RU2077573C1 - Strain of yeast hansenula species - a producer of food biomass - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: invention relates to new industrial nonpathogenic strain of yeast Hansenula species Y-2133 that is tested in fermenter (working volume is 20 l) on mineral medium Rider where carbon source is molasses (concentrations - 3.75 and 5.0%), medium pH = 4.5-7.0, growth temperature - 30-37 C. Yield of dry biomass Hansenula species Y-2133 under conditions of periodic culturing is 72-73% of molasses level, consumption coefficient is 1.36-1.4, crude protein content in biomass is 54-56%. At accumulation stage in periodic culture yield is 96.0%, consumption coefficient is 1.04. At continuous culturing process productivity of the strain H. species Y-2133 is 6.2 g/l/h (dilution coefficient D = 0.2 h^-1) and 7.1 g/l/h (D = 0.25 h^-1). Strain H. species Y-2133 is tested in model experiments on Rider medium with 2% glucose and furfurol (0.02-0.5 vol.%). Strain can be grown on nutrient medium with 0.02-0.04 vol.% furfurol. EFFECT: enhanced productivity of new yeast strain. 2 tbl

Description

 The present invention relates to the field of microbiological, hydrolysis, yeast and food industry and is a new industrial non-pathogenic strain of Hansenula Species VSB-943 yeast producer of fodder biomass, characterized by high productivity when cultivated on molasses and wood hydrolyzate.

 The intensification of the process of production of fodder yeast and improving the quality of the finished product is largely determined by the properties of the industrial producer strain.

 Known yeast strains used as producer cultures for the production of feed biomass on a sugar-containing substrate, in particular molasses (ed. Certificate of the USSR N 1620478 C 12 NN 1/16, C 12 R 1/72; N 1622389; N 1643606; N 1677057; N 1595900; N 1571061; N 1532580; N 1463755; N 1411335; N 1419149 Particleboard; N 1427820 Particleboard; N 1507788 Particleboard; GDR patent N 124658 C 12 N 1/16; GDR patent N 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 Candida. This reduces their consumer ability. In addition, the use of yeast of the genus Candida as a producer culture requires the complication of production technology by the obligatory introduction of the heat treatment stage of the resulting feed biomass to ensure the absence of living cells in the finished product.

 Known strains of an imperfect yeast-like fungus of the genus Trichosporon, which are also used as producer cultures for producing fodder biomass (ed. Certificate of the USSR N 1482941; N 803472 DSP; N 632201 DSP; N 606518 DSP; patent GDR N 2788354). The disadvantages of these strains are low growth rate, low technology (poor separation), as well as morphological instability of cultures, which consists in dimorphism of cells, which reduces the productivity of the culture and the quality of the finished product.

 From a biological point of view, yeast strains are more promising, according to the systematic position, they belong to perfect fungi. For example, strains of saccharomycetes known for the production of fodder biomass are known (French patent N 2646435; French patent N 2646436; ed. Certificate of the USSR N 1677057; N 1742320; N 1575570 chipboard; US patent N 4379845). The disadvantage of these strains is the lack of ability to actively grow in a continuous cultivation process.

 The closest analogue of the proposed strain is a strain of yeast Saccharomyces cerevisiae VKPM U-760 source of protein and biologically active substances. (Auth. St. USSR N 1575570 C 12 N 1/16). The strain S. cerevisiae VKPM U-760 is grown in a continuous process in the pH-stat mode on a medium with sucrose, or molasses, or hydrolyzate at a concentration of 8 g / l (calculated as sugar), and a biomass concentration of 3.92 is obtained 3.95 g ASB / l, productivity 2.07 2.58 g ASB / l • hour (determined by the gravimetric method). The disadvantage of the strain S. cerevisiae VKPM U-760 is the ability to grow at a low concentration of the substrate in the medium, which is non-technological and reduces the economic efficiency of the cultivation process. Another disadvantage of the strain is its low productivity and high expenditure coefficient (about 2), which reduces the performance of the strain. The disadvantages of the strain, based on its systematic position, include the ability to ferment various sugars (glucose, galactose, sucrose, maltose and raffinose), which can lead to bloating when feeding animals.

 In connection with the stated objective of the present invention was the selection of an active non-pathogenic strain of yeast, according to the systematic position related to higher fungi and characterized by production properties: high productivity, manufacturability and favorable biological properties (lack of fermentability) with growth on molasses and wood hydrolysates.

