KR20200019082A - Method for producing anthocyanin oligomer using viscozyme l - Google Patents

Abstract

The present invention relates to a method for producing an anthocyanin oligomer using Viscozyme L, which comprises the following steps: (1) preparing an anthocyanin monomer; (2) mixing the anthocyanin monomer with distilled water to prepare an anthocyanin monomer solution; (3) adding Viscozyme L to the anthocyanin monomer solution to prepare a mixture; (4) stirring and fermenting the mixture to prepare a fermented material; and (5) filtering and freeze-drying the fermented material. According to the present invention, an anthocyanin oligomer is synthesized with a high synthesis rate by using an anthocyanin monomer and Viscozyme L, and thus, it is possible to economically produce an anthocyanin oligomer without using expensive glucosidase.

Description

Method for producing anthocyanin oligomer using biscozyme L {METHOD FOR PRODUCING ANTHOCYANIN OLIGOMER USING VISCOZYME L}

The present invention relates to a method for producing anthocyanin oligomer using biscozyme L, and more particularly, to a method for producing anthocyanin oligomer by fermenting an anthocyanin monomer (monomeric Anthocyanin) and biscozyme L (Viscozyme L).

In addition to its function as a natural pigment, anthocyanins exhibit various physiological activities, such as antioxidant, cholesterol lowering, vision improvement, vascular protection, arteriosclerosis, heart disease prevention, anti-ulcer function, anticancer, anti-inflammatory, diabetes suppression, and protection from ultraviolet rays. It shows physiological activity and is actively used in medicines, so it is being highlighted as a new target for the creation of health foods and new medicines.

Anthocyanins are unstable in neutral or alkaline solutions, and even when exposed to light in acidic solutions, color fades slowly and is considered one of the structurally unstable substances. In particular, factors affecting the stability of anthocyanin pigments include chemical structure of anthocyanin, concentration of pigment, pH of solution, temperature, presence of coexisting pigments, metal ions, enzymes, oxygen, ascorbic acid and sugars. The difference in these factors may indicate the maintenance of chromaticity, ie structural stability. Due to this structural instability, many difficulties arise in active use as food and pharmaceuticals, and thus researches to improve stability are being conducted.

Anthocyanins contained in most foods are monomeric forms, which have the disadvantage of being unstable in neutral and alkaline and unstable in light and heat. Polymer forms are present in foods in small amounts, but they are more functional and stable than monomers, and their typical antioxidant activity is doubled. Recently, studies of anthocyanin oligomers that improve subjective symptoms and contrast sensitivity in the problems of myopia and amblyopia have been reported.

Related prior arts include Korean Patent Registration No. 10-1182630 (2012.09.07.) And Korean Patent Application Publication No. 10-2012-0079040 (2012.07.11.).

Aspergillus genus is a useful microorganism that produces enzymes, organic acids, and metabolites with pharmacological activity, and has been used in food, liquor, and pharmaceutical industries as well as in agriculture. Is recognized. In addition, because fungi have many exo-enzymes to be discharged to the outside and vary in function, this can be used to economically produce useful substances using natural enzymes. Previous studies have been reported on the production of anthocyanin oligomers using Aspergillus genus filamentous fungi, but using the strain directly has the disadvantage that contamination occurs in the production of anthocyanin oligomers.

In order to check whether anthocyanin oligomer can be produced using an enzyme that is discharged to the outside without using Aspergillus spp. Directly, the present inventors have extracted coenzyme from a culture medium cultured with black fungus (Aspergillus niger, Aspergillus niger). We devised a manufacturing method using glucosidase and Viscozyme L enzyme obtained by analyzing coenzyme and confirmed the effect. Among them, by further confirming the efficacy appearing in the difference between the fermentation method and the production method according to the temperature, the present invention was completed by devising a more industrially available method.

Republic of Korea Patent No. 10-1756123 (2017.07.04) Republic of Korea Patent Registration No. 10-1182630 (2012.09.07.) Republic of Korea Patent Publication No. 10-2012-0079040 (2012.07.11.)

An anthocyanin monomer (Monomeric Anthocyanin) and biscozyme L (Viscozyme L) by using an enzyme to synthesize an anthocyanin oligomer, to provide a production method for producing the anthocyanin oligomer economically.

In order to achieve the above object, the present invention comprises the steps of (1) preparing an anthocyanin monomer, (2) mixing the anthocyanin monomer with distilled water to prepare an anthocyanin monomer solution, (3) a biscozyme L ( Preparing a mixture by adding Viscozyme L), (4) stirring and fermenting the mixture to prepare a fermentation product, and (5) filtering and freeze-drying the fermentation product. to provide.

According to the present invention as described above, by synthesizing the anthocyanin oligomer using an anthocyanin monomer and Viscozyme L enzyme, there is an effect of producing an anthocyanin oligomer economically without using expensive glucosidase.

