KR101480209B1 - Method for preparation of extracts of berries and colored potatos comprising anthocyanin-based pigments using Novozym 33095 - Google Patents

Abstract

The present invention relates to a method for preparing an extract of berries or color potatoes having an increased anthocyanin pigment content by pretreating an enzyme of Novozym 33095 (Novozyme 33095, Novozyme), specifically a) Lt; / RTI > And b) extracting the raw material treated with the novozyme 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, extract; Or a composition containing the same, and a method for separating anthocyanin from an extract prepared by the method.

Description

FIELD OF THE INVENTION The present invention relates to a method for preparing an extract of berries or colored potatoes containing anthocyanin-based pigments using NOVOZYM 33095,

The present invention relates to a method for preparing an extract of berries or color potatoes having an increased anthocyanin pigment content by pretreating an enzyme of Novozym 33095 (Novozyme 33095, Novozyme), specifically a) Lt; / RTI > And b) extracting the raw material treated with the novozyme 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, extract; 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 technique capable of separating large quantities 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 novozyme 33095 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 color potatoes having an increased anthocyanin pigment content, comprising the step of pre-treating Novozymes 33095 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 potatoes having an increased anthocyanin pigment content, comprising the step of pre-treating novozyme 33095 to provide.

A) treating novozyme 33095 enzyme in berries or colored potatoes at a temperature of 20 ° C to 60 ° C for 1 hour to 2 hours; And b) extracting the raw material treated with Novozymes 33095 enzyme with water, an alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof.

The step a) is a step of treating novozyme 33095 enzyme in order to improve extraction yield of anthocyanin pigment in the production of berries or color potato extract.

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 brambles refer to fruits that can increase the extraction of anthocyanin-based pigments upon pre-treatment of Novozymes 33095 enzyme. In addition, the above-mentioned Odi is a deciduous mulberry ( Morus alba L.) as a fruit of a variety of physiological activities such as flavonoids, stilbenes, prenylflavonoids, coumarin, deoxynoijirimycin and rutin It is known to contain substances. 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 pigment is increased by pretreatment with a Novozyme 33095 enzyme to a color potato such as berries such as brambles or audi or self-smearing or red flour 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 " Novozym 33095 enzyme "in the present invention is a pectinolytic enzyme preparation of Novozymes, which has a pectin lyase activity. In the present specification, the Novozymes 33095 can be used in combination with Novozyme® 33095. In one embodiment of the present invention, the optimized treatment conditions of the Novozymes 33095 enzyme were elucidated in order to extract a high content of anthocyanin pigment from bokbunja, audi, selfish or Hongyoung.

Specifically, in the case of pretreatment of Novozymes 33095 enzyme to bokbunja, treatment temperature and treatment time have a greater effect on the anthocyanin content than the concentration considering the enzyme activity. Therefore, the treatment of the Novozymes 33095 enzyme can be performed preferably at a temperature of 20 to 60 캜 for 1 hour to 2 hours, more preferably at a temperature of 20 to 40 캜 for 1.5 to 2 hours .

In an embodiment of the present invention, when the Novozyme 33095 enzyme is treated with an enzyme concentration of 0.25 mL / L, an enzyme treatment temperature of 20 ° C and an enzyme treatment time of 1.5 hours in the bokbunja, the anthocyanin content of the enzyme can be increased by 7 times or more (Experimental Examples 2-3 and Table 8). It was also confirmed that when the Novozyme 33095 enzyme was subjected to an enzyme concentration of 0.15 mL / L, an enzyme treatment temperature of 60 DEG C, and an enzyme treatment time of 2 hours, the polyphenol content could be enhanced as compared with the control group (Experimental Example 2 -6 and Table 12). In the case of flavonoid, the highest flavonoid content was obtained at the enzyme concentration of 0.25 mL / L, the enzyme treatment temperature of 40 ℃ and the enzyme treatment time of 1 hour. In addition, DPPH radical scavenging activity and SOD-like activity were increased when Novozymes 33095 enzyme was pretreated before bacteriological extraction, suggesting that it could be useful for preparing antioxidant-active extracts (Table 13).

In addition, in the case of pretreatment of ODIENOBOZYME 33095 enzyme, the treatment temperature has a great influence on the anthocyanin content rather than the concentration considering the enzyme activity and the treatment time. Accordingly, the treatment of the Novozymes 33095 enzyme can be performed at a temperature of preferably 20 ° C to 60 ° C, more preferably 30 ° C to 40 ° C, but is not limited thereto. The treatment of the Novozymes 33095 enzyme can be carried out preferably at a temperature of 20 ° C to 40 ° C and for 1 hour to 2 hours, more preferably at a temperature of 30 ° C to 40 ° C and for 1 hour to 1.5 hours, It does not.

In an embodiment of the present invention, when the Novozyme 33095 enzyme was treated with an enzyme at an enzyme concentration of 0.25 mL / L, an enzyme treatment temperature of 40 DEG C, and an enzyme treatment time of 1 hour, the anthocyanin content was increased to about 6 times that of the control group (Experimental Examples 3-3 and Table 16). In addition, DPPH radical scavenging activity and SOD-like activity were increased when Novozymes 33095 enzyme was pretreated before ODI extraction, suggesting that it can be useful for preparing antioxidant-active extracts (Table 21).

In addition, in the case of pretreatment of Novozymes 33095 enzyme for self-administration, the treatment temperature and the treatment time have a greater influence on the anthocyanin content than the concentration considering the enzyme activity. Therefore, the treatment of the Novozymes 33095 enzyme can be carried out preferably at a temperature of 20 ° C to 60 ° C for 1 hour to 2 hours, more preferably at a temperature of 30 ° C to 50 ° C for 1.5 hours to 2 hours .

In one embodiment of the present invention, when the novozyme 33095 enzyme was treated with an enzyme concentration of 0.05 mL / L, an enzyme treatment temperature of 40 ° C and an enzyme treatment time of 2 hours, the anthocyanin content was increased to 3.1 times that of the control group (Experimental Example 4-3 and Table 24). In addition, it was confirmed that the content of polyphenol can be increased when Novozyme 33095 enzyme was carried out at an enzyme concentration of 0.25 mL / L, an enzyme treatment temperature of 40 ° C, and an enzyme treatment time of 1 hour. In the case of flavonoid, the highest flavonoid content was shown at the enzyme concentration of 0.15 mL / L, the enzyme treatment temperature of 20 ° C and the enzyme treatment time of 1 hour (Experimental Examples 4-6 and Table 28). In addition, when the Novozymes 33095 enzyme was pretreated before self-extraction, the SOD-like activity was increased, suggesting that it could be useful for preparing antioxidant-active extracts (Table 29).

