RU2233325C1 - Method for preparing complex lipase and lipoxygenase enzyme preparation - Google Patents

Abstract

FIELD: biotechnology, biochemistry, microbiology, baking industry.

SUBSTANCE: method for preparing enzyme preparation involves submerged culturing the strain-producer Rhizopus chinensis VKM F-1062 in nutrient medium of the following composition: soybean oil cake, sunflower oil, yeast extract, sodium dihydrogen phosphate, potassium hydrogen phosphate and distilled water followed by isolation of enzyme preparation. Invention provides synthesis of enzyme preparation with high lipase and lipoxygenase activities, to reduce cost in producing baked goods and duration of dough fermentation process, to improved dough and ready articles quality, to delay their staling.

EFFECT: improved preparing method.

3 tbl, 5 ex

Description

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

A known method of producing a lipase enzyme by culturing Rhizopus japonicus 1403 on a composition medium (%): soybean meal 1.7; oleic acid 0.35; corn extract 0.7; (NH ₄ ) ₂ HPO ₄ 0.4. The initial pH of the medium is 6.8-7.0, the growth temperature is 30 ° C, the cultivation time is 60 hours. The lipase activity in the culture fluid is 1350 units / cm ³ . (Optimization of lipase biosynthesis conditions / S. A. Shelamova, N. A. Zherebtsov, V. V. Schegolev // Enzyme and alcohol industry. - 1984 - No. 4. - P.26-28).

A known method of producing a lipoxygenase enzyme by deep cultivation of the producers of Aspergillus orysae 3-9-15 or Aspergillus awamori 466 on a nutrient medium containing soybean oil, soybean flour, sodium nitrate, magnesium sulfate, monosubstituted potassium phosphate, an initial pH of 6.0-7, 0. Cultivation is carried out under aeration of the medium, at a temperature of 28-32 ° C for 48 hours. The activity of lipoxygenase determined by the carotene oxidase method is 20000-50000 conventional units per 1 cm ^{3 of} culture fluid (a.s. 888542 USSR, MKI C 12 N 9/02 / Method for producing an enzyme preparation of lipoxygenase / R.R. Yarovenko, V.L. Kretovich, E.V. Budnitskaya; No. 2949321 / 