09 ^a 4.26 + - 0.01 ^c Audi 73.45 ± 0.59 ^b 76.65 ± 0.78 ^a 2.88 ± 0.14 ^d Self-help 14.09 ± 1.56 ^d 35.65 ± 1.25 ^b 5.97 ± 0.02 ^a Hongyoung 21.78 ± 1.42 ^c 40.47 ± 8.63 ^b 4.69 ± 0.01 ^b

As a result, as shown in Table 3, the content of total polyphenols and flavonoids was higher than that of color potatoes such as self potatoes or black potatoes. In the case of DPPH radical scavenging activity and SOD-like activity, as shown in Table 4, in the case of DPPH radical scavenging ability, color potatoes self-irritation and redness were higher than those of bokbunja and odi, and there was a significant difference at the level of 5% . Also, the SOD-like activity was the highest in the bokbunja and the 5% level showed no significant difference. As shown in Table 5, in the case of the hydroxy radical, the hydrogen peroxide scavenging activity and the elastase inhibitory activity, the scavenging activity was much higher than that of the black potato, Potatoes were higher than bokbunja and audi.

Experimental Example  2: using bokbunja Pectinex Ultra SP -L Confirmation of effect by enzyme pretreatment

Experimental Example  2-1: Using bokbunja Pectinex Ultra SP -L of Enzyme pretreatment  Experimental design of process

In order to confirm the pretreatment effect of Pectinex Ultra SP-L enzyme using bokbunja and to derive optimal process, the experiment of the process as shown in Table 6 was designed.

Experimental Design of Pectinex Ultra SP-L Enzyme Pretreatment Process Using Bokbunja Experiment number X _One X ₂ X ₃ Conc. of enzymatic activity (mL / 100 L) Temperature (° C) Time (hr) One 0 -One One 1.0 20 8 2 0 One -One 1.0 60 2 3 0 0 0 1.0 40 5 4 0 -One -One 1.0 20 2 5 -One 0 One 0.5 40 8 6 -One -One 0 0.5 20 5 7 -One 0 -One 0.5 40 2 8 0 0 0 1.0 40 5 9 One One 0 1.5 60 5 10 0 0 0 1.0 40 5 11 One 0 -One 1.5 40 2 12 One 0 One 1.5 40 8 13 One -One 0 1.5 20 5 14 0 One One 1.0 60 8 15 -One One 0 0.5 60 5

Experimental Example  2-2: Using Rubus Pectinex Ultra SP -L of Enzyme pretreatment  Measurement of post-yield and freeze-dried yield

The yield and lyophilization yield after enzyme pretreatment were measured based on the above experimental design, and the results are shown in Table 7 below.

The yield and freeze-drying yield of Pectinex Ultra SP-L after pretreatment with enzyme Experiment number X _One X ₂ X ₃ Yield after enzyme pre - treatment (%) Yield after freeze  dehydration treatment (%) One 0 -One One 30.63 6.36 2 0 One -One 31.76 4.93 3 0 0 0 29.46 5.77 4 0 -One -One 28.71 5.26 5 -One 0 One 28.01 5.95 6 -One -One 0 30.06 5.09 7 -One 0 -One 33.15 6.05 8 0 0 0 28.56 6.18 9 One One 0 29.64 4.55 10 0 0 0 32.63 6.32 11 One 0 -One 32.09 4.66 12 One 0 One 28.67 5.32 13 One -One 0 26.83 5.85 14 0 One One 25.93 4.04 15 -One One 0 29.12 4.34

As a result, as shown in Table 7, the yield after enzyme treatment was in the range of 25.93 to 33.15%, and the level after freeze-drying was 4.04 to 6.36%.

Experimental Example  2-3: using bokbunja Pectinex Ultra SP Measurement of total anthocyanin content after enzyme pretreatment of -L

Total anthocyanin content was measured after Pectinex Ultra SP-L enzyme pretreatment, and the results are shown in Table 8 below.

Total Anthocyanin Content after Pretreatment of Pectinex Ultra SP-L with Bokbunja Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 5.29 ± 0.08 2 0 One -One 5.77 ± 0.18 3 0 0 0 3.43 ± 0.09 4 0 -One -One 3.98 ± 0.09 5 -One 0 One 2.93 + 0.04 6 -One -One 0 6.61 ± 0.16 7 -One 0 -One 4.50 + - 0.20 8 0 0 0 4.40 + 0.02 9 One One 0 6.08 ± 0.01 10 0 0 0 3.30 ± 0.01 11 One 0 -One 8.77 ± 0.27 12 One 0 One 3.81 ± 0.03 13 One -One 0 5.08 ± 0.12 14 0 One One 4.49 + 0.14 15 -One One 0 5.51 + - 0.12

As a result, as shown in the above table, the treatment conditions of the enzyme with the highest total anthocyanin content were an enzyme (Pectinex Ultra SP-L) concentration of 1.5 mL / 100 L, an enzyme treatment temperature of 40 ° C and an enzyme treatment time of 2 hours. Total anthocyanin content was 8.77%. According to the enzyme treatment conditions, the total anthocyanin content ranged from 2.93% to 8.77%, which was slightly higher than the untreated 1.10% (Table 2). When the pretreatment process of the present invention was used, the anthocyanin content was maximized 8.7 times as much.

Experimental Example  2-4: Using Bokbunja Pectinex Ultra SP Analysis of variance of total anthocyanin content after -L enzyme treatment

The analysis of variance of total anthocyanin content, the regression coefficient of the secondary polynomial regression curve, and the fitness test of the secondary polynomial regression equation were performed after treatment with Pectinex Ultra SP-L enzyme using bokbunja, and the results are shown in Tables 9 to 11, respectively.

Analysis of the distribution of total anthocyanin content after Pectinex Ultra SP-L enzyme treatment using bokbunja Factor Bokbunja F value Pr> F Conc. of enzymatic activity (mL / 100 L) 2.97 0.0446 Temperature (° C) 4.88 0.0065 Time (hr) 4.52 0.0092

As shown in Table 9, in the three factors, the concentration of anthocyanin in the enzyme pretreatment was affected in the order of treatment temperature, treatment time and enzyme activity, and the results showed that the temperature and the treatment time were influenced by the enzyme activity And anthocyanin content was significantly higher than that of control group.

