RU2233324C1 - Method for preparing lipoxygenase enzyme preparation - Google Patents

Abstract

FIELD: biotechnology, technical biochemistry, microbiology.

SUBSTANCE: invention can be used in baking industry. Method for preparing the lipoxygenase enzyme preparation involves submerged culturing the strain-producer of lipoxygenase Rhizopus oryzae VKM F-605 in nutrient medium of the following composition: soybean oil cake, sunflower oil, corn extract, ammonium dihydrophosphate, sodium hydrophosphate and water followed by isolation of the enzyme preparation. Invention provides synthesis of enzyme preparation showing high lipoxygenase and low carotene oxidase activity, and improved qualitative indices of bread.

EFFECT: improved preparing method.

2 tbl, 5 ex

Description

 The invention relates to biotechnology and can be used in the baking industry.

The closest in technical essence and the achieved effect is a method of producing an enzyme preparation of lipoxygenase by deep cultivation of its producers - fungi of the genus Aspergillus: A. oryzae 3-9-15 or A. awamori 466 on a nutrient medium containing soy flour and mineral as a carbon source salts of nitrogen, phosphorus, potassium and magnesium; and soybean oil as an inductor. The activity of lipoxygenase, determined by the carotene oxidase method, is 20,000-50000 conv. units per 1 cm of culture fluid (USSR AS No. 88542, MKI C 12 N 9/02 / Method for the preparation of the enzyme preparation of lipoxygenase / PP Tokareva, E. A. Dvadtsatova, M. V. Solovyova, V. L. Yarovenko, V.L. Kretovich, E.V. Budnitskaya; No. 2949321 / 28-13; claimed 30.05.80; publ. 15.08.83, Bull. No. 30).

The disadvantage of this method is the high carotene oxidase activity of the enzyme preparation, in which the biologically active substances of the flour — carotenoids — are destroyed.

An object of the invention is to obtain an enzyme preparation of lipoxygenase with high activity of formation of hydroperoxides and low carotene oxidase activity.

The technical problem is achieved by the fact that in the method for producing an enzyme preparation of lipoxygenase, including preparation of a nutrient medium, deep cultivation of a producer strain, it is new that Rhizopus oryzae 605 is used as a producer strain of the enzyme preparation of lipoxygenase, and they are introduced into the preparation of a nutrient medium , wt.%: soybean meal 2.0-2.5 and sunflower oil 0.7-0.9 as organic nutrients, corn extract 0.8-1.0 as a source of growth factors, mineral salts - sodium hydrogen phosphate 0 , 4-0.6, ammonium dihydrogen phosphate 0.8-1.2, the initial pH of the medium is 7.0.

The technical result of the invention is expressed in the synthesis of an enzyme preparation with high lipoxygenase and low carotene oxidase activity, which allows to obtain high quality bakery products.

The method is implemented as follows.

The producer strain Rhizopus oryzae 605 was obtained from the All-Russian Collection of Microorganisms of the Russian Academy of Sciences, where it is stored under the number VKM F-605.

Pure culture is grown on an agarized wort with a solids content of 4% for 3-4 days at a temperature of (30 ± 1) ° С.

The preparation of the nutrient medium is as follows. Ammonium dihydrogen phosphate mineral salts are dissolved in water in an amount of 0.8-1.2 wt.% And sodium hydrogen phosphate 0.4-0.6 wt.%; add, wt.%: corn extract 0.8-1.0, soybean meal 2.0-2.5, sunflower oil 0.7-0.9. The initial pH of the medium is adjusted, if necessary, to 7.0. Then the medium is sterilized for 1 h at 0.1 MPa, and then cooled.

Thus prepared nutrient medium containing soybean meal, sunflower oil, corn extract, ammonium dihydrogen phosphate, sodium hydrogen phosphate and water are inoculated with spores of the producer strain Rhizopus oryzae 605 and grown at a temperature of 30-32 ° С and aeration for 72 hours.

After growing the producer strain, the culture fluid is separated from the biomass by filtration. The enzyme preparation of lyoxygenase is obtained by precipitation with isopropyl alcohol (its concentration is 62 vol.%) At minus 2 - minus 4 ° C. The precipitate of the enzyme preparation is dried under vacuum.