 The proposed new strain of yeast obtained in the process of industrial selection from the number of isolates isolated from the biocenosis of industrial fermenters of the Volga hydrolysis plant. For this, the yeast suspension selected from the fermenters was scattered in Petri dishes with wort agar and isolated colonies (isolates) were selected, which were then tested for growth activity in flasks on a rocking chair, using furfural-free wood hydrolyzate as a nutrient substrate containing 1 reducing substance 1 , 5% More than 100 isolates were investigated, the growth activity of which was 1.5 to 7.5 g / l. Of these, 8 of the most active isolates with a growth activity of 6.0 7.5 g / L were selected. The selected 8 yeast isolates were tested under continuous cultivation in an intensive mass transfer apparatus on a Reader synthetic medium with 2% glucose and on a wood hydrolyzate with a content of reducing substances of 1.8 2.0%, which made it possible to obtain the desired strain, indicated in the museum's collection under the number VSB-943.

 Determination of the systematic position of the selected strain of VSB-943, carried out in accordance with the yeast determinant Kreger-van-Rai (The Yeast. A taxonomyc etudy. Ed. By Kreger-van-Rij. N.I. W. Amsterdam 1984), showed its belonging to Hansenula species.

 Determining the degree of pathogenicity of the selected strain of Hansenula species VSB-943 confirmed the absence of pathogenicity.

 Characterization of the strain of yeast Hansenula sp. VSB-943.

 Morphological signs: the cells on the malt wort of the three-day culture are oval, short-oval, round, single and with small branches and chains. Ascospores are hat-shaped.

 Cultural traits: colonies on malt wort agar are off-white in color, convex with a smooth and slightly wavy edge, smooth, slightly shiny. The line on the malt wort agar (30-day culture) is smooth with a smooth or slightly wavy edge, matte, grayish-beige. On liquid malt wort rings and films does not form; the precipitate is dense, light.

 Physiological and biochemical characteristics: assimilates glucose, sucrose, maltose, cellobiose, trehalose, melecitosis, xylose, ribose, rhamnose, glycerin, dulcite, ribite, mannitol, sorbitol, α methyl-D-glucoside, salicin, erythritol, weak and lemon ) acid. Does not assimilate galactose, sorbose, lactose, melibiosis, raffinose, soluble starch, L- and D-arabinose, inositol, succinic acid. Sugar does not ferment. It does not need vitamins and other additional growth factors.

 The strain Hansenula species VSB-943 was deposited in the All-Union Collection of Industrial Microorganisms of the All-Union Scientific Research Institute of Genetics and Selection of Microorganisms under the number U-2133.

The strain Hansenula species VSB-943 was tested in laboratory conditions in a fermenter with a volume of 40 l (working volume 20 l) on a Reder mineral medium, in which molasses from the Ufa baking yeast plant at a high substrate concentration of 3.75 and 5.0 served as a carbon source % pH of the medium was 5.0 5.9, growth temperature 34 and 37 ^o C.

Inoculum biomass was prepared by growing a one-two-day-old yeast culture in rocking flasks with Reader mineral medium, in which 2% glucose or sucrose or 5% molasses served as a carbon source (growth temperature 32 34 ^° , pH 5.5).

 For comparative evaluation, studies were also conducted with the following yeast strains: Saccharomyces cerevisiae (prototype), Candida utilis VSB-726, C. valida VSB-825, and C. tropicalis VSB-936. The last three strains are used as producer cultures at the Bashkir BHC in the production of paprina and apriina.

According to the data obtained (table 1) during periodic cultivation of the strain of Hansenula sp. VSB-943 had a yield of biomass (absolutely dry biomass) from molasses 72 73%, a consumption coefficient of 1.36 1.4, a crude protein content of 54 56.0. The strain of S. cerevisiae prototype under the same conditions had the following indicators: AFM yield 49.0%, consumption coefficient 2.04, crude protein content 45.4%
For other studied strains, the biomass yield was 57.6 66.8%; consumption coefficient 1.4 1.8; crude protein content 50.5 52.7%
Hansenula sp. VSB-943, which showed the best results in office conditions, was tested in an industrial fermenter of the pure culture department of the Bashkir Biochemical Plant. At the same time, at the accumulation stage in a periodic culture, a yield of 96.0% was achieved with a expenditure coefficient of 1.04. In the continuous mode of the cultivation process, the productivity of the strain Hansenula sp. VSB-943 amounted to 6.2 and 7.1 g / l / hour (table 2).

