RU1817471C - Strain of yeast candida ufilis - a producer of protein biomass - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: strain of yeast Candida ufilis is obtained by selection. Productivity of strain is 4.36 g/l x hr, specific growth rate is 0.48 h^-1 at continuous cultivation at PH-state regime. At cultivation on ethanol-containing medium biomass accumulation is 10.2-13.2 g/l, biomass yield of ethanol dose is 71.8-77%, protein content is 58.1-63.9%. EFFECT: improved properties of strain. 11 tbl

Description

 The present invention relates to the microbiological industry and is a new strain of yeast that can be used to obtain feed yeast based on ethanol, wine materials, as well as hydrolysates of agricultural waste (wood, cotton husk, etc.).

The purpose of the invention is the selection of highly non-pathogenic new yeast strain, the systematic position belonging to Candida ufilis, competitive on international market and actively growing at a temperature of 32-34 ^{° C,} which is a scheduled for fodder yeast production technology, t. E. A strain having mesophilic properties, but characterized by a high yield of biomass and the high protein content in the final product when cultured at a temperature of 32-40 ^{° C} on a medium with ethanol and otlichayuschegos I have relatively large cell sizes, which helps to reduce losses in the allocation of biomass, as well as high competitiveness. The statement of this problem is due to modern toughened requirements aimed at ensuring environmentally friendly production, one of the guarantees for this is the use of mesophilic yeast strains, which are more stable in maintaining a sign of the absence of pathogenicity in the conditions of long non-sterile processes of continuous cultivation.

Truly thermotolerant strains having an optimum growth temperature elevated (37-42 ° ^C) have greater mutabelnosti that is more likely to lead to the formation of pathogenic yeast forms. In addition, conducting the cultivation process at elevated temperatures is accompanied by the development in the biocenosis of the concomitant bacterial microflora of the sanitary-hygienic group, which is not permissible according to existing sanitary standards and the technological regulations in force in factories.

The invention consists in the fact that the proposed strain was obtained during the selection process when testing about 100 yeast strains of the museum collection of the Institute of the All-Russian Research Institute of Synthesis of Proteins under the conditions of a continuous process of cultivation on an environment with ethanol in the mode of chemostat and pH-stat with the subsequent testing of the strain in model, chamber and pilot industrial conditions having the optimum growth temperature of 32-34 ^{° C,} but growth characterized by the ability in a wide range of temperatures (32-40 ^{° C)} while preserving the performance of the growth rate, the accumulation biome assy and raw protein content.

 The proposed strain can be used to obtain protein biomass not only based on ethanol, but also on the basis of wood hydrolysates and wine materials.

As a result of the studies, it was revealed that the growth activity of the strain VSB-726, i.e., the accumulation of biomass in model experiments (in rocking flasks) on a mineral medium with 1% ethanol, with the addition of growth factors of yeast autolysate and without it at a growth temperature of 32-40 ^about 18 hours after cultivation, was 6.0-6.3 g / l; for the prototype strain VSB-825 under the same conditions 4.8-3.5 g / l; for the known mesophilic strain of Candida ufilis VSB-651 and the thermo-tolerant strain of Candida krusei VSB-896, 4.1-5.6 and 3.6-1.9 g / l, respectively (see table 1).

 We draw attention to the fact that the new strain of VSB-726 does not need additional growth factors (yeast autolysate), while the growth of the strain of the prototype VSB-825 depends on the presence of additional growth factors in the medium. The latter is essential for the conditions of production, since it reduces the cost of the product due to lower costs of scarce reagents.

 Tests of strain VSB-726 in model experiments with a high ethanol content in the medium, i.e. in conditions of high population density. The latter is extremely important for industrial fermentation, since the cultivation process with a high concentration of biomass in the fermenter will allow for a significant increase in biomass removal per unit volume.

 According to the table. 2 the new strain VSB-726 is able to grow at a population density of 23-28.8 g / l (by dry weight of yeast), providing the highest biomass yield of 73.3-84.6% when processing 2.5-4.5% ethanol in environment. The strain VSB-651 under similar conditions (2.5-4.5% ethanol) had a population density of 12.9-26.4 g / l (i.e., 2.4-10 g / l less than the strain VSB-726 ) with a biomass yield of 66.2-76.2% (i.e., 6.9-8.4% less than the strain VSB-726).

