RU2270868C1 - METHOD FOR PREPARING LICOPIN, PHOSPHOLIPIDS, FATTY ACIDS AND ERGOSTEROL BY COMBINED CULTURING (+)= AND (-)-STRAINS OF FUNGUS Blakeslea trispora - Google Patents

Abstract

FIELD: biotechnology, mycology.

SUBSTANCE: invention relates to a method for producing vitamins and antioxidants, in particular, producing licopin, phospholipids, fatty acids and ergosterol. Method involves the combined submerged culturing pair of fungus Blakeslea trispora of strains B-1(-) and B-2(+) in mineral medium with glucose. In 26 h culturing the accumulation of licopin ACB is up to 40 mg/g, and in medium with hydrol and maize extract in 30 h after onset of culturing - up to 50 mg/g of licopin ACB. For preparing biologically active compounds (licopin, ergosterol, phospholipids and fatty acids) biomass is separated from cultural fluid and dried and lipids from biomass are extracted with a hydrophobic solvent. Prepared extract is separated from biogrist by its hot filtration. Licopin is crystallized in medium the same organic solvent as used in the extraction process. Licopin crystals are separated by filtration. The yield of crystalline licopin by this method is 70-72% of its content in biomass. After separation of licopin crystals the following stages are carried out sequentially: isolation and purification of phospholipids; isolation and purification of fatty acids, and isolation and purification of ergosterol. The advantage of invention is the enhanced yields of the end products, simplifying and declining cost of the extraction process.

EFFECT: improved preparing method.

1 tbl, 4 ex

Description

The invention relates to the field of biotechnology and relates to the production of vitamins and antioxidants, in particular the production of lycopene, ergosterol, phospholipids, fatty acids.

It has now been established that biologically active compounds, such as lycopene, ergosterol, phospholipids, fatty acids, etc., are successfully used for the treatment and prevention of various diseases.

Lycopene belongs to the class of natural compounds - carotenoids. This is a pigment that gives a healthy red color to fruits and vegetables, such as tomatoes, watermelons, pink grapefruits, sea buckthorn, etc. Recent studies show that lycopene, along with the coloring function, has an independent value as a dietary supplement. Lycopene has a general strengthening effect on the body and has a large set of valuable pharmacological properties. Suppressing free radical oxidation in the body, lycopene stabilizes the body’s immune status, improves the course of several important biological processes in the body, including normalizes blood glucose, lipid metabolism, vision and controls cell proliferation (neoplasm). It was found that lycopene is highly effective in the treatment of diseases of the prostate gland, lungs, stomach, cataract, coronary heart disease, and atherosclerosis [1-3]. The biological activity of lycopene is associated primarily with its antioxidant properties, i.e. ability to inhibit free radical processes in cells. It was shown that lycopene has a more pronounced ability to eliminate the harmful effects of singlet oxygen compared to β-carotene [4].

Ergosterol is a precursor of vitamin D ₂ , which is obtained by irradiating ergosterol with ultraviolet rays. D ₂ plays an important role in providing the body with calcium, primarily due to the proper use of calcium from food. With a lack of vitamin D ₂ in the body, there is a violation of mineralization in the process of bone formation, which leads to serious changes in the skeleton.

Any liver disease, regardless of the etiology (the effect of alcohol, drugs, industrial and household toxins, malnutrition, viral infections, etc.) causes damage to cell membranes with the loss of phospholipids, which are the main structural components of the membranes. Loss of intrinsic phospholipids leads first to inactivation, and then to the death of liver cells. Phosphatidylcholine (lecithin), which is part of phospholipids, is the main structural component of all cell membranes. When administered orally, phosphatidylcholine restores the integrity of cell membranes, primarily the liver. Phosphatidylcholines are used in many industries due to their excellent dispersing and emulsifying properties.

Fatty acids have a wide range of applications in perfumery, pharmacology, medicine. The most valuable are polyunsaturated fatty acids. They must be ingested with food. With their insufficiency, a growth lag is observed, characteristic lesions from the skin and hair cover develop.

Numerous methods are known for producing individual biologically active compounds, in particular lycopene, ergosterol, phospholipids, fatty acids using various producer microorganisms.

