SU1602868A1 - Method of producing pectolytic enzymic preparation - Google Patents

Abstract

The invention relates to biotechnology and relates to a method for producing a pectolytic enzyme preparation, which is used in extracting juice from fruit and vegetable gruel and in cleaning fruit and vegetable juices. The purpose of the invention is to increase the activity of the target product and its heat resistance. The method of preparing the pectolytic preparation consists in using the mutant strain ASPERGILLUS NIGER - 8МХ-41, registered in DIKLS under N8, for cultivation in the fermentation nutrient medium, after which it is concentrated from the fermentation liquid and dried at a temperature of 30-38 ° C. The advantages of the method are higher thermal stability of the preparation (50 ° C optimum effect), three times higher biosynthetic activity with respect to polygalacturonase and five times higher biosynthesis of pectinoesterase, which reflects positively on the economic efficiency of the production process, and high cleansing activity of the preparation that determines its low consumption and reduced depectinization time.

Description

The invention relates to biotechnology and relates to a method for producing a pectolytic enzyme preparation with a mutant} line of Aspergillus niger, which is used in extracting juice from fruit and vegetable porridge and in clarifying fruit and vegetable juices.

A known method for the production of a pectolytic preparation by means of submerged cultivation of a strain of the species Aspergillus niger Van Tieghera 8M-68 DIKLS on an aqueous nutrient medium containing dry beetroot

pulp, wheat bran, ammonium sulphate, dibasic potassium phosphate and magnesium sulphate at optimum temperature and aeration, providing culture liquids with an activity of 1500 U / cm.

The disadvantages of the method are the unsatisfactory level of pectolytic activity of culture liquids and the insufficiently high temperature optimum, 45 C, which negatively affects the economic results of production and use.

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The purpose of the invention is to increase the activity of the target product and its heat resistance.

The invention begins to ferment with Aspergil-1US niger-8MX-41 culture, registered in No. 8, obtained after natural selection and exposure to A. niger-8M conidia with ultraviolet rays, followed by multiple screening based on the enzyme activity realized during laboratory fermentations. A producer has the following characteristics.

Cultural and morphological features.

On the malt agar, Aspergillus ni-ger-8MX-41, at 72 o'clock, forms round colonies with a smooth edge, a fallen center and thick radial folds with a yellow-brown central part, with d 1.7 cm and a white periphery of width 1, 2 cm. By the 7th day the diameter of the colony reaches 9 cm. The color is black. The center continues to be sunken, and the radial folds are more prominent. Culture does not release pigment in the medium. The reverse side of the colony of pale cream ..

On potato agar, by the 72nd hour, Aspergillus niger-8MX-41 forms round colonies with a light yellowish central part with d 0.5 cm, followed by a second with d 2.2 cm, with black pigment and a peripheral white band pshriny 3 mm. By the 7th day, the colony diameter reaches 7 cm. The light center remains, followed by a circumferential black zone 2.5 cm wide and to the very end is a white strip 0.5 cm wide.

On agar of čapek-doksa Asp. The niger-8MX-41 forms, by the 72nd hour, round colonies with d 1.1 cm, having a white center and a lightly pigmented surface in a light cream color. By the 7th colony, the diameter of the colony reaches 3-4 cm with a white center, followed by a ring with d 0.7 yellow-green color, behind which there is a 2 centimeter zone: with black pigment and white peripheral zone width

2 .--- 3 MMo:

Culture forms conidiophores with a length of 250–500 (U and 2–3,5 W thick, terminating in spherical crabs with d - HW - 200 W and spherical conidies with d 1.5 - 2.2fU,

five

0

painted in dark brown to black color and covered with thorns 0,2 - 0,5Shch.

On solid nutrient Asp. The niger-8MX-41 decorates colonies with well-defined aerial mycelium and a weak substrate mycelium constructed from septate Hythaeus.

The culture is maintained on a canopy, non-doped, agarized wort extract with 6% dry matter.

Differences Aspergillus niger-8MH-41 from the used production culture Asp. niger-8M is expressed in significantly higher biosynthetic enzyme activity. By the 144th hour of its development in an environment with an optimized composition, the culture of 81-Ж-41 accumulates in a ml of fermentation liquid 4000 ± 300 E polygalacto- rhose activity and 20 i 4 E pectin esterase activity, and production - 1500 ± respectively. 20 U / ml PHA and 4 +0.5 U / ml PEA. .

After conducting a multifactor experiment 2 and the following mathematical modeling, it was established that the fermentation should be carried out in an environment having the following composition,%:

Dry beet pulp4,5. - 5.5

Wheaten bran 0.75 - 1.5 Ammonium sulfate 0.35 - 0.75 Sodium phosphate

disubstituted 0.125 - 0.250 Starch, 0.5 -1.5 Magnesium sulfate 0.025 - 0.05 Potassium chloride 0.025 - 0.05 Water Others

After the separation of the mycelium, the fermentation liquid is filtered to eliminate mechanical impurities, free from low molecular weight soluble impurities by ultrafiltration, the volume is reduced by 7-9 times, and the dry matter increases from 1.2 - 1.5 to. 1.75 - 2%. It should be 8-fold vacuum concentration at a temperature below 36 ° C and lilophilic or vacuum drying at a temperature. Not exceeding 38 C.

The resulting enzyme preparation has a higher thermal stability (50 ° C optimum action), a threefold higher biosynthetic activity with respect to polyalignonuronase and five times more advanced

synthesis of pectinesterase, which reflects positively on the economic efficiency of the production process and the high clarification activity of the preparation, which determines its small consumption and reduced time of depectinization.

