RU2112808C1 - Method of crystalline beta-carotene preparing - Google Patents

Abstract

FIELD: biotechnology, food industry. SUBSTANCE: method involves successive carrying out stages of carotene extraction from biomass with nonpolar organic solvent, separation of obtained extract, its purification with alcohol, carotene crystallization, separation of obtained crystals, their washing out and drying. Extraction is begun in reactor at 95-100 C at ratio (1: 1.5)-(1:3) for 15-60 min and finished on filter with the same solvent heated to 60-90 C. Then saturated extracts are washed out with polar solvent at ratio (10: 1)-(3:1) (by volume) at 40-55 C, crystallized at 10-20 C for 1-3 days followed by heating the obtained suspension to 40-60 C. Crystals are filtered, washed out with polar solvent at ratio (1:3)-(1:25) at 50-55 C. EFFECT: improved purity of carotene, decreased cost. 2 cl, 1 tbl

Description

 The invention relates to the microbiological, food and medical industries, and in particular to methods for producing natural food dyes and medicines of different protective effects containing carotenoid plants and microorganisms - beta-carotene.

Beta-carotene (C ₄₀ H ₅₆ ) is a carotenoid widely distributed in the world of plants (fruits of carrots, pumpkins) and microorganisms.

 Carotene solutions absorb light in the range 420–470 nm and are therefore used to color food products yellow. Carotene-containing preparations (solutions, emulsions, powders) that are recommended for coloring dairy, fish products, muffins, jellies, puddings, juices, corn sticks, etc. are widely represented on the food coloring market.

 The use of carotene in medicine is based on its ability to neutralize free radicals and an oxygen singlet, which, as you know, react with the genetic material of human cells, contributing to malignant neoplasms, impaired immunity, and the development of aging processes. It is noted that a diet enriched in carotene helps to reduce the risk of malignant neoplasms, increase immunity, and increase life expectancy.

 Currently, two methods are known for producing crystalline carotene: by chemical synthesis and by extraction with organic solvents from natural sources (vegetables, fruits, biomass of microorganisms) [1, 2, 3, 4].

 The disadvantage of these methods is the use of raw materials with a low concentration of carotene and, as a result, a high specific consumption of solvents and energy.

 Of all the possible microorganisms producing beta-carotene, only the microscopic mushroom Blakeslea trispora is used on an industrial scale, as able to accumulate carotene per unit mass tens of times more than known plants and other microorganisms [5].

Closest to the claimed method according to the final form of the product (crystals) and feedstock (biomass of the mushroom Blakeslea trispora) is a method for producing beta-carotene, comprising:
- drying biomass with alcohol;
- multistage extraction of carotene from unmilled biomass using non-polar organic solvents - H-alkanes and their chlorine derivatives;
- distillation of the solvent;
- cooling the extract and crystallization of carotene;
- dilution of the resulting suspension of crystals with a polar solvent (acetone, methanol, ethanol) playing the role of a salting out agent, separating the crystals, washing and drying them under vacuum (prototype) [6].

 It is known that carotene, due to its unique chemical structure, is easily oxidized and isomerized, losing A-provitamin activity.

 The quality of beta-carotene is determined by the correspondence of the geometry of the absorption spectrum of the solution to the standard. Impurities and carotene isomers absorb light in the region of 341 nm. Therefore, the greater the quotient of the optical densities of a solution of a beta-carotene sample in hexane at 451 nm (main absorption maximum) to 341 nm, the higher the degree of purification of beta-carotene. It is believed that the quality of carotene is quite high if the ratio of optical densities of the solution of the analyzed sample (see further in the text D451nm / D341nm) is more than 9.0.

The implementation of this method allows to obtain crystals of beta-carotene with a melting point of 179-181 ^o C, with the ratio of the optical density of the solutions D451 / D341 = 8.0 and with a yield of 27% (see example 1 experiment 6).

The disadvantages of the method are:
- extraction of carotene from unmilled biomass, and as a result, a relatively low degree of extraction of carotene from biomass into the extract;
- high ratio of solvent / extractable biomass used by the author (5: 1 at each stage), which leads to a significant consumption of extractant;
- crystallization of carotene at a low temperature with simultaneous salting out without additional heating before filtration, which contributes to the deposition of impurities of microbial lipids of biomass simultaneously with carotene, and as a result, the relatively low quality of the obtained crystals;
- the use of solvents (chloro-H-alkanes, methanol), unacceptable in food products, as well as the need to create a distillation facility for their regeneration.

 The aim of the present invention is to improve the quality of the drug and reduce its cost, subject to the use of raw materials permitted in the food industry.