A known method of producing ergosterol from yeast (Auth. Certificate. USSR No. 465812) [5]. A strain of the microscopic fungus Hypomyces rosellus is described, which is used to obtain lipids with a high degree of unsaturation of fatty acids (RF patent No. 2020155) [6]. A known strain of Streptomyces chrostomyceticus, which accumulates 20-50 mg / kg of lycopene on a mineral medium (Italian patent No. 343773) [7]. A bacterial strain Mycobacterium rubrum is described, which accumulates up to 7.05 mg / g of total carotenoids on a medium with molasses and corn extract. Moreover, in the pigment complex of bacteria, in addition to lycopene, α, β, and γ-carotene, auroxanthin, lutein, cryptoxanthin, violoxanthin, zeaxanthin, anteraxanthin, rubixanthin, leprotein, neoxanthin were found (AS USSR No. 1071636) [8].

The most active lycopene producers are Phycomyces blakesleanus and Blakeslea trispora. In the fungus Phycomyces blakesleanus, depending on the characteristics of the strain and cultivation conditions, the level of lycopene accumulation varies in the range of 192-600 μg / g ASV [9].

Known methods for producing lycopene from the biomass of the fungus Blakeslea trispora [10-12]. In accordance with the described methods, the yield of crystalline lycopene is 31-56% of its amount in biomass.

According to the invention described in the patent of the Russian Federation No. 2102416, strains of the fungus Blakeslea trispora A-732-3 (+) and A-731-3 (-) or 8A (+) and 8A (-) are grown on corn-soybean medium with the addition of as stimulants of lycopene formation of compounds of the class of aminomethylpyridines or tobacco chips. The cultivation of (+) and (-) strains is carried out for 48 hours, the resulting seed is used in a ratio of 1: 9 for subsequent fermentation. Obtaining the target product is carried out by extraction with sunflower oil, crystallization using ethanol and toluene and subsequent column chromatography in the n-hexane - acetone 50: 1 system.

In accordance with the described method, the yield of lycopene is 0.7 g / l of medium. The ratio of lycopene and β-carotene is 11: 1 or 92% and 8%, respectively. Unfortunately, the patent does not indicate the level of accumulation of the biomass of the fungus per unit volume of the nutrient medium, which makes it impossible to evaluate the lycopsynthesis activity of the producer culture. In addition, for the accumulation of lycopene by the culture of the Blakeslea trispora fungus, the indicated strains add stimulants to the medium: aminomethylpyridine or tobacco chips. At the same time, tobacco crumb is a weaker stimulant compared to aminomethylpyridine, so when it is added to the culture medium, the level of lycopene accumulation by Blakeslea trispora fungus is 0.4 g / l, which is 1.7 times lower compared to aminomethylpyridine. The addition of pyridine derivatives to the culture medium despite their low concentrations (0.01-0.005%) is undesirable.

In all of the above methods for producing biologically active compounds, as a rule, one specific strain of a microorganism is used - a producer of one or another type of compound.

Closest to the claimed invention in technical essence and the achieved result is a method for producing lycopene described in Eurasian patent No. 002468 [13] and a method for producing antioxidants and biologically active compounds of lipid nature described in Eurasian patent No. 003212 [14]. According to the invention [13], lycopene is obtained by co-growing strains of the heterotallic fungus Blakeslea trispora VSB-129 (-) VSB-130 (+). Cultivation is carried out according to a 2-stage cultivation scheme on a mineral medium with glucose or corn-soybean medium.

On the mineral medium with glucose in the 1st stage, a (-) strain of the fungus is grown for 20 hours, then at the 2nd stage 3% glucose and (+) the strain of the fungus are added to the culture medium. Co-cultivation of (+) and (-) strains is carried out for 36 hours. In this case, up to 35 mg of lycopene is accumulated in the biomass in terms of 1 g of absolutely dry weight (ASV) of the biomass.

In the corn-soybean medium at the 1st stage, a (-) strain is grown for 32 hours, then at the 2nd stage a (+) strain of the fungus is introduced into the culture medium. Co-cultivation is carried out for 48 hours. In this case, up to 30 mg of lycopene is accumulated in the biomass per 1 g of absolutely dry weight (ASW) of the biomass.

Extraction from the biomass of the fungus is carried out with a mixture of solvents ethanol: hexane in a ratio of 4: 1 in 3 steps at a temperature of 60 ° C for 30 minutes per step. The ratio of biomass and solvent is 1: 5. Crystallization of lycopene is carried out in ethyl alcohol at a temperature of 5 ° C for 20 hours. The yield of lycopene is 55% of its content in biomass. The proportion of the main substance is 90%.