In the present invention, the enzyme preparation of lipoxygenase is evaluated by lipoxygenase and carotene oxidase activity (Oganesyan N.A., Borisova I.G., Solomatin D.A., Budnitskaya E.V. Isozyme composition and activity of LOs of some wheat varieties of the species Triticum aestivum // Dokl. . USSR Academy of Sciences. 1983. T.269. No. 4. S.1002-1005).

The method for determining lipoxygenase activity is based on measuring the amount of formed conjugated hydroperoxides of fatty acids recorded at a wavelength of 234 nm on a spectrophotometer and are the final products of substrate oxidation. The composition of the reaction mixture: 0.2-0.3 cm ³ of a substrate solution, 0.5 cm ^{3 of} an enzyme solution, 5 cm ^{3 of} 0.05 M phosphate buffer. The duration of the reaction is 1-2 minutes, it stops when diluting 1 cm ^{3 of the} reaction mixture in 5 cm ^{3 of} 60% ethyl alcohol. To prepare a control sample, ethyl alcohol was added to 1 cm ^{3 of the} reaction mixture after 5-10 s from the start of the reaction.

Linoleic acid was used as a substrate.

For a unit of lipoxygenase activity, the change in extinction was taken to be 0.001 per 1 min, referred to 1 g of the drug.

A method for determining the conjugated oxidation of β-carotene in the presence of linoleic acid, based on measuring the optical density of the reaction mixture containing β-carotene, at a wavelength of 440 nm, records the degree of destruction of carotene by intermediate products of fatty acid oxidation with the participation of lipoxygenase. To obtain a solution of β-carotene to 40 mg add 150 cm ^{3 a} mixture of ethanol and acetone, taken in a ratio of 1: 1. The reaction mixture consists of 4 ml of 0.05 M phosphate buffer, 0.2-0.3 cm ³ of carotene solution, 0.2-0.3 cm ³ of substrate solution (Oganesyan N.A., Borisova I.G., Solomatin D.A., Budnitskaya E.V. Isoenzyme composition and LO activity of some wheat varieties of the Triticum aestivum species // Dokl. Academy of Sciences of the USSR. 1983. T. 269. No. 4. S.1002-1005), 0.2-0, 5 cm ³ of the enzyme solution. The reaction is carried out for 60 s; then added to the reaction medium 1.0 cm ³ 1 M NaOH solution. In the control, alkali is introduced into the reaction medium before the introduction of the enzyme.

The unit of carotene oxidase activity was the change in optical density by 0.001 in 1 min.

The peculiarity of the producer strain and the composition of the nutrient medium for its cultivation provide the biosynthesis of lipoxygenase with high lipogenesis and low carotene oxidase activity.

The method is illustrated by examples.

Example 1. Prepare a nutrient medium of the following composition, wt.%:

Soybean Cake 1.8

Sunflower oil 0.5

Corn extract 0.6

NH ₄ H ₂ PO ₄ 0.6

Na ₂ HPO ₄ 0.2

Water Else

For inoculation of the medium, an aqueous-spore suspension of the producer strain Rhizopus oryzae 605 is prepared. For this, the bevel of a 4-day-old culture in 18 × 180 mm test tubes is filled with 10 cm ^{3 of} sterile water and the spores are washed off. In Erlenmeyer flasks with a capacity of 750 cm ³ contribute 100 cm ³ nutrient medium, which is inoculated with a spore suspension in an amount of 1% by volume of medium. Producer cultivation is carried out on a rocking chair at a speed of 1.7-1.8 s ^-1 , temperature (30 ± 2) ° C for 72 hours. Then, the mycelium and suspended particles are separated from the culture fluid by filtration.

The enzyme preparation is obtained by precipitation with isopropanol with a concentration of 62 vol.% At a temperature of minus 2 minus 4 ° C. The drug is dried under vacuum.

The activity of lipoxygenase is 285700 units / g, carotenoxidase - 19800 units / g.

