SU1430402A1 - Strain of trichosporon cutaneum yeast as source of protein biomass and method of producing same - Google Patents

Abstract

This invention relates to the microbiological industry and concerns a new yeast strain — a source of protein biomass and a method for producing it using hydrolyzed lignin as a carbon source. The aim of the invention is the strain of the yeast Trichosporon cutaneijra VKPM U-495, which makes it possible to obtain a higher biomass yield with an increased protein content in it during the cultivation process. The invention consists in the fact that the strain T.cutaiim EKCHHM U-495 is grown on a nutrient medium containing 7.5-12.5 g / l of hydrolyzed lignin, 1-2 g / l of ammonium phosphate dibasic, and water base is added to 1 liter of decantate. post-yeast brew, which is a waste of hydrolyzed yeast production. After 48 h of cultivation of the VKPM U-495 strain on the proposed nutrient medium, the yield of ligno-biomass is 13 g / l. The content of raw protein is 19-21 g / l, Klason lignin is 37-40%. 2 sec. f-ly, 4 tab. S cl 4 with

Description

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This invention relates to the microbiological industry and concerns a new yeast strain — a source of protein biomass and a method for producing it using hydrolyzed lignin as a carbon source.

The aim of the invention is a strain of the yeast Trichosporon cutaneum, which makes it possible to obtain a higher biomass code with a higher content of protein in it during cultivation.

The invention in part of the Tricho sporon cutaneum strain is as follows.

The strain T. cutaneum was obtained on the strain T. cutaneum 70 as follows.

An aqueous suspension of a T.cutaneum 70 culture was seeded into Petri dishes with an agarized Czapek-Doks medium containing finely divided hydrolyzed lignin as a carbon source. Achieve separate colonies. After 4 days of cultivation, the largest colonies were used to inoculate a liquid nutrient medium with hydrolytic lignin. Deep cultivation was carried out in flasks on a shaker for 3 days, the lignobiomass was determined to keep Klason protein and lignin. Behind

From this deep culture, a passage was made into Petri dishes with agar medium and homogeneous colonies were selected, which were re-grown under deep conditions. After a 3-fold passage, a strain was accumulated accumulating up to 22% of crude protein and decomposing up to. 25% Klason lignin. The strain is stored on wort agar at 10-15 ° C. Reseeding was done once every 3 months.

When stored and browned on wort, the level of biosynthetic activity of the strain does not decrease.

Strain. T. cutaneum D-46 is deposited in the All-Union Collection of Industrial Microorganisms under the number BKPM U-495 and has the following characteristics.

Cultural and morphological features.

In a one-day malt wort culture, the cells are oval, elongated-oval, assembled in chains and single (sometimes irregularly shaped), measuring 2.4-6.4-4.2-2.1 μm.

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In a 10-day culture, a well-developed mycelium disintegrating into arthrospores forms on wort agar, a pseudo-small cell is found. Additional outgrowths often occur on the hyphae.

Wort - agar. The stroke is plentiful, dry, dull, eminent, wrinkled, covered with light fluff. For a 7-day culture, grayish colonies are characteristic, the white suede remains only in the center, the beginning of colony colonization is noticeable. The surface is lumpy, the base is translucent.

Malt wort agar. The stroke is ample, grayish-cream with a large folded surface, the profile is convex, the edge is festooned, the consistency is dense. For a 7-day culture, the colonies are greyish-cream, okrugly-round, convex, with a rising folded center. Land edge. The base of the colony is less pubescent, almost transparent.

Agar Chapek-Doksa with hydrolyzed lignin. Stroke whitish color, folded, slightly convex, the surface of the mat, velvety. For a 7-day culture, whitish-colored colonies are rounded in shape, dull, slightly convex, scalloped. At the top of the colonies is a bezel with a hollow.

Physiological and biochemical signs.