In addition, in the case of pretreatment of Novozymes 33095 enzyme to Hongyoung, the treatment temperature and the treatment time have a greater effect on the anthocyanin content than the concentration considering the enzyme activity. Accordingly, the treatment of the Novozymes 33095 enzyme can be carried out preferably at a temperature of 20 ° C to 60 ° C for 1 hour to 2 hours, more preferably at a temperature of 40 ° C to 60 ° C for 1.5 hours to 2 hours .

In one embodiment of the present invention, when the Novozyme 33095 enzyme was treated with an enzyme concentration of 0.15 mL / L, an enzyme treatment temperature of 60 ° C and an enzyme treatment time of 2 hours, the anthocyanin content was increased to about 3 times that of the control group (Experimental Examples 5-3 and Table 32). The highest polyphenol content was obtained when Novozyme 33095 enzyme was used at an enzyme concentration of 0.05 mL / L, an enzyme treatment temperature of 40 ° C, and an enzyme treatment time of 1 hour. The enzyme concentration was 0.15 mL / L, 60 ° C, and enzyme treatment time of 1 hour, respectively (Experimental Examples 5-6 and Table 36). In addition, DPPH radical scavenging activity and SOD-like activity were increased when Novozyme 33095 enzyme was pretreated before the red-leaf extract, suggesting that it could be useful for preparing antioxidant-active extracts (see Table 37) .

In step b), berries or color potatoes pretreated with Novozyme 33095 enzyme are extracted with water, an alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof to prepare an extract of berries or color potatoes having an increased anthocyanin pigment content . In the case of extracting the raw material pretreated with the Novozymes 33095 enzyme in 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 polar solvent having 1 to 4 carbon atoms, or a mixture ratio of these solvents of about 1: 0.1 to 1:10, But it is preferable that the concentration of ethanol is 60 to 90% as ethanol.

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 berries or color potato by pretreating the Novozyme 33095 enzyme of the present invention results in a significant increase in the content of the anthocyanin-based natural pigment in the preparation of the extract, the present inventors have found that anthocyanin- And can be usefully used in medicine, food, or cosmetics.

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 reaction surface contours of total anthocyanin content after treatment with Novozym 33095 enzyme using brambles.
4 is a graph showing the reaction surface contour of total anthocyanin content after Novozym 33095 enzyme treatment using an olive oil.
FIG. 5 is a graph showing reaction surface contours of total anthocyanin content after Novozym 33095 enzyme treatment using self-administration. FIG.
FIG. 6 is a graph showing reaction surface contours of total anthocyanin content after treatment with Novozym 33095 enzyme using red sea bream.
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, Novozym (R) 33095 from Novozyme (Bagsvaerd, Denmark) was used as a pretreatment process before extraction of anthocyanin with ethanol to improve the yield during anthocyanin extraction. Specifically, Novozym® 33095 is an enzyme preparation comprising pectin lyase.

The enzymatic pretreatment of Novozym 33095 (Novozym 33095) was carried out by adding 8 times water to lyophilized brambles, olive oil, green alginate, and red lemon, and the suspension was adjusted to pH 4. The concentration was adjusted to Novozym 33095 ) Was added and reacted at 250 rpm in a shaking incubator (Si-900R, Jeio Tech, Korea) at a treatment temperature (X ₂ ) of 20-60 ° C and a treatment time (X ₃ ) of 1-2 hours. After enzyme treatment, the enzyme was removed by centrifugation at 4000 rpm for 10 minutes with a centrifuge (UNION 55R, Hanil, Korea). 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. Specifically, the treatment conditions of the enzyme pretreatment process of Novozym 33095 are 0.05-0.25 (ml / L) in terms of concentration (X ₁ ) considering the enzyme activity for the sample, 20 ° C-60 ° C at the treatment temperature (X ₂ ) (X ₃ ) was 1-2 hours (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.

Independent variables Symbol Levels -One 0 One Conc. of enzymatic activity (mL / L) X ₁ 0.05 0.15 0.25 Temperature (° C) X ₂ 20 40 60 Time (min) X ₃ 60 90 120

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 resulting filtrate was concentrated under reduced pressure at 40 ° C using a vacuum rotary condenser (Buchi R-114, SWISS) and lyophilized (Ilshin Biobase, KOREA). In addition, the analysis method was analyzed by 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 scavenging activity and elastase inhibitory activity are shown in Tables 3 - 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 Novozym  33095 Confirmation of effect by enzyme pretreatment

Experimental Example  2-1: Using bokbunja Novozym  33095 of Enzyme pretreatment  Experimental design of process

In order to determine the optimum pretreatment effect of Novozym 33095 enzyme pretreatment using bokbunja, the following composition was designed as shown in Table 6 below.

Experimental Design of Novozym 33095 Enzyme Pretreatment Process Using Bokbunja Experiment number X _One X ₂ X ₃ Conc . of enzymatic  activity ( mL / L) Temperature (° C) Time ( min ) One 0 -One One 0.15 20 120 2 0 One -One 0.15 60 60 3 0 0 0 0.15 40 90 4 0 -One -One 0.15 20 60 5 -One 0 One 0.05 40 120 6 -One -One 0 0.05 20 90 7 -One 0 -One 0.05 40 60 8 0 0 0 0.15 40 90 9 One One 0 0.25 60 90 10 0 0 0 0.15 40 90 11 One 0 -One 0.25 40 60 12 One 0 One 0.25 40 120 13 One -One 0 0.25 20 90 14 0 One One 0.15 60 120 15 -One One 0 0.05 60 90

Experimental Example  2-2: Using Rubus Novozym 33095 Enzyme pretreatment  Measurement of post-yield and freeze-dried yield

Based on the above design conditions, the yield and lyophilization yield after pretreatment of Novozym 33095 enzyme using bokbunja were measured and the results are shown in Table 7 below.

Yield and lyophilization yield of Novozym 33095 after enzyme pretreatment using bokbunja Experiment number X _One X ₂ X ₃ Yield after enzyme pre - treatment (%) Yield after freeze  dehydration treatment (%) One 0 -One One 26.79 4.45 2 0 One -One 32.59 5.08 3 0 0 0 28.67 5.23 4 0 -One -One 29.58 5.38 5 -One 0 One 28.90 5.02 6 -One -One 0 29.03 6.01 7 -One 0 -One 28.02 5.82 8 0 0 0 28.04 4.78 9 One One 0 30.88 3.96 10 0 0 0 29.33 5.21 11 One 0 -One 29.13 4.38 12 One 0 One 29.49 5.15 13 One -One 0 28.61 4.21 14 0 One One 33.45 3.85 15 -One One 0 28.89 4.82

As a result, the yields after enzyme treatment were 26.79-33.45% and 3.85-6.01% after freeze-drying, respectively.