 The strain prototype VSB-825 when processing 4.5-6.5% ethanol in the medium dramatically reduces growth activity to 17.7-8.3 g / l (i.e. by 11.1-17.9 g / l less strain VSB-726) with a biomass yield of 56.2-11.8% (i.e., 25.9-39.7% less strain VSB-726).

 Similar results were obtained when testing cultures in a chemostat mode at an ethanol concentration of 4 and 6% (see table 3).

Yeast strain VSB-726 tested in a cameral sterile process conditions of continuous cultivation in a pH stat mode, in comparison with the prototype and known mesophilic and thermotolerant yeast strains at the optimum temperature for growth of each strain 34 ± 0,5 39 ± 0,5 and ^C) and the concentration of ethanol in the mineral medium 10-12 g / l.

According to the results presented in Table 4, the new strain VSB-726 had the highest productivity (4.36 g / l) and biomass yield (56%) compared with strain VSB-825, for which these indicators were 2.93- 3.1 g / l h and 50-52%, respectively. Compared with thermo-tolerant strains, including the prototype, the new strain of VSB-726 was characterized by maintaining almost the same high specific growth rate (0.48 h ^-1 ) as the strains of VSB-825 (0.52 h ^-1 ) and VSB -896 (0.5 h ^-1 ). The known mesophilic strain VSB-651 had a lower specific growth rate (0.43 h ^-1 ).

Yeast strain VSB-726 tested in batch culture laboratory conditions at a temperature of 32-40 ° ^C. In this case identified (Table 4), that a new strain of VSB-726 actively growing over a wide temperature range (32-40 ° ^C), providing accumulation biomass at the level of 10.2-13.2 g / l, the biomass yield from a given ethanol of 71.8-77.0% with a crude protein content of biomass of 58.1-63.9%, the specific strain growth rate was 0.33- 0.38 h ^-1 . The mesophilic strain VSB-651 under these cultivation conditions had the following indicators: biomass accumulation of 1.5-9.1 g / l, biomass yield of 40-74% crude protein content 56-60% specific growth rate of 0.13-0.33 h ^-1 , i.e. even at the optimum growth temperature, the strain VSB-651 had worse performance compared to the strain VSB-726. With increasing temperature, the cultivation process to ^about 37-39 C strain VSB-651 dramatically reduces the parameters listed at the growth temperature of 40 ^C. FAB-651 strain substantially does not increase.

 The known thermotolerant strain C. krusei VSB-896 under these conditions of the cultivation process also had lower rates compared to the new strain VSB-726 (see table 5).

Comparative Test 3 times cameral biological replicates in sterile conditions and continuous culture in a chemostat mode process at a temperature of 32-34 ^{° C,} pH 4.2, a flow rate of 0.25 h ^-1, the concentration of ethanol was 2% As seen from table 6, the proposed strain VSB-726 provided a biomass yield of a given ethanol of an average of 73% with a protein content (crude) in biomass of 62.3%. The mesophilic strain VSB-651 under these cultivation conditions had a biomass yield of 70.3% crude protein content in biomass 58.3%
Strain VSB-726 was also tested on other carbon sources of wine materials and wood hydrolyzate.

According to the table. 7, a new strain of VSB-726 in model experiments in shake flasks at a growth temperature of 32-34 ^{° C} in a mineral medium with winematerials after 24 h and 36 h cultivation provided biomass accumulation 8,1-9,1 rel. units opt.pl. The mesophilic strain of VSB-651 under these conditions had 6.8-6.9 rel. units opt. pl. known thermotolerant strain VSB-896 6.1 and 6.6 rel. opt.pl.

VSB-726 strain was further tested in non-sterile laboratory conditions of continuous cultivation process in chemostat mode in the apparatus "Abiteks" at 34 ± 0,5 ^{° C,} the growth rate 0,2ch ^-1 on a mineral medium with winematerials, ethanol and mixtures of wine and ethanol, balanced in terms of absolute alcohol, amounting to 24.0%. It was shown that the highest biomass yield (83.5%) was achieved in the processes of cultivation on wine materials, the smallest (61.2%) on ethanol rectified. On a substrate representing a mixture of wine materials and ethanol, the biomass yield is 63-68.0% depending on the amount of ethanol added (75-90%) (see table 8).