1 is a graph confirming the results of the synthesis of the anthocyanin oligomer prepared according to the preparation method of Example 2 through the ESI mass.
Figure 2 is a graph confirming the resulting synthesis of the anthocyanin oligomer prepared according to the preparation method of Example 5 through the ESI mass.
Figure 3 is a graph confirming the resulting synthesis of the anthocyanin oligomer prepared according to the preparation method of Example 8 through the ESI mass.
Figure 4 is a graph confirming the resulting synthesis of the anthocyanin oligomer prepared according to the preparation method of Example 11 through the ESI mass.
5 is a graph comparing hydroxy radical scavenging ability of anthocyanin oligomers prepared according to the preparation methods of Examples 2, 5, 8 and 11.
6 is a graph comparing the hydroxy radical scavenging ability of the anthocyanin oligomer prepared according to Example 7 to Comparative Example 9.

Hereinafter, the present invention will be described in detail.

An anthocyanin oligomer manufacturing method of one embodiment of the present invention is provided.

The anthocyanin oligomer manufacturing method comprises the steps of (1) preparing an anthocyanin monomer, (2) mixing the anthocyanin monomer with distilled water to prepare an anthocyanin monomer solution, and (3) a biscozyme L (Viscozyme L) in the anthocyanin monomer solution. Adding to prepare a mixture, (4) stirring and fermenting the mixture to prepare a fermentation product, and (5) filtering and lyophilizing the fermentation product.

The anthocyanin monomer (Monomeric Anthocyanin) is an anthocyanin monomer powder based on grape skin extract.

The anthocyanin monomer solution may be prepared by mixing anthocyanin monomer and distilled water in a mass ratio of 1: 8 to 1:15, and preferably, may be prepared by mixing anthocyanin monomer and distilled water in a mass ratio of 1:10.

The mixture may be prepared by mixing the anthocyanin monomer solution and the biscozyme L in a mass ratio of 300: 1 to 1000: 1. Preferably, the anthocyanin monomer solution and the biscozyme L may be 500: 1. It can manufacture by mixing by mass ratio.

The stirred fermentation may be characterized in that the fermentation with stirring for 3 to 5 days at a rate of 30 to 150 RPM at a temperature of 25 to 35 ℃. Preferably at a temperature of 32 ℃ 40 to 80 RPM can be incubated for 4 days. It was confirmed that the amount of anthocyanin oligomer synthesis decreased below 40 PRM, and the increase in the amount of anthocyanin oligomer synthesis slowed down to 80 RPM or higher.

The filtration is a filter using a filter paper, and the filter paper may be filtered using a tubular, capillary, coiled spiral, and place membrane. In addition, the filtration can be filtered under reduced pressure.

The freeze drying may be carried out at minus 50 to minus 80 ℃. Preferably it can be prepared at 60 to 65 ℃.

Anthocyanin oligomers can be prepared with glucosidase instead of biscozyme L, and the production of anthocyanin oligomers can be made with glucosidase. This has the disadvantage of being so high. In order to secure this, the present invention has devised a method for increasing the synthesis rate of anthocyanin oligomer of biscozyme L.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1.

Anthocyanin monomer and distilled water are mixed in a 1:10 mass ratio to prepare anthocyanin mono solution. A mixture is prepared by mixing Viscozyme L in an anthocyanin monomer solution at a 500: 1 mass ratio. The mixture is mixed to mix small and then fermented by stirring at a speed of 20 RPM. Fermentation temperature is 32 ℃, fermentation period is carried out for 4 days. After the fermentation is completed, anthocyanin oligomer is prepared by lyophilization at 55 ° C. after filtration under reduced pressure using a filter paper.

Example 2.

The process was carried out in the same manner as in Example 1, but was performed at a freeze-drying temperature of minus 65 ° C.

Example 3.

The process was carried out in the same manner as in Example 1, but was performed at a freeze-drying temperature of minus 75 ° C.

Example 4.

The process was carried out in the same manner as in Example 1, but when the fermentation was performed at 40 RPM.

Example 5.

The process was carried out in the same manner as in Example 4, but was performed at a freeze-drying temperature of minus 65 ° C.

Example 6.

The process was carried out in the same manner as in Example 4, but was performed at a freeze-drying temperature of minus 75 ° C.

Example 7.

The process was carried out in the same manner as in Example 1, but when the fermentation was carried out by stirring at a speed of 60 RPM.

Example 8.

The process was carried out in the same manner as in Example 7, but was performed at a freeze-drying temperature of minus 65 ° C.

Example 9.

The process was performed in the same manner as in Example 7, except that the freeze-drying temperature was set to minus 75 ° C.

Example 10.

The process was carried out in the same manner as in Example 1, but when the fermentation was performed at 80 RPM.