Attitude to carbohydrates. Sahlra is not. fermenting. It assimilates glucose, galactose, sucrose, fructose, lactose, raffinose, xylose, D-arabinose, rhamnose, and sorbose, trehalose, melibiosis, and melicytosis more weakly. Breaks down starch, cellulose.

Attitude to

cerin, dulcite,

alcohols.

Absorbs mannitol, sorbitol.

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ethanol.

Relation to acids. Absorbs lactic, succinic, citric, acetic, formic, fumaric, block, oxalic acid.

Relation to nitrogen sources. Uses ammonium nitrogen, nitrate, nitrogen asparagine, peptone, urea. Does not absorb nitrite and potassium nitrate.

Growth-promoting vitamins are tropamine and biotin. The culture grows well on hydrolyzed lignin, being the destructor of this substrate.

Relation to temperature. The optimum growth at 30-35 ° C, can withstand temperatures up to 40 C.

Attitude to oxygen. Aerobe.

Grows in a wide range of pH 3.5-8.3 with an optimum of 4.5-5.5.

Toxicological studies carried out at the Belarusian Scientific Research Institute of Veterinary Medicine showed the harmlessness of both the staff itself and the product obtained by deep cultivation of a culture on hydrolytic lignin.

The method for producing protein biomass is as follows.

The composition of the nutrient medium containing 7.5-12.5 g / l (by dry weight) of hydrolytic lignin, 1-2 g / l of ammonium phosphate disubstituted, as a water base is added up to 1 l of decantate of the yeast brew, which is hydrolyzed yeast production (Table 1)

From the data table. As can be seen in Figure 1, higher or lower concentrations of the components lead either to a sharp decrease in the yield of ligno-biomass, or to a deterioration of its qualitative composition due to a decrease in the content of crude protein and an increase in Klason lignin, which is equally economically unprofitable.

The post-fermented brew is the main component of the wastewater of the Bob-Rui hydrolysis plant and has the following composition: reducing substances 0.09-0.2%, organic acids 0.12-0.2% (in terms of hydrocarbons

hydrochloric acid), volatile acids 0,015-0,03% nitrogen 90-120 mg / l, P 0550-70 mg / l.

The need for decanting the brew before adding to the nutrient media is due to the presence of a residual amount of yeast biomass in it, distorting the true process of destruction of hydrolytic lignin by the T. cutaneum strain VKPM U-495. The decant of the mash contains all its basic soluble components and is an additional source of easily digestible carbon, nitrogen, phosphorus in the medium.

The protein product Biogil obtained after cultivation with CMPM U-495 on the proposed medium has the chemical composition given in table.

Compared with the initial substrate, Biogil is enriched with phosphorus, it contains 10–13 times more protein and

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There are only few less hydrolizable components — Klason lignin and cellulose. In this connection, the digestibility of J. lignobiomass is almost 4 times higher than the digestibility of hydrolytic lignin and reaches 40%. According to the content of nucleic acids (up to 1.2%), Biogil approaches the usual food products (mous, liver), and its use in feed rations does not create the danger of disturbing the purine metabolism in animals. The nutritional value of lignobiomass is increased due to the presence of easily digestible fat, which is dominated by non-fatty acids, including the indispensable ones - linoleic, linolenic and arachidonic, which has F-vitamin activity.

The fatty acid composition of the LIP1-yov product BIOGIL following 11% of the total:

40

77.60

3.6: 1 For comparison

Unsaturated

acids

Unsaturated /

/ saturated 5 Example 1

lignolytic (determined by the decrease of Klason lignin in lignobiomas.-sa) and protein-synthesizing active-; According to the proposed and known 0 strains, the cultivation of T.cutaneum VKPM U-495 is carried out in 300 ml Erlenmeyer flasks containing 100 ml of medium on a rocking chair (200 rpm)

within 48 hours at 30 ° C on a known nutrient medium of the following composition, g / l: crushed hydrolysis lignin 10 (by dry mass); , 1, MgS040,5, corn extract 4; tap water to 1-liter.