Regression coefficients of secondary polynomial regression curves of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L using bokbunja Parameter Estimate Bokbunja Intercept 3.710000 X ₁ ^{1 )} 0.525000 X ₂ ^{2 )} 0.109375 X ₃ ^{3 )} -0.813125 X ₁ * X ₁ 1.113750 X ₂ * X ₁ 0.526250 X ₂ * X ₂ 0.997500 X ₃ * X ₁ -0.848750 X ₃ * X ₂ -0.647500 X ₃ * X ₃ 0.177500 ¹⁾ Conc. of enzymatic activity (mL / 100 L) ²⁾ Temperature (℃) ³⁾ Time (hr)

Fertility of secondary polynomial regression equation of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L using bokbunja Regression Bokbunja R ² F value Pr  > F Linear 0.2375 4.59 0.0134 Quadratic 0.244 4.64 0.0128 Cross product 0.1773 3.42 0.0369 Total regress 0.6548 4.22 0.0035

Experimental Example  2-5: Using bokbunja Pectinex Ultra SP -L of enzyme surface treatment

In addition to the above analysis, the reaction surface contour of total anthocyanin content was examined after treatment with Pectinex Ultra SP-L enzyme using bokbunja. The results are shown in Fig. As a result, as shown in Fig. 3, the treatment temperature (X ₂ ) and the treatment time (X ₃ ) were higher than the concentration (X ₁ ) considering the enzyme activity in the reaction surface curve by the reaction surface analysis method Respectively. In addition, the influences were in the order of treatment temperature, treatment time, concentration in terms of enzyme activity.

Experimental Example  2-6: Using Bokbunja Pectinex Ultra SP -L of Enzyme pretreatment  After total polyphenol and flavonoid content; And DPPH Radical Scatters  And SOD - like  Activity investigation

The total polyphenol and flavonoid content of Pectinex Ultra SP-L was measured after enzyme pretreatment using bokbunja. The results are shown in Table 12 below.

Total polyphenol and flavonoid content after enzyme pretreatment of Pectinex Ultra SP-L using bokbunja Experiment number X _One X ₂ X ₃ Total polyphenol  contents ( mg / g) Total flavonoid  contents ( mg / mL ) One 0 -One One 8.81 ± 0.01 4.97 ± 0.21 2 0 One -One 9.96 + 0.04 4.17 ± 0.22 3 0 0 0 8.51 + 0.02 4.27 ± 0.61 4 0 -One -One 8.49 + 0.03 3.79 ± 0.24 5 -One 0 One 9.49 + 0.02 4.26 ± 0.52 6 -One -One 0 7.89 ± 0.03 3.92 + 0.16 7 -One 0 -One 8.44 + 0.03 4.09 ± 0.21 8 0 0 0 12.34 + 0.02 4.45 + 0.47 9 One One 0 8.08 ± 0.01 3.90 + 0.14 10 0 0 0 8.36 ± 0.01 4.38 ± 0.42 11 One 0 -One 7.47 ± 0.00 4.17 ± 0.10 12 One 0 One 6.33 ± 0.00 3.19 ± 0.17 13 One -One 0 7.94 ± 0.00 4.02 + -0.31 14 0 One One 6.30 ± 0.00 3.34 0.11 15 -One One 0 9.38 ± 0.14 4.48 + 0.04

As a result, as shown in Table 12, the enzyme pretreatment process with the highest total polyphenol content was an enzyme concentration of 1.0 mL / 100 L, an enzyme treatment temperature of 40 ° C, and an enzyme treatment time of 5 hours. The total polyphenol content The average value was 10.47 mg / g. On the other hand, total flavonoids had the highest enzymatic pretreatment rate of 1.0 mL / 100 L of enzyme concentration, enzyme treatment temperature of 20 ° C, and enzyme treatment time of 8 hours. The total flavonoid content in the process was 4.97 mg / mL. The total polyphenol content and the total flavonoid content were 8.25 mg / g and 4.42 mg / mL, respectively (Table 3), and thus the polyphenols and flavonoids can be increased by the enzyme pretreatment of the present invention Respectively.

DPPH radical scavenging activity and SOD-like activity were measured after pre-treatment with Pectinex Ultra SP-L enzyme using bokbunja. The results are shown in Table 13 below.

DPPH radical scavenging activity and SOD-like activity after enzyme pretreatment of Pectinex Ultra SP-L using bokbunja Experiment number X _One X ₂ X ₃ DPPH radical  scavenging activity (%) SOD - like activity (%) One 0 -One One 33.10 + - 4.49 9.03 + - 0.65 2 0 One -One 51.52 ± 4.09 8.77 ± 0.65 3 0 0 0 48.88 ± 1.06 8.25 ± 1.75 4 0 -One -One 51.39 ± 2.85 9.16 + - 0.46 5 -One 0 One 49.50 ± 0.18 10.32 + 0.09 6 -One -One 0 55.27 + - 2.70 12.90 + - 0.61 7 -One 0 -One 51.14 + - 2.49 38.32 + - 0.56 8 0 0 0 42.74 1.54 9.03 + - 0.65 9 One One 0 56.52 ± 2.00 10.19 ± 0.82 10 0 0 0 46.23 + - 1.62 8.51 ± 0.65 11 One 0 -One 61.16 ± 2.91 8.90 ± 1.20 12 One 0 One 60.27 1.52 9.68 ± 0.46 13 One -One 0 52.14 ± 3.91 8.26 ± 0.29 14 0 One One 65.17 ± 2.23 12.00 ± 0.80 15 -One One 0 49.50 ± 1.59 10.32 ± 0.27

As shown in the above table, DPPH radical scavenging activity and SOD-like activity were 11.76% and 1.10%, respectively (Table 4), but DPPH radical scavenging ability after enzyme pretreatment of the present invention was 33.10-65.17% SOD-like activity increased from 8.26% to 38.32% up to 38-fold. Thus, the above results suggest that the enzyme pretreatment process of the present invention can significantly increase the efficiency of anthocyanin-based natural pigment extraction.

Experimental Example  3: Using Audi Pectinex Ultra SP -L Confirmation of effect by enzyme pretreatment

Experimental Example  3-1: Using Audi Pectinex Ultra SP Experimental design of enzyme pretreatment process of -L

In order to confirm the pretreatment effect of Pectinex Ultra SP-L enzyme using ODI and to derive the optimum conditions, the experiment of the process as shown in the following table was designed.

Experimental design of enzyme pretreatment process of Pectinex Ultra SP-L Experiment number X _One X ₂ X ₃ Conc . of enzymatic  activity ( mL / 100L) Temperature (° C) Time ( hr ) One 0 -One One 1.0 20 8 2 0 One -One 1.0 60 2 3 0 0 0 1.0 40 5 4 0 -One -One 1.0 20 2 5 -One 0 One 0.5 40 8 6 -One -One 0 0.5 20 5 7 -One 0 -One 0.5 40 2 8 0 0 0 1.0 40 5 9 One One 0 1.5 60 5 10 0 0 0 1.0 40 5 11 One 0 -One 1.5 40 2 12 One 0 One 1.5 40 8 13 One -One 0 1.5 20 5 14 0 One One 1.0 60 8 15 -One One 0 0.5 60 5

Experimental Example  3-2: Using Audi Pectinex Ultra SP Measurement of yield and freeze-drying yield after enzyme pretreatment of -L

The yield and lyophilization yield after enzyme pretreatment were measured based on the above experimental design, and the results are shown in the following table.

