RU2126806C1 - Method of crystalline licopin preparing - Google Patents

Abstract

FIELD: microbiological, food and medicinal industries. SUBSTANCE: method involves extraction of preliminary dried biomass with a nonpolar organic solvent followed by licopin crystallization from saturated extract and filtration. Licopin crystals are washed out with an organic solvent. Method is based on using optimal technological parameters of extraction, crystallization and purification of crystals from impurities by washing out with organic solvents. EFFECT: high yield and quality of the end product. 6 cl, 5 tbl, 7 ex

Description

 The invention relates to the food, microbiological and medical industries.

 A known method of producing crystalline lycopene, including the extraction of carotenoids from the raw biomass of the fungus Phycomyces blakesleeanus NRRL 1555 (-) with a solvent mixture of methanol: hexane (1: 1), the separation of the extract with water, the allocation of impurities and waste products of the producer fungus in the aqueous layer and the isolation of lycopene crystals filtrations from the residue obtained after evaporation of the hexane layer (Shlomai - P; Ben-Amotz-A; Margalith-p, Production of carotene stercoisomers by Phycomyces blakesleeanus Appl. Microbiol, Biotechnol; 1991, 34, 4, 458-62).

 The closest technical solution to the claimed is a method for producing lycopene from the mycelium of the fungus Blakeslea trispora. The mushroom biomass is obtained, triturated, acetone is added, passed through a filter, the destroyed biomass is separated, and a bright orange acetone solution is obtained, followed by extraction of lycopene from it with n-hexane (a more non-polar solvent). Then it is evaporated and a saturated solution is obtained, which is subjected to further purification. (Journal of Microbiology, 1994, v. 63, issue 6, pp. 1111-1116).

 The disadvantage of this method is the low yield of crystalline lycopene, the use of a mixture of solvents and, as a consequence, the relatively high cost of the final product.

 The technical result consists in increasing the yield of lycopene, improving the quality and lowering the cost.

 The high yield and quality of the target product is achieved through the use of pre-dried biomass with a moisture content of not more than 20%, the use of optimal technological parameters for extraction, crystallization and purification of lycopene crystals from impurities by washing with organic solvents, for example, ethanol or acetone.

 Lycopene is dissolved and extracted only with non-polar solvents. Water contained in the biomass of microorganisms prevents the penetration of non-polar extract into the cells. Therefore, in order to achieve high extraction efficiency, a polar solvent, for example alcohol, acetone or tetrahydrofutane, is added to the system, which solvates water molecules and promotes the formation of a homogeneous system: non-polar extractant - water.

 Using in the proposed method dried to a moisture content of not more than 20% biomass allows you to extract lycopene with one non-polar solvent. This, firstly, facilitates the extraction process, and secondly, eliminates the stage of regeneration of ethyl alcohol or other polar solvent from an aqueous solution, which ultimately leads to a significant reduction in the specific consumption of biomass, solvents and energy.

 In the proposed method, the purification of lycopene is carried out by washing the crystals with an organic solvent, for example ethanol or acetone, which allows avoiding both chromatographic purification and the use of a mixture of solvents as an extractant.

 The developed limits of the technological process allow to obtain crystals, the content of 90-95% of lycopene.

The invention consists in the following. Extraction of lycopene is carried out with a non-polar organic solvent, for example, chladone-11 at a temperature of from 20 to 26 ^o C, ethyl acetate from 40 to 65 ^o C. With sunflower oil from 80 to 95 ^o C, from pre-dried to a moisture content of not more than 20% biomass Blakeslea trispora fungus in the form of (+) and (-) strains 8A; then lycopene is crystallized from the saturated extract at 10-22 ^o C for 1-5 days. After filtration, the lycopene crystals are washed with an organic solvent at a temperature of 45-78 ^o C in the ratio (mass parts): for one part of crystals from 3 to 35 parts of the solvent, and dried.

