EA002468B1 - A pair of strains of heterothallic fungus "blakeslea trispora" vsb-129 (-) and vsb-130 (+), producing lycopene and process for lycopene production - Google Patents

Abstract

1. A pair of strains of heterothallic fungus "Blakeslea trispora" VSB-129 (-) and VSB-130 (+) producing lycopene. 2. A process for lycopene production comprising growing a pair of strains of of heterothallic fungus "Blakeslea trispora" producing lycopene in a nutrient medium, containing sources of carbon, nitrogen, antioxidant with subsequent recovering lycopene, characterized in that a pair of strains of heterothallic fungus "Blakeslea trispora" VSB-129 (-) and VSB-130 (+) is produced, wherein (-) strain is grown first after which (+) strain is added to the grown (-) strain and further they are grown together and lycopene is recovered.

Description

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

Lycopene is a red pigment of tomato fruits, belongs to the class of natural compounds, namely carotenoids. It is widely used in the manufacture of food as a food coloring. Its value is especially great in the production of sausages and ham products, where it can replace sodium nitrite.

In recent years, it has been shown that lycopene has a more pronounced ability to eliminate the harmful effects of singlet oxygen compared with β-carotene and other carotenoids [1]. Activity to inhibit the growth of cancer cells in lycopene is also higher compared with β-carotene [2]. In addition, the positive effect of lycopene in the complex treatment of patients with diabetes mellitus has been established [3].

A method of obtaining lycopene from the skin of tomatoes by extraction with methylene chloride in the presence of an adsorbent [4]. However, the yield of lycopene is insignificant, since the content of lycopene in tomatoes is small. This makes this method economically inexpedient. In addition, the seasonality of obtaining raw materials for the implementation of this method can also be attributed to the disadvantages. The more promising methods for obtaining lycopene include biotechnological methods for producing lycopene, based on growing microorganisms producing pigment and isolating it from biomass with organic solvents.

The ability to synthesize lycopene is found in various types of microorganisms of bacteria, fungi, actinomycetes. A known strain of 81crc1oscuss5 sytoyotusse Bsik, which accumulates 20-50 mg / kg of lycopene on the mineral medium [5]. The bacterial strain Musobilus rumen is described, which on a medium with molasses and corn extract accumulates up to 7.05 mg / g of total carotenoids. At the same time, in addition to lycopene, α-, β-, and γ-carotene, auroxanthin, lutein, cryptoxanthin, violoxanthin, zeaxanthin, anteraxanthin, rubiksanthin, leprotein, neoxanthin were found in the pigment complex of bacteria [6].

The most active producers of lycopene are the fungi P11usotuss5 L1ax51Sapi5 and BaccaDea Hirrosov. Depending on strain characteristics and cultivation conditions, the level of accumulation of lycopene varies in the range of 192–600 μg / g DIA [7].

There are known methods for producing lycopene from the biomass of the fungus Blake§lea I1515rog [8, 9]. In accordance with the described methods, the yield of crystalline lycopene is 31-56% of its quantity in biomass. The closest to the claimed invention to the technical essence and the achieved result is a method for producing lycopene, described in the patent of the Russian Federation No. 2102416 [10]. According to the present invention, the strains of the fungus Blake§lea (threshold A-732-3 (+) and A-731-3 (-) or 8A (+) and 8A (-)) are grown on corn soybean medium with the addition of the following compounds as stimulants of lycopene formation Aminomethylpyridine class or tobacco chips. Growing (+) and (-) strains is carried out for 48 h, the obtained seed is used in a ratio of 1: 9 for subsequent fermentation. Obtaining the desired product is carried out by extraction with sunflower oil, crystallization using ethanol and toluene and subsequent column chromatograph iey in the system n-geksanatseton 50: 1.

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 does not allow one to estimate the lycopene-synthesizing activity of the producer-culture. In addition, for the accumulation of lycopene by the culture of the fungus Blake§lea (the threshold for these strains is added to the environment by stimulants: aminomethylpyridine or tobacco chips. At the same time, tobacco chips are a weaker stimulant compared to aminomethylpyridine, so when it is added to the culture medium, the level of accumulation of lycopene by the mushroom B1 §1еа (threshold is 0.4 g / l, which is 1.7 times lower than aminomethylpyridine. The addition of pyridine derivatives to the cultivation medium, despite their low concentrations (0.010.005%), is undesirable.

The objective of this invention is to obtain a new pair of strains heterotallichnogo fungus VIAKEBEAA TYAKROYAA BCE-129 (-) and BAB-130 (+), producing lycopene, and the development of a more effective way to obtain lycopene using the specified pair of strains.