Yield and lyophilization yield of Pectinex Ultra SP-L by enzyme pretreatment Experiment number X _One X ₂ X ₃ Yield after enzyme pre - treatment (%) Yield after freeze  dehydration treatment (%) One 0 -One One 29.69 8.89 2 0 One -One 30.57 5.74 3 0 0 0 29.19 8.97 4 0 -One -One 27.15 8.52 5 -One 0 One 25.97 7.16 6 -One -One 0 27.94 8.40 7 -One 0 -One 31.33 8.80 8 0 0 0 28.34 7.98 9 One One 0 24.02 7.55 10 0 0 0 31.27 8.78 11 One 0 -One 31.25 8.86 12 One 0 One 29.36 8.61 13 One -One 0 26.91 8.72 14 0 One One 27.66 7.59 15 -One One 0 23.61 7.42

As shown in the above table, when the Pectinex Ultra SP-L enzyme was treated with ODI, the yield was 23.61-31.33% and the yield after lyophilization was 5.74-8.89%. As shown in Experimental Example 2, when Pectinex Ultra SP-L enzyme was treated with bokbunja, the yield was 25.93-32.63%. However, in the case of bokbunja, the value was 4.04-6.36% after lyophilization, which was rather small (Table 8).

Experimental Example  3-3: Using Audi Pectinex Ultra SP Measurement of total anthocyanin content after enzyme pretreatment of -L

After the Pectinex Ultra SP-L enzyme was pretreated, the total anthocyanin content was measured. The results are shown in the table below.

Total anthocyanin content after enzyme pretreatment of Pectinex Ultra SP-L using ODI Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 5.76 + - 0.32 2 0 One -One 3.43 + 0.14 3 0 0 0 6.23 ± 0.39 4 0 -One -One 6.51 ± 0.61 5 -One 0 One 4.42 0.55 6 -One -One 0 5.94 + - 0.86 7 -One 0 -One 5.41 0.35 8 0 0 0 3.01 + - 0.42 9 One One 0 2.55 + - 0.20 10 0 0 0 5.38 + - 0.40 11 One 0 -One 3.46 ± 0.29 12 One 0 One 3.26 ± 0.15 13 One -One 0 3.80 ± 0.02 14 0 One One 2.82 + 0.17 15 -One One 0 2.74 ± 0.04

As shown in the above table, the enzyme treatment conditions with the highest total anthocyanin content were an enzyme concentration of 1.0 mL / 100 L, an enzyme treatment temperature of 20 ° C., and an enzyme treatment time of 2 hours. The total anthocyanin content in this process was 6.51% Respectively. As shown in the above table, the total anthocyanin content ranged from 2.74 to 6.51% depending on the enzyme treatment conditions, and the difference was somewhat large. On the other hand, the total anthocyanin content of Pectinex Ultra SP-L was 0.92% (Table 2). Therefore, the pretreatment process of the present invention for treating Pectinex Ultra SP-L in Audi shows that the total anthocyanin content can be enhanced by about 7.1 times, and the pretreatment process of the present invention can reduce the total anthocyanin content of berries or color potatoes Suggesting that it can be used very usefully to promote the

Experimental Example  3-4: Using Audi Pectinex Ultra SP Analysis of the distribution of total anthocyanin content after enzyme treatment of -L

Analysis of variance of total anthocyanin content, regression coefficient of the secondary polynomial regression curve, and fitness of the secondary polynomial regression equation were performed after the Pectinex Ultra SP-L enzyme treatment using ODI, and the results are shown in the table below.

Analysis of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L Factor Audi F value Pr  > F Conc. of enzymatic activity (mL / 100 L) 0.70 0.6036 Temperature (° C) 10.62 <.0001 Time (hr) 5.06 0.0056

As shown in the above table, the effect of the enzyme pretreatment on the anthocyanin extract content in the order of treatment temperature, treatment time, and enzyme activity was influenced by three factors.

Regression coefficients of secondary polynomial regression curves of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L using ODI Parameter Estimate Audi Intercept 4.870000 X ₁ ^{1 )} -0.678750 X ₂ ^{2 )} -1.311250 X ₃ ^{3 )} -0.317500 X ₁ * X ₁ -0.801875 X ₂ * X ₁ 0.488750 X ₂ * X ₂ -0.309375 X ₃ * X ₁ 0.198750 X ₃ * X ₂ 0.033750 X ₃ * X ₃ 0.070625 ¹⁾ Conc. of enzymatic activity (mL / 100 L) ²⁾ Temperature (℃) ³⁾ Time (hr)

Adequacy of total anthocyanin content after enzymatic treatment of Pectinex Ultra SP-L Regression Audi R ² F value Pr  > F Linear 0.6261 17.15 <.0001 Quadratic 0.0922 2.52 0.0867 Cross product 0.0384 1.05 0.3919 Total regress 0.7566 6.91 0.0002

Experimental Example  3-5: Using Audi Pectinex Ultra SP -L of enzyme surface treatment

In addition to the above analysis, the reaction surface contour of the total anthocyanin content after the treatment with Pectinex Ultra SP-L enzyme using ODI was investigated. The results are shown in FIG. As shown in FIG. 5, in the case of Audi, the treatment surface temperature (X ₂ ) rather than the concentration (X ₁ ) and the treatment time (X ₃ ) .

Experimental Example  3-6: Using Audi Pectinex Ultra SP -L of Enzyme pretreatment  After total polyphenol and flavonoid content; And DPPH radical Scatters  And SOD - like activity  Research

After the Pectinex Ultra SP-L enzyme was pretreated, the total polyphenol and flavonoid contents were examined. The results are shown in the following table.

Total polyphenol and flavonoid content after enzyme pretreatment of Pectinex Ultra SP-L using ODI Experiment number X _One X ₂ X ₃ Total polyphenol  contents ( mg / g) Total flavonoid  contents ( mg / ml ) One 0 -One One 4.67 ± 0.13 3.05 ± 0.10 2 0 One -One 4.74 ± 0.13 3.43 ± 0.05 3 0 0 0 5.92 + - 0.32 3.50 ± 0.16 4 0 -One -One 5.15 + 0.47 3.17 ± 0.25 5 -One 0 One 5.13 ± 0.17 3.21 ± 0.15 6 -One -One 0 5.51 + - 0.41 3.39 ± 0.24 7 -One 0 -One 5.79 ± 0.63 3.23 ± 0.31 8 0 0 0 4.26 + 0.03 3.22 + 0.02 9 One One 0 5.71 + - 0.32 3.44 ± 0.01 10 0 0 0 5.74 + 0.14 3.64 ± 0.21 11 One 0 -One 5.38 + 0.04 3.28 0.30 12 One 0 One 5.35 0.20 3.22 ± 0.16 13 One -One 0 4.38 + 0.03 2.97 ± 0.26 14 0 One One 4.26 + 0.04 3.44 ± 0.04 15 -One One 0 5.48 ± 0.32 3.59 + 0.02

As shown in the above table, the enzyme pretreatment process with the highest total polyphenol content was 1.0 mL / 100 L enzyme concentration, the enzyme treatment temperature was 20 ° C, and the enzyme treatment time was 2.0 hours. The average value of the total polyphenol content in the process was 5.83 mg / g. Total flavonoids The highest enzyme pretreatment was 1.0 mL / 100 L enzyme concentration, enzyme treatment temperature was 40 ℃, enzyme treatment time was 5.0 hours, and the total flavonoid content in the process was 3.64 mg / mL. The polyphenol content and the flavonoid content at the non-treatment were 5.64 mg / g and 3.08 mg / mL, respectively (Table 3).