 Additional strain studies were performed to study growth activity on hydrolysis media.

It is known that hydrolysis media are characterized by a complex chemical composition, including, in addition to carbohydrates (glucose, mannose, galactose, fructose, xylose and arabinose), various volatile neutral substances and various volatile and non-volatile organic acids, the combination of which determines the quality of the nutrient hydrolysis medium and the productivity of the culture producer. Among the volatile neutral substances in the hydrolyzate there are substances such as furfural, methyl furfur, oxymethyl furfural, formaldehyde, which have a strong inhibitory effect. Strain Hansenula sp. VSB-943 was tested in model acute experiments on the effect of furfural on the biomass accumulation activity. For this, the culture was grown in flasks on a rocking chair at a temperature of 30 34 ^o C on mineral medium Reader with autolysate of 0.5% vol. and 2% glucose when the content of furfural in the amount of 0.02 to 0.5% (vol.). The control was yeast growth on the same medium without furfural. The seed material was yeast suspension of a one-day culture on wort agar at an initial optical density of 0.5 rel. units The duration of the growing process was 24 hours. As a result of the studies, the possibility of the growth of a strain of Hansenula sp. VSB-943 on a nutrient medium with 0.02 0.04% vol. furfural: biomass accumulation on a medium with 0.02% vol. furfural amounted to 6.9 7.2 rel. units opt. pl. at a concentration of furfural 0.04% vol. 3.6 4.0, in the control 7.9 8.2.

 Information confirming the possibility of carrying out the invention.

Example 1
The strain of yeast Hansenula species VSB-943 was cultivated in batch culture in a laboratory fermenter with a volume of 40 l (working volume 20 l) on a mineral medium of the following composition (g / l): (NH ₂ ) ₂ SO ₄ - 3, MgSO ₄ 0.7, NaCl 0.5, Ca (NO ₃ ) ₂ 0.4, KH ₂ PO ₄ 1.0, K ₂ HPO ₄ 0.1. the pH of the medium is 5.0 5.9, the growth temperature is 37 ^o C. Molasses of the Ufa baking yeast plant with the following characteristics served as a substrate: specific gravity 1.34, pH 6.4, solids content 82% sucrose content 51 52% SO ₃ - 0.06% nitrogen 1.6 1.7, K ₂ O 3 5% The microbial number of molasses was 10 ⁴ cells / ml. Benign quality of molasses 62%. Molasses concentration in the medium 3.75%. Inoculum biomass was prepared by growing a one-day culture in rocking flasks with the above mineral medium and 2% glucose at a growth temperature of 37 ^o C and a pH of 5.5.

1,36, время накопления 9 час, содержание сырого протеина в биомассе 54,3% доминирование штамма в биоценозе 80%
Штамм S.cerevisiae Сар.-12 (прототип) в тех же условиях при оптимальной температуре роста 30^oC и содержании мелассы в среде 5% имел следующие показатели: максимальное накопление биомассы 24,5 г/л, выход от мелассы 49,0% выход от сахарозы 95,0% расходный коэффициент 2,04, время выращивания 8 час, содержание сырого протеина 45,4% доминирование штамма в биоценозе 70%
Пример 2
Штамм Hansenula species ВСБ-943 выращивали по примеру 1, но при температуре роста 34^oC и концентрации мелассы в среде 5,0%
Получены следующие показатели: максимальное накопление биомассы 36,2 г/л, выход АСМ от мелассы 72,3% выход АСМ от сахарозы 141,8% расходный коэффициент 1,4, время выращивания 9,5% содержание сырого протеина 56,0% доминирование штамма в биоценозе 90%
Показатели штамма S. cerevisiae Сар.-12 (прототип) приведены в примере 1.The following indicators were obtained: maximum biomass accumulation (AFM) 29.0 g / l, biomass yield (AFM) from molasses 73.3% biomass yield (AFM) from sucrose 143.9% expenditure coefficient

1.36, accumulation time 9 hours, crude protein content in biomass 54.3% strain dominance in biocenosis 80%
The strain S. cerevisiae Sar.-12 (prototype) under the same conditions at an optimum growth temperature of 30 ^o C and a molasses content of 5% in the medium had the following indicators: maximum biomass accumulation of 24.5 g / l, molasses yield 49.0% yield from sucrose 95.0% consumption coefficient 2.04, growing time 8 hours, crude protein content 45.4%, the dominance of the strain in the biocenosis 70%
Example 2
The strain Hansenula species VSB-943 was grown according to example 1, but at a growth temperature of 34 ^o C and a molasses concentration in the medium of 5.0%
The following indicators were obtained: maximum biomass accumulation 36.2 g / l, AFM yield from molasses 72.3% AFM yield from sucrose 141.8% expenditure coefficient 1.4, growing time 9.5% crude protein content 56.0% dominance strain in the biocenosis of 90%
The indicators of the strain S. cerevisiae Car.-12 (prototype) are shown in example 1.