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The hydrolytic lignin is obtained., At the Bobruisk hydrolysis plant and has the following composition,%: extractives 1 water-soluble 3J easily-hydrolysable lignin Claso at 57; mineral substances 6.

Nutrient medium in flasks is boiled for 15 minutes, cooled and seeded with a 2-day culture of T.cutaneum VKGTM U-495 grown on beer wort (4 according to Balling) in flasks on a rocking chair (200 rpm) at 30 ° C. material 10ob.

Comparison of the main indicators of the cultivation of the proposed cultivar And known strains - Hydrolyzed lignin destructors are given in mpl. 3

The results obtained (lbl. 4) indicate that compared with the known strain - destructuring of hydrolytic lignin, the proposed producer is distinguished by a higher productivity of protein biomass synthesis and provides a more complete destruction of lignin with a shorter cultivation time. On Klason.

Example 2. In order to optimize and cheapen the nutrient medium for growing the strain T. cutaneum EKHGM U-495, crushed hydrolysis lignin 10 g / l (NH) J IRO, 1, 5 g / l is introduced into its composition as the main carbon source and decantate post-yeast brew up to 1 l, pH 4.5-5.5. Cultivation conditions, as in Example 1, the yield of lignobiomass is 14 g / l, the content of crude protein in it is 21%, and Klason lignin is 40%.

Example 3. Cultivation was carried out in semi-sterile conditions in a fermenter (useful volume 10 l) at 30 ° C, stirring speed 300 rpm and intensity of aeration 1 l / l "min.

The method of preparing the inoculum as in example 1. The nutrient is medium as in example 2.

In order to improve the processability of the process, preparation of the nutrient medium includes thorough mixing of all components, adjusting the pH and subsequent filtration of the suspension through a sieve with 1 mm hole diameter, which eliminates the pre-B stage: drying and grinding.

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no hydrolytic lignin. The amount of hydrolyzed lignin applied is adjusted for the initial moisture and losses by separating the coarse fraction during filtration. Then the medium is boiled for 15 minutes, cooled and seeded with a 2-day culture of T.cutaneiom VKGTM U-495. The amount of seed 25 vol.% Ensures the dominant development of the proposed strain over extraneous microflora in the initial period of semi-sterile fermentation.

In tab. Figure 4 shows the dynamics of growth and consumption of nutrients by the T. cutahexm V1SPM U-495 strain on a medium with hydrolyzed lignin and mash.

For 24-32 hours of cultivation, the yield of lignobiomass is. 13- 4.3 g / l, crude protein content 2f-23%, Klason lignin content is about 40%. The total decrease in the Klason lignin concentration in the culture fluid for 72 hours of the fermentation process is 26.3% compared to the initial medium.

Claims (2)

1. Yeast strain Trichosporon cutaneum VKPM U-495 - a source of protein biomass. 2. A method of obtaining protein biomass, which involves preparing a liquid nutrient medium containing hydrolytic lignin sources of nitrogen and phosphorus as a carbon source, an aqueous base, and growing a producing microorganism under optimal conditions to maximize the accumulation of protein biomass, characterized in that The purpose of increasing the biomass yield, as a water base, is used decantate of the post-yeast brew, which is a waste of hydrolysis-yeast production, while the components of the nutrient medium are next form a ratio, g / l: Hydrolytic lignin 7.5-12.5 Ammonium phosphate disubstituted I- 2 Decant of the post-yeast brew up to 1 liter, and Trichosporon cutaneuni VKPM U-495 is used as a yeast strain. Table I Optimization of nutrient medium for deep cultivation of the strain Trichosporon cutaneum VKPM U-495 Note: As a liquid phase in all variants, with the exception of the latter, a decanter of post-yeast brew is used. Hydrolytic lignin2.0-2.6 0.8-1.2 Viogil 21, 10.2-12.5 Initial PitaTable 2 4,5-5,0 Traces 57-5926,6-28,0 ) 5.5-6.0 1.5-2.0 36.4-39.7 12.6-14.1 .Table 3 Continuation of table 4