SU1030409A1 - Method for recovering complex of protolytic enzymes from sea products - Google Patents

Abstract

METHOD Mark COMPLEX PROTEOYAITICHESKIH ENZYMES FROM Seafood by homogenization sf and deposition of enzymes, characterized in that, in order rasshnreni resource base and increase the yield of desired product, as a raw material Using T pyloric appendages flounder, and deposition of enzymes are atsetonsm at a ratio homogenate acetone 1:

Description

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The invention relates to a technology for the production of proteolytic enzymes and can be used in the enzymatic, chemical and food processing, in particular, to accelerate the maturation processes of saline production from fish and the production of conservators.

Currently, enzyme preparations are widely used in various industries of the industry. O l guide |) olysis of protein substrates from raw materials of animal and vegetable origin.,

 The increased needs of the national economy in enzyme preparations 15 necessitate the search for new sources of raw materials for their production and the development of effective methods for the production of enzymes.

Known methods for the preparation of enzyme preparations mainly from raw materials of animal origin LI}

Such methods, based on the treatment of crushed raw materials from animal organs, the isolation of enzymes 25 by sedimentation and multistage purification of the enzyme fraction, are economically disadvantageous due to the low yield of enzyme preparations from expensive raw materials.

Cheaper methods for obtaining proteolytic enzymes from microbiological raw materials are known, based on growing highly productive strains of microorganisms and isolating enzymes from them by precipitating proteins, fractioning them, and purifying the enzyme fractions {2J.

However, the complexes obtained by this method are suitable only for deeper hydrolysis of proteins (to amino acids), and not to low molecular weight peptides; which limits their use in obtaining fish products.

The closest technical essence to the proposed method is the method of isolating a complex of proteolytic enzymes from seafood, including the homogenization of Tikhrokee-Squid hepatopancreas; hp and krill of New Zealand in aqueous solution at a ratio of fabric; water equal to (1: 1) - (1: 1.3); Purification of the obtained liquid phase from impurities by introducing ammonium sulfate to saturation 0.8; separation of the precipitate by trifugation at 8000 rpm; the dissolution of Ermedov in distilled water; dialysis and lyophilization of the target NW product:

The disadvantage of method 60 is that the yield of the desired product is not high enough. In the process of isolation, some of the enzymes are lost and the qualitative composition of the resulting enzyme complex is deteriorating. Cro 65

Ie Trgo, the need to harvest raw materials far into the sea and its long-term transportation causes a loss of enzyme activity.

The aim of the invention is the expansion of the raw material base, an increase in the yield of enzymes that are included in the complex and which have a wide spectrum of action. .

The goal is achieved by the method of isolating a complex of proteolytic enzymes from seafood by means of homogenizing raw materials and precipitating enzymes, using pyloric appendages of flounder as raw materials, and enzymes are precipitated with acetone at a ratio of homogenate: acetone 1: (1-2.3). .

The use of seafood waste - pyloric appendages of flounder as a raw material for carrying out the proposed method is economically advantageous, since it is available in sufficient quantity in connection with the plans of the USSR fishing industry and the work of fish factories in the country.

It has been found that the complex to be isolated is a valuable set of enzymes, in particular, alkaline, neutral and acidic proteases, aminopeptidases and carboxypeptidases.

The combination of these conditions makes it possible to obtain from waste seafood using a simple technology an enzyme complex that has a valuable set of enzymes that retain their native activity in high yield.

For example 1. as a source of raw materials used pyloric. appendages of the plaice of the species HyjSpoglosoides alassodon. 1 kg of the appendages isolated from fresh flounder is hemogenised on a micro-minimizer tissue. WG-2 (MRTU-42-1505-bZ) at a speed of 5000 rpm at 2 ° C until a homogeneous mass is obtained.

Two volumes (2 liters) of acetone, cooled to, are added to 1 kg of the cooled material with rapid stirring. . .

After 15 min, the resulting material is squeezed and dried thoroughly, removing traces of acetone.

From 1 kg of pyloric appendages flounder receive 200 g of the drug. The output caseinolytic activity - 60%.