Example 3
Strain Hansenula sp. VSB-943 was grown in batch culture in an industrial fermenter V 200 l (working volume 100 l) of the pure culture department of the Bashkir BHK. Mineral nutrition was a solution of ammophos and potassium containing nitrogen 11.0 12.5 g / l, phosphorus 41.2 46.9 g / l P ₂ O ₅ , potassium 11 20 g / l, and a solution of trace elements with an iron content of 4 , 0 g / l, zinc 0.9 1.1 g / l, manganese 0.1 0.13 g / l, magnesium 3.0 3.3 g / l. Maintaining the pH level of 7.0 was provided with ammonia water with a content of 24.5 to 26.1% ammonia. Growth temperature 35 37 ^o C.

 The following indicators were obtained: maximum biomass accumulation (AFM) 48 g / l, AFM yield from molasses 96.0% AFM yield from sucrose 149.0% expenditure coefficient 1.04.

 The strain S. cerevisiae Sr.-12 (prototype) in the same cultivation conditions does not grow, alkalization of the nutrient medium occurs.

Example 4
The strain of yeast Hansenula species VSB-943 was grown according to example 3, but under the conditions of a continuous cultivation process (18 hours) with a dilution factor of the medium D of 0.2 h ^-1 (flow rate 20 l / h). pH 5.8 7.0, growth temperature 35 37 ^o C.

 The following indicators were obtained: the biomass concentration (AFM) was 31.2 g / l, the AFM yield from molasses 62.4%, the AFM yield from sucrose was 122.4%, the consumption coefficient was 1.6, and the productivity was 6.24 g / l / hour.

 The growth characteristic of the strain S. cerevisiae Car.-12 (prototype) is the same as in example 3.

Example 5
Hansenula sp. VSB-943 was grown according to example 3, but under the conditions of a continuous cultivation process (46 hours) with a dilution factor D of 0.25 hour ^-1 (flow rate 25 l / hour). the pH of the medium is 5.0 - 6.9, a growth temperature of 32 35 ^o C.

 The following indicators were obtained: biomass concentration (AFM) 28.4 g / l, AFM yield from molasses 56.8% AFM yield from sucrose 111.3% expenditure coefficient 1.7, productivity 7.1 g / l / hour.

 The growth characteristic of the strain S. cerevisiae Car.-12 (prototype) is the same as in example 3.

Thus, on the basis of the conducted studies, we can conclude that the proposed strain of Hansenula species VSB-943 is a new carbohydrate-assimilating strain, characterized by the following distinctive features from the prototype S. cerevisiae Sr.-12:
actively grows on a nutrient medium with molasses, ensuring the yield of absolutely dry biomass from a given substrate 72 96%
actively growing on wood hydrolyzate with furfural content 0.02 - 0.04%
actively growing in a wide range of pH 5.0 7.0 and a growth temperature of 30 37 ^o C;
actively growing under the conditions of a continuous cultivation process, providing productivity at an optimal growth rate (D 0.2 0.25 hour _-1 ) at the level of 6.2 7.1 g / l / hour;
it is not able to ferment sugar, which is important when feeding animals with fodder yeast (does not cause bloating) on the one hand and, on the other hand, simplifies the production of fodder yeast using the proposed strain, as it does not require a guaranteed absence of producer cells in the finished product;
competitive, providing high dominance in the biocenosis of fermenters in the conditions of non-sterile cultivation process;
It is stable in its morphological-cultural and physiological properties, which is important for an industry characterized by possible fluctuations in the composition of the nutrient medium and physico-chemical growth parameters.

 Based on the data from the studies, the Hansenula species strain VSB-943 is recommended for industrial introduction as a producer culture at the Bashkir Biochemical Plant for the production of fodder yeast in molasses.

Claims (1)

1 Hansenula species yeast strain VKPM U-2133 producer of fodder biomass.

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