FR2550219A1 - Method for preparing biologically active substances from ginseng tissue culture biomass - Google Patents

Abstract

The invention relates to microbiology. The method indicated comprises the culture of the seed material in a nutrient medium containing kinetin, the culture of the biomass in the Murashige and Scooge nutrient medium containing 1.5-2.5 mg/l of nicotinic acid, the harvesting of the biomass and its drying at a temperature of 51-54 DEG C to a moisture content of 7-9%, with subsequent extraction of the biologically active substances from the said biomass. The proposed invention may be used in the perfume industry, in medicine and in the food industry.

Description

 The present invention relates to microbiology, particularly methods for obtaining biologically active substances from the biomass of ginseng tissue culture, substances which can be used in various branches of industry, food, medical and perfumery.

A process is known for preparing biologically active substances from the biomass of ginseng tissue culture ("Fitokhimichesky analyz shtamma cultury tkaci kornya zhenshenya i standartizatsia ego preparatov", RG Butenko, TI Khretonova at. Et al.,
Rastitelnye resursy, v.XV, p.356, 1979).

 The method includes the following operations. First, a Murashige and Scooge nutrient medium containing kinetin is prepared. The culture flasks are then prepared: the flasks are washed by hand with detergents, the flasks are dried in a drying oven at 140 ° C. for 2 hours. The nutrient medium is poured into culture flasks of 250 ml capacity at a rate of 50 ml. The culture flasks are sterilized with the nutrient medium in an autoclave, at a temperature of 116 ° C. for 15 minutes. The tissue is seeded on the nutrient medium in a box and the tissue is grown locally with air conditioning. The biomass is harvested 30-60 days later. Part of the biomass is used as seed material (inoculum) , that is to say it is sown on a fresh nutritive medium of the same composition. The raw biomass is dried by lyophilization. Extractive substances are obtained by extraction. Extraction is carried out with ethanol at 20 and 40 = 0.

 The yield of extracted substances then constitutes 40.88 0 and 40.32 S respectively.

 We also know a process for the preparation of substances extracted from the biomass of the ginseng culture described in the work "Kultura tkani zhenshenya", communication I, by R.I. Vysotskaya, L.I. Slepyan, "Rastitelnye resursy", c. XVI; fasc. 1, p.123, 1980. This process for obtaining the substances extracted from the biomass of the ginseng tissue culture comprises the same operations as the process described above, however the drying is carried out in an oven at the temperature of 50 OC. The yield of extracted substances constitutes 40.32 S of the initial quantity.

 The above-mentioned methods are only carried out under laboratory conditions. The disadvantage of the methods indicated is due to a low yield of extracted substances, since the biomass after drying by lyophilization is in the form of a swollen and hygroscopic powder which makes extraction difficult. Drying in the oven does not ensure regular drying of the biomass up to the required humidity. The moisture resulting from the desiccated product gradually accumulates inside the oven until the biomass begins to acidify, and form lumps, which makes extraction conditions more difficult. more, the process does not ensure complete sterilization of all operations, it results in cases of possible contamination of the biomass with a foreign microflora.

 In the patent literature, information on the industrial culture of ginseng biomass is absent.

 It has therefore been proposed in the process for obtaining biologically active substances from the biomass of the ginseng culture, to develop a technology which would make it possible to obtain biologically active substances from the biomass of the culture of ginseng tissue by an industrial process.

The solution consists in proposing a process for obtaining biologically active substances from the biomass of the culture of ginseng tissue, which consists in the cultivation of sowing material on a nutritive medium containing kinetin, followed by the subsequent culture of the ginseng biomass, the sterile separation, drying and extraction of the biologically active substances, in accordance with the invention, the culture of the biomass is carried out on the nutrient medium of
Murashige and Scooge, containing 1.5-2.5 mg / l of nicotinic acid, with the subsequent harvesting of the biomass under sterile conditions and its drying at a temperature of 51 to 54 OC up to a humidity of 7 to 9% .

 As seed material is used a seed obtained from the mixed biomass of the culture of ginseng tissue by selection after heat treatment of the tissue at a temperature of 34 to 38 OC, this material is kept as grafted culture in ln darkness, at a temperature of 26 ° C., at a humidity of 70%, the culture cycle is 30 days, and containing calusses cells with dimensions of 15 to 20 and 25 to 30 microns polydiploides, capable of division .

 It is advantageous in order to protect the biomass against contamination by a foreign microflora, to subject it before its extraction to ultraviolet radiation for 20-30 minutes.