The essence of the invention lies in the fact that as a result of multi-stage selection and selection, a pair of strains of the heterotallic fungus Blake§lea 1g1kroga BWA-129 (-) and BWA-130 (+) was obtained, which, when co-cultivated in depth on a mineral medium with glucose, after 36 h cultivating accumulates up to 35 mg of lycopene per 1 g of absolutely dry weight (DIA) of biomass (or up to 0.8 g / l of culture medium); on the corn soybean medium after 48 hours from the start of cultivation - up to 30 mg of lycopene per 1 g of absolutely dry weight (DIA) of biomass (or up to 1.5 g / l of culture medium). In addition, lycopene stimulators are not added to the cultivation medium. Lycopene content is 92-96% of the amount of carotenoids. Lycopene-containing biomass is separated from the culture fluid. The extraction is carried out with a mixture of organic solvents hexane-ethanol. Crystallization of lycopene is carried out in ethanol. The yield of crystalline lycopene is 50-55% of its content in biomass.

A pair of strains of the fungus B1Ake§lea 1g1xrg BWA-129 (-) and BWA-130 (+) obtained from (+) and (-) forms of the collection strain B1Ake§1e1r15roh as a result of multi-stage selection using UV rays and и-methyl-nitro -Y-nitrosoguanidine and benzylpenicillin as mutagenic factors and sieving on Petri dishes with a wort agar nutrient medium (7 ° Balling). After 18-24 h of cultivation, the isolated colonies were screened in tubes on beveled wort agar. Colonies were selected after 8–10 days of cultivation in test tubes at 26 ° C on the basis of the greatest pigmentation with subsequent determination of the productivity of the isolated variants. The strains of BWA-129 and BWA-130 have the following characteristics.

Wort agar. The strain BAB-130 (+). The growth of the strain is intense. Aerial mycelium is developed uniformly, fluffy, beige color. Substrate mycelium of fawn color. Sporulation is active.

The strain BAB-129 (-). Growth is less active compared to the strain BAB-130. Aerial mycelium is underdeveloped. Sporulation scanty. Substrate mycelium strain bright orange color.

With the joint growth on the wort agar medium at the point of contact (+) and (-) forms, no characteristic band of black zygospores is formed. After contact with the (+) form, the entire surface of the mycelium (-) of the form is colored maroon-red.

The yield of lycopene during the joint cultivation of strains of BWA-129 and BWA120 on mineral medium with glucose after 36 hours of cultivation accumulates up to 35 mg of lycopene per 1 g of absolutely dry weight (DIA) of biomass (or up to 0.8 g / l of culture medium) ; on corn-soy medium after 48 hours from the start of cultivation - up to 30 mg of lycopene per 1 g of absolutely dry weight (DIA) of biomass (or up to 1.5 g / l of culture medium).

Example 1

The strains of the fungus VakekeChe 1g15roga BWA-129 and BWA-130 are grown on the jambs wort agar for 12 days. The strain BWA-1 29 seeded in katalochnye flasks on pre-hydrolyzed corn-soy environment of the following composition:

soy flour - 4.5%;

corn flour - 2.3%;

KN ₂ PO ₄ - 0.05%;

thiamine chloride - 2x10 ^-4 %.

The hydrolysis of flour is as follows: corn and soy flour is diluted with tap water, 2.8 ml of concentrated sulfuric acid are added and sterilized at 1 atm for 1.5 hours. After sterilization, the pH is adjusted to 6.5-6.3 and KH ₂ PO _{4 is} added. Growing (-) strain is carried out at 26-27 ° C on a rocking chair at 200 rpm for 48 hours. The grown culture of the BWA strain 129 is used as seeding material for seeding flasks with corn soybean medium of the composition described above. Maize environment for the main fermentation is not subjected to hydrolysis. The flasks seeded with VSB-129 are grown on a rocking chair for 32 hours at 26-27 ° C at 200 rpm. After 32 hours from the beginning of cultivation, antioxidant and pre-grown on the corn soybean medium (+) strain BWA-130 are introduced into flasks with grown culture of BWA-129. The number of introduced (+) strain is 1.5% of the volume of the medium. Joint cultivation of strains are 48 h under the above conditions. After 48 h, the biomass concentration is 50 g / l, the biomass content in the biomass is 30 mg / g DIA biomass, the total lycopene yield is 1.5 g / l of culture medium. Lycopene and β-carotene are 92 and 8%, respectively.

