RU2435862C1 - Method for producing high-polymer rna from used beer yeast - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: what is offered is a method for producing high-polymer yeast RNA of used beer yeast. Used beer yeast concentrated by centrifugation is suspended in an aqueous solution of oleic acid titrated by alkali to pH 7-8. The suspension is kept at 98-102C for 40-60 minutes. The hot lysate is settled for 20-24 h at room temperature. A supernatant is poured out with using a siphon. Then NaCl is added to the poured out liquid to the concentration 2-3 M. The suspension is kept for 20-96 h at room temperature. Then it is centrifuged without cooling in a low-speed centrifuge. The prepared cakes are consistently washed out in the NaCl solution 2-3 M and 92-96% ethanol by resuspension at room temperature and low-speed centrifugation. ^ EFFECT: invention promotes spread-out of the raw-material base and simplification of the high-polymer RNA technology. ^ 1 ex

Description

The invention relates to the field of biotechnology, in particular to the selection of physiologically active compounds.

Known methods for producing RNA from yeast allow the isolation of low polymer RNA, for example [1, 2]. It has long been used in medicine in the treatment of a wide range of diseases: from viral infections to memory disorders [3]. However, this drug is always administered to patients in gram quantities orally and the mechanism of its action most likely includes the supply of monomers for the synthesis of endogenous nucleic acids. The mechanism of action of high polymer yeast RNA is fundamentally different. It is much less toxic than low polymer, and is injected into animals in milligram quantities by injection, which leads to selective induction of the synthesis of certain proteins, such as hemoglobin [4].

Closest to the proposed is a method of producing a high polymer RNA from baker's yeast by suspending them in an aqueous 0.3-1.2 M solution of 2-ethylhexanoic acid containing 0.1-0.5 M NaCl (pH 7.0-7, 5), the suspension is maintained at 92-98 ° C for 10-40 min, cooled to room temperature, centrifuged at 4000 rpm for 15 min at 0-4 ° C, ethanol is added to the supernatant to a content of 50-55 vol.%, centrifuged (4000 rpm, 15 min, 0-4 ° C), the resulting precipitate total RNA is dissolved in water to a content of 150-200 OE ₂₆₀ / ml The solution is clarified by centrifugation at 20,000 rpm for 20 minutes at 0-4 ° C. Then, NaCl was added to the supernatant to a concentration of 2 M, the mixture was kept at 0-4 ° C for 1-2 h, and the precipitate of high-polymer RNA was separated by centrifugation, dissolved to 150-200 OE ₂₆₀ / ml, after centrifugation of the resulting solution (20,000 r / min, 20 min, 0-4 ° C) NaCl is added to the supernatant to a content of 1.5 M and ethanol to a content of 50-55 vol.%. The precipitate of sodium salt of RNA is collected by centrifugation at 0-4 ° C, NaCl up to 0.1-0.5 M and ethanol are added to the supernatant to a content of 50-55 vol.%, The resulting precipitate of high-polymer RNA is separated and dried by conventional methods [5] .

This method involves the use of high-quality raw materials - baker's yeast, which significantly limits the ability to scale it and the unnatural, rare and highly toxic lytic agent - 2-ethylhexanoic acid, which requires deep cleaning of the final product, as a result of which this method is not economical and practically not scalable.

The aim of the invention is the expansion of the raw material base for the production of biologically active high-polymeric RNA, namely the replacement of high-quality raw materials with beer wastes and the replacement of an unnatural, rare and highly toxic lytic agent with a large-tonnage food one, which significantly reduces the cost of the process, since it makes it practically waste-free.

The goal is achieved in that the spent brewer's yeast is concentrated by centrifugation (2500-3500 g, 5-15 minutes, without cooling), suspended in an aqueous 1-2% solution of oleic acid, titrated with alkali to pH 7-8, the suspension is maintained at 98 -102 ° C for 40-60 minutes, the hot lysate is defended for 20-24 hours at room temperature, NaCl is added to the drained supernatant with a siphon to a concentration of 2-3 M; centrifuged (2500-3500 g, 5-15 min, without cooling), the precipitate is washed with 2 portions of 2-3 M r NaCl solution and 5 portions of 92-96% ethanol by sequentially resuspending it at room temperature and centrifuging (2500-3500 g, 5-15 min, without cooling). From the obtained meal, the high polymer RNA is extracted with distilled water and dried in the usual way. Along the way, a feed protein concentrate and low polymer RNA are obtained.

With a decrease in centrifugal acceleration below 2500 g and a centrifugation time of less than 5 min, the precipitate of high-polymeric RNA forms friable and reaches for the decanted supernatant, and an increase in acceleration of more than 3500 g and centrifugation time of more than 15 min does not lead to its additional densification.

An example implementation of the proposed method

RNA extraction. 1 kg of concentrated brewer's yeast concentrated by centrifugation (3000 g, 10 min, without cooling) (Rodnik OJSC, Novosibirsk; Tinkoff LLC, Novosibirsk; Petrovich LLC, Stavropol) was suspended in portions for 20 min in 2 l of boiling water containing 45 g of oleic acid (Kupavnareaktiv CJSC, qualification “h” according to TU 6-09-5290-86), titrated with 20 ml of 2.5 M NaOH so that the temperature of the suspension does not drop below 98 ° C. The suspension was boiled for 40 min at 98-102 ° C with frequent stirring. As evaporated, boiling water up to 3 l was added to it. After 10, 20 and 30 minutes after suspending the last portion of yeast, 10 ml of 2.5 M NaOH were added to the boiling suspension. At the end of the extraction, boiled water up to 4.5 L was added to the suspension, mixed, transferred to a 5 L narrow cylindrical vessel made of heat-resistant glass, in which it settled at room temperature for 22 hours.

