RU2092549C1 - Yeast strain endomycopsis fibuligera - a producer of the protein biomass - Google Patents

Abstract

FIELD: biotechnology, technical microbiology. SUBSTANCE: new industrial nonpathogenic yeast strain Endomycopsis fibuligera VSB-12 - a producer of protein biomass. The proposed strain is obtained by method of continuous culturing on agricultural waste in the presence of hydrocarbon additions. Yeast strain E. fibuligera VSB-12 is tested under chamber conditions in fermenter (volume is 10 l) where the following substrates were used: molasses (rye bran hydrolyzate), mixture of agriculture waste, hydrocarbons (n-alkanes) and pig manure flows. EFFECT: increased output, yield and crude protein. 1 tbl

Description

 The invention relates to microbiology and can be used in hydrolysis-yeast and food industries, as well as in agriculture and is a new industrial non-pathogenic strain of yeast Endomycopsis fibuligera VSB-12 producer of protein biomass, characterized by high productivity when cultured on a carbohydrate, hydrocarbon and a mixture of these substrates .

 The effectiveness of the biotechnological process is largely determined by the properties of the industrial producer strain.

 Known yeast strains used as producer cultures for the production of fodder biomass on a carbohydrate-containing substrate, in particular molasses (A.S. USSR NN 1620378; 1622389; 1643606; 1677057; 1595900; 1571061; 15232580; 1507788; Pat. GDR NN 124658 ; 275070). The disadvantage of these strains is that, according to a systematic position, they belong to imperfect yeast-like conditionally pathogenic microorganisms of the genus Condida. This reduces their consumer value.

 Known strains of an imperfect yeast-like fungus of the genus Trichosporon, which are also used as producer cultures for protein production (A.S. USSR NN 632201; 606518; 803472; 1482941; Pat. GDR N 2788354).

 The main disadvantage of these strains is the low specific growth rate, as well as morphological instability of the cultures.

 Strains related to perfect fungi are also known, for example, saccharomycetes used in the production of feed protein (A.S. USSR NN 1742320; 1677057; US Pat. France NN 2646435; 2646436, US Pat. No. 4,379,845). The disadvantage of these strains is their low specific growth rate, as well as instability in continuous cultivation.

The closest analogue of the proposed strain is a strain of the yeast Socchoromyces cerevisiae VKPM U-760 producer of protein and biologically active substances (AS USSR N 1575570 C 12 1/16). The strain S.arevisiae VKPM U-760 is grown under continuous cultivation conditions on carbohydrates (sucrose, or molasses, or hydrolyzate) at a concentration of 8 g / l (calculated on PB), and a biomass concentration of 3.9 g ACB is obtained in the culture medium / l The productivity of the process is 2.07-2.57 g ACB / m ³ hour. The disadvantage of the strain S. cerevisiae VKPM-U-760 is its ability to grow at low concentrations of the substrate in the medium, which does not allow to achieve high productivity of the process at a sufficiently high flow rate (2.05 kg / kg).

 Currently, the urgent problem is to obtain protein biomass from renewable raw materials of plant origin, such as molasses and agricultural waste, such as manure, bran, etc. with their simultaneous disposal.

In this regard, the purpose of the invention was the selection of non-pathogenic mycelial strain of yeast, which is actively growing on molasses, agricultural waste and agricultural waste with the addition of hydrocarbons (C ₈ C ₂₂ ). The addition of hydrocarbons is used to enrich the substrate with carbon, which will increase the productivity of the process.

 The technical result consists in increasing productivity, increasing the biomass yield from the substrate, increasing the protein content, as well as in economic feasibility, since along with the production of protein biomass, the problem of recycling agricultural waste is solved.

 The proposed new yeast strain was obtained by continuous cultivation on agricultural waste with the addition of hydrocarbons in laboratory conditions at the Laboratory of Industrial Biosynthesis Technologies, State Research Institute of Synthesis Protein. To do this, agricultural waste containing 0.5-1.0% hydrocarbons was introduced into the Reader medium and grown in flasks on a rocking chair. After five passages into a new nutrient medium, the culture fluid was dispersed into Petri dishes with wort agar and isolated colonies (isolators) were selected, which were then individually tested (17 isolates) for growth activity in flasks on a rocking chair in a liquid mineral nutrient medium, where Agricultural waste (RW 2.0%) and hydrocarbons (0.5%) were used as a substrate. The activity of isolates was determined by the concentration of biomass, which ranged from 3.0-11.0 g / L. Of these, 3 of the most active yeast isolates with growth activity from 9-11 g / l were selected.