According to the invention [14] biologically active compounds of a lipid nature from the biomass of Blakeslea trispora fungus are obtained by extraction with a polar solvent (eg, acetone or ethanol) or a mixture of polar and non-polar solvents (eg, acetone: hexane or ethanol: hexane) at a temperature of 40-70 ° C in three steps with a biomass: extractant ratio of 1: 3.5 in the first step, up to 1: 1.5 in the last. The extract obtained is separated from bio-meal by hot filtration at 50-60 ° C. After which the extract is placed on the crystallization of carotenoids in a mixture of polar and non-polar solvents at 5-20 ° C for 3-24 hours with gentle stirring. Precipitated carotenoid crystals were separated by filtration and washed with ethanol. After separation of the carotenoid crystals from the lipid solution, phospholipids are isolated at a temperature of 5-20 ° C for 1-5 hours with gentle stirring. Crystallization of phospholipids is carried out in acetone or a mixture of acetone and a non-polar solvent (e.g. hexane). The obtained phospholipids are sent for further fractional separation in a solution of ethanol in order to isolate the lecithin fraction. The resulting solution of neutral lipids is saponified in acetone in an aqueous solution of potassium hydroxide at 50-70 ° C for 20-60 minutes. Then the saponified fraction is diluted with water and hydrolyzed with sulfuric acid to obtain a mixture of fatty acids. The unsaponifiable fraction is evaporated, redissolved in hexane and ergosterol is crystallized at 0-20 ° C for 4 hours at a hexane: unsaponifiable fraction ratio of 1: 3-1: 50, and then the ergosterol obtained during crystallization is filtered off. Ergosterol on the filter is washed with an organic solvent or, if necessary, recrystallization is carried out. Next, ergosterol is cleaned from the filter and dried at 60 ° C.

The objective of the invention is to obtain new highly effective strains producing biologically active compounds, in particular lycopene, ergosterol, phospholipids, fatty acids, developing a more effective method for their preparation, expanding the number of producers, simplifying and reducing the cost of the extraction process.

The essence of the invention lies in the fact that as a result of multi-stage selection and selection, a pair of Blakeslea trispora B-1 (-) and B-2 (+) fungus is obtained, which, when combined with the cultivation in a mineral medium with glucose, accumulates up to 40 after 26 hours of cultivation mg / g ASB lycopene, on a medium with hydrol and corn extract (waste starch production) after 30 hours from the start of cultivation, up to 50 mg / g ASB lycopene. The lycopene content is 92-96% of the total carotenoid content.

The strains of the fungus B-1 (-) and B-2 (+) were obtained from the strains of the fungus Blakeslea trispora VKPM F - 414 (-) and VKPM F - 414 (+) as a result of multi-stage selection with sequential use of ultraviolet rays, N-methyl- N-nitro-N-nitrosoguanidine and diphenylamine as mutagenic factors and sieving on Petri dishes with wort-agar nutrient medium (7 ° B). After 18-24 hours of cultivation, isolated colonies were seeded into test tubes on mown wort agar. Colonies were selected after 8-10 days of growing them in test tubes at 26 ° C according to the sign of the highest pigmentation with subsequent determination of the productivity of the selected variants. Strains B-1 and B-2 have the following morphological characteristics:

Wort agar. Strain B-2 (+) form. The growth of the strain is intense. Aerial mycelium is poorly developed. The substrate mycelium is well developed, dense, yellowish-beige. Spore formation is weak.

Strain B-1 (-) form. Growth is less active compared to strain B-2. Aerial mycelium is absent. Spore formation in this environment is not detected. The substrate mycelium of the strain is reddish-orange.

With joint growth on the wort-agar medium at the point of contact of (+) and (-) strains, a characteristic band of black zigospores does not form. After contact with the (+) strain, the entire surface of the mycelium of the (-) strain becomes burgundy red.

The strains are stored in the collection of the company Biosynthesis-Mt LLC, the authors of the strains are a team of authors Avchieva PB, Butorova I.A. and etc.

To obtain biomass with the highest content of fat-soluble biologically active compounds - lycopene, phospholipids, fatty acids and ergosterol, the cultivation of Blakeslea trispora strains B-1 (-) and B-2 (+) is carried out on specially selected nutrient media and growing conditions. The nutrient medium should contain readily available sources of carbon and nitrogen, as well as trace elements.