Experimental Example  2-3: using bokbunja Novozym  Measurement of total anthocyanin content after enzyme pretreatment of 33095

In addition, total anthocyanin content was measured after pre-treatment of Novozym 33095 enzyme using bokbunja. The results are shown in Table 8 below.

Anthocyanin content after enzymatic pretreatment of Novozym 33095 with rubella Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 5.67 ± 0.09 2 0 One -One 6.87 ± 0.27 3 0 0 0 3.78 ± 0.12 4 0 -One -One 5.28 ± 0.18 5 -One 0 One 6.13 ± 0.32 6 -One -One 0 6.57 ± 0.26 7 -One 0 -One 5.78 + - 0.10 8 0 0 0 5.98 ± 0.09 9 One One 0 5.87 ± 0.14 10 0 0 0 5.19 ± 0.18 11 One 0 -One 6.79 ± 0.39 12 One 0 One 5.27 ± 0.29 13 One -One 0 7.52 + - 0.12 14 0 One One 7.39 ± 0.29 15 -One One 0 5.76 ± 0.25

As shown in the above results, the anthocyanin content was only 1.10% in the untreated state (Table 2), but when Novozym 33095 was treated, it showed an increased anthocyanin content of 3.78-7.52% 7 times higher than that of the control group.

In particular, the enzyme treatment conditions with the highest total anthocyanin content were 0.25 mL / L of enzyme concentration, the enzyme treatment temperature was 20 ° C., and the enzyme treatment time was 1.5 hours. At this time, the total anthocyanin content in the process was 7.52%.

Experimental Example  2-4: Using Bokbunja Novozym  Analysis of variance of total anthocyanin content after enzyme treatment

In addition, Novozym 33095 enzyme using bokbunja was pretreated, and the total anthocyanin content was analyzed and the results are shown in the following table.

Factor Bokbunja F value Pr  > F Conc. of enzymatic activity (mL / 100 L) 1.85 0.1407 Temperature (° C) 3.63 0.0141 Time (hr) 2.80 0.0405

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 concentration considering enzyme activity was shown.

In addition, analysis of variance of total anthocyanin content and compatibility results were performed after enzymatic treatment of Novozym 33095 using bokbunja. The results are shown in the following table.

The regression coefficient of the secondary polynomial regression curve of anthocyanin content after enzymatic treatment of Novozym 33095 using bokbunja Parameter Estimate Bokbunja Intercept 4.981111 X ₁ ^{1 )} 0.151250 X ₂ ^{2 )} 0.106250 X ₃ ^{3 )} -0.306670 X ₁ * X ₁ 0.569028 X ₂ * X ₁ -0.207500 X ₂ * X ₂ 0.880694 X ₃ * X ₁ -0.465000 X ₃ * X ₂ 0.031667 X ₃ * X ₃ 0.441528 ¹⁾ Conc. of enzymatic activity (mL / L) ²⁾ Temperature (℃) ³⁾ Time (hr)

Conformity of total anthocyanin content after enzymatic treatment of Novozym 33095 using bokbunja Regression Bokbunja R ² F value Pr  > F Linear 0.0215 0.44 0.7290 Quadratic 0.3228 6.54 0.0013 Cross product 0.0796 1.61 0.2043 Total regress 0.4239 2.86 0.0122

Experimental Example  2-5: Using bokbunja Novozym  33095 Reaction surface contour irradiation after enzyme treatment

In addition, the reaction surface contour of the total anthocyanin content after enzymatic treatment of Novozym 33095 using brambles was confirmed, and the results are shown in Fig. As a result, it was shown that the treatment temperature (X ₂ ) and the treatment time (X ₃ ) had an effect on the yield improvement in the extraction of anthocyanin rather than the concentration (X ₁ ) considering the enzyme activity in the reaction surface curve by the reaction surface analysis method.

Experimental Example  2-6: Using Bokbunja Novozym  33095 Enzyme pretreatment  After total polyphenol and flavonoid content; And DPPH Radical Scatters  And SOD - like  Activity investigation

Total polyphenol and flavonoid contents were measured after pretreatment with Novozym 33095 enzyme using bokbunja, and the results are shown in Table 9 below.

Total Polyphenol and Flavonoid Content after Pretreatment of Novozym 33095 with Bokbunja Experiment number X _One X ₂ X ₃ Total polyphenol  contents ( mg / g) Total flavonoid  contents ( mg / ml ) One 0 -One One 8.91 ± 0.01 3.82 ± 0.32 2 0 One -One 9.59 ± 0.01 4.26 ± 0.40 3 0 0 0 9.93 + - 0.01 3.86 ± 0.42 4 0 -One -One 9.67 ± 0.00 4.01 + - 0.47 5 -One 0 One 11.05 + 0.02 4.33 + - 0.50 6 -One -One 0 8.08 ± 0.01 3.47 ± 0.40 7 -One 0 -One 10.08 ± 0.00 3.99 + - 0.38 8 0 0 0 10.28 ± 0.00 4.46 + 0.14 9 One One 0 9.17 ± 0.01 4.10 ± 0.22 10 0 0 0 9.12 ± 0.01 3.95 + - 0.43 11 One 0 -One 11.79 ± 0.00 4.98 ± 0.20 12 One 0 One 7.24 ± 0.00 3.36 + - 0.42 13 One -One 0 8.62 ± 0.35 3.58 ± 0.21 14 0 One One 11.34 ± 0.22 4.90 + - 0.34 15 -One One 0 8.65 ± 0.71 3.36 0.30

As shown in the table, the enzyme pretreatment process with the highest total polyphenol content was 0.15 mL / L of enzyme concentration, the enzyme treatment temperature was 60 ° C, and the enzyme treatment time was 2 hours. The total polyphenol content in the process was 11.34 mg / g Respectively. In contrast, total flavonoids showed the highest enzyme pretreatment at 0.25 mL / L of enzyme concentration, enzyme treatment temperature of 40 ℃, enzyme treatment time of 1 hour, and the total flavonoid content in the process was 4.98 mg / mL. The total polyphenol content and the total flavonoid content in the untreated samples were 8.25 mg / g and 4.42 mg / mL, respectively (Table 3). Thus, the results show that the Novozym 33095 enzyme pretreatment of the present invention significantly increased polyphenol and flavonoid contents This is a result indicating that

DPPH radical scavenging activity and SOD-like activity were measured after enzymatic pretreatment of Novozym 33095 using bokbunja, and the results are shown in the following table.