The selected 3 yeast isolates were studied under continuous cultivation under aerobic conditions with intensive mass transfer on a mineral medium Reader with agricultural waste (PB 10%) and hydrocarbons (1.5%) at a flow rate of 0.2-0.3 hour ^-1 , which allowed to obtain the desired strain, indicated in the museum's collection of the institute under the number VSB-12.

 Determination of the systematic position of the selected strain VSB-12, carried out in accordance with the determinant of the yeast Kreger-Van-Rai (The yeast taxonomye sfady. Ed.by Kreger-van-Riy. N.Y.W. Amsferdam, 1984) showed its belonging to Endomycopsis fibuligera.

 Determining the degree of pathogenicity of the selected strain of Endomycopsis fibuligera VSB-12 confirmed the absence of pathogenicity.

 Characterization of the strain of yeast Endomycopsis fibuligera VSB-12.

 Cultural and morphological characters. Colonies with a size of 4-10 mm round, with a flat edge, form a convex profile with a fleecy surface, light brown in color, form a homogeneous structure, a dense consistency. Stroke on malt wort agar (3-day-old culture) is fleecy with a slightly wavy edge, light brown in color.

Three-day culture cells grown on a liquid nutrient medium: molasses substrate, agricultural waste substrate, agricultural waste substrate + hydrocarbons at pH 4.5-5.5, temperature 30-40 ^o C, single, oval, short-oval, round, have septic mycelium. Ascospores are oval. Rings and films do not form on a liquid nutrient medium; the sediment is dense and light.

Physiological and biochemical characteristics. The strain Endomycopsis fibuligera VSB-12 assimilates carbohydrate components, glucose, soluble starch, arabinose, ferments glucose, maltose. Non-vitamin dependent. Uses organic and inorganic nitrogen. It grows at pH 3.5-6.5 (optimum 4.5-5.5) and a temperature of 20-40 ^o C (optimum 30-34 ^o C). The effect of stimulants on strain growth has not been studied.

The strains are stored on jambs in vitro on wort agar at a temperature of 0 to +10 ^o C or in a leophilized-dried state.

The strain Endomycopsis fibuligera VSB-12 was tested in laboratory conditions in a 10 L fermenter in a Reader mineral medium, in which molasses, agricultural waste, a mixture of agricultural waste and hydrocarbons served as a carbon source, the pH of the medium was 4.5-5.5, the temperature was 30- 34 ^o C.

 Inoculum biomass was prepared by growing a one-two-day yeast culture in rocking flasks with Reader mineral medium, in which 5% molasses served as a carbon source.

 For comparative evaluation, studies were performed with yeast strains: Saccharomyces arevisiae (prototype) and Candida afilis BSC 726.

The cultivation was carried out in laboratory conditions in a 10 L fermenter with intensive mass transfer (stirrer revolutions 800 rpm, air flow 1 rpm rpm), medium pH 4.5-5.5, temperature 30-34 ^o C, flow rate 0 25 h ^-1 .

 The obtained research results are presented in table. 1. The invention is illustrated by examples of implementation.

Example 1. The strain of yeast Endomycopsis fibuligera VSB-12 was cultivated continuously in a fermenter with a volume of 10 l on a mineral medium of the following composition (g / l): (H ₄ ) ₂ O ₄ 3.0; MoO ₄ 0.7; aCl - 0.5; Ca (O ₃ ) ₂ 0.4; KH ₂ PO ₄ 1.0; K ₂ HPO ₄ 0.1. the pH of the medium is 4.5-5.5, the temperature is 30-34 ^o C, the molasses substrate, the solids content is 83% sucrose 51-52% The flow rate was 0.25 h ^-1 . At a molasses concentration to the duct of 5%, the following results were obtained: biomass concentration of 29.6 g / l ACB; yield from substrate 59.1%; productivity 7.4 kg / m ³ h; crude protein content 58%. Under the same conditions, continuous cultivation of Saccharomyces cerevisiae yeast was carried out (prototype) and the following results were obtained: biomass concentration of 24.0 g / l ACB; yield from the substrate 48.0% productivity 6.0 kg / m ³ h, crude protein content 52%. The data obtained show that a high concentration of the substrate inhibits the culture of S. cerevisiae yeast, which affects the biomass yield from the substrate and the productivity of the process. The results of the cultivation of yeast Candida ufilis VSB-726 are presented in table. 1. The results are close to those of the new strain Endomycopsis fibuligera VSB 12, however, it should be noted that the strain C. ufilis is conditionally pathogenic.