Sowing material is prepared by separately growing B-1 (-) and B-2 (+) forms on jambs with wort agar for 10 days at a temperature of 26-28 ° C. Next, the strains are separately seeded in rocking flasks with sterile media containing corn and soy flour in an amount of 1-10% of each component, KH ₂ PO ₄ from 0.01 to 1.00%, thiamine chloride - 2 × 10 ^-5 - 2 × 10 ^-4 %, the optimal pH for crop growth is pH 6.0-8.0. In this medium (-), the strain of the fungus Blakeslea trispora B-1 is grown for 60-92 hours at a temperature of 26-28 ° C, with a swing of 150-250 rpm. (+) The strain of the fungus is grown under the same conditions, but for 36-72 hours. The biomass obtained as a result of separate cultivation is further used for the joint fermentation of B-2 (+) and B-1 (-) strains of the hornbeam Blakeslea trispora. Co-fermentation is carried out under sterile conditions on a mineral medium containing readily available sources of carbon and nitrogen, as well as trace elements, or on media containing starch production waste (hydrol and corn extract), at the same pH, temperature and rocking speed, and first the (-) strain of the fungus is added to the culture medium, and only after 12-36 hours from the start of cultivation is the (+) strain added in the amount of 0.1-20% (volume) of the originally introduced volume of the (-) strain. Co-cultivation of (+) and (-) forms is carried out within 24-72 hours.

Methods for obtaining the biomass of the fungus Blakeslea trispora, containing lycopene, phospholipids, fatty acids and ergosterol, are illustrated in examples 1 and 2.

To obtain biologically active compounds (lycopene, ergosterol, phospholipids, fatty acids), the biomass of co-grown (+) and (-) strains is separated from the culture fluid and dried. The extraction of lipids from biomass (humidity 10-15%) is carried out by any hydrophobic solvent at a temperature of 40-65 ° C in several stages with a ratio of biomass: extractant from 1: 1 to 1:20 (wt.). The most suitable extractants are: nefras, hexane, carbon tetrachloride, chloroform. The extract obtained is separated from the bio-meal by hot filtration at 50-60 ° C.

Crystallization of lycopene is carried out at 5-20 ° C for 3-24 hours with gentle stirring in the medium of the same organic solvent as the extraction process. This technical solution allows the use of the same organic solvent in the first two stages of the technological process, which simplifies the hardware design and reduces the cost of producing the target products. The precipitated lycopene crystals are separated by filtration. The yield of crystalline lycopene with a purity of 92-96% as a result of the proposed process is 70-72% of the content of this carotenoid in the biomass. After separation of the lycopene crystals, the following steps are carried out sequentially:

- isolation and purification of phospholipids;

- isolation and purification of fatty acids;

- isolation and purification of ergosterol.

The isolation and purification of the above substances is carried out by methods proposed by us earlier [15, 16], which are illustrated by examples 3 and 4.

Example 1

Seed preparation:

The strains of the fungus Blakeslea trispora B-1 and B-2 are grown on jambs of wort agar for 10 days. Next, the strains are seeded in rocking flasks on a corn-soybean medium of the following composition:

Soya flour - 4.5% Corn flour - 2.3% KN ₂ PO ₄ - 0.05% Thiamine chloride - 2 × 10 ^-4 % pH of the medium - 6.8

The cultivation of (-) strain is carried out at 26-27 ° C on a rocking chair at 200 rpm for 72 hours, (+) the strain is grown for 48 hours. The grown culture of the fungus B-1 (-) was used as an inoculum for basic fermentation.

Fermentation is carried out on a mineral medium with glucose of the following composition:

Glucose - 20 g / l (NH ₄ ) ₂ SO ₄ - 9.0 g / l NaH ₂ PO ₄ - 5.8 g / l K ₂ HPO ₄ - 2.5 g / l MgSO ₄ - 0.34 g / l Peptone - 20 g / l Thiamine chloride - 2 × 10 ^-4 % pH of the medium - 6.8

On the mineral medium (-), the strain of the fungus Blakeslea trispora B-1 is grown for 18 hours at 27-28 ° C. Then, a fungal strain in the amount of 1% of the medium volume pre-grown on corn-soybean medium for 48 hours is added to the culture medium, and the strains are then grown together for 28 hours. After 26 hours from the start of co-cultivation, biomass accumulates 40 mg of lycopene per 1 gram of absolutely dry biomass.

Example 2

The same as in example 1, but for the main fermentation using a medium based on waste starch production (hydrol and corn extract) of the following composition:

hydrol - 6.5 g / l corn extract - 81 g / l (NH ₄ ) ₂ SO ₄ - 4.1 g / l K ₂ HPO ₄ - 1.28 g / l pH of the medium - 6.8

In this medium (-), the strain of the fungus Blakeslea trispora B-1 is grown for 20 hours at 27-28 ° C. Then, a fungal strain in the amount of 1% of the medium volume preliminarily grown on corn-soybean medium is added to the cultivation medium for 48 hours in the amount of medium and then the strains are co-grown for 30 hours. After 30 hours from the start of co-cultivation in biomass, 50 mg of lycopene is accumulated per 1 gram of absolutely dry biomass.