This goal is achieved by the fact that in the method for producing beta-carotene, which provides for the extraction of carotene from biomass with a non-polar organic solvent, separation of the obtained extract, purification of the extract with a polar solvent, crystallization of carotene, separation of the obtained crystals, washing and drying them, it is proposed, according to the invention, to pre-extract chopped biomass start in the reactor at a temperature of 95-100 ^o C in a ratio of 1: 1.5 - 1: 3 (wt.) for 15-60 minutes and complete on the filter with the same solvent, heated m to 60-90 ^o C, then wash the saturated extracts with a polar solvent in a ratio of 10: 1 - 3: 1 (vol.) at a temperature of 40-55 ^o C, crystallize at 10-20 ^o C for 1-3 days then the resulting suspension is heated to a temperature of 40-60 ^o C, filter the crystals, rinse them with a polar solvent in a ratio of 1: 3 - 1:25 (wt.) at a temperature of 50-55 ^o C.

 It was proposed to use vegetable oil as a non-polar organic solvent, as well. polar - ethyl alcohol.

The process of obtaining carotene in such technological parameters allows you to obtain a product using solvents permitted in the food industry, with a yield of 25 - 27%, a given level of quality (mass fraction of carotene - more than 90%, melting point 179-181 ^o C, optical density ratio D451nm / D341nm solution of at least 9.0) and reduce production costs.

The method is as follows. First, sunflower oil is loaded into the apparatus, it is heated to a temperature of 95 -100 ^o C, and then pre-crushed biomass in an amount that provides an extractant / mycelial mass ratio of 1: 1.5 - 1: 3. The first extraction is carried out with stirring for 15-60 minutes

 The choice of these extraction intervals is based on the fact that a decrease in the oil / biomass ratio below 1.5 leads to the fact that the extraction mass becomes sufficiently thick, which sharply reduces the efficiency of mass transfer processes and makes it difficult to transport the suspension through technological communications. An increase in the ratio of more than 1: 3 leads to an unsaturated extract and a decrease in the yield of crystals.

 The resulting suspension is transferred to a filter press, separating the saturated extract of carotene in oil.

The residual carotenoids in the spent biomass are extracted with vegetable oil heated to 60 - 90 ^o C in two doses, first with the amount taken to obtain a saturated extract, and then until the carotenoids completely transfer from the solid phase to the extract.

 The resulting saturated extracts are combined and sent to the alcohol purification step. Unsaturated extracts are used in subsequent extraction operations or as commercial food coloring.

The conduct of the second extraction process directly on the filter at 60-90 ^o C is necessary and sufficient so that, with minimal losses, more than 90% of carotene passes into the extract, loss of carotene in meal <10% of the original in biomass.

 The use of more severe temperature conditions is not justified, because inevitably leads to a decrease in carotene quality.

Saturated extracts after treatment with alcohol are transferred to a crystallizer. The process is carried out at a temperature of 10-20 ^o C for 1-3 days. It was established by experiments that a decrease in temperature leads to an increase in the yield of crystals, but to a deterioration in their quality due to coprecipitation of impurities. With increasing temperature, the opposite pattern was observed. The duration of crystallization of more than 3 days is not rational, because increased energy and material costs are not offset by an increase in the yield of crystals.

 In the process of crystallization, impurities of microbial biosynthesis (phospholipids, sterols, etc.) pass into the extract from biomass simultaneously with carotene and then precipitate simultaneously with carotene crystals.

 Extraction of impurities from saturated extracts and washing the crystals with alcohol are substantially intensified at elevated temperatures. Therefore, a compromise between the process of temperature destruction of carotene and an increase in the degree of transition of impurities to the alcohol phase was the basis for choosing the temperature parameters for the purification of the saturated extract and crystals.

It is known that phospholipids are poorly soluble in alcohol. Therefore, in the inventive method, an additional purification of crystals of substances of this class by dissolving them is proposed. To do this, the suspension of crystals before filtration is heated to 40-60 ^o C. Impurities dissolve and are easily separated from the precipitate of crystals by filtration. The technological parameters of this operation are justified experimentally. Experiments have established that conducting the process at elevated temperature leads to a decrease in the yield of crystals due to their dissolution. Lowering the temperature below 40 ^o C does not allow to obtain crystals with the quality of a given level.

 Example 1

 Tests N 1 to N 5 were carried out according to the claimed method.

 The carotene-containing biomass of Blakeslea trispora fungus was preliminarily ground in a roller mill and then extracted with vegetable oil with stirring. The extraction mass was filtered, and then an additional extraction of the solid phase was carried out on the filter with the same solvent with stirring. Saturated extracts were washed with ethanol and sent for crystallization.