DPPH radical scavenging activity and SOD-like activity after enzymatic pretreatment of Novozym 33095 using bokbunja Experiment number X _One X ₂ X ₃ DPPH radical  scavenging activity (%) SOD - like activity  (%) One 0 -One One 56.53 + - 3.06 12.64 + - 0.98 2 0 One -One 46.25 + - 3.34 10.06 + - 0.64 3 0 0 0 47.26 + - 5.83 10.06 + - 0.64 4 0 -One -One 50.26 + - 2.66 17.16 + - 0.34 5 -One 0 One 45.50 ± 2.98 13.16 ± 0.24 6 -One -One 0 55.65 ± 3.23 14.71 ± 0.23 7 -One 0 -One 53.64 ± 1.80 11.09 + - 0.63 8 0 0 0 45.24 + 1.92 9.03 + - 0.65 9 One One 0 47.48 + - 4.45 14.58 ± 0.32 10 0 0 0 48.61 ± 3.91 14.19 ± 0.13 11 One 0 -One 39.74 ± 3.65 8.11 ± 5.22 12 One 0 One 59.38 + - 4.19 9.67 ± 1.55 13 One -One 0 55.75 + - 4.04 14.96 ± 0.96 14 0 One One 42.86 ± 1.01 16.51 ± 0.58 15 -One One 0 58.29 ± 5.20 14.06 + - 0.42

As shown in the above table, DPPH radical scavenging activity and SOD-like activity were 11.76% and 1.10%, respectively, in the untreated control (Table 4), but significantly increased to 39.74-59.38% and 8.11-17.16% when treated with Novozym 33095 Respectively.

Experimental Example  3: Using Audi Novozym  33095 Confirmation of effect by enzyme pretreatment

Experimental Example  3-1: Using Audi Novozym 33095  Experimental design of enzyme pretreatment process

Novozym 33095 using ODI. In order to confirm the pretreatment effect of the enzyme and to derive the optimal conditions, the experiment of the process as shown in the following table was designed.

Experimental Design of Novozym 33095 Enzyme Pretreatment Process Using Oddi Experiment number X _One X ₂ X ₃ Conc . of enzymatic  activity ( mL / L) Temperature (° C) Time ( min ) One 0 -One One 0.15 20 120 2 0 One -One 0.15 60 60 3 0 0 0 0.15 40 90 4 0 -One -One 0.15 20 60 5 -One 0 One 0.05 40 120 6 -One -One 0 0.05 20 90 7 -One 0 -One 0.05 40 60 8 0 0 0 0.15 40 90 9 One One 0 0.25 60 90 10 0 0 0 0.15 40 90 11 One 0 -One 0.25 40 60 12 One 0 One 0.25 40 120 13 One -One 0 0.25 20 90 14 0 One One 0.15 60 120 15 -One One 0 0.05 60 90

Experimental Example  3-2: Using Audi Novozym  33095 Measurement of yield and freeze-drying yield after enzyme pretreatment

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 Novozym 33095 after pre-treatment with enzyme Experiment number X _One X ₂ X ₃ Yield after enzyme pre - treatment (%) Yield after freeze  dehydration treatment (%) One 0 -One One 28.33 7.35 2 0 One -One 31.80 7.41 3 0 0 0 27.44 7.32 4 0 -One -One 30.72 7.48 5 -One 0 One 27.19 7.68 6 -One -One 0 29.84 6.46 7 -One 0 -One 26.72 6.81 8 0 0 0 27.28 6.89 9 One One 0 30.86 6.23 10 0 0 0 28.66 7.19 11 One 0 -One 28.23 7.17 12 One 0 One 28.33 4.38 13 One -One 0 30.38 6.38 14 0 One One 28.80 3.64 15 -One One 0 32.39 7.28

As shown in the table, the yield after enzyme treatment was 27.19-32.39%, and the yield after freeze drying was 3.64-7.68%.

Experimental Example  3-3: Using Audi Novozym  33095 Total anthocyanin content measurement after enzyme pretreatment

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

The total anthocyanin content after pretreatment of Novozym 33095 with the enzyme Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 4.34 0.66 2 0 One -One 4.74 ± 0.78 3 0 0 0 3.67 ± 0.11 4 0 -One -One 3.91 ± 0.21 5 -One 0 One 3.36 ± 0.21 6 -One -One 0 5.15 + - 0.43 7 -One 0 -One 5.36 ± 0.36 8 0 0 0 4.76 ± 0.05 9 One One 0 5.33 + - 0.54 10 0 0 0 5.16 ± 0.44 11 One 0 -One 5.60 ± 0.65 12 One 0 One 3.74 ± 0.29 13 One -One 0 4.77 + - 0.45 14 0 One One 3.92 ± 0.25 15 -One One 0 5.38 ± 0.36

As shown in the above results, the enzyme treatment conditions with the highest total anthocyanin content were an enzyme concentration of 0.25 mL / L, an enzyme treatment temperature of 40 ° C and an enzyme treatment time of 1 hour, and the total anthocyanin content was 5.60% . Also, the total anthocyanin content ranged from 3.36 to 5.60% depending on the enzyme treatment conditions. The total anthocyanin content was only 0.92% (Table 2), suggesting that pre-treatment of Novozym 33095 may increase the anthocyanin content by approximately 6-fold and thus may be useful for increasing the anthocyanin content of the Ahi extract .

Experimental Example  3-4: Using Audi Novozym  Analysis of variance of total anthocyanin content after enzyme treatment

Analysis of variance of total anthocyanin content, the regression coefficient of the quadratic polynomial regression curve, and the fitness test of the secondary polynomial regression equation were performed after Novozym 33095 enzyme treatment using ODI, and the results are shown in the table below.