The specific activity expressed in units / mg of protein is: 514 (the substrate BTME-benzoyl-L-tyrosine methyl ester); 222 (substrate BJAE-benzoyl-L-arginine ethyl ester); 0.76 (casein substrate).

When dried, the proteolytic activity of the enzyme preparation is fully preserved. Example 2. As a raw material, pyloric appendages of the Cleisthenes herzenstein type flounder are used. 1 kg of the pyloric appendages of the flatfish are homogenized at 6000 rpm until a homogeneous mass is obtained. . To 1 kg of the cooled homogenate, add 2.3 liters of acetone cooled to -20 ° C with stirring. After 20 minutes, the precipitate is squeezed and then dried at 25 ° C, removing traces of acetone. The resulting enzyme complex contains alkaline, neutral and acidic proteinase aminopeptidases, carboxypeptidases. From 1 kg of pyloric appendages of flounder 210 g of the drug. The yield of caseinolytic activity is 66%. Froze The raw material used is the pyloric appendages of the clan Bulma of the species Limanda herzensteini. . 1 kg of pyloric appendages of the flounder are homogenized at 5,500 rpm, until a homogeneous mass is obtained. To 1 kg of the cooled homogenate was added 2.0 L of acetone cooled to -15 ° C with stirring. After 15 min, the precipitate is wrung out and dried at 25 ° C. The resulting enzyme complex contains alkaline, neutral, and proteinases, aminopepe, car-, boxypeptidases. From 1 kg of pyloric appendages flounder receive 205 g of the drug. The yield of caseinolytic activity is 65%. Comparative tests of the activity of the enzyme preparation obtained from the pyloric appendages of the flounder, proposed and known methods. The yield of the drug on the total caseinolytic activity is proved in the table.

From pyloric appendages of flounder (on proposed sposb 200 15200: 12600 From hepatopancreas squid and krill (according to 1120, 1575 to a known method) 28 The obtained results indicate that from 1 kg of raw material (pyloric appendages of flounder) it is possible to obtain a proteolytic complex from which the enzyme activity is an order of magnitude higher (proteolytic - pH 7.8; 6.0; 3.0), several orders higher (ester-like: chymotrypsin- and trypsin-like; peptidase) as compared to the lime method of producing a complex of proteolytic enzymes. Set the x of proteolytic enzymes from the pyloric appendages of the cambium contains more trypsin- and chymotrypsin-like proteinases (substrates - casein - pH 7.8 / 0.76 protein); BAEE-M-benzoyl-C6-arginine-ethyl ester (22 U / mg protein); BTME-flbenzoyl-oC-tyrosine methyl ester (514 units / mg protein); neutral proteases (pH 6.0), which may include trypsin and chymotrip 705010794000 4662000 588000 378000 107243176 23320 8400 traces of syn (substrate hemoglobin - 0.60 u / mg protein); acid proteases - substrate hemoglobin (pH 3.0) - 0.35 u / mg protein; neopeptidases - substrate leucyl-hl, isyl-glycine (23 units / mg of protein); carboxypeptidae - substrate-N-Kapobenzoxy-glycine-leucine (18 u / mg protein). The proposed method is simple, it allows using a small number of stages to obtain a proteolytic complex, possessing a valuable set of enzymes, which is especially effective when used in practice. 200 g of the preparation (20.6 g of protein) containing 152000 units were obtained from 1 kg of raw material. total caseinolytic activity, and by a known method, the activity is 1120 units. In addition, chymotrypsin- and trypsin-like activity is higher than

51030409

with a known method of producing com- due to the use of acetone

a complex of proteolytic enzymes. more complete removal is achieved

A fe {1entny preparation with sushikelids, thereby improving the product does not practically lose the caseinolytic form of the enzyme preparation, scrape activity, and the specific fish smell.

Claims (1)

METHOD FOR ISOLATING A COMPLEX OF PROTEOLYTIC ENZYMES FROM SEAFOOD by homogenizing raw materials and precipitating enzymes, characterized in that, in order to expand the raw material base and increase the yield of the target product, pyloric flounders are used as raw materials, and the precipitation of enzymes is done with acetone! ” with a homogenate-acetone Is ratio (2-2.3). СО с с 3> 1 1030409