KR950004772B1 - Preparation of anthocyan-type red pigment via tissue cultivation of rhaphanus sativus l. - Google Patents

Abstract

The antocyanin in pigments production method from callus of radish such as Rhaphanus sativus L. (I) comprises the steps of (1) culturing seeds of (I) in artificial soils for 15 days, (2) cutting the cotyledons and the roots of cultured (I) on pigment production medium (II) at 24-26'C, below 10,000 lux light at 8 hrs/day for 20 days, (3) extracting pigment as malvidin- 3,5-diglucoside from callus with 1% HCl- ethanol. The callus contains pigments 16.4% in dried weight. The composition of (II) is ammonium nitrate, boric acid, calcium chloride, cobalt chloride, cupric sulfate, solium EDTA, ferrous sulfate, Mg sulfate, Mn sulfate, molybdic acid, potassium iodide, potassium nitrate, potassium phosphate, zinc sulfate, inositol, thiamin, malt extract, sucrose, 2,4-D, kinetin.

Description

Production method of anthocyanin-based red pigment by tissue culture of blue skin red radish

Figure is a comparison of absorption curves under visible light of callus and plant roots according to the present invention.

The present invention provides a method for producing anthocyanin-based red pigment that can be used for food, cosmetics, and the like, for culturing callus (Callus) derived from the tissues of blue radish (Rhaphanus sativus L) which is one of several varieties. The purpose is.

In general, various synthetic or natural pigments are used as food coloring agents to enhance aesthetics, etc. in foods, but synthetic pigments are limited in their use due to their toxicity and carcinogenicity. Pigments used in foods, cosmetics, etc. are very important in terms of the safety of the human body, and in recent years, the use of vegetable natural pigments has been impaired.

However, these natural plant pigments are constrained by the natural environment such as seasons, climate, latitude, soil, etc., and thus have the drawback that the supply and demand of raw materials are unstable and expensive.

Therefore, the present invention has been made for the smooth supply and supply of vegetable natural pigments, which are emerging as such a conventional problem, and changes the components and concentration in the culture medium through the tissue culture technique of cheongpi red radish, By artificially adjusting the culture environment to increase the pigment productivity as described in detail through the production process and Examples as follows.

production process

First Step (Preparation for Seeds): Prepare the seeds in optimal condition for growth by sterilizing and cleaning the seeds of red skin radish.

Second process (cultivation process in agar medium): After seeding prepared in the first process on agar medium, light irradiation with 5000-10000Lux for 20-18 ℃ with 23-28 ℃ temperature and 14-18 hours Cotyledons and roots are formed when cultured for about 15 days in a combined culture environment of cancer condition for 24 hours a day for 6-10 hours under temperature conditions.

Third step (cutting step): The cotyledons and roots cultured in the second step are taken and sterilized using a sterile scalpel, in the case of cotyledon, about 0.5-1Cm × 0.5-1Cm, and in the case of root, about 1-2Cm. Cut.

4th process (Calus induction process by culture): Culture medium (Murashing-skog base medium or anthocyanin production medium (APM, etc.) modified from the cotyledons and roots cut in the third process After being injured, the red callus is induced after about 20 days by incubating at a temperature of 24-26 ° C. and a constant temperature of 0-10,000Lux for 24 hours a day under 8-24 hours.

5th process (pigment extraction process): The red callus obtained by the said 4th process is immersed in 1% hydrochloric acid-ethanol, it is left to stand in 3-5 degreeC cow for about 24 hours, and a supernatant is isolate | separated by centrifugation, This supernatant contains the anthocyanin pigment which this invention requires.

6th process (concentration process) The supernatant (containing anthocyanin pigment) separated by the said 5th process is decompressed and concentrated by rotary evaporator at about 30 degreeC-35 degreeC.

Seventh step (pigment elution step): The concentrated solution obtained in the sixth step is dipped in a thin layer of cellulose cellulose (TLC cellulose) and developed on the upper layer of 1-butanol-acetic acid-water (4: 1: 1) solvent, and then the red part is removed. An aliquot is dissolved in 0.1% hydrochloric acid-ethanol and the pigment is eluted to complete the production of the anthocyanin-based red pigment.