Biomass accumulation new strain VSB-726 on wood hydrolyzate when tested in model experiments in shake flasks at ³⁷ ° C and the content of mineral substances redutsiruyushih medium (CM) was 1.45% 7.7 g / l; strain VSB-651 3.1 g / l (see table 9).

 The cultural-morphological and physiological-biochemical properties of the strain VSB-726 were studied. Morphological signs: cells in a one-day culture on a glucose-peptone medium are single, oval or elongated-oval and form twigs and short chains. The size of the cells in a one-day culture in liquid wort is 3.5-7.0 x 7.0-13 microns. Budding is polar. In industrial continuous cultivation conditions on a mineral medium with ethanol, the average cell size is 5.0-9.0 microns. It does not form ascospores. On corn agar, it forms an underdeveloped primitive pseudomycelium, consisting of branching chains, short pseudohyphae, and oval cells. In malt wort (3-30 days), the ring and the film does not form. The precipitate is a cloudy, creamy color. Cultural traits: colonies on malt wort agar (7 days) are large, slightly elevated with a smooth surface, even edge, grayish. The center of the colony is often raised in the form of a tubercle; colonies with a fine-wrinkled surface may appear. The line on the malt wort agar is smooth, grayish with time to cream, slightly shiny, and may be slightly folded. Physiological signs: assimilates glucose, sucrose, maltose, cellobiose, trehalose, raffinose, melicitosis, inulin, xylose, glycerin, mannitol, salicin, methyl-D-glucose (weakly), acetic, lactic, succinic and citric acids. Fermentation: glucose, sucrose, raffinose. Does not ferment: maltose, galactose, lactose, melecitosis, cellobiosis. It uses nitrate and ammonium salts as nitrogen (for example, sodium nitrate, ammonium sulfate, etc.). It grows well on standard synthetic nutrient media without the addition of growth factors (medium Reader, Miller, Saburo, etc.). As non-traditional sources of carbon and energy, it uses ethanol, water-alcohol condensate, wine materials, post-alcohol distillery stillage, acetic acid, agricultural waste hydrolysates, as well as metabolic products of hydrocarbon-oxidizing yeast.

The strain is stored in a lyophilized state or on wort agar under a layer of liquid paraffin at room temperature. The frequency of reseeding in the latter case is 3-4 times a year. Working cultures of yeast strain is maintained by inoculating in tubes (jambs) on wort agar, cultured at a temperature of 32-34 ^{° C} for two days, followed by storage at room temperature.

 Biochemical composition of the yeast biomass of strain VSB-726: crude protein content of 59-63% lipids 7.1-7.4% ash 8.7-9.2, nucleic acids 7.4-9.9% moisture 2.4-4 , 3. The amino acid composition of the true protein is presented in table 10.

The sum of saturated and unsaturated fatty acids in the composition of lipids is 18.9 and 72.2 rel. respectively (including C ₁₆ and C ₁₈ 18.9 and 72.2%).

EXAMPLE EXAMPLE 1 The yeast Candida ufilis VSB-726 was grown in intensive mass transfer apparatus 5 liter mineral medium without growth factors at a temperature of 32-23 ^{° C} and pH 4.0-4.3 in a continuous mode. Ethanol in an amount of 3 vol. Was used as a carbon source. the flow rate was 0.20 h ^-1 .

The composition of the nutrient medium, g per 1 l: Potassium chloride 0.7 Phosphoric acid (70%) 0.7 Ammonium sulfate 1.0 Magnesium sulfate 0.4 Iron sulfate 0.04 Zinc sulfate 0.02 Manganese sulfate 0.02
The yield of absolutely dry biomass from a given ethanol is 75%; the crude protein content in biomass is 62%; productivity is 3.51 g / l ^. h

PRI me R 2. Yeast Candida ufilis VSB-726 was grown in a apparatus with a capacity of 30 l under the conditions described in example 1. Ethanol was used as a carbon source in an amount of 2-2.5 vol. the flow rate was 0.33 h ^-1 ; pH 5.5 The yield of absolutely dry biomass from a given ethanol was 73%; the crude protein content in biomass 62%; productivity 3.76 g / l h.