The DPPH radical scavenging activity and the SOD-like activity were measured after Pectinex Ultra SP-L enzyme pretreatment using an oudy, and the results are shown in the following table.

DPPH radical scavenging activity and SOD-like activity after enzyme pretreatment of Pectinex Ultra SP-L using ODI Experiment number X _One X ₂ X ₃ DPPH radical  scavenging activity (%) SOD - like activity  (%) One 0 -One One 52.50 ± 2.29 21.54 ± 1.08 2 0 One -One 45.99 ± 1.27 16.26 ± 0.58 3 0 0 0 52.00 ± 0.52 18.45 + - 1.11 4 0 -One -One 52.25 + - 0.52 18.71 + - 0.74 5 -One 0 One 47.86 + - 3.56 19.48 ± 0.37 6 -One -One 0 51.00 ± 1.23 19.35 + - 0.55 7 -One 0 -One 52.13 + - 0.34 20.25 ± 0.73 8 0 0 0 48.23 + - 3.38 19.61 + - 0.55 9 One One 0 46.97 ± 5.16 14.45 ± 0.50 10 0 0 0 49.25 + - 0.88 22.32 ± 0.71 11 One 0 -One 48.74 ± 2.31 19.61 + - 0.55 12 One 0 One 51.87 ± 1.76 18.19 ± 0.75 13 One -One 0 52.62 ± 3.17 18.58 ± 0.20 14 0 One One 40.09 ± 3.26 20.00 ± 0.36 15 -One One 0 38.83 + - 3.62 18.19 ± 1.11

As a result, DPPH radical scavenging activity and SOD-like activity were 34.17% and 0.88%, respectively, when Pectinex Ultra SP-L was treated with ODI (Table 4), but after enzyme pretreatment, 38.83-52.62% The results are shown in Fig.

Experimental Example  4: Self-employed Pectinex Ultra SP -L Confirm enzyme pretreatment effect

Experimental Example  4-1: Self-employed Pectinex Ultra SP Experimental design of enzyme pretreatment process of -L

In order to confirm the pretreatment effect of Pectinex Ultra SP-L enzyme using self-administration, the experiment of the process as shown in the following table was designed in order to derive the optimum condition process.

Experimental design of Pectinex Ultra SP-L enzyme pretreatment process Experiment number X _One X ₂ X ₃ Conc . of enzymatic  activity ( mL / 100L) Temperature (° C) Time ( hr ) One 0 -One One 1.0 20 8 2 0 One -One 1.0 60 2 3 0 0 0 1.0 40 5 4 0 -One -One 1.0 20 2 5 -One 0 One 0.5 40 8 6 -One -One 0 0.5 20 5 7 -One 0 -One 0.5 40 2 8 0 0 0 1.0 40 5 9 One One 0 1.5 60 5 10 0 0 0 1.0 40 5 11 One 0 -One 1.5 40 2 12 One 0 One 1.5 40 8 13 One -One 0 1.5 20 5 14 0 One One 1.0 60 8 15 -One One 0 0.5 60 5

Experimental Example  4-2: Using self-help Pectinex Ultra SP Measurement of yield and freeze-drying yield after enzyme pretreatment of -L

The yield and lyophilization yield after enzyme pretreatment were measured based on the above experimental design, and the results are shown in the following table.

Pectinex Ultra SP-L enzyme pretreatment yield and freeze-drying yield Experiment number X _One X ₂ X ₃ Yield after enzyme pre - treatment (%) Yield after freeze  dehydration treatment (%) One 0 -One One 21.33 1.71 2 0 One -One 16.86 2.10 3 0 0 0 18.92 2.37 4 0 -One -One 19.39 1.47 5 -One 0 One 16.79 1.53 6 -One -One 0 21.50 2.10 7 -One 0 -One 21.40 2.04 8 0 0 0 18.66 1.81 9 One One 0 10.42 0.26 10 0 0 0 20.42 2.21 11 One 0 -One 19.95 1.92 12 One 0 One 18.44 2.67 13 One -One 0 21.14 2.33 14 0 One One 16.56 1.45 15 -One One 0 19.90 1.70

As a result, the yield after enzyme treatment was in the range of 10.42-21.50% and 0.26-2.37% after freeze drying, as shown in the table above.

Experimental Example  4-3: Using self-help Pectinex Ultra SP Measurement of total anthocyanin content after enzyme pretreatment of -L

The total anthocyanin content after enzyme pretreatment was measured and the results are shown in the table below.

Total anthocyanin content after pretreatment with Pectinex Ultra SP-L enzyme Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 1.39 ± 0.26 2 0 One -One 1.28 + 0.07 3 0 0 0 1.38 + 0.09 4 0 -One -One 1.51 + 0.09 5 -One 0 One 0.89 ± 0.06 6 -One -One 0 1.72 ± 0.05 7 -One 0 -One 1.23 + 0.04 8 0 0 0 1.06 ± 0.01 9 One One 0 0.75 + - 0.01 10 0 0 0 1.15 ± 0.08 11 One 0 -One 0.96 + - 0.05 12 One 0 One 0.71 ± 0.00 13 One -One 0 0.96 + 0.07 14 0 One One 1.14 + 0.03 15 -One One 0 1.39 + 0.04

As a result, as shown in the table, the enzyme treatment conditions with the highest total anthocyanin content were 0.5 mL / 100 L of enzyme (Pectinex Ultra SP-L) concentration, enzyme treatment temperature of 20 ° C and enzyme treatment time of 5 hours. Total anthocyanin content was 1.72%. The total anthocyanin content of untreated individuals was only 0.56% (Table 2), suggesting that total anthocyanin content can be significantly increased to 3-fold or more when pretreated with Pectinex Ultra SP-L enzyme.

Experimental Example  4-4: Using self-help Pectinex Ultra SP Analysis of the distribution of total anthocyanin content after enzyme treatment of -L

The analysis of variance of total anthocyanin content, the regression coefficient of the secondary polynomial regression curve, and the fitness test of the secondary polynomial regression equation were performed after the Pectinex Ultra SP-L enzyme treatment using self-administration, and the results are shown in the table below.