Analysis of total anthocyanin content after enzymatic treatment of Novozym 33095 Factor Audi F value Pr  > F Conc. of enzymatic activity (mL / 100 L) 0.83 0.5243 Temperature (?) 0.94 0.4629 Time (hr) 4.05 0.0145

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

Regression coefficient of secondary polynomial regression curve of total anthocyanin content after enzymatic treatment of Novozym 33095 Parameter Estimate Audi Intercept 4.530000 X ₁ ^{1 )} 0.023750 X ₂ ^{2 )} 0.152500 X ₃ ^{3 )} -0.531250 X ₁ * X ₁ 0.455625 X ₂ * X ₁ 0.081250 X ₂ * X ₂ 0.170625 X ₃ * X ₁ 0.036250 X ₃ * X ₂ -0.311250 X ₃ * X ₃ -0.471875 ¹⁾ Conc. of enzymatic activity (mL / L) ²⁾ Temperature (℃) ³⁾ Time (hr)

The suitability of total anthocyanin content after enzymatic treatment of Novozym 33095 Regression Audi R ² F value Pr  > F Linear 0.2726 3.81 0.0261 Quadratic 0.2038 2.85 0.0633 Cross product 0.0467 0.65 0.5908 Total regress 0.5231 2.44 0.0466

Experimental Example  3-5: Using Audi Novozym  33095 Reaction surface contour irradiation after enzyme treatment

In addition to the above analysis, the surface contour of the total anthocyanin content after Novozym 33095 enzyme treatment with ODI was investigated. The results are shown in FIG. 4, the effect on the concentration (X ₁₎ to the processing temperature (X ₂₎ Processing time (X ₃₎ the yield during anthocyanin extraction rather than considering the enzymatic activity in the response surface curve of the case of the audio response surface methodology .

Experimental Example  3-6: Using Audi Novozym  33095 Enzyme pretreatment  After total polyphenol and flavonoid content; And DPPH radical Scatters  And SOD - like activity  Research

Also, total polyphenol and flavonoid contents were investigated after pretreatment of Novozym 33095 with olive oil. The results are shown in the table below.

Total polyphenol and flavonoid content after enzyme pretreatment of Novozym 33095 with Odi Experiment number X _One X ₂ X ₃ Total polyphenol  contents ( mg / g) Total flavonoid  contents ( mg / ml ) One 0 -One One 5.98 + - 0.36 3.48 0.11 2 0 One -One 6.44 ± 0.61 3.32 0.31 3 0 0 0 5.67 ± 0.25 3.66 ± 0.10 4 0 -One -One 5.56 ± 0.56 3.15 ± 0.37 5 -One 0 One 5.77 ± 0.36 3.22 + - 0.38 6 -One -One 0 5.48 ± 0.15 3.27 ± 0.14 7 -One 0 -One 6.22 ± 0.18 3.69 ± 0.09 8 0 0 0 4.70 + 0.09 2.92 + 0.04 9 One One 0 6.57 ± 0.91 3.44 ± 0.04 10 0 0 0 5.43 + 0.18 3.45 ± 0.10 11 One 0 -One 6.11 + 0.07 3.45 + - 0.42 12 One 0 One 5.35 ± 0.21 3.39 ± 0.09 13 One -One 0 6.09 ± 0.38 3.51 + - 0.25 14 0 One One 4.12 + 0.03 3.07 ± 0.00 15 -One One 0 5.40 ± 0.15 3.22 ± 0.10

As shown in the above results, the total polyphenol and flavonoid contents were 4.12-6.44% and 2.92-3.69%, respectively.

DPPH radical scavenging activity and SOD-like activity were measured after Novozym 33095 enzyme pretreatment using an olive oil.

DPPH radical scavenging activity and SOD-like activity after enzyme pretreatment of Novozym 33095 using ODI Experiment number X _One X ₂ X ₃ DPPH radical  scavenging activity (%) SOD - like activity  (%) One 0 -One One 56.52 ± 1.64 18.32 + - 0.17 2 0 One -One 54.77 ± 3.40 18.83 + - 1.65 3 0 0 0 43.22 + - 4.83 18.45 + 0.38 4 0 -One -One 52.27 ± 2.67 15.60 ± 2.59 5 -One 0 One 52.52 + - 3.39 23.36 + - 0.76 6 -One -One 0 53.13 + - 1.04 18.32 ± 0.20 7 -One 0 -One 54.64 0.32 19.61 ± 0.92 8 0 0 0 59.00 + - 5.43 19.10 ± 0.19 9 One One 0 54.87 + - 4.57 18.96 + 1.83 10 0 0 0 48.48 ± 5.15 20.77 ± 1.09 11 One 0 -One 58.40 + - 0.41 20.00 ± 0.36 12 One 0 One 49.37 ± 1.78 19.74 ± 0.37 13 One -One 0 51.75 ± 0.87 21.54 ± 1.08 14 0 One One 52.87 ± 2.82 20.13 + - 0.55 15 -One One 0 41.46 + 4.14 18.19 ± 1.48

As shown in the above results, DPPH radical scavenging activity and SOD-like activity were 41.46-59.00% and 15.60-23.36%, respectively, and the values of DPPH radical scavenging activity and SOD-like activity were 34.17% and 0.88% ) And exhibited significantly improved scavenging activity and SOD-like activity.

Experimental Example  4: Self-employed Novozym  33095 Confirmation of enzyme pretreatment effect

Experimental Example  4-1: Self-employed Novozym  33095 Experimental design of enzyme pretreatment process

Novozym 33095 enzyme pretreatment using autogenous enzyme was investigated in order to determine the optimum process conditions.

Experimental design of Novozym 33095 enzyme pretreatment process using autogenous Experiment number X _One X ₂ X ₃ Conc . of enzymatic  activity ( mL / L) Temperature (° C) Time ( min ) One 0 -One One 0.15 20 120 2 0 One -One 0.15 60 60 3 0 0 0 0.15 40 90 4 0 -One -One 0.15 20 60 5 -One 0 One 0.05 40 120 6 -One -One 0 0.05 20 90 7 -One 0 -One 0.05 40 60 8 0 0 0 0.15 40 90 9 One One 0 0.25 60 90 10 0 0 0 0.15 40 90 11 One 0 -One 0.25 40 60 12 One 0 One 0.25 40 120 13 One -One 0 0.25 20 90 14 0 One One 0.15 60 120 15 -One One 0 0.05 60 90

Experimental Example  4-2: Using self-help Novozym  33095 Measurement of yield and freeze-drying yield after enzyme pretreatment

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 of Novozym 33095 after enzyme pretreatment 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 19.36 2.11 2 0 One -One 19.08 2.63 3 0 0 0 17.82 2.17 4 0 -One -One 21.49 4.07 5 -One 0 One 17.78 2.45 6 -One -One 0 22.75 4.92 7 -One 0 -One 17.63 3.55 8 0 0 0 17.14 3.71 9 One One 0 18.92 2.45 10 0 0 0 18.20 1.37 11 One 0 -One 17.28 2.09 12 One 0 One 17.29 2.72 13 One -One 0 20.35 1.42 14 0 One One 14.11 2.33 15 -One One 0 18.80 1.31

As a result, as shown in the above table, the yield after enzyme treatment was 14.11-22.75% and the yield after freeze drying was 1.31-4.92%.