 The process of obtaining crystalline lycopene in the specified technological parameters allows to obtain crystals containing more than 90% lycopene with a yield of 31 to 56% of the amount of lycopene in the initial biomass (see table 5, experiments NN 1, 2, 3).

 The exit from the boundary of the indicated parameters of the process in comparison with the proposed leads either to a decrease in the yield of crystals and their quality, or to an unjustified increase in the specific consumption of raw materials and solvents (see table 5, experiments NN 4, 5, respectively).

 Example 1

 In order to determine the limiting value of the biomass moisture at which the lycopene yield to the extract would be more than 90%, experiments were carried out in which the lycopene-containing biomass was dried under vacuum to a moisture content of 25, 20, 10, and 3%.

Equal samples of biomass of the indicated moisture are placed in flasks with refined sunflower oil taken in a ratio of 3: 1 by weight of the biomass, and lycopene is extracted at 95 ^° C for 30 minutes. The meal is separated by filtration and the lycopene content is analyzed. The data presented in table No. 2 indicate that an increase in biomass moisture of more than 20% significantly reduces the release of lycopene in the extract, and a further decrease in humidity, associated with significant energy costs, gives a slight increase in the outputs of lycopene in the extract.

 Example 2

Lycopene-containing biomass is loaded into a flask containing sunflower oil in a ratio of 1: 4 by weight and extraction is carried out with stirring, at a temperature of 90 ^o C for 30 minutes In order to determine the optimal crystallization conditions, a saturated extract containing 0.41% lycopene is separated from the meal on the filter, divided into four equal parts, which are sent to crystallization at temperatures of 25-28 ^o C, 18-22 ^o C, 6-8 ^o C, 0 - +4 ^o C, respectively. In all cases, after 0.5, 2, 5, 7 days, the precipitated lycopene crystals are filtered off, analyzed and the yield of lycopene crystals from the loaded in saturated extract is determined.

 The results are presented in table 2.

 Example 3

A lycopene-containing biomass and ethyl acetate were charged into the flask in a ratio of 1: 2.5 (by weight). The extraction is carried out with stirring, at a temperature of 60 ^o C for 30 minutes. The meal is filtered off with suction. The saturated extract is divided into four equal parts, which are sent to crystallization at temperatures of 25-28 ^o C, 18-22 ^o C, 6-8 ^o C, 0 - +4 ^o C, respectively. In all cases, after 0.5, 1, 5, 7 days, the precipitated lycopene crystals are filtered off, analyzed, and the yield of lycopene crystals from the loaded in saturated extract is determined.

 The results are presented in table 3.

 Example 4

75 g of biomass and 1175 g of freon 11 are charged into the flask. The extraction is carried out with stirring at a temperature of 24-26 ^° C for 45 minutes. The meal is filtered off under vacuum. In order to completely remove the pigment from the meal, a second extraction is carried out, under conditions similar to the first one at a ratio of 1: 9. As a result, 29.9 g of meal, containing 0.027% lycopene and 1177.5 g of extract, were obtained. The extracts are combined and sent to distillation of the freon, which is carried out at a temperature of 20-28 ^o C. Evaporated extract is left to crystallize for 66 hours at a temperature of 18-22 ^o C. The precipitated crystals are filtered off, vacuum on the filter for 1 hour. The yield of crystals from the biomass loaded in the biomass is 55.64%, the losses of lycopene in the meal and from the destruction are 1.96% and 23.28%, respectively.

 Example 5

 Determination of the conditions for the purification of lycopene crystals is carried out in model experiments. The raw materials for the experiments were samples of crystalline lycopene with a mass fraction of the main substance of 16%, obtained as a result of extraction from biomass with sunflower oil (see Experiment 7 in table 2).

 The crystals were washed with ethanol for 30-45 minutes with stirring.

In the experiments, the ratio of crystals: ethanol and the washing temperature from 35 to 78 ^o C (the boiling point of ethanol) are varied. The data presented in table 4 indicate that washing with ethanol, in a ratio of 1: 3-1: 35 (in mass parts), at a temperature of 45 - 78 ^o C allows you to get crystals with a mass fraction of the main substance from 90 to 95%.