Analysis of the distribution of total anthocyanin content after Pectinex Ultra SP-L enzyme treatment Factor Self-help F value Pr  > F Conc. of enzymatic activity (mL / 100 L) 2.30 0.0945 Temperature (° C) 11.61 <.0001 Time (hr) 5.70 0.0032

The results showed that the enzyme pretreatment affects the anthocyanin extract content in the order of treatment temperature, treatment time, and enzyme concentration.

Regression coefficients of secondary polynomial regression curves of total anthocyanin content after Pectinex Ultra SP-L enzyme treatment Parameter Estimate Self-help Intercept 1.195000 X ₁ ^{1 )} 0.230625 X ₂ ^{2 )} -0.126250 X ₃ ^{3 )} -0.108125 X ₁ * X ₁ -0.185000 X ₂ * X ₁ 0.028750 X ₂ * X ₂ 0.196250 X ₃ * X ₁ 0.022500 X ₃ * X ₂ -0.008750 X ₃ * X ₃ -0.062500 ¹⁾ Conc. of enzymatic activity (mL / 100 L) ²⁾ Temperature (℃) ³⁾ Time (hr)

Compliance of total anthocyanin content after Pectinex Ultra SP-L enzyme treatment using self-administration Regression Self-help R ² F value Pr  > F Linear 0.5397 17.97 <.0001 Quadratic 0.2553 8.50 0.0008 Cross product 0.0047 0.16 0.9242 Total regress 0.7997 8.87 <.0001

Experimental Example  4-5: Using self-help Pectinex Ultra SP -L of enzyme surface treatment

In addition to the above analysis, the surface contour of the total anthocyanin content was examined after Pectinex Ultra SP-L enzyme treatment using self-administration, and the result was shown in FIG. As shown in FIG. 5, the extraction temperature (X ₂ ) and extraction time (X ₃ ) affect the yield improvement of anthocyanin extraction, rather than the concentration (X ₁ ) considering the enzyme activity in the reaction surface curve by the reaction surface analysis appear. The influences were in the order of treatment temperature, treatment time, and enzyme activity.

Experimental Example  4-6: Self-employed Pectinex Ultra SP -L of Enzyme pretreatment  After total polyphenol and flavonoid content; And DPPH radical Scatters  And SOD - like activity  Research

The total polyphenol and flavonoid content of Pectinex Ultra SP-L after pretreatment with enzyme was also investigated. The results are shown in the table below.

Total polyphenol and flavonoid content after pretreatment with Pectinex Ultra SP-L enzyme Experiment number X _One X ₂ X ₃ Total polyphenol  contents ( mg / g) Total flavonoid  contents ( mg / ml ) One 0 -One One 3.50 + - 0.23 2.59 ± 9.85 2 0 One -One 3.41 ± 0.01 2.66 + 0.02 3 0 0 0 3.12 ± 0.01 2.65 + 0.04 4 0 -One -One 3.14 ± 0.00 2.82 + 0.07 5 -One 0 One 3.24 ± 0.02 2.95 + - 7.45 6 -One -One 0 3.49 + 0.02 3.08 ± 0.10 7 -One 0 -One 4.08 ± 0.06 3.16 ± 0.14 8 0 0 0 3.09 ± 0.20 2.80 0.20 9 One One 0 4.07 ± 0.18 3.17 ± 0.15 10 0 0 0 3.06 ± 0.05 2.82 + - 0.10 11 One 0 -One 3.16 ± 0.06 2.86 ± 0.07 12 One 0 One 3.34 ± 0.18 2.75 ± 0.05 13 One -One 0 3.07 ± 0.12 2.81 ± 0.16 14 0 One One 2.37 ± 0.01 2.44 ± 0.01 15 -One One 0 2.69 ± 0.01 2.65 ± 0.21

As shown in the above table, the enzyme pretreatment process with the highest total polyphenol content was 0.5 mL / 100 L enzyme concentration, the enzyme treatment temperature was 40 DEG C, and the enzyme treatment time was 2 hours. In this case, the total polyphenol content in the process was 4.08 mg / g level. On the other hand, total flavonoids had the highest enzyme pretreatment rate of 1.5mL / 100L of enzyme concentration, enzyme treatment temperature of 60 ℃, and enzyme treatment time of 5 hours. Total flavonoid content in the process was 3.17mg / mL. Considering that the total polyphenol content and total flavonoid content of untreated were 3.74 mg / g and 2.26 mg / mL, respectively, (Table 3), the above results show that the process of the present invention for treating Pectinex Ultra SP- Polyphenol, and flavonoid content.

DPPH radical scavenging activity and SOD-like activity were measured after pretreatment with Pectinex Ultra SP-L enzyme using self-administration. The results are shown in the following table.

DPPH radical scavenging activity and SOD-like activity after pre-treatment with Pectinex Ultra SP-L enzyme Experiment number X _One X ₂ X ₃ DPPH radical  scavenging activity (%) SOD - like activity  (%) One 0 -One One 31.06 + - 4.74 7.93 + - 0.53 2 0 One -One 36.70 ± 4.88 8.69 ± 0.53 3 0 0 0 28.44 + - 2.46 9.57 ± 0.36 4 0 -One -One 30.43 + - 4.92 8.56 0.71 5 -One 0 One 28.67 + - 8.13 10.20 ± 0.18 6 -One -One 0 28.31 ± 1.93 7.93 ± 0.18 7 -One 0 -One 28.93 + - 5.29 8.19 ± 0.53 8 0 0 0 26.56 ± 3.00 10.71 ± 0.18 9 One One 0 36.71 + - 2.40 6.93 + - 0.53 10 0 0 0 24.93 + - 2.13 7.56 ± 0.36 11 One 0 -One 36.97 + 1.15 9.32 ± 0.71 12 One 0 One 35.59 + - 0.61 9.07 + - 0.71 13 One -One 0 31.85 + - 4.48 9.95 + 0.89 14 0 One One 24.67 + - 4.96 10.20 ± 0.89 15 -One One 0 25.43 + - 1.06 12.47 ± 0.18

DPPH radical scavenging activity and SOD-like activity were 50.55% and 0.66%, respectively (Table 4), but DPPH radical scavenging activity and SOD-like activity after enzyme pretreatment were 24.67-36.97% And 6.93-12.47%, respectively, which showed an increase in SOD-like activity.

Experimental Example  5: Hongyoung  Used Pectinex Ultra SP -L Confirm enzyme pretreatment effect

Experimental Example  5-1: Hongyoung  Used Pectinex Ultra SP Experimental design of enzyme pretreatment process of -L

In order to confirm the effect of pretreatment with Pectinex Ultra SP-L enzyme using Hongyeong, the process experiment as shown in the following table was designed to derive the optimum condition process.