Experimental Example  4-3: Using self-help Novozym  33095 Total anthocyanin content measurement after enzyme pretreatment

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

Total anthocyanin content after enzyme pretreatment of Novozym 33095 using self-administration Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 1.30 + 0.04 2 0 One -One 1.38 ± 0.05 3 0 0 0 1.03 + - 0.01 4 0 -One -One 0.73 + 0.04 5 -One 0 One 1.59 + 0.08 6 -One -One 0 0.75 + 0.09 7 -One 0 -One 0.84 + 0.02 8 0 0 0 1.02 + - 0.01 9 One One 0 1.23 + 0.03 10 0 0 0 1.47 ± 0.05 11 One 0 -One 0.99 ± 0.02 12 One 0 One 0.71 ± 0.00 13 One -One 0 1.18 ± 0.14 14 0 One One 0.86 ± 0.03 15 -One One 0 1.36 + 0.07

As a result, as shown in the above table, the enzyme treatment condition with the highest total anthocyanin content was 0.05 mL / L of enzyme concentration, the enzyme treatment temperature was 40 ° C., and the enzyme treatment time was 2 hours. At this time, the total anthocyanin content in the process was 1.59% . (Table 2), suggesting that the total anthocyanin content can be increased up to 3 times when Novozym 33095 is self-administered.

Experimental Example  4-4: Using self-help Novozym  Analysis of variance of total anthocyanin content after enzyme treatment

Analysis of variance of the 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 Novozym 33095 enzyme treatment using self - administration. The results are shown in the following table.

Analysis of the distribution of total anthocyanin content after enzymatic treatment of Novozym 33095 using self-administration Factor Self-help F value Pr  > F Conc. of enzymatic activity (mL / 100 L) 5.46 0.0039 Temperature (° C) 6.91 0.0012 Time (hr) 9.28 0.0002

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 enzymatic treatment of Novozym 33095 Parameter Estimate Self-help Intercept 1.175000 X ₁ ^{1 )} -0.053125 X ₂ ^{2 )} 0.109375 X ₃ ^{3 )} 0.065000 X ₁ * X ₁ -0.040625 X ₂ * X ₁ -0.141250 X ₂ * X ₂ -0.005625 X ₃ * X ₁ -0.257500 X ₃ * X ₂ -0.272500 X ₃ * X ₃ -0.101875 ¹⁾ Conc. of enzymatic activity (mL / L) ²⁾ Temperature (℃) ³⁾ Time (hr)

Conformity of total anthocyanin content after enzymatic treatment of Novozym 33095 using self-administration Regression Self-help R ² F value Pr  > F Linear 0.1291 2.97 0.0567 Quadratic 0.036 0.83 0.4947 Cross product 0.5448 12.52 <.0001 Total regress 0.7098 5.44 0.0008

Experimental Example  4-5: Using self-help Novozym  33095 Reaction surface contour irradiation after enzyme treatment

In addition to the above analysis, the reaction surface contour of total anthocyanin content after Novozym 33095 enzyme treatment using self-administration was investigated, and the result is shown in FIG. As shown in FIG. 5, the extraction time (X ₃ ) and the extraction temperature (X ₂ ) influence the yield enhancement upon extraction of anthocyanin rather than the concentration (X ₁ ) considering the enzyme activity in the reaction surface curve by the reaction surface analysis Respectively.

Experimental Example  4-6: Self-employed Novozym  33095 Enzyme pretreatment  After total polyphenol and flavonoid content; And DPPH radical Scatters  And SOD - like activity  Research

The total polyphenol and flavonoid contents of Novozym 33095 after enzyme pretreatment were also examined. The results are shown in the table below.

Total polyphenol and flavonoid content after enzyme pretreatment of Novozym 33095 using self-administration Experiment number X _One X ₂ X ₃ Total polyphenol  contents ( mg / g) Total flavonoid  contents ( mg / mL ) One 0 -One One 2.67 ± 0.15 2.21 ± 0.12 2 0 One -One 3.45 ± 0.36 2.85 ± 0.06 3 0 0 0 2.67 ± 0.14 2.62 + 0.14 4 0 -One -One 3.19 ± 0.50 2.95 + 0.14 5 -One 0 One 2.57 + 0.03 2.17 ± 0.01 6 -One -One 0 2.17 ± 0.59 1.70 + - 0.10 7 -One 0 -One 1.99 + - 0.13 1.50 ± 0.05 8 0 0 0 2.53 + - 0.32 2.12 + 0.02 9 One One 0 2.69 ± 0.04 2.18 ± 0.01 10 0 0 0 2.99 + 0.03 2.39 ± 0.06 11 One 0 -One 3.45 ± 0.34 2.92 ± 0.11 12 One 0 One 3.11 + - 0.38 2.88 ± 0.37 13 One -One 0 3.08 ± 0.47 2.81 ± 0.21 14 0 One One 2.35 ± 0.21 1.85 ± 0.10 15 -One One 0 3.36 0.30 2.63 ± 0.12

As a result, as shown in the above table, the enzyme treatment conditions with the highest total polyphenol content were 0.25 mL / L enzyme concentration, the enzyme treatment temperature was 40 ° C, and the enzyme treatment time was 1 hour. At this time, the total polyphenol content in the process 3.45 mg / g. In addition, enzyme treatment conditions with the highest total flavonoid contents were 0.15 mL / L enzyme concentration, enzyme treatment temperature 20 ℃, enzyme treatment time 1 hour, and the total flavonoid content in the process was 2.95 mg / mL level.

DPPH radical scavenging activity and SOD-like activity were measured after pretreatment of Novozym 33095 enzyme using self-administration, as shown in the following table.