The proposed invention is implemented in the following manner: a nutritive medium of the following composition is prepared
macro elements from Murashige and Scooge
micro-elements after Murashige and Scooge
sucrose 30,000 mg / l
thiamine chloride 0.4 mg / l
meso-inositol 80 mg / l
casein hydrolyzate 500 mg / l
kinetin 1 mg / l
naphthiaciacetic acid 2 mg / l
agar-agar 600 mg / l
Fe-chelate 5 mg / l
To increase the biomass yield, it is desirable to select the seed beforehand.

 The seed is obtained from the mixed biomass of the culture of ginseng tissue by selection after heat treatment of the tissue at a temperature of 34 to 38 ° C., seed which is stored as a graftable culture in the dark at the temperature of 26 oC and humidity of 70 So, with a 30-day culture cycle, containing calusses cells, whose size is from 15 to 20 and from 25 to 30 microns polydiploids capable of division.

 It is not rational to carry out the heat treatment at a temperature below 38 ° C., since the treatment at this temperature causes the loss of the tissue.

 The sowing material obtained is seeded so that 1/10 of the total volume is reseeded on a fresh nutritive medium of the same composition, and 9/10 of the biomass are reseeded on a modified nut-ritive medium, which is distinguished from the medium nutritive mentioned above by the fact that instead of kinetin 1.5-2.5 mg / l of nicotinic acid was introduced. After 30 days of culture, the biomass is harvested under sterile conditions, it is dried at a temperature of 51 to 54 ° C. to a humidity of 7-9% by mass and the biologically active substances are extracted from this biomass. To protect the biomass from contamination by a foreign microflora, it is subjected to ultraviolet radiation for 20-30 minutes before extraction. The biomass obtained is in the form of irregular porous pieces colored in light yellow which disintegrate easily during crushing.

The humidity of the biomass is not more than 7-9%.

 Cultivation on the modified nutrient medium increases the yield of biomass by 30%, simultaneously with the increase in the yield of biologically active substances. The drying of the biomass to a humidity of 7-9 7o ensures total extraction.

 Other characteristics and advantages of the invention will be better understood on reading the description which follows of several concrete embodiments.

EXAMPLE 1
Preparation of a nutrient medium, containing
micro-elements after Murashige and Scooge
macro elements from Murashige and Scooge
sucrose 30,000 mg / l
thiamine chloride 0.4 ma / l
meso-inositol 80 mg / l
casein hydrolyzate 500 mg / l
kinetin 1 mg / l
naphthylacetic acid 2 mg / i
agar-agar 6oe mg / l
Fe-chelate 5 mg / i
The nutrient medium is poured into carefully washed culture flasks with a capacity of 250 ml, at a rate of 50 ml per flask.

The previously closed bottles are sterilized to destroy the microorganisms. The biomass of the culture of ginseng tissue is reseeded in a sterile box using sterile instruments. The flasks are capped and transported to the culture chamber, where a temperature of 26 ° C. and a humidity of 70 ° C. are kept steadily protected from light. The culture cycle lasts 30 days. Part of the cultivated biomass is used as seed material
Another part of the biomass is re-seeded on a modified nutritive medium, containing the abovementioned components, but instead of kinetin, nicotinic acid is introduced at the rate of 1.5 mg / l. The culture is carried out in a conditioned room for 30 days. The augmented biomass is harvested under sterile conditions. Drying is carried out in a drying machine at a temperature of 51 ° C. for 3 hours. The final humidity of the product is then 9 S. The extraction is carried out with ethanol at 40 70 at a ratio of the raw material and the extractant of 1:10.

 The result is small, irregular, light yellow porous pieces of dry biomass. The humidity of the biomass is 9 Vo, the yield of biomass according to the dry weight is 10.3 g / l and in biologically active substances of 4.15%, the content of extracted substances is 46.1 So, the biomass productivity being 427.5 mg / l.

EXAMPLE 2
The procedure is as in Example 1, with the difference that drying is carried out for 4 hours. The residual moisture of the product constitutes 8.6%. The content of extracted substances is 49.7%.

EXAMPLE 3
The process of Example 1 is carried out, with the difference that the drying is carried out for 4.5 hours. The residual humidity of the product is 8.4. The content of extracted substances constitutes 50.2 Uo EXAMPLE 4
The process is carried out as in Example 1, with the difference that the nutritive medium contains 2 mg / l of nicotinic acid, drying is carried out at the temperature of 53 ° C. for 3 hours. The residual humidity of the product is then 8.33 S. The content of substances extracted is 51.7 SO. The yield of dry biomass is 11.8 g / l, of biologically active substances is 493 mg / l.