 Example 6

 Of all the solvents allowed for use in the food industry, lycopene is sparingly soluble in ethyl acetate, chladone-11 (trichlorofluoromethane), and vegetable oil.

 Due to the fact that the solubility of lycopene in this series decreases, then each solvent has its own optimal extraction parameters (see the column "extraction" in table 5). Therefore, the selection of technological regimes for the extraction of lycopene from biomass by the entire class of nonpolar organic solvents is carried out by determining the minimum and maximum points from a series of experiments that provide a given yield and quality of lycopene (see table 5 experiments NN 1, 2, 3).

 In experiments NN 1 and 4, lycopene is extracted from the biomass of the fungus (+) (-) 8A Blakeslea trispora with sunflower oil at elevated temperature and constant stirring. Saturated extracts are filtered off and sent to crystallization. The crystals are filtered off and washed with ethanol.

 Run No. 2 was also carried out as run No. 1, except that ethyl acetate (ethyl acetate, ethyl acetate) was used as the extractant, and extraction was carried out twice. Extracts combine. The solvent is distilled off in vacuo. The crystals precipitated from the bottom residue are filtered off and then washed with ethanol.

 Run NN 3 and 5 were carried out similarly to Run N 2, except that Freon-11 was used as the extractant.

 Experiments NN 4 and 5 are carried out outside the claimed method.

 The data presented in table 5 indicate that the experiments carried out within the stated limits (experiments NN 1, 2, 3) make it possible to obtain crystalline lycopene with a yield of 31 to 56% and a basic substance content of 90 to 94%. In experiments conducted outside the scope of the claimed method, either a decrease in the crystal yield was observed due to insufficient extraction efficiency (see Loss in meal experiment No. 4) or a relatively high solvent consumption with a constant process efficiency (experiment No. 5).

 Example 7

300 kg of biomass coarse (+) (-) 8A Blakeslea trispora, containing 6.7 kg of lycopene, dried to a moisture content of 7%, and 1200 kg of vegetable oil, are loaded into a reactor equipped with a jacket and a stirrer. The extraction mass is heated to a temperature of 90 ^o C, transferred to the filtration for frame filtration process. The saturated extract is directed to crystallization, which is carried out at a temperature of 22 ^o C for 5 days, then the mass is heated to 40 ^o C. The precipitated crystals of lycopene are filtered on a filter and washed with 85 kg of ethanol with stirring and a temperature of 50 ^o C for 10 min The washed lycopene crystals are filtered off and dried under vacuum at a moisture content of not more than 0.5%. The result is 2.31 kg of lycopene crystalline lycopene with a mass fraction of the main substance of 90%.

Claims (6)

1. A method of obtaining crystalline lycopene, including the extraction of lycopene from the lycopene-containing biomass of Blakeslea trispora fungus with a non-polar organic solvent and obtaining a saturated extract, characterized in that the extraction of lycopene with a non-polar organic solvent is carried out from a pre-dried biomass, after obtaining a saturated extract, lycopene is crystallized at 10 - 12 ^o C for 1 - 5 days, filtering the crystals, washing at a temperature of 45 - 78 ^o C in a ratio of 1 part of crystals of lycopene, 3 - 35 astey organic solvent.  2. The method according to p. 1, characterized in that use lycopene-containing biomass of the fungus Blakeslea trispora obtained by growing (+), (-) strains 8A.  3. The method according to claim 1, characterized in that the biomass is used with a moisture content of not more than 20%.  4. The method according to claim 1, characterized in that as a non-polar organic solvent use either sunflower oil, or freon-11, or ethyl acetate, or acetone.  5. The method according to claim 1, characterized in that ethanol or acetone is used to rinse the lycopene crystals. 6. The method according to claim 4, characterized in that the extraction with Freon-11 is carried out at a temperature of from 20 to 26 ^o C, ethyl acetate from 40 to 65 ^o C, sunflower oil from 80 to 95 ^o C.

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