Strain of micromycete aspergillus oryzae-producer of proteolytic and amylolytic ferments to be used in food industry

FIELD: biotechnologies.

SUBSTANCE: strain Aspergillus oryzae RCAM01135 is a producer of proteolytic and amylolytic ferments, which has ability to produce proteolytic and amylolytic ferments. It was deposited at GNU VNIISKHM with the following registration number: RCAM01135. It can be used at production of different food products (fermented spices, additives, and drinks).

EFFECT: invention allows increasing the growth speed and intensity of spore formation.

4 tbl, 2 ex

 

The invention relates to biotechnology, in particular the production of strain - producer of proteolytic and amylolytic enzymes, and can be used for hydrolysis of vegetable and animal substances in food industry, fermentation industry, agriculture.

It is known that the filamentous fungus Aspergillus oryzae has wide application in the food industry, as they do not have pathogenic properties. Biosynthesis of proteases and amylases, characteristic for representatives of Aspergillus oryzae, used, for example, in the manufacture of certain food products (fermented seasonings, fermented drinks)when you want the hydrolysis of proteins and carbohydrates (EN 2361914, 2009, EN 2187947, 2002).

A known strain of Aspergillus oryzae VKPM F-369, with the ability to produce complex proteases exhibiting maximum activity in the acidic and weakly acidic zone pH, α-amylase. However, the main disadvantage of this strain is the lack of synthesis of related hydrolases, as well as the long period of growth and development of micromycete; strain intensively produces α-amylase and protease in liquid media only on the fourth - fifth day (SU 1440922, 1988).

A known strain of Aspergillus oryzae VKPM F-683, producing complex acidic and weakly acidic protease, α-amylase and related xylanase, glucanase and cites. It is known that deep cultivated and this strain produces a complex of highly active enzymes. The resulting enzyme complexes used in the food industry.

However, the application of strain is limited due to a lack of high growth rate and specific requirements for nutrient environments, as it is growing on media with solids content of not more than 18% (EN 2070921, 1996).

The task of the invention consists in obtaining a strain of Aspergillus oryzae producing protease and α-amylase with high growth rate, mainly for solid-phase cultivation.

As the invention proposes a new strain of micromycete Aspergillus oryzae 37-53, obtained by the method of multi-stage selection and mutagenesis deposited in the GSI research Institute for agricultural Microbiology in the Departmental collection of useful microorganisms for agricultural purposes RAAS (RCAM) under registration number RCAM01135. The strain is a producer of protease and α-amylase, has a high growth rate, mainly for solid-phase cultivation and intensive sporoobrazovanie.

The technical result of the invention is a higher growth rate of the proposed strain of Aspergillus oryzae RCAM01135.

In particular, in environments with soybean meal highs proteoliticheskoi and amylolytic activities occur on the third day of cultivation, and the intensity of sporoobrazovanie was the most high is also some on the third day. This allows to consider the claimed strain as a promising tool for use in the food industry for processing of agricultural raw materials, such as soybean, with the aim of obtaining various food flavoring additives, fermentation production, the baking industry.

A strain of Aspergillus oryzae RCAM01135 characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural morphological traits:

(a) the nature of growth (pubescence, the edge of the colony). Colony fluffy, rounded, smooth edge, white mycelium, spore-forming, colony color from olive to dark green. Vegetative mycelium septate, well-branched with large swellings, the thickness of the hyphae 6-12 µm; conidia are formed exogenously, the surface of conidia smooth, rounded form, the diameter of the spores 5-6 μm; forms a conidial heads with single-stage sterigmate;

b) the size of the colony (at a certain time of incubation). 3 days of growth on wort-agar the colony diameter 50×51 mm;

b) aerial mycelium (availability, color, features of sporulation). Plentiful sporification dark green color. On the edge of the colony white fluffy mycelium 3 mm;

g) the color of the substrate mycelium. White;

d) the color of the reverse side of the colony. Light brown;

e) other signs (pigment, the exudate is so). The pigments are not, exudate absent;

g) changing the culture as we grow older. The color of the colony darkens with age, becoming brownish-green;

C) behavior in other environments. On Chapek's medium with glucose: 3 day growth colony color - green, surface smooth; the diameter of the colony 32×34 mm

Studies of the morphological characteristics of the culture identification of strain in accordance with the determinant of filamentous fungi (Bilai VI, Koval AS Aspergilli. The determinant. - Kiev: Nauk. Dumka, 1988, - 204 C.).

Physiological and biochemical characteristics

Relation to oxygen - aerobe. Optimum growth temperature is 28-30°C. the Maximum temperature is 50°C. the Minimum temperature is 18°C. the Optimum pH value for growth of the fungus is 5.5. Fungus growth is observed in the pH range from 2.5 to 10.0.

As a source of carbon to the fungus uses starch, glucose, sucrose, xylose, maltose, mannitol, glycerol and lactose. Assimilates nitrate, ammonium and amine nitrogen, proteins. The strain is not pathogenic. In the mode of deep and solid state cultivation was established ability to synthesis of enzymes, amylolytic and proteolytic action.

The strain is stored on agar malt wort 8% CB; pH environment - natural; storage temperature +20-25°C.