DPPH radical scavenging activity and SOD-like activity after enzymatic pretreatment of Novozym 33095 using self-administration Experiment number X _One X ₂ X ₃ DPPH radical  scavenging activity (%) SOD - like activity  (%) One 0 -One One 28.44 + - 2.81 7.68 ± 0.53 2 0 One -One 32.33 + - 0.83 6.93 + - 0.53 3 0 0 0 31.57 ± 2.61 8.94 ± 0.18 4 0 -One -One 40.34 ± 2.90 5.04 + 1.42 5 -One 0 One 39.34 + 1.49 8.94 ± 0.18 6 -One -One 0 25.56 ± 1.24 5.54 + - 0.36 7 -One 0 -One 31.20 ± 2.08 8.82 ± 0.36 8 0 0 0 36.59 ± 0.61 6.93 + 0.18 9 One One 0 38.84 + 0.79 8.06 + - 0.36 10 0 0 0 42.98 + 1.64 6.80 + - 0.71 11 One 0 -One 48.49 + - 0.90 6.05 ± 0.71 12 One 0 One 39.10 + 0.08 4.79 ± 1.07 13 One -One 0 33.34 ± 0.59 8.06 + - 0.71 14 0 One One 20.43 + - 0.32 6.80 ± 1.42 15 -One One 0 24.93 + - 2.48 5.48 ± 0.53

As shown in the above table, the maximum values of DPPH radical scavenging activity and SOD-like activity after enzyme pretreatment were 48.49% and 8.94%, respectively, and the DPPH radical scavenging ability was remarkably improved especially in the case of no treatment (Table 4) Respectively.

Experimental Example  5: Hongyoung  Confirmation of enzyme pretreatment effect

Experimental Example  5-1: Hongyoung  Used Novozym  33095 Experimental design of enzyme pretreatment process

In order to confirm the effect of pretreatment of Novozym 33095 enzyme using Hongyoung, the experiment of the process as shown in the following table was designed.

Experimental design of Novozym 33095 enzyme pretreatment process using Hongyoung Experiment number X _One X ₂ X ₃ Conc . of enzymatic  activity ( mL / L) Temperature (° C) Time ( min ) One 0 -One One 0.15 20 120 2 0 One -One 0.15 60 60 3 0 0 0 0.15 40 90 4 0 -One -One 0.15 20 60 5 -One 0 One 0.05 40 120 6 -One -One 0 0.05 20 90 7 -One 0 -One 0.05 40 60 8 0 0 0 0.15 40 90 9 One One 0 0.25 60 90 10 0 0 0 0.15 40 90 11 One 0 -One 0.25 40 60 12 One 0 One 0.25 40 120 13 One -One 0 0.25 20 90 14 0 One One 0.15 60 120 15 -One One 0 0.05 60 90

Experimental Example  5-2: Hongyoung  Used Novozym  33095 Measurement of yield and freeze-drying yield after enzyme pretreatment

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 of Novozym 33095 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 20.03 1.69 2 0 One -One 13.74 1.79 3 0 0 0 16.41 2.26 4 0 -One -One 20.89 1.83 5 -One 0 One 16.29 2.03 6 -One -One 0 20.64 2.15 7 -One 0 -One 16.32 2.07 8 0 0 0 15.84 1.90 9 One One 0 17.78 1.95 10 0 0 0 17.46 2.28 11 One 0 -One 16.82 1.58 12 One 0 One 17.58 2.72 13 One -One 0 20.54 1.59 14 0 One One 15.62 1.77 15 -One One 0 10.93 1.61

As shown in the above table, the yield after enzyme treatment was 10.93-20.89%, and after lyophilization, the yield was 1.58-2.28%.

Experimental Example  5-3: Hongyoung  Used Novozym  33095 Total anthocyanin content measurement after enzyme pretreatment

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

Total anthocyanin content after enzyme pretreatment of Novozym 33095 using Hongyoung Experiment number X _One X ₂ X ₃ Total  anthocyanin (%, dry weight ) One 0 -One One 1.27 + - 0.46 2 0 One -One 1.03 + - 0.01 3 0 0 0 1.01 + 0.02 4 0 -One -One 1.16 ± 0.00 5 -One 0 One 0.99 ± 0.02 6 -One -One 0 0.84 ± 0.01 7 -One 0 -One 1.00 + - 0.01 8 0 0 0 1.47 ± 0.01 9 One One 0 1.02 + 0.09 10 0 0 0 0.92 + 0.03 11 One 0 -One 1.50 + 0.03 12 One 0 One 0.74 + 0.03 13 One -One 0 1.52 + 0.02 14 0 One One 1.81 + 0.06 15 -One One 0 1.33 + 0.04

As a result, as shown in the above table, the enzyme treatment conditions with the highest total anthocyanin content were an enzyme concentration of 0.15 mL / L, an enzyme treatment temperature of 60 ° C, and an enzyme treatment time of 2 hours. At this time, the total anthocyanin content in the process was 1.81% , But only 0.61% at no treatment (Table 2). Therefore, it was shown that treatment of Hongyoung with Novozym 33095 significantly increased the total anthocyanin content.

Experimental Example  5-4: Hongyoung  Used Novozym  Analysis of variance of total anthocyanin content after 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 carried out after Novozym 33095 enzyme treatment using Hongyoung. The results are shown in the following table.

Analysis of the distribution of total anthocyanin content after enzymatic treatment of Novozym 33095 using Hongyoung Factor Hongyoung F value Pr  > F Conc. of enzymatic activity (mL / 100 L) 1.96 0.1402 Temperature (?) 3.52 0.0248 Time (hr) 3.69 0.0209

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

Regression coefficients of secondary polynomial regression curves of total anthocyanin content after enzymatic treatment of Novozym 33095 using Hongyoung Parameter Estimate Hongyoung Intercept 1.133333 X ₁ ^{1 )} 0.076875 X ₂ ^{2 )} 0.049375 X ₃ ^{3 )} 0.015000 X ₁ * X ₁ -0.109792 X ₂ * X ₁ -0.251250 X ₂ * X ₂ 0.152708 X ₃ * X ₁ -0.190000 X ₃ * X ₂ 0.165000 X ₃ * X ₃ 0.033958 ¹⁾ Conc. of enzymatic activity (mL / L) ²⁾ Temperature (℃) ³⁾ Time (hr)

Conformity of total anthocyanin content after enzymatic treatment of Novozym 33095 using Hongyoung Regression Hongyoung R ² F value Pr  > F Linear 0.0496 0.69 0.5688 Quadratic 0.1046 1.45 0.2571 Cross product 0.3661 5.09 0.0089 Total regress 0.5204 2.41 0.0486

Experimental Example  5-5: Hongyoung  Used Novozym  33095 Reaction surface contour irradiation after enzyme treatment

In addition to the above analysis, the reaction surface contour of the total anthocyanin content after Novozym 33095 enzyme treatment using Hongyeong was investigated, and the result is shown in Fig. As shown in FIG. 6, in the reaction surface curve by the reaction surface analysis, the extraction time (X ₃ ) and the extraction temperature (X ₂ ) rather than the concentration (X ₁ ) Respectively.