EXAMPLE 5
The procedure is as in Example 1, but the nutritive medium contains 2 mg / l of nicotinic acid, and drying is carried out at the temperature of 53 ° C. for 4 hours.

The residual humidity of the product is 8.0 0. The content of extracted substances is 55.5%.

EXAMPLE 6
The procedure is as in Example 1, but the nutritive medium contains 2 mg / l of nicotinic acid, and the drying is carried out at a temperature of 53 ° C. for 4.5 hours. The residual humidity of the product is 7.8%. The content of extracted substances is 52.0 = 0.

EXAMPLE 7
The procedure is as in Example 1, but the nutritive medium contains 2.5 mg / l of nicotinic acid, and drying is carried out at the temperature of 54 ° C. for 3 hours. The residual humidity of the product is 7.8%. The content of extracted substances is 52.8%. The biomass yield on dry weight constitutes 11.65 g / l, in biologically active substances 4.05%, the productivity of the biomass is 471.8 mg / l.

EXAMPLE 8
The procedure is as in Example 1, but the nutritive medium contains 2.5 mg / l of nicotinic acid, and the drying is carried out at the temperature of 54 ° C. for 4 hours.

The residual humidity of the product is 7.4%. The content of extracted substances is 51.0%.

EXAMPLE 9
The procedure is as in Example 1, but the nutritive medium contains 2.5 mg / l of nicotinic acid, and drying is carried out at the temperature of 54 ° C. for 4.5 hours. The residual humidity of the product is 7.0 <0, the content of extracted substances is 50.6 flo.

EXAMPLE 10
To obtain the seed material, use is made of the mixed culture of the biomass of ginseng on the nutrient medium indicated in Example 1. The vials with the biomass are placed for one day after the culture chamber in a thermostatically controlled chamber, where the temperature is raised to 26-34 OC. Then the flasks with the culture are transported under normal culture conditions the temperature is -26 oC, the humidity of 70%, and cultivation is continued for 35 days. The inhibition of growth and hydration of the tissue is observed around the 30th day of culture.

 The biomass yield relative to the initial weight of the inoculate of 20 g / l on dry weight, constitutes 340 g / l. Repeated treatment under the same conditions has produced similar results.

 The seed material obtained is cultivated on the nutrient medium indicated in Example 1.

 Biomass is obtained from the aforementioned seed material on a modified medium containing instead of kinetin nicotinic acid. We then proceed as in Example 1.

 The biomass yield constitutes 475 mgZl.

EXAMPLE it
To obtain seed material, the mixed culture of the growing ginseng biomass is iridescent on the nutritive medium indicated in example 1. The flasks with the biomass are placed after the culture chamber in the thermostatically controlled chamber for one day, where the temperature is gradually increased from 26 to 36 OC, then the flasks are transported with the culture under normal culture conditions: the temperature is 26 OC, the humidity is 70%, protected from light and the culture still believes 35 days.

  Around the 30th day of culture, the essential mass of the tissue turns brown, hydration is observed. Around the 35th day, actively growing free zones appear, they are separated from the essential tissue and transplanted onto a fresh nutritive medium of the same composition. The biomass yield, in the case of the initial weight of the inoculate of 20 g / l, constitutes around the 30th day 466 g / l.

 The seed material obtained is cultivated on the nutritive medium indicated in Example 1. The biomass is obtained from the abovementioned seed material on the modified nutritive medium, containing nicotinic acid instead of kinetin. Then proceed as in Example I, The biomass yield constitutes 500.3 mg / l.

Claims (3)

R E V E N D I C A T I O N S  1 - Process for the preparation of Diologically active substances from the biomass of the culture of ginseng tissue, consisting in the culture of seed material on a nutritive medium containing kinetin with subsequent culture of the biomass of ginseng, its separation, drying and extraction therefrom of the biologically active substances, characterized in that one carries out the culture of the biomass on the nutritive medium of Murashige and Scooge containing 1.5-2.5 mg / l nicotinic attack, with the subsequent harvesting of the increased biomass under sterile conditions and its drying at a temperature of 51-54 OC up to a humidity of 7-9, 1,  2 - Process according to claim 1, characterized in that as seed material is used the seed obtained from the mixed biomass of the culture of ginseng tissue by selection after heat treatment of the tissue at a temperature of 34 at 38 C, seed preserved as a graftable culture at a temperature of 26 OC and a humidity of 70%, with a culture cycle of -30 3 days, containing callus cells whose size is 15-20 and 25-50 microns polydiploides, capable of division.  3 - Process according to claim 1, characterized in that to protect the biomass against contamination by a foreign microflora, it is subjected before extraction ultraviolet radiation for 20-30 minutes.