Example 2. Prepare a nutrient medium of the following composition, wt.%:

Soybean meal 2.0

Sunflower oil 0,7

Corn Extract 0.8

NH ₄ H ₂ PO ₄ 0.8

Na ₂ HPO ₄ 0.4

Water Else

The cultivation of the producer strain Rhizopus oryzae 605: preparation of a spore suspension, inoculation of seed, growth, separation of mycelium, precipitation of the enzyme preparation, determination of lipoxygenase activity is carried out analogously to example 1.

The resulting enzyme preparation has a lipoxygenase activity of 336400 units / g, carotenoxidase activity is 22600 units / g.

Example 3. Prepare a nutrient medium of the following composition, wt.%:

Soybean meal 2.5

Sunflower oil 0,9

Corn Extract 1.0

NH ₄ H ₂ PO ₄ 1.2

Na ₂ HPO ₄ 0.6

Water Else

Cultivation of the producer: preparation of a spore suspension, inoculation of seed, growth, separation of mycelium, precipitation of the enzyme preparation, determination of lipoxygenase activity is carried out analogously to example 1.

The resulting enzyme preparation has a lipoxygenase activity of 356800 units / g, carotenoxidase 26400 units / g.

Example 4. Prepare a nutrient medium of the following composition, wt.%:

Soybean meal 2.7

Sunflower oil 1,2

Corn Extract 1.2

NH ₄ H ₂ PO ₄ 1.4

Na ₂ HPO ₄ 0.8

Water Else

Cultivation of the producer: preparation of a spore suspension, inoculation of seed, growth, separation of mycelium, precipitation of the enzyme preparation, determination of lipoxygenase activity is carried out analogously to example 1.

In the obtained enzyme preparation, the activity of lipoxygenase is 315200 units / g, carotene oxidase activity - 19100 units / g.

As can be seen from the examples, when cultivating Rhizopus oryzae 605 on a nutrient medium composition, wt.%: Soybean meal 2.0-2.5; sunflower oil 0.7-0.9; corn extract 0.8-1.0; (NH ₄ ) ₂ HPO ₄ 0.8-1.2; Na ₂ HPO ₄ 0.4-0.6, gives the maximum effect (examples 2 and 3). Compared with the known method, the activity of lipoxygenase is higher, and the oxidation of β-carotene occurs to a lesser extent.

Thus, the proposed method allows to obtain an enzyme preparation of lipoxygenase with high activity of the formation of hydroperoxides and low oxidation activity of β-carotene, which is important for improving the physical properties of the dough in bakery and at the same time preserves biologically valuable substances of flour.

Example 5. The use of the drug lipoxygenase in the technology of making mayonnaise bread. The formulation and mode of preparation of the test are presented in table 1.

The enzyme preparation is dosed in an amount of 0.015% of flour in the dough. Previously, it is dissolved in water and introduced into the dough when kneading. Quality indicators of May bread are presented in table. 2.

The use of such a preparation in bread baking improves the physical properties of the dough - viscosity increases, adhesion decreases. Improving such quality indicators of bread as a specific volume of 18-25%; shape stability by 11-15%; porosity by 7-10% (table. 2).

Especially important is the use of the drug in the processing of flour with weak gluten. According to the main characteristics, gluten, washed from the test with the introduction of lipoxygenase, passes from a satisfactory weak discharge to a satisfactorily strong discharge. At the same time, biologically active substances of flour - carotenoids - are preserved.

Claims (1)

A method of producing an enzyme preparation of lipoxygenase, providing for deep cultivation of the producer strain on a nutrient medium containing organic nutrients, a source of growth factors, mineral salts and water, followed by isolation of the enzyme preparation, characterized in that they use the producer strain Rhizopus oryzae VKM F-605, cultivation is carried out on a nutrient medium containing soybean cake, sunflower oil as organic nutrients, and corn as a source of growth factors extract, mineral salts - ammonium dihydrogen phosphate, sodium hydrogen phosphate in the following components, wt.%: soybean meal 2.0-2.5 sunflower oil 0.7-0.9 corn extract 0.8-1.0 sodium hydrogen phosphate 0.4-0.6 ammonium dihydrogen phosphate 0.8-1.2 water rest