Experimental design of Pectinex Ultra SP-L enzyme pretreatment process using Hongyoung Experiment number X _One X ₂ X ₃ Conc . of enzymatic  activity ( mL / 100L) Temperature (° C) Time ( hr ) One 0 -One One One 20 8 2 0 One -One One 60 2 3 0 0 0 One 40 5 4 0 -One -One One 20 2 5 -One 0 One 0.5 40 8 6 -One -One 0 0.5 20 5 7 -One 0 -One 0.5 40 2 8 0 0 0 One 40 5 9 One One 0 1.5 60 5 10 0 0 0 One 40 5 11 One 0 -One 1.5 40 2 12 One 0 One 1.5 40 8 13 One -One 0 1.5 20 5 14 0 One One One 60 8 15 -One One 0 0.5 60 5

Experimental Example  5-2: Hongyoung  Used Pectinex Ultra SP Measurement of yield and freeze-drying yield after enzyme pretreatment of -L

The yield and lyophilization yield after enzyme pretreatment were measured based on the above experimental design, and the results are shown in the following table.

Yield and freeze-drying yield after pretreatment with Pectinex Ultra SP-L enzyme Experiment number X _One X ₂ X ₃ Yield after enzyme pre - treatment (%) Yield after freeze  dehydration treatment (%) One 0 -One One 21.24 2.18 2 0 One -One 28.35 2.55 3 0 0 0 32.58 2.74 4 0 -One -One 19.11 2.70 5 -One 0 One 16.06 2.39 6 -One -One 0 21.22 2.64 7 -One 0 -One 18.39 2.06 8 0 0 0 18.29 2.75 9 One One 0 16.03 3.17 10 0 0 0 20.98 3.17 11 One 0 -One 19.91 1.98 12 One 0 One 18.81 2.74 13 One -One 0 20.33 2.74 14 0 One One 13.66 2.31 15 -One One 0 10.14 2.56

As a result, the yield after enzyme treatment was 10.14-32.58%, and after lyophilization, it was 1.98-3.17%.

Experimental Example  5-3: Hongyoung  Used Pectinex Ultra SP Measurement of total anthocyanin content after enzyme pretreatment of -L

After the Pectinex Ultra SP-L enzyme was pretreated, the total anthocyanin content was measured. The results are shown in the table below.

Total anthocyanin content after pretreatment with Pectinex Ultra SP-L enzyme using Hongyeong Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 1.79 + 0.04 2 0 One -One 1.95 + - 0.12 3 0 0 0 1.42 + 0.17 4 0 -One -One 1.65 0.36 5 -One 0 One 2.28 + 0.04 6 -One -One 0 1.52 + 0.15 7 -One 0 -One 1.95 + 0.03 8 0 0 0 2.10 ± 0.11 9 One One 0 1.42 ± 0.09 10 0 0 0 2.11 + 0.07 11 One 0 -One 2.09 ± 0.28 12 One 0 One 2.15 + - 0.10 13 One -One 0 1.46 ± 0.16 14 0 One One 1.66 + 0.09 15 -One One 0 1.44 ± 0.05

As a result, as shown in the above table, the enzyme treatment conditions with the highest total anthocyanin content were 0.5 mL / 100 L of enzyme (Pectinex Ultra SP-L) concentration, enzyme treatment temperature of 40 ° C. and enzyme treatment time of 8 hours. Total anthocyanin content was 2.28%. On the other hand, the total anthocyanin content was 0.61% in the untreated (Table 2), indicating that the treatment with Pectinex Ultra SP-L significantly improved the total anthocyanin content by 3.7 times.

Experimental Example  5-4: Hongyoung  Used Pectinex Ultra SP Analysis of the distribution of total anthocyanin content after enzyme treatment of -L

The analysis of variance of total anthocyanin content, the regression coefficient of the secondary polynomial regression curve, and the fitness test of the secondary polynomial regression equation were performed after treatment with Pectinex Ultra SP-L enzyme using Hongyeong. The results are shown in the following table.

Analysis of the distribution of total anthocyanin content after treatment with Pectinex Ultra SP-L enzyme Factor Hongyoung F value Pr  > F Conc. of enzymatic activity (mL / 100 L) 0.23 0.9182 Temperature (° C) 5.59 0.0035 Time (hr) 3.19 0.0351

As shown in the above table, among the three factors, the effect on the anthocyanin extract content was shown in the order of treatment time, treatment temperature, and enzyme activity in the order of enzyme pretreatment.

Regression coefficients of secondary polynomial regression curves of total anthocyanin content after treatment with Pectinex Ultra SP-L enzyme Parameter Estimate Hongyoung Intercept 1.878333 X ₁ ^{1 )} -0.009375 X ₂ ^{2 )} 0.008125 X ₃ ^{3 )} 0.027500 X ₁ * X ₁ -0.031042 X ₂ * X ₁ 0.013750 X ₂ * X ₂ -0.386042 X ₃ * X ₁ -0.070000 X ₃ * X ₂ -0.107500 X ₃ * X ₃ 0.270208 ¹⁾ Conc. of enzymatic activity (mL / 100 L) ²⁾ Temperature (℃) ³⁾ Time (hr)

Adequacy of total anthocyanin content after treatment with Pectinex Ultra SP-L enzyme Regression Hongyoung R ² F value Pr  > F Linear 0.0049 0.09 0.9643 Quadratic 0.5929 11.05 0.0002 Cross product 0.0445 0.83 0.4929 Total regress 0.6423 3.99 0.0048

Experimental Example  5-5: Hongyoung  Used Pectinex Ultra SP -L of enzyme surface treatment

In addition to the above analysis, the reaction surface contour of total anthocyanin content was examined after treatment with Pectinex Ultra SP-L enzyme using red pepper. The results are shown in FIG. As shown in FIG. 6, it was shown that the temperature (X ₂ ) and time (X ₃ ) rather than the concentration (X ₁ ) in the reaction surface curve according to the reaction surface methodology had an effect on yield improvement upon extraction of anthocyanin .

Experimental Example  5-6: Hongyoung  Used Pectinex Ultra SP -L of Enzyme pretreatment  After total polyphenol and flavonoid content; And DPPH radical Scatters  And SOD - like activity  Research

In addition, total polyphenol and flavonoid contents were investigated after pretreatment with Pectinex Ultra SP-L using red pepper. The results are shown in the following table.