Salting out high polymer RNA and collecting protein feed concentrate. The settled supernatant (~ 2.9 L) was poured using a siphon into a 5 L glass container. A jelly-like yeast sludge collected at the bottom of the vessel and containing up to 70% of the denatured protein was dried at 58-62 ° C in the usual way. 620 g of NaCl was added to the supernatant (FSUE of the SIBSOL plant, Usolye-Sibirsky, Irkutsk Region, GOST R 51574-2000) and freshly boiled water up to 3.5 l. The suspension was intensively mixed until the salt was completely dissolved and left to form a precipitate at room temperature for 22 hours.

Rinsing of salted high-polymeric RNA with 3 M NaCl solution and ethanol and precipitation of 3 M NaCl solution of low-polymer RNA with concentrated HCl from waste. Then, the salted meal-cake containing high-polymer RNA was separated from the supernatant by centrifugation (3000 g, 10 min, without cooling), washed with 2 portions of 0.8 L of a 3 M NaCl solution and 5 portions of 200 ml of 94% ethanol (OJSC Alcohol Plant, Mariinsk, Kemerovo Region, GOST R 51652-2000); while the meal was separated from a 3 M solution of NaCl and ethanol by centrifugation (3000 g, 10 min, without cooling). The spent alcohol was regenerated by double distillation. The low polymer RNA contained in the supernatant (~ 3.5 L) from under the high polymer RNA was precipitated by adding 7 ml of concentrated HCl. The precipitate of low polymer RNA formed for 2 days at a temperature of 5-25 ° С was isolated by centrifugation (3000 g, 10 min, without cooling), washed with 2 portions of 400 ml of 94% ethanol, and dried in air at room temperature.

Commodity high-polymeric RNA was obtained by extracting it from washed meal with distilled water, clarifying the extract by centrifugation (3000 g, 10 min, without cooling) and final lyophilization.

From 1 kg of spent brewer's yeast concentrated by centrifugation, up to 4.0 g of high polymer RNA was obtained with the following characteristics: color of the lyophilized preparation is white; solubility in water is satisfactory; weight extinction, OE ₂₆₀ / mg - 16-18; CRF content,% ≤7.5; an increase in CRF for 1 day at 20-25 ° C in distilled water,% ≤3.0; spectral ratios: D ₂₃₀ / D ₂₆₀ - 0.40-0.55; D ₂₅₀ / D ₂₆₀ - 0.90-0.94; D ₂₈₀ / D ₂₆₀ - 0.50-0.60.

When comparing the characteristics of the high-polymeric RNA isolated by the proposed method with the same characteristics of Polyribonate (according to TU 9291-002-44040845-2004 for grade A Polyribonate: weight extinction, ОЕ ₂₆₀ / mg ≥ 16; CRF content,% ≤ 3; spectral ratios: D ₂₃₀ / D ₂₆₀ - no data; D ₂₅₀ / D ₂₆₀ - 0.86-0.95; D ₂₈₀ / D ₂₆₀ - 0.44-0.50), isolated by the prototype method [5], it is clear that they differ in mainly the content of CRF. The high polymer RNA isolated by the proposed method, this indicator is higher. However, as was shown in [4] on Polyribonate, the great importance of CRF for high-polymer RNA is not a critical obstacle to the manifestation of its biological activity.

Information sources

1. RF patent No. 2103365.

2. RF patent No. 1708845.

Z. Zemskov V.M., Lidak M.Yu., Zemskov A.M., Mikstays U.Ya. // Low molecular weight RNA: production, hydrolysis and use in medicine. - Riga: Zinatne, 1985 .-- 191 p.

4. Yamkova T.V., Yamkova V.I., Panin L.E. The biological activity of various preparations of RNA from baker's yeast. // Bulletin of the Siberian branch of RAMS. - Novosibirsk, 2006. - No. 3 (121). - C.117-121.

5. Copyright certificate SU 936701.

Claims (1)

A method of producing high-polymer RNA from yeast by suspending them in a boiling solution of a lytic agent, followed by prolonged boiling of the suspension, settling the hot lysate at room temperature, precipitating high-polymer RNA from the supernatant with sodium chloride and washing the precipitate with sodium chloride solution and ethanol by successive resuspension - centrifugation and isolation the final product from the obtained meal by extraction with water, characterized in that concentrated centrifuges The spent brewer's yeast is suspended in an aqueous solution of oleic acid with titrated alkali, the suspension is kept at 98-102 ° C for 40-60 minutes, the hot lysate is maintained for 20-24 hours at room temperature, NaCl is added to the drained supernatant to a concentration of 2 -3 M, the suspension is incubated for 20-96 hours at room temperature, centrifuged in a low speed centrifuge without cooling, the resulting meal is washed successively with 2-3 M NaCl solution and 92-96% ethanol by resuspension at room temperature and low-speed centrifugation.