Example 2

The method for producing lycopene is carried out in the same manner as described in Example 1, but the corn-soy medium for the main fermentation is subjected to hydrolysis. On the hydrolyzed medium (-) strain BWA-1, 29 is grown for 24 hours, then suspension (B) of the BWA-130 strain in the amount of 1.0% of the medium volume and antioxidant are added to the grown culture of the (-) strain. Joint cultivation of (+) and (-) strains are 40 h under the above conditions. After 40 h the content of lycopene in biomass is 28 mg / g DIA biomass, the total yield of lycopene is 1.4 g / l of culture medium. Lycopene and β-carotene are 94 and 6%, respectively.

Example 3

The method of producing lycopene is carried out in the same manner as described in Example 1, but after growing on corn-soy medium, the BWA-1 strain 29 is seeded into the mineral medium with glucose. The composition of the medium:

glucose-15 g / l;

(ΝΗ ₄ ) ₂ 8Ο ₄ - 5.8 g / l; NaH ₂ PO ₄ - 5.2 g / l;

K ₂ NRA ₄ - 5.0 g / l;

MASS - 0.34 g / l;

peptone -10 g / l;

thiamine chloride - 2x10 ^-4 %.

On the mineral medium (-) the strain BWA-129 is grown for 20 h at 26-27 ° С on a rocking chair with

200 rpm Then, an antioxidant, 3% glucose, and a BAB-130 strain preliminarily grown on corn soybean medium (+) are added to the culture medium. The amount of the introduced (+) strain is 1% of the volume of the medium. Joint cultivation of (-) and (+) strains is carried out under the above conditions for 36 hours. 36 hours after the start of joint cultivation of strains, biomass accumulation is 23 g / l, lycopene content in biomass is 35 mg / g biomass DIA, total lycopene yield - 0.8 mg / l of culture medium. Lycopene and β-carotene are 96 and 4%, respectively.

Example 4

The method of producing lycopene is carried out as described in Example 3, but the (-) BAB-129 strain is grown on a mineral medium with 2% glucose as a carbon source and energy for 28 hours at a temperature of 26 ° C in flasks with bump stops to create optimal aeration mode. After 28 hours of growing the (-) strain, 1.5% glucose, 1.0% suspension of the (+) strain BWA-130 and antioxidant are added to the flasks with the grown culture of the (-) strain. Co-cultivation of (+) and (-) strains is carried out for 40 hours under the conditions described above. The content of lycopene in biomass is 33 mg / g DIA biomass, the total yield of lycopene per unit volume of the nutrient medium is 940 mg / l. Lycopene and β-carotene are 96 and 4%, respectively.

Example 5

The flask is charged with lycopene-containing biomass and a mixture of solvents hexane-ethanol in a 4: 1 ratio. The ratio of biomass and solvent is 1: 5. Extraction is carried out in 3 steps at 1 = 60 ° C for 30 minutes at each step. Next, the meal is filtered and the saturated extract is directed to crystallization in ethyl alcohol at (= 5 ° C for

h. The output of lycopene is about 55% of its content in biomass. The proportion of the main substance - 92%.

Bibliography

1. Masyu Ρ.Ό., Esoa ^ schauat, T.P.A., Ka1gseg 8., 81e§ N. || Vusyet. 8os Tgap8asIop8.-1990ν.18. I.6.r. 1055-1056.

2. Application for patent EP 0600544.

3. And a tepap trademark about £ 1999; 149: 168-176.

4. RF patent number 2039775.

5. Patent 1T number 343773.

6. The USSR AS No. 1071636.

7. V.E. Me1Ia. E.Seg6a-O1te6o || Arr1 M1stoYo1. Vu! Eipo1.-1995.-№ 42-p. 836-838.

8. RF patent 2112747.

9. RF patent № 2126806.

10. RF patent № 2102416.

Claims (2)

CLAIM 1. A pair of strains of the heterotallic fungus Blake§lea (threshold BSR-129 (-) and BWA-130 (+), producing lycopene. 2. A method of producing lycopene, which includes growing a pair of strains of the heterotallic fungus B1 ake§lea Ic15rog producing lycopene on a nutrient medium containing sources of carbon, nitrogen, an antioxidant, followed by release of lycopene, characterized by the fact that a pair of strains of heterotallic fungus is grown as a lycopene producer B1a§§ea 1g15rog BWA-129 (-) and BWA-130 (+), the (-) strain is first grown, then the (+) strain is added to the grown culture (-) strain, then the strains are grown together and lycopene is isolated.