Total polyphenol and flavonoid content after pretreatment with Pectinex Ultra SP-L enzyme using Hongyeong Experiment number X _One X ₂ X ₃ Total polyphenol  contents ( mg / g) Total flavonoid  contents ( mg / mL ) One 0 -One One 3.48 ± 0.06 2.88 ± 0.09 2 0 One -One 3.52 + 0.21 2.84 ± 0.12 3 0 0 0 3.34 ± 0.07 2.63 ± 0.05 4 0 -One -One 3.48 0.12 3.00 ± 0.16 5 -One 0 One 3.46 ± 0.01 2.76 ± 0.06 6 -One -One 0 3.87 ± 0.10 3.00 + 0.07 7 -One 0 -One 3.31 ± 0.01 2.88 ± 0.06 8 0 0 0 3.57 ± 0.09 3.22 ± 0.19 9 One One 0 3.29 ± 0.14 2.97 ± 0.24 10 0 0 0 3.44 ± 0.17 2.81 ± 0.09 11 One 0 -One 3.15 + 0.02 2.71 ± 0.09 12 One 0 One 4.33 ± 0.09 3.06 ± 0.14 13 One -One 0 3.90 + 0.13 2.91 ± 0.12 14 0 One One 4.09 + 0.03 2.77 ± 0.05 15 -One One 0 3.28 ± 0.03 2.67 ± 0.06

As a result, as shown in the above table, the enzyme pretreatment process with the highest total polyphenol content was an enzyme concentration of 1.5 mL / 100 L, an enzyme treatment temperature of 40 ° C and an enzyme treatment time of 8 hours, and the total polyphenol content in the process was 4.33 mg / g, while it was 3.83 mg / g in the non-treatment (Table 3).

DPPH radical scavenging activity and SOD-like activity were measured after pretreatment with Pectinex Ultra SP-L enzyme using Hongyeong.

DPPH radical scavenging activity and SOD-like activity after pretreatment with Pectinex Ultra SP-L enzyme Experiment number X _One X ₂ X ₃ DPPH radical  scavenging activity (%) SOD - like activity  (%) One 0 -One One 44.61 + - 0.75 13.48 ± 0.53 2 0 One -One 42.47 ± 2.71 13.10 ± 0.00 3 0 0 0 40.47 ± 2.37 14.74 ± 0.18 4 0 -One -One 38.58 + - 4.33 14.99 ± 0.18 5 -One 0 One 38.70 + - 4.87 12.47 ± 0.18 6 -One -One 0 42.47 + - 4.48 13.98 ± 0.53 7 -One 0 -One 40.59 ± 3.26 12.47 ± 0.18 8 0 0 0 29.68 + - 4.57 13.48 ± 0.53 9 One One 0 38.60 ± 0.08 15.11 + - 0.36 10 0 0 0 35.84 + - 0.10 9.19 ± 0.18 11 One 0 -One 37.08 + - 4.70 12.47 ± 0.18 12 One 0 One 37.58 ± 3.99 14.36 ± 0.36 13 One -One 0 40.23 + - 0.11 11.21 ± 1.25 14 0 One One 51.49 + - 3.00 15.99 ± 0.18 15 -One One 0 28.06 ± 1.57 5.67 ± 0.18

As shown in the table, DPPH radical scavenging activity and SOD-like activity after enzyme pretreatment were 28.06-51.49% and 5.67-15.99%, respectively. Especially, SOD-like activity was increased compared with no treatment (Table 4) The results are shown.

Experimental Example  6: Development of optimal extraction process of anthocyanin-based natural pigments

Experimental Example  6-1: Experimental design of ethanol extraction process

Pectinase Ultra SP-L from Novozyme (Bagsvaerd, Denmark) was used as the pretreatment enzyme treatment in the present invention, and ethanol was used as a solvent. Response surface methodology (RSM) was used to optimize the ethanol extraction process for selected mushroom and red mullet. The central composite design was used for the experimental design of the extraction process and MINITAB statistical software was used for the reaction surface analysis. The factors and levels, each material / solvent ratio of the ethanol extraction process (X ₁₎ 10-20% extraction temperature (X ₂₎ 30-70 ℃, extraction time (X ₃₎ was 1-5 hours (Table 38). These independent variables were coded as 3 units (-1,0,1) and set according to the central synthesis plan (Table 39). The analytical method was determined in accordance with the method of screening the raw material of the anthocyanin-based natural pigment of Example 1 above.

Independent variables Symbol Levels -One 0 One Ratio of sample to solvent (%) X ₁ 10 15 20 Temperature (° C) X ₂ 30 50 70 Time (hr) X ₃ One 3 5

Experiment number X _One X ₂ X ₃ Ratio of sample  to solvent (%) Temperature (° C) Time ( hr ) One 0 -One One 15 30 5 2 0 One -One 15 70 One 3 0 0 0 15 50 3 4 0 -One -One 15 30 One 5 -One 0 One 10 50 5 6 -One -One 0 10 30 3 7 -One 0 -One 10 50 One 8 0 0 0 15 50 3 9 One One 0 20 70 3 10 0 0 0 15 50 3 11 One 0 -One 20 50 One 12 One 0 One 20 50 5 13 One -One 0 20 30 3 14 0 One One 15 70 5 15 -One One 0 10 70 3

Experimental Example  6-2: Determination of total anthocyanin content according to ethanol extraction conditions of bokbunja

The total anthocyanin content was measured according to the ethanol extraction conditions of the bokbunja, and the results are shown in the following table.

Total anthocyanin content of bokbunja according to ethanol extraction conditions Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 5.20 ± 0.02 2 0 One -One 2.68 ± 0.02 3 0 0 0 4.10 ± 0.01 4 0 -One -One 5.24 + 0.04 5 -One 0 One 3.34 ± 0.04 6 -One -One 0 4.04 + 0.02 7 -One 0 -One 3.23 ± 0.05 8 0 0 0 3.80 ± 0.02 9 One One 0 3.11 ± 0.00 10 0 0 0 4.14 + 0.04 11 One 0 -One 3.46 ± 0.00 12 One 0 One 4.23 + 0.04 13 One -One 0 2.81 ± 0.01 14 0 One One 3.55 ± 0.05 15 -One One 0 3.27 ± 0.01

As a result, as shown in the above table, among the fifteen test points, the condition of the largest anthocyanin content in the ethanol extraction was the sample to solvent ratio of 15% (w / v), extraction temperature 30, extraction time 1 hour.

Experimental Example  6-3: Analysis of variance of total anthocyanin content of bokbunja ethanol extract

Analysis of variance of total anthocyanin content, regression coefficient of secondary polynomial regression curve, and fitness of secondary polynomial regression equation were performed after ethanol extraction from bokbunja. The results are shown in the following table.

Analysis of variance of total anthocyanin content after extraction of ethanol from bokbunja Factor Bokbunja F value Pr  > F Ratio of sample to solvent (%) 3.77 0.0192 Temperature (° C) 6.24 0.0020 Time (hr) 1.61 0.2104

As shown in the above table, among the three factors, the extraction temperature, the ratio of the solvent to the solvent to the solvent, and the extraction time showed the effect on the anthocyanin content in the ethanol extraction.

