RU2028382C1 - Yeast strain rhodotorula gracillis - a producer of carotinoids - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: strain of yeast Rhodotorula gracillis is deposited in VKPM at number 1724. Dry biomass of strain prepared at culturing on wort-agar for 7 days at 28 C contains, %: crude protein 47.5-49; total nitrogen 7.3-7.8; amine nitrogen 3.9-4.5; ash 5.1-5.3; lipids 3.5-3.8, and carotinoids (5820 mcg/l medium), pantothenic acid and other vitamins. EFFECT: increased efficacy of yeast strain.

Description

 The invention relates to the search for producers of biologically active substances of microbial origin, more specifically to the production of a new strain of yeast that synthesizes carotenoids, and can be used in agriculture to increase feed efficiency by enriching them with environmentally friendly carotene-containing protein-vitamin concentrates.

 Interest in carotenoid pigments is due to their vital functions. In particular, the lack of carotene and vitamin A in food and feed negatively affects the growth and formation of epithelial cells of the animal body, disrupts the glandular part of the pituitary gland, causes a decrease in visual acuity, eye disease.

 The ever-growing needs of feed production, medicine and veterinary medicine for carotene can be met to a large extent with the help of microbiological synthesis. In this regard, the search and selection among microflora of active strains of carotene-forming yeast favors the solution of this problem.

 Various types of microorganisms are known that are widely found as epiphytes on grassy fodder plants that synthesize carotenoids: Blakeslea trispora, Sporobolomyces pararoseus, Rhodotorula aurantiaca. Rh. glutinis.

 The described yeast strains are characterized by a relatively low content of carotenoids and β-carotene, they use unconventional raw materials, including peat hydrolysates (ed. St. N 391174), ethanol, oil hydrocarbons (ed. St. N 5311844). However, these nutrient substrates (although easily absorbed by breeding cultures) can be potential sources of carcinogen synthesis by a yeast cell.

 The closest analogue (prototype) of the present invention is a strain of yeast Rhodotorula glutinis 214, forming carotenoids and β-carotene. The strain (prototype) was obtained artificially using a ten-minute exposure to an electric current of industrial frequency with a voltage of 23 kV / cm on the ET-6 mutant, which was selected by UV and ethyleneimine. Culture (prototype) synthesizes the optimal amount of carotenoids and β-carotene on media with H-alkanes.

 The disadvantage of this strain is its low efficiency in the release of carotenoids and β-carotene.

 The purpose of the invention is to obtain a more effective strain of Rhodotorula gracillis 1934 with a high content of carotenoids and β-carotene.

 The proposed strain of Rhodotorula gracillis 1934 forms the aforementioned target products in higher concentrations when the culture grows on substrates with carbohydrates (glucose, sucrose, etc.), on the basis of which the cells synthesize environmentally friendly metabolites.

 A producer of biologically active substances, which can practically be used to increase feed efficiency, should be characterized by a certain growth rate, a high level of biosynthetic activity, not possess toxigenic, toxic, virulent properties for humans and farm animals, and not pollute the environment with potentially carcinogenic products.

The yeast strain Rhodotorula gracillis 1934 is stored in the culture collection of the Department of Microbiology and Virology of the Ukrainian Order of Red Banner of Labor Agricultural Academy on peptone-glucose agar (PGA) and wort agar (CA ^about 7 by Balling) at 4 ° ^C. The strain deposited under the PMBC N U-1724. The strain Rhodotorula gracillis 1934 isolated from fresh alfalfa hay and has the following characteristics:
Morphological signs. The daily culture cells on SA are oval, elongated, single, or in short chains. Cell sizes 9.5-10 x 3.5-4 microns. The dispute and pseudomycelia does not form.

 Cultural signs. In CA and PGA, the colonies are boundedly growing, convex, smooth, shiny with a smooth edge, orange-red in color. On liquid media, after 48-96 hours, the culture forms a film, ring, and precipitate.

Physiological signs. Optional aerob. The maximum growth at 28-30 ^about C, pH 5.5-6.0.

 Attitude to carbohydrates. It assimilates well glucose, sucrose, maltose, mannose, galactose, fructose, trehalose, raffinose, xylose. Weak - sorbose and dextrin.

 Attitude to alcohols. Absorbs ethyl alcohol, glycerin, mannitol, sorbitol.

 Attitude to acids. It uses succinic and malic acids well.

 Attitude to nitrogen sources. Absorbs nitrate and ammonium salts of nitrogen.

 Pathogenic properties. It does not possess pathogenic (toxigenic, toxic, virulent) and carcinogenic properties when it is administered orally and intraperitoneally to white mice and rats in living or killed cells at a dose of 500 million. Coagulase, hyaluronidase, lecithinase activity, necrotic and fibrinolytic properties are absent.

 To obtain such a strain, carotene-synthesizing properties were studied in 220 yeast isolates of different origin: museum, freshly isolated strains from the rhizosphere of fodder plants, plant residues, different types of feed - hay, straw, grain, grain forage, silage and other substrates.

The study of the biosynthetic activity of yeast was carried out by conventional methods. The proteolytic properties were determined by the Anson method (Samper et al., 1948), the protein content in cells and culture fluids was determined according to Lowry (Zakordonets, 1982). When calculating crude protein, a coefficient of 6.25 was used. To assess the dynamics of growth and accumulation of pigments, samples were taken every 24 hours after inoculation of the medium. Biomass concentration was determined by weight and nephelometric methods, yeast dry matter - drying the sample at a temperature of 105 ^{° C} to constant weight.

 The determination of carotenoids in yeast cells was carried out according to the method of Odintsova (Odintsova, 1959). For quantitative accounting of β-carotene, a spectrophotometric method was used to detect this group of pigments in the extract (Evening, Kulikova, 1969). Pantothenic acid and other vitamins were determined according to the method described by Shcherbina) Shcherbina, 1982).

Comparison of the values of several samples of the carotene formation intensity of the studied strains was carried out using Student's criterion. The significance of differences between the results was at 0.1% significance level t _fact = = 3.48> t _table. = 2.28.

Dry biomass Rhodotorula gracillis 1934 strain obtained by growth on the surface SA or PHA method for 7 days, at ²⁸ ° C represents the dry red-brown substance, which contains in its composition 47,5-49% crude protein, total nitrogen 7.3-7.8%, amine nitrogen 3.9-4.5%, ash 5.1-5.3%, lipids 3.5-3.8%. carotenoids, pantothenic acid, other vitamins - 5820 μg / l of the medium, which in terms of 1 g of absolutely dry mass of yeast cells is 2983-3720 μg (34%) of carotenoids and 2000-2100 μg (66%) of β-carotene.

 Biological studies of the products obtained in laboratory animals for a month indicate the absence of toxic impurities in them. Animals willingly ate food containing metabolites of Rhodotorula gracillis 1934, grew well and developed during the entire period of testing, gained weight (30-40%) compared with the control.

 The resulting strain can be recommended for the development of effective methods for enriching feed with vitamins, enzymes, protein and other biologically active products of microbial origin.

Claims (1)

 STRAIN OF YEAST PHODOTORULA GRACILLIS - PRODUCER OF CARATINOIDS.  The yeast strain RHODOTORULA GRACIL LIS VKPM U-1724 is a producer of carotenoids.