Example 2. The strain of yeast Endomycopsis fibuligera VSB-12 was grown under the same conditions as in example 1, but the substrate was agricultural waste hydrolyzed rye bran with a content of 1.1% PB. The following indicators were obtained: biomass concentration of 13.2 g / l ASB ; yield from substrate 120.2%; productivity 3.3 kg / m ³ h; crude protein content 54.2%. Under the same conditions, S. cerevisiae and C.ufilis yeast were grown. The results are presented in table. 1. Higher technological parameters of E.fibuligera yeast are due to the amylase activity of this strain and, as a result, the deep utilization of the substrate.

Example 3. The strain of yeast Endomycopsis fibuligera VSB-12 was grown under the same conditions as in example 1, but the substrate was a mixture of agricultural waste (rye bran) and hydrocarbons (n-alkanes) in concentrations of 0.8% RV and 0.9% GW respectively. The results are obtained: the biomass concentration of 18.7 g of ASB / l; output from the substrate 110% productivity 4.7 kg / m ³ h; crude protein content of 59%. Under the same conditions, S. cerevisiae yeast was cultured (prototype). The results are obtained (table. 1): biomass concentration of 8.9 g ASB / l; yield from substrate 56.3% productivity 2.25% kg / m ³ ; crude protein content of 54% As can be seen from the data presented, the culture of S. cerevisiae yeast practically does not utilize hydrocarbons. The results of growing yeast Candida ufilis are presented in table. one.

Example 4. The strain of yeast Endomycopsis fibuligera VSB-12 was grown in a fermenter with a volume of 10 l, stirrer revolutions 900 rpm air flow 1 rpm. min, pH 4.5-5.0, temperature 30-32 ^o C. Pig-breeding manure runoff with a solids concentration of 2% served as a substrate; monosubstituted sodium phosphate in the amount of 0.5% served as a mineral feed Two-day seed culture in an amount of 10% of the volume of the medium. The following results were obtained: a decrease in BOD of 88% nitrogen 90%. The crude protein content in the finished product was 47.9%. Yeast cultures: Saccharomyces cerevisiae (prototype) and Candida ufilis VSB-726 did not grow under similar conditions on this substrate.

Thus, on the basis of the studies it can be concluded that the proposed strain Endomycopsis fibuligera VSB-12 is a new carbohydrate-hydrocarbon assimilating strain, characterized by the following distinctive features from the prototype Saccharomyces cerevisiae VKPM-U-760:
actively grows on a nutrient medium with a high concentration of molasses, providing a productivity of more than 7 kg / m ³ h and a yield of absolutely dry biomass from the substrate of more than 59%
actively growing on a nutrient medium on a mixture of substrates carbohydrates (agricultural waste) and hydrocarbons (n-alkanes), providing a process productivity of 4.7 kg / m ³ h, 110% yield from the substrate and 59% crude protein content
actively grows on a nutrient medium with agricultural waste, providing an exit from a substrate (RV) of more than 120;
actively growing on pig manure runoff, providing a BOD reduction of 88% nitrogen by 90% when obtaining protein biomass with a crude protein content of 47.9%
competitive, providing high dominance in the biocenosis of fermenters in a non-sterile cultivation process.

 Based on the data of desk and experimental studies, the yeast strain Endomycopsis fibuligera VSB-12 is recommended for industrial introduction in the production of protein biomass on agricultural waste.

Claims (1)

 The yeast strain Endomycopsis fibuligera VKPM Y-2173 producer of protein biomass.

Images