The regression coefficient of the secondary polynomial regression curve of total anthocyanin content after ethanol extraction of bokbunja Parameter Estimate Bokbunja Intercept 4.011777 X ₁ ^{1 )} -0.032162 X ₂ ^{2 )} -0.585637 X ₃ ^{3 )} 0.214574 X ₁ * X ₁ -0.650762 X ₂ * X ₁ 0.266897 X ₂ * X ₂ -0.052643 X ₃ * X ₁ 0.162250 X ₃ * X ₂ 0.228495 X ₃ * X ₃ 0.205613 ¹⁾ Ratio of sample to solvent (%) ²⁾ Temperature (℃) ³⁾⁾ Time (hr)

Conformity of total anthocyanin content after extraction of bokbunja ethanol Regression Bokbunja R ² F value Pr  > F Linear 0.3838 7.99 0.0011 Quadratic 0.2224 4.63 0.0129 Cross product 0.0737 1.53 0.2363 Total regress 0.6799 4.72 0.0019

Experimental Example  6-4: Reaction surface contour irradiation after extracting bokbunja ethanol

The reaction surface contour lines of total anthocyanin content were examined after extracting bokbunja ethanol, and the results are shown in Fig. As shown in Fig. 7, the optimum extraction conditions of the reaction surface analysis method were sample-to-solvent ratio of 14.6% (w / v), extraction temperature of 30 ° C, and extraction time of 1 hour. The anthocyanin content in this extract was 4.80 (%, dry weight), suggesting that the above condition is an excellent extraction condition.

Experimental Example  6-5: Hong Young  Determination of total anthocyanin content by ethanol extraction conditions

Total anthocyanin content was measured according to the ethanol extraction conditions of Hongyoung, and the results are shown in the following table.

Total anthocyanin content of ethanol extract of Hongyoung Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 1.85 ± 0.05 2 0 One -One 1.48 ± 0.05 3 0 0 0 1.85 ± 0.00 4 0 -One -One 1.72 + - 0.01 5 -One 0 One 1.38 + 0.07 6 -One -One 0 2.02 + 0.02 7 -One 0 -One 1.54 ± 0.01 8 0 0 0 1.86 ± 0.02 9 One One 0 1.55 ± 0.01 10 0 0 0 1.51 + 0.02 11 One 0 -One 2.01 ± 0.00 12 One 0 One 2.17 ± 0.05 13 One -One 0 1.41 ± 0.00 14 0 One One 1.63 + - 0.01 15 -One One 0 1.65 ± 0.01

As a result, as shown in the above table, the anthocyanin content of the 15 ethanol extracts was highest at the sample to solvent ratio of 20% (w / v), the extraction temperature was 50 ° C, and the extraction time was 5 hours.

Experimental Example  6-6: Hongyoung  Analysis of the distribution of total anthocyanin content in ethanol extracts

Analysis of variance of total anthocyanin content, regression coefficient of secondary polynomial regression curve, and fitness test of secondary polynomial regression equation were performed after ethanol extraction from Hongyoung. The results are shown in the following table.

Analysis of the distribution of total anthocyanin content after ethanol extraction of Hongyoung Factor Hongyoung F value Pr> F Ratio of sample to solvent (%) 2.72 0.0399 Temperature (° C) 3.37 0.0161 Time (hr) 0.82 0.5177

As a result, among the three factors as shown in the above table, the extraction temperature, the ratio of the solvent to the solvent to the solvent, and the extraction time showed the effect on the anthocyanin content in the ethanol extraction.

Regression coefficients of secondary polynomial regression curves of total anthocyanin content after ethanol extraction of Hongyoung Parameter Estimate Hongyoung Intercept 1.737056 X ₁ ^{1 )} 0.067497 X ₂ ^{2 )} -0.083939 X ₃ ^{3 )} 0.034512 X ₁ * X ₁ 0.014264 X ₂ * X ₁ 0.125814 X ₂ * X ₂ -0.097837 X ₃ * X ₁ 0.078543 X ₃ * X ₂ 0.008178 X ₃ * X ₃ 0.030157 ¹⁾ Ratio of sample to solvent (%) ²⁾⁾ Temperature (° C) ³⁾⁾ Time (hr)

The suitability of total anthocyanin content after ethanol extraction Regression Hongyoung R ² F value Pr> F Linear 0.1266 2.96 0.0409 Quadratic 0.0519 1.22 0.3139 Cross product 0.1092 2.56 0.0657 Total regress 0.2877 2.24 0.034

Experimental Example  6-4: Hongyoung  Ethanol extraction and reaction surface contour irradiation

The reaction surface contour lines of the total anthocyanin content were investigated after ethanol extraction of Hongyoung, and the results are shown in Fig. As shown in FIG. 8, the optimum extraction conditions of the reaction surface analysis method were as follows: sample-to-solvent ratio of 20% (w / v), extraction temperature of 55.4 ° C, and extraction time of 5 hours. Anthocyanin content was 1.97 (%, dry weight) in this extraction condition, suggesting that the above condition is an excellent extraction condition.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (13)

a) treating the Berry or color potato with a Pectinex enzyme; And
b) extracting the raw material treated with the Pectinex enzyme with water, an alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof.
The method according to claim 1, wherein the berries are selected from the group consisting of olive, bergamot and blueberry, and the color potato is self-pot or red-spot.
The method according to claim 1, wherein the pectinex enzyme is Pectinex Ultra SP-L (Novozyme) enzyme.
The method according to claim 1, wherein the alcohol is ethanol.
[2] The method according to claim 1, wherein the step a) comprises treating the vertebrate or the color potato with a Pectinex enzyme at a temperature of 20 ° C to 60 ° C for 2 to 8 hours.
3. The method according to claim 2, wherein the treatment of the bacterium with pectinex enzyme is performed at a temperature of 30 to 50 DEG C for 2 to 3 hours.
3. The method according to claim 2, wherein the treatment of the pectinex enzyme with the oti is performed at a temperature of 20 to 40 DEG C for 2 to 5 hours.
3. The method according to claim 2, wherein the treatment of the self-pectinex enzyme is performed at a temperature of 20 to 40 DEG C for 2 to 3 hours.
3. The method according to claim 2, wherein the treatment of the pectinex enzyme with the red pepper is carried out at a temperature of 30 to 50 DEG C for 6 to 8 hours.
The method according to claim 4, wherein the ethanol extraction in step b) is performed at a sample to solvent ratio of 10% (w / v) to 20% (w / v) at 30 ° C to 70 ° C for 1 hour to 5 hours Lt; / RTI &gt;
The method according to claim 2, wherein the extraction of the brambles in step (b) is performed using ethanol as an extraction solvent at a temperature of 30 ° C to 50 ° C at a sample to solvent ratio of 10% (w / v) to 15% Lt; / RTI &gt; for 3 hours to 5 hours.
3. The method according to claim 2, wherein ethanol extraction is carried out at a temperature of 40 to 60 DEG C in a ratio of 15% (w / v) to 20% (w / v) Lt; / RTI &gt; for 3 hours to 5 hours.
12. A method for producing anthocyanin comprising separating anthocyanin from berries or colored potato extract comprising anthocyanin produced by the method of any one of claims 1 to 12.

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