 The resulting suspension was heated and filtered; the crystals were washed with ethanol and dried.

 Experiment 1 was carried out in the parameters beyond the lower limits of the claimed method. In this case, crystals with a mass fraction of carotene of 75.7% were obtained, the ratio D451 / D341 = 7.8, the yield of crystalline carotene from the biomass loaded in the biomass was 20.1%.

 Experiment 5 was carried out in the parameters beyond the upper limits of the claimed method. In this case, crystals with a mass fraction of carotene of 84.4% were obtained, the D451 / D341 ratio was 7.2, the yield of crystalline carotene from the biomass loaded in the biomass was 27.0%.

 Experiments 2, 3, 4 were carried out in the intervals of the claimed method. The obtained crystals with a purity of more than 90%, the ratio of D451 / D341 more than 9; crystal yield 25 - 27% (see table).

 The data presented in the table indicate that the intervals of the claimed production method are necessary and sufficient to ensure the mass fraction of carotene in the obtained crystals - more than 90% with a ratio of D451 / D341 more than 9.0.

 Reducing or increasing the parameters of the process away from the stated limits leads either to a decrease in the yield of crystals and their quality, or to an increase in the specific consumption of raw materials (see table experiments NN 1 and 5 in comparison with NN 2-4).

 Experience No. 6 was carried out in the conditions stated by the prototype.

The carotene-containing biomass of Blakeslea trispora fungus was preliminarily dried with ethanol and then was extracted with carbon tetrachloride with stirring in a ratio of 1: 5 (wt.) And a temperature of 75 ^o C. The extraction mass was filtered and additional solid phase extraction was carried out with the same solvent under the same conditions. The extracts were combined, evaporated. Ethanol was added to the obtained residue in a ratio of 1: 3 (wt.) And crystallization was carried out for 24 hours at a temperature of 5 ^° C. The crystalline carotene was filtered off, washed with ethanol, and dried under vacuum.

As a result, crystalline beta carotene was obtained with a yield of 27%, with a basic substance content of 90%, a melting point of 179-181 ^o C, with a ratio of optical densities of solutions D451 / D341 = 8.0.

 The test results of the claimed method for producing beta-carotene in comparison with the prototype (see experiments NN 2-4 table. In comparison with experiment N 6) indicate that conducting the process within the stated limits allows to obtain crystals with a yield and quality level at the level prototype, but with a lower specific consumption of raw materials and with the use of substances permitted in the food industry.

Example 2. In a reactor with a capacity of 2 m ³ containing 600 l of sunflower oil heated to 90 ^° C, 300 kg of Blakeslea trispora mushroom biomass twice ground in a roller mill were loaded with a concentration of 30 g of carotene per 1 kg of mass.

The extraction was carried out with stirring and 95-100 ^o C for 30 minutes The extraction mass was filtered, and then additional extraction was carried out on the filter with the same solvent heated to 90 ^o C. Saturated extracts were washed with ethanol in a ratio of 3: 1 at 45 ^o C and sent to crystallization for 3 days at 15 - 18 ^o C.

The resulting suspension was heated to a temperature of 45 ^o C, filtered; the crystals were washed with ethanol in a ratio of 1:25 at 55 ^° C. for 30 minutes and dried.

 The result is 2.48 kg of crystalline carotene with a mass fraction of the main substance of 92.8%, the ratio of D451 / D341 = 10.8. The yield of crystalline carotene from the biomass loaded was 25.1%.

Claims (2)

1. A method of obtaining crystalline beta-carotene, including extraction of carotene from biomass with a non-polar organic solvent, separating the obtained extract, purifying the extract with a polar solvent, crystallizing carotene, separating the obtained crystals, washing and drying them, characterized in that the extraction of the pre-ground biomass begins in the reactor at 95 - 100 ^o C in a ratio of 1: 1.5 - 1: 3 (wt.) for 15-60 minutes and complete on the filter with the same solvent heated to 60 - 90 ^o C, then the saturated extracts are washed with the polar solvent in a ratio of 10: 1 - 3: 1 (vol.) at 40 - 55 ^o C, crystallized at 10 - 20 ^o C for 1 to 3 days, after which the resulting suspension is heated to 40 - 60 ^o C, filtered crystals washed with a polar solvent in a ratio of 1: 3 - 1: 25 (wt.) at 50 - 55 ^o C.  2. The method according to claim 1, characterized in that vegetable oil is used as a non-polar organic solvent, and ethyl alcohol is used as a polar.

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