Example 11.

The process was carried out in the same manner as in Example 10, but was performed at a freeze-drying temperature of minus 65 ° C.

Example 12.

The process was carried out in the same manner as in Example 10, but was performed at a freeze-drying temperature of minus 75 ° C.

Table 1 summarizes the anthocyanin oligomer preparation conditions of Examples 1 to 12.

Monomer: Distilled Water
(Mass ratio) Monomer solution: Biscozyme L
(Mass ratio) Stirring speed Fermentation temperature Fermentation period Freeze drying temperature Example 1 1:10 500: 1 20 RPM 32 ℃ 4 days -55 ℃ Example 2 1:10 500: 1 20 RPM 32 ℃ 4 days -65 ℃ Example 3 1:10 500: 1 20 RPM 32 ℃ 4 days -75 ℃ Example 4 1:10 500: 1 40 RPM 32 ℃ 4 days -55 ℃ Example 5 1:10 500: 1 40 RPM 32 ℃ 4 days -65 ℃ Example 6 1:10 500: 1 40 RPM 32 ℃ 4 days -75 ℃ Example 7 1:10 500: 1 60 RPM 32 ℃ 4 days -55 ℃ Example 8 1:10 500: 1 60 RPM 32 ℃ 4 days -65 ℃ Example 9 1:10 500: 1 60 RPM 32 ℃ 4 days -75 ℃ Example 10 1:10 500: 1 80 RPM 32 ℃ 4 days -55 ℃ Example 11 1:10 500: 1 80 RPM 32 ℃ 4 days -65 ℃ Example 12 1:10 500: 1 80 RPM 32 ℃ 4 days -75 ℃

Measurement Example 1.

It was confirmed whether the resultant synthesis of the anthocyanin oligomer prepared according to the preparation method of Example 2, Example 5, Example 8 and Example 11 by ESI mass measurement, the measurement result graph is shown in Figure 1-4.

1 to 4, it can be seen that the peak is increased in the vicinity of 600, 900 and 1200. This means that the anthocyanin monomer was converted into anthocyanate oligomers such as dimers, trimers, and tetramers as a result of fermentation, and 40 RPM and 60 RPM than FIG. 2 and 3 showed a higher or more peaks in the vicinity of 600, 900 and 1200 of the stirred culture as shown in FIG. By comparison, you can see that there is little difference.

Measurement Example 2.

The hydroxyl radical scavenging ability of the anthocyanin oligomers prepared according to the preparation methods of Example 2, Example 5, Example 8 and Example 11 was measured, and a graph comparing the measured values is shown in FIG. 5. The hydroxy radical scavenging ability is a measure for comparing the efficacy of the anthocyanin oligomer, the intermediate concentration (inhibitory concentration) according to the concentration of the anthocyanin oligomer obtained through Examples 2, 5, 8 and 11 IC). The higher the value of the graph shows a higher inhibitory effect at the measured concentration, the lower the value of IC ₅₀ , a 50% inhibitory concentration, the higher the inhibitory activity can be confirmed.

Referring to FIG. 5, the IC ₅₀ value of 50% inhibiting hydroxy radical generation showed that stirring at 60 RPM showed the highest inhibitory activity at 0.264 mg / mL.

Measurement Example 3.

The hydroxy radical scavenging ability of the anthocyanin oligomer prepared according to the preparation method of Examples 7 to 9 was measured, and a graph comparing the measured values is shown in FIG. 6.

Referring to Figure 6, the IC ₅₀ value of the anthocyanin oligomer obtained by varying the freeze-drying temperature of the fermented product by stirring at 60 RPM, the anthocyanin oligomer lyophilized at 65 ℃ shows the highest inhibitory activity as 0.264 mg / mL I could confirm it.

As mentioned above, specific parts of the present invention have been described in detail, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (2)

(1) preparing an anthocyanin monomer;
(2) mixing the anthocyanin monomer with distilled water to prepare an anthocyanin monomer solution;
(3) preparing a mixture by adding Viscozyme L to the anthocyanin monomer solution;
(4) stirring the fermentation of the mixture to produce a fermentation product; And
(5) filtering and freeze drying the fermentation product;
In the anthocyanin monomer solution of step (2), the anthocyanin monomer and distilled water are mixed in a mass ratio of 1: 8 to 1:15,
In the mixture of step (3), the anthocyanin monomer solution and biscozyme L are mixed in a mass ratio of 1000: 1 to 300: 1,
The freeze drying of step (5) is an anthocyanin oligomer production method, characterized in that carried out at a temperature of minus 50 to minus 80 ℃.
The method of claim 1,
The stirring fermentation of the step (4) is an anthocyanin oligomer production method characterized in that performed for 3 to 5 days while stirring at a temperature of 30 to 150 RPM at a temperature of 25 to 35 ℃.

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