Example 3

The biomass of Blakeslea trispora fungus in an amount of 110 g (humidity 10.0%) is extracted in three steps with hexane (biomass: extractant ratio - 1: 6) at 60.0 ° C. Then the obtained extracts are combined. The result is 29.7 grams of lipids having the presented fractional composition (see table 1).

Table 1.
Fractional composition of lipids of the fungus Blakeslea trispora (hexane extractant). * Components Content in% of total lipids Phospholipids 28.8 Monoglycerides 4.0 Diglycerides 4.0 Ergosterol 4.7 Free fatty acids 23,2 Triglycerides 18,4 Lycopene 11.7 Hydrocarbons 5.2 * the composition is presented on lipids purified from an organic solvent.

The solution of lipids in hexane is cooled to + 5 ° C and maintained for 3 hours with gentle stirring, the precipitated lycopene crystals are filtered off. The obtained lycopene in the amount of 2.9 g is washed with ethyl alcohol, as a result, crystals with a basic substance content of at least 92-96% are obtained. After separation of lycopene from the lipid solution, phospholipids are isolated at -5 ° C, for which a triple volume of acetone is added to the lipid solution. Phospholipids crystallize within 2.5 hours. As a result of the process, 6.1 g of phospholipids are obtained. The lipid solution in acetone was evaporated, and then redissolved in ethanol and saponified in a 25% aqueous potassium hydroxide solution at 50 ° C for 20 minutes. The saponified fraction is diluted with 200 ml of water and hydrolyzed with H ₂ SO ₄ . The resulting fatty acids are extracted with hexane, resulting in 4.8 grams of a mixture of fatty acids. The unsaponifiable fraction is extracted with hexane in three steps of 150 ml each, the extracts are combined, ergosterol is partially evaporated and crystallized at 0 ° C and a hexane: lipid ratio of 1: 3. The precipitated ergosterol is filtered, washed and dried at 60 ° C, resulting in 0.9 g of ergosterol. The content of the main substance in the ergosterol sample is 85.0%.

Example 4

The same as in example 3, but the extraction of biomass is carried out in nephras medium (t boiling. = 70-80 ° C) with a ratio of biomass: extractant equal to 1: 3, at a temperature of 65 ° C, in four steps, lasting 40 minutes each. When the combined extracts are cooled, a precipitate is collected on the filter containing a complex fraction of phospholipids and lycopene in an amount of 11.8 g. The obtained precipitate is the final product of the technology, since a significant phospholipid content protects lycopene from oxidation, i.e. increases its shelf life.

Bibliography.

1. Minthy L., Steven J. Shwartz // Lycopene: Chemical and Biological Properties. Foodtechnology, 1999, vol. 53, No. 2, p. 33-43.

2. Patent EP 00544.

3. American Journal of Epidemiology 1999; 149: 168-176.

4. MacioP.D., Devasagayam T.P.A., Kaiser S., Sies H. // Biochem. Soc. Transactions. - 1990 - v. 18. N6. - p.1055-1056.

5. A.s. USSR No. 465812.

6. RF patent No. 2020155.

7. Italian patent No. 343773.

8. A.S. USSR No. 1071636.

9. B.J. Mehta. E.Cerda-Olmedo // Appl. Microbiol. Biotechnol. - 1995. - No. 42. - p. 836-838.

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11. RF patent No. 2126806.

12. RF patent No. 2102416.

13. Prototype Eurasian patent No. 002468.

14. Prototype Eurasian patent No. 003212.

15. Deev S.V., Butorova I.A., Avchieva P.B. // Biotechnology. - 2000 - No. 5 - p. 36-40.

16. Deev S.V., Butorova I.A., Avchieva P.B. // Biotechnology. - 2001 - No. 4 - C.22-31.

Claims (1)

A method of obtaining biologically active compounds of lipid nature, namely lycopene, phospholipids, fatty acids and ergosterol, by co-culturing two strains of heterotallic fungus Blakeslea trispora in a nutrient medium containing carbon, nitrogen, trace elements, followed by sequential isolation of lycopene fungus, phospholipids from biomass fatty acids and ergosterol by extraction and crystallization, characterized in that as a producer of biologically active compounds using a pair of strains of gr ba Blakeslea trispora B-1 (-) and B-2 (+), and the processes of extraction of lycopene, phospholipids, fatty acids, ergosterol and crystallization of lycopene are carried out in the same hydrophobic organic solvent.