Example 1

Deep and solid phase kultivirovala strain Aspergillus ryzae RCAM01135

Deep cultivation of the strain Aspergillus oryzae 37-53 was performed on various complex natural environments tailored to the physiological needs of the producer. When designing nutrient media took into account the fact that for the synthesis of protease and α-amylase favorable starch, proteins, therefore, the main components of the environment was grain raw materials. As a source of phosphate nutrition for micromycete applied potassium phosphate. Thus, for submerged cultivation of a strain of Aspergillus oryzae RCAM01135 were used nutrient medium of the following composition, %:

1. Barley flour - 3,0, wheat bran - 3.0, starch - 2,0, KH2PO4- 1,5;

2. Wheat flour is 6.0, bran - 2,0, KH2PO4- 1,5%;

3. Soy flour - 6,0, barley flour - 5,0, KH2PO4- 1,5;

4. Soy flour is 6.0, bran - 2,0, KH2PO4- 1,5;

Cultivation of a strain of Aspergillus oryzae RCAM01135 was carried out in Erlenmeyer flasks with a volume of 750 cm3containing 50 cm3sterile nutrient medium, the pie rocking chair with a speed of 220 rpm at 30°C. the results Obtained are shown in table 1.

Table 1
Deep cultivated the e fungus Asp.oryzae RCAM01135 natural nutrient media
No. environmentDayGrowth (density, color QOL)pHRSW, %Farms, the act of the terrain, units/cm3
PSAU
11++beige porridge4,744,0--
2+khaki (yellowish) porridge4,82,81,50,64
3+khaki (yellow) liquidare 5.362,21,80,82
21++++beige porridge5,093,0--
2++khaki (yellowish) porridge5,2 2,01,30,93
3++khaki light liquid5,62,21,51,10
31+++++very thick beige porridge5,695,0--
2dark beige porridge7,65,00,050,35
3++++khaki dark porridge8,46of 5.40,030,56
41+++++thick beige porridge7,105,0--
2dark beige porridge8,33a 4.9 0,060,35
3+++dark khaki porridge8,73of 5.40,020,12

As can be seen from table 1, the selected strain of Aspergillus oryzae RCAM01135 on nutrient media containing barley or wheat flour and wheat bran (environments 1 and 2), showed the ability to synthesis of protease and α-amylase: the total proteolytic activity on the 3rd day of cultivation amounted to 1.5 to 1.8 units PS/cm3, amylolytic 0,82-1,10 unit ° C/cm3. On Wednesday, which consisted soeva flour (medium 3 and 4), the level of activity of synthesized enzymes were decreased in 2-3 times.

Solid-phase cultivation of a strain of Aspergillus oryzae RCAM01135 carried out on wheat bran with humidity 52,4% and soybean meal with humidity to 51.8% at 30°C in stationary conditions. The results are presented in table 2.

Table 2
Surface cultivation of the fungus A.oryzae RCAM01135 in various environments
The medium for cultivation
of
The characteristic growth of the fungus, the day Enzymatic activity, units/g
247PSAU
BranWhite mycelium, the beginning of sporoobrazovanie, dispute color greenAbundant sporulation, the spores dust, color green with olive tintUnchanged43,59,0
Soybean meal12341,0to 12.0
White myceliumAbundant sporulation, dispute color green with olive tintAbundant sporulation, the spores olive green

The results of in-depth and solid-phase cultivation confirm biosynthetic ability of a selected strain of Aspergillus oryzae RCAM01135 against proteolytic and amylolytic enzymes, which is characteristic of fungi of the genus Aspergillus oryzae species.

Thus, the strain grows well on liquid and solid who's nutrient media, synthesizes enzymes proteolytic and amylolytic activities and identified as the filamentous fungus Aspergillus oryzae.

Example 2

Cultivation of a strain of Aspergillus oryzae RCAM01135 in environments with soybean meal,

The accumulation of the experimental samples selected culture of Aspergillus oryzae RCAM01135 carried out on medium containing soybean meal and soybean meal with the addition of 10% barley with different humidity for 3 days at 30°C (table 3).

Table 3
Biochemical characteristics of experimental samples of the fungus A.oryzae RCAM01135
The medium for cultivation
of
Humidity surface. culture %Biochemical indicators of fungus
Protein by Kjeldahl method, % ASVProtein by Lowry, mg/g ASVnh2+
mg%/g ASV
Soybean meal58,743,035,0317,0
With the new meal +10% barley 51,341,052,8309,7
62,444,358,3322,5

Experimental samples of the fungus A.oryzae RCAM01135 were obtained as a result of rapid growth of micromycete at surface cultivation on soyabean meal, rich in protein, amino nitrogen and can be used for further processing in the production of fermentation products soybeans.

Selected strain of Aspergillus oryzae RCAM01135 and accumulated experimental samples of the fungus A.oryzae RCAM01135 were investigated in a Test lab techno-chemical control and arbitration methods of analysis of the GSI VNIIBT Rosselhozakademii.

The results of the tests for the identification of lags showed that in pure culture micromycete Aspergillus oryzae RCAM01135 transgenic sequence no (test report dated 23 March 2012).

In the analysis of heavy metals in the experimental samples of the fungus A. oryzae RCAM01135 it was found that their content does not exceed maximum permissible concentrations (minutes dated April 18, 2012). Table 4 shows the results of the analysis on the content of heavy metals in the substrate - soybean meal and superficial culture of the fungus A.oryzae RCAM01135. Ana is ulichnye results were obtained in all studied experimental samples.

Table 4
The content of heavy metals in surface culture of the fungus A.oryzae RCAM01135
The analyzed objectThe content of heavy metals, mg/kg
Lead PbArsenic AsCadmium CdMercury Hg
Soybean meal0,00520,00340,00270,0009
(MPC for soybean)1(1SanPiN 2.3.2.1078-01 "Hygienic requirements for safety and nutrition value of food products")0,50,30,10,02
Superficial fungal culture A.oryzae 37-530,00650,00470,00290,0014

Strain micromycete Aspergillus oryzae deposited in the GSI ARRIAM (Departmental collection of useful microorganisms for agricultural purposes - RCAM) under registration the first room RCAM01135, producing proteolytic and amylolytic enzymes for use in the food industry.



 

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