Example. To 45 liters of fermentation medium sterilized and cooled to 27 ° C, having the composition,%: dry beet pulp 4.5; wheat bran 0.75; ammonium sulfate 0.35; sodium phosphate disubstituted; 0.125; starch 0.5; magnesium sulfate 0.025; potassium chloride 0.025; add 1.5 l 24-: hour inoculum from Asp. niger-8MX- -41 No. 8 DIKLS, prepared in the medium, has a composition identical to 2, |) up to 6.0, with activity optimally occurring at pH 4.5. The optimum temperature is at pH 4.5 - 5. The drug retains 90% of its activity at 50 ° C for 60 minutes.

Formula of invention

ten

A method for producing a pectolytic enzyme preparation, which involves the submerged cultivation of Aspergillus niger product on an aqueous nutrient medium containing dry beet pulp, wheat bran, ammonium sulfate, disubstituted phosphoric acid salt, and magnesium sulfate at the optimum temperature for bio- xi-ivtvii iii / rj 1 c; iMiit:; | jci 1 JlJln.

fermentation medium, on a circular 20 enzyme synthesis and aeration to maxi-eccentric agitator from 220 rpm at 27.5 + 0.5 ° C.

Fermentation in the fermentor proceeds at 27.5 + 0.5 C, aeration with sterile air at a flow rate of 0.8 - 1.1 l / l of the medium per 1 minute, with a peripheral speed of the stirrer 2.4 - 3.2 m / s, for 144+ ± 8 h. At the end of the process, 4000 cm + 300 U of polygalacturonase and 20 ± 4 U of pectinesterase activity accumulate in 1 cm of fermentation liquid. After elimination of the micelle, insoluble components of the medium and fine filtration or separation, 35 - 37 l of fermentation G) are obtained, which is subjected to membrane ultrafiltration, reducing the volume by about 8 times, 8-fold vacuum concentration at a temperature below 38 ° C and vacuum or lyophilized at temperatures below 38 ° C. 300 to 350 g of the preparation with an activity of 400,000 U / g of PHA and 2000 U / g of PEA are obtained. After standardization with perlite or diatomite, about 2 kg of the preparation with 60000 U / g of PGA and 300 U / g of PEA are obtained. ..

The resulting p-tolytic agent acts in wide pH ranges.

25

thirty

maximum accumulation of activity, separation of the mycelium, concentration of cultured ultrafiltration and vacuum concentration and drying, characterized in that, in order to increase the activity of the target product and its heat resistance, Aspergillus niger strain DIXEL fc 8 is cultivated in an aqueous medium, additionally containing starch, potassium chloride, and phosphate bidisubstituted sodium as a disubstituted phosphoric acid salt in the following ratio, wt.%:

Beetroot dry

bagasse

Wheat bran

Starch

Ammonium sulfate

Phosphate

disubstituted

sodium

Magnesium sulfate

Potassium chloride

Water

40

4.5 0.75 0.5 0.35

-5,5

-1.5

-1.5

-0.75

45

0.125 - 0.250 0.25 - 0.05 0.025 - 0.05 Else

wherein the evacuation and drying is carried out at 30 -.

1602868

from 2, |) to 6.0, with the activity optimally occurring at pH 4.5. The optimum temperature is at pH 4.5 - 5. The drug retains 90% of its activity at 50 ° C for 60 minutes.

Formula of invention

A method for producing a pectolytic enzyme preparation, which involves deep cultivation of the producer Aspergillus niger on an aqueous nutrient medium containing dry. beet pulp, wheat bran, ammonium sulphate, the disubstituted phosphoric acid salt and magnesium sulphate, at the optimum temperature for the bio- "i-ivtvii iii / rj 1 c; iMiit:; | jci 1 JlJln.

enzyme synthesis and aeration to maxi25

0

maximum accumulation of activity, separation of the mycelium, concentration of cultured ultrafiltration and vacuum concentration and drying, characterized in that, in order to increase the activity of the target product and its heat resistance, Aspergillus niger strain DIXEL fc 8 is cultivated in an aqueous medium, additionally containing starch, potassium chloride, and phosphate bidisubstituted sodium as a disubstituted phosphoric acid salt in the following ratio, wt.%:

Beetroot dry

bagasse

Wheat bran

Starch

Ammonium sulfate

Phosphate

disubstituted

sodium

Magnesium sulfate

Potassium chloride

Water

0

4.5 0.75 0.5 0.35

-5,5

-1.5

-1.5

-0.75

five

0.125 - 0.250 0.25 - 0.05 0.025 - 0.05 Else

wherein the evacuation and drying is carried out at 30 -.

Claims (1)

The claims 10 A method of obtaining a pectolytic enzyme preparation, providing deep cultivation of the producer Aspergillus niger in aqueous 15 nutrient medium containing dry beet pulp, wheat bran, ammonium sulfate, a disubstituted salt of phosphoric acid and magnesium sulfate, at the optimum temperature for bio- 20 synthesis of the enzyme and aeration to the maximum accumulation of activity, separation of mycelium, concentration of the culture fluid by ultrafiltration and vacuum concentration and drying, 25 characterized in that, in order to • increase the activity of the target product and its heat resistance, Aspergillus niger DIXL No. 8 strain is used as a producer, cultivation is carried out in an aqueous medium additionally containing starch, potassium chloride and sodium phosphate disubstituted as double-chipped chips salts of phosphoric acid in the following ratio of components, wt.%: Dry beetroot bagasse 4,5 - 5.5 Wheat bran 0.75 - 1.5 Starch 0.5 - 1.5 Ammonium sulfate 0.35 - 0.75 Phosphate bicameral sodium 0.125 - 0.250 Magnesium sulfate 0.25 - 0.05 Potassium chloride 0,025 - 0.05 Water Rest moreover, evacuation and drying are carried out at 30 - 38 ° C.