Experimental Example  5-6: Hongyoung  Used Novozym  33095 total polyphenol and flavonoid content after enzyme pretreatment; And DPPH radical Scatters  And SOD - like activity  Research

The total polyphenol and flavonoid contents of Novozym 33095 were investigated after pretreatment with enzyme.

Total polyphenol and flavonoid content after enzyme pretreatment of Novozym 33095 using Hongyoung Experiment number X _One X ₂ X ₃ Total polyphenol  contents ( mg / g) Total flavonoid  contents ( mg / ml ) One 0 -One One 6.52 ± 0.16 4.62 + 0.14 2 0 One -One 6.99 ± 0.09 5.66 ± 0.10 3 0 0 0 7.82 + 0.14 5.24 ± 0.15 4 0 -One -One 6.91 + 0.15 4.74 ± 0.01 5 -One 0 One 5.25 ± 0.53 3.26 ± 0.15 6 -One -One 0 5.08 0.30 3.37 ± 0.14 7 -One 0 -One 8.04 + 0.13 5.35 ± 0.17 8 0 0 0 6.59 ± 0.27 4.39 ± 0.16 9 One One 0 6.11 ± 0.18 4.16 + 0.06 10 0 0 0 6.74 ± 0.49 4.44 0.26 11 One 0 -One 7.13 + - 0.24 4.82 ± 0.20 12 One 0 One 5.89 0.41 3.57 ± 0.14 13 One -One 0 4.36 + 0.11 3.41 + - 0.14 14 0 One One 7.28 ± 0.39 4.22 + 0.14 15 -One One 0 5.64 + - 0.12 3.79 ± 0.06

As a result, enzyme treatment conditions with the highest total polyphenol content were 0.05 mL / L of enzyme concentration, enzyme treatment temperature of 40 ° C. and enzyme treatment time of 1 hour, and the content in the process was 8.04 mg / g level . In addition, enzyme treatment conditions with the highest total flavonoid contents were 0.15 mL / L enzyme concentration, enzyme treatment temperature 60 ℃, and enzyme treatment time 1 hour, respectively, and the content in the process was 5.66 mg / mL. On the other hand, in the untreated samples, 3.83 mg / g and 2.47 mg / mL, respectively (Table 3), showed that pre-treatment of Novozym 33095 before extraction of anthocyanin pigment from Hongyoung significantly increased polyphenol and flavonoid .

DPPH radical scavenging activity and SOD-like activity were measured after pretreatment with Novozym 33095 enzyme using Hongyeong. The results are shown in the following table.

DPPH radical scavenging activity and SOD-like activity after enzyme pretreatment of Novozym 33095 using Hongyoung Experiment number X _One X ₂ X ₃ DPPH radical  scavenging activity (%) SOD - like activity  (%) One 0 -One One 45.61 + - 1.80 19.48 ± 0.73 2 0 One -One 33.96 + 1.13 17.93 + - 0.75 3 0 0 0 51.63 + - 0.37 19.35 + - 0.55 4 0 -One -One 44.87 ± 1.03 18.31 + - 2.39 5 -One 0 One 60.40 ± 0.28 22.06 + 0.02 6 -One -One 0 57.13 + - 3.49 18.19 ± 1.11 7 -One 0 -One 38.48 ± 1.16 17.16 + - 0.39 8 0 0 0 51.25 + - 0.88 20.39 ± 0.19 9 One One 0 57.15 ± 1.11 17.42 ± 0.75 10 0 0 0 57.90 ± 1.83 20.77 ± 1.82 11 One 0 -One 52.76 ± 1.61 19.22 ± 0.37 12 One 0 One 62.28 + - 0.97 18.44 + 1.47 13 One -One 0 54.38 ± 1.03 20.13 + - 0.55 14 0 One One 51.89 ± 2.49 20.13 + - 0.18 15 -One One 0 36.33 ± 3.29 21.68 + 0.17

As a result, as shown in the above table, the maximum DPPH radical scavenging activity and SOD-like activity after enzyme pretreatment were 62.28% and 22.06%, respectively, compared with 57.90% and 0.88% DPPH radical scavenging activity and SOD-like activity were all increased.

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

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

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 of the ethanol extraction process were 10-20% of raw material / solvent ratio (X ₁ ), 30-70 ° C of extraction temperature (X ₂ ), and 1-5 hours of extraction time (X ₃ ) (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 (12)

a) treating the Berry or colored potato with Novozym 33095 (Novozym 33095, Novozyme) enzyme; And
b) extracting the raw material treated with Novozymes 33095 enzyme with water, an alcohol having 1 to 6 carbon atoms or a mixed solvent thereof,
Wherein the berries are selected from the group consisting of olive, bergamot, and blueberry, and the color potato is self-made or red-berry.
delete 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 berry or the color potato with Novozymes 33095 enzyme at a temperature of 20 to 60 ° C for 1 to 2 hours.
The process according to claim 1, wherein the treatment of nobozyme 33095 enzyme with respect to the bacterium is carried out at a temperature of 20 ° C to 40 ° C for 1.5 hours to 2 hours.
2. The process according to claim 1, wherein the treatment of the Novozymes 33095 enzyme against the otic is performed at a temperature of 30 to 40 DEG C for 1 to 1.5 hours.
The process according to claim 1, wherein the treatment of Novozymes 33095 enzyme against autogenous is carried out at a temperature of 30 ° C to 50 ° C for 1.5 hours to 2 hours.
2. The method according to claim 1, wherein the treatment of Novozymes 33095 enzyme with respect to the iris is carried out at a temperature of 40 to 60 DEG C for 1.5 to 2 hours.
The method according to claim 3, wherein the ethanol extraction in step b) is 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 &Lt; / RTI &gt;
The method according to claim 1, wherein the extraction of the brambles in step (b) uses ethanol as an extraction solvent, and the ratio of the sample to the solvent is 10% (w / v) to 15% (w / v) Lt; / RTI &gt; for 3 hours to 5 hours.
The method according to claim 1, wherein ethanol extraction is carried out at a temperature of 40 to 50 DEG C in a ratio of 15% (w / v) to 20% (w / v) Lt; / RTI &gt; for 3 hours to 5 hours.
A process for the preparation of anthocyanins, which comprises separating anthocyanins from berries or colored potato extracts prepared by the process of any one of claims 1 to 11.

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