PRI me R 3. Yeast Candida ufilis VSB-726 was grown under the conditions described in example 1, in the apparatus with a capacity of 5 l As a carbon source used wine materials in an amount of 3 vol. (in terms of ethanol). The flow rate was 0.3 h ^-1 . The biomass yield from a given ethanol was 75%; the crude protein content in biomass was 63%; the productivity was 5.27 g / l ^. h

PRI me R 4. The yeast Candida ufilis VSB-726 was grown in a column type apparatus with a capacity of 145 l on a mineral medium according to example 1 with a double salt concentration at a temperature of 37 ^about C and a pH of 4.2-4.5. Ethanol in an amount of 4 vol. Was used as a carbon source. The flow rate was 0.25 h ^-1 ; biomass yield from a given ethanol 74% crude protein content in biomass 62% productivity 5.77 g / l ^. h

PRI me R 5. Yeast Candida ufilis VSB-726 was grown under the conditions described in example 4. As a carbon source used wine materials in an amount of 6 vol. (in terms of ethanol). The flow rate was 0.35 h ^-1 , the biomass yield from the given ethanol 75%, the crude protein content in the biomass 61%
EXAMPLE Example 6 Yeast Candida ufilis VSB-726 were grown in flasks on a shaker in a mineral medium at a temperature of 34 ^{° C} and pH 5,5-4,2 medium for 30 hours. As the carbon source used was 2 vol . (in terms of ethanol) of water-alcohol condensate. The yield of dry biomass from a given substrate was 71%; the crude protein content in biomass was 60%
EXAMPLE Example 7 Yeast Candida ufilis VSB-726 were grown in flasks on a shaker (220 rev / min) in a mineral medium according to Example 1 at a temperature of 32 ^{° C,} pH 5.5, at high population densities achievable processing high concentration of ethanol (4.5 vol.) supplied fractionally (1.5 vol. at sowing and 1 vol. daily). The yield from the target ethanol was 82.1%
PRI me R 8. The yeast Candida ufilis VSB-726 was grown under sterile conditions of a continuous cultivation process in the mode of pH auxostat at a growth temperature of 34 ± 0.5 ^about C0 pH 4.0, the concentration of ethanol 10-12 g / L. Process parameters were supported automatically. The following growth indicators were obtained: the yield from the given and consumed ethanol was 56 and 78%, respectively, the specific growth rate was 0.48 h ^-1 , and the biomass productivity was 4.36 g / l ^. h, protein productivity 2.35 g / l ^. h

The VSB-825 prototype strain had the following characteristics: yield from given and consumed ethanol 52 and 90%, respectively, specific growth rate of 0.52 h ^-1 , biomass productivity of 3.1 g / l ^. h, protein productivity 1.64 g / l ^. h

Thus, the proposed new strain of yeast Candida ufilis VSB-726 has the following distinctive features from the well-known thermotolerant ethanolassimilating strain of yeast Candida valida VSB-825:
strain VSB-726 differs from strain VSB-825 in its ability to grow on a mixture of ethanol and wine materials (a different substrate), and in terms of ethanol, this nutrient medium is characterized by a high concentration;
strain VSB-726 differs from strain ABSB-825 in its ability to grow on a wood hydrolyzate; an alternative substrate for producing fodder yeast; the growth of strain VSB-825 on the wood hydrolyzate is practically absent;
the strain VSB-726 is more competitive in comparison with the strain VSB-825 in the temperature regime of cultivation of 32-38 ^о С, which was revealed in tests at the Bashkir BHK (see table 11);
characterized by a stable crude protein content of mainly 60-62% (strain prototype VSB-825 48-62%);
strain VSB-726 grows on a medium with a high concentration of ethanol (at 4.5% with a biomass yield of 82.1%), the prototype strain VSB-825 under the same conditions has a biomass yield of 56.2% i.e. 25.9% less;
strain VSB-726 has a large cell size (4.1-7.0 x 6.3-10.4 microns); the size of the cells of the strain VSB-825: 2.0-6.2 x 3.0-2.0 μm (this feature is of great industrial importance, since large-cell yeast is better separated, which significantly reduces the loss of biomass at the separation stage, leads to reduce the expenditure coefficient for the supplied substrate, i.e. economically more profitable);
strain VSB-726 differs from strain VSB-825 in a number of physiological and biochemical properties: strain VSB-726 assimilates a number of carbohydrates (maltose, cellobiose, etc.), potassium nitrate, ferments sucrose, raffinose, which can serve as a test sign for the development of microbiological control of production to assess the dominance of the strain in the biocenosis of industrial fermenters.

Claims (1)

 Candida ufilis yeast strain VKMP U-263-producer of protein biomass.

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