In the above production process, the callus induced in the fourth step between the fourth step and the fifth step is placed on an anthocyanin production medium (APM) and repeated passage in the same culture conditions as the fourth step, red callus is It has been shown to actively proliferate.

In addition, anthocyanin production medium (APM), which is one of the culture medium used in the present invention, has a composition as shown in Table 1, which is capable of actively promoting callus proliferation and pigmentation by modifying known Murashige-Skoog medium. New media were invented.

TABLE 1

Hereinafter, described in detail through the embodiment according to the production process as follows.

Example

Step 1): Cheongpi red radish seeds were soaked in 70% ethanol for 30 seconds, immersed in Yuhanlax solution adjusted to effective chlorine concentration 2% for 15 minutes to complete sterilization, and then washed three times with sterile distilled water. The remaining bactericide was removed.

2nd step): Sterilized and washed seeds are agar plated in agar medium and mixed in a culture environment of 26 ℃, 16 hours, 5000Lux light irradiation and 24 ℃, 8 hours dark 24 hours a day. Incubate for days to form plants.

Third step): The cultured cotyledons and roots were harvested, and the cotyledons were cut into 1Cm × 1Cm size, and the roots were cut into 1.5Cm size.

Step 4): Calli was induced after cutting the cotyledons and roots cut into the anthocyanin production medium (APM) shown in Table 1) above, and passaged to anthocyanin production medium (APM) at 30 day intervals. .

The callus was carried out while varying the culture environment as shown in Table 2.

TABLE 2

5th step): The callus obtained in the 4th step was immersed in 1% hydrochloric acid-ethanol, and left to stand at 4 ° C. for 25 hours to extract the pigment, and centrifuged at about 500 × g to obtain a supernatant. The supernatant was measured for absorbance at 530 nm using a spectrophotometer, and the anthocyanin pigment content was measured based on the standard curve of Malvidin-3,5 diglucoside, respectively. The pigment content (%) per callus dry weight according to each culture condition as shown in Table 3).

TABLE 3

That is, it can be seen in Table 3) that callus of blue skin red radish has the maximum pigment content in the B culture condition, which is the light-blocked cow. It turns out that you can expect.

6th process) The supernatant liquid obtained by the 5th process was concentrated under reduced pressure at 30 degreeC-35 degreeC with the rotary evaporator.

Step 7): The concentrated solution was added to a thin layer of cellulose cellulose, and then developed on top of a 1-butanol-acetic acid-water (4: 1: 1) solvent, and then the red portion was aliquoted into 0.1% hydrochloric acid-ethanol. After melting, the pigment was eluted to complete the preparation.

As a result of measuring the absorption curve under visible light with a spectrophotometer, the dissolution pigment obtained from callus induced by artificial culture using the seeds of the red skin red radish and the plant roots grown in the natural environment was 530 nm. The peak (peak) in the vicinity showing the absorption wavelength peculiar to anthocyanin was confirmed to have the same properties.

Further, in the relationship between the pigment obtained from the callus according to the present invention and the pigment obtained through the same pigment extraction process in the plant root, the pigment content per dry weight in the callus according to the present invention was 16.4%, and the pigment content obtained from the plant root was 6.5 It was confirmed that it is%.

Therefore, the pigment produced according to the present invention can artificially produce stable anthocyanin-based red pigments obtained in nature without limitations of seasons, climate, soil, etc. and obtained from plant beaks due to natural growth in the amount of red pigment extraction. It is superior to the production efficiency, so that natural pigments can be smoothly supplied in various fields.

Claims (3)

It is characterized by a series of processes of artificially cultivating the green radish seeds for about 15 days, extracting and cutting cotyledons and roots, and inducing red callus by subculture for 20 days in culture medium to produce red pigment extraction, concentration and elution. Method for producing anthocyanin-based red pigment by tissue culture of blue skin red radish. The method of producing an anthocyanin-based red pigment by tissue culture of blue skin red radish according to claim 1, characterized in that the medium is cultured by subcultured cut cotyledons and roots. The method of producing an anthocyanin-based red pigment by tissue culture of red skin red radish according to claim 1, characterized in that it is a culture condition for 8-24 hours under 24-26 ° C. and 0-10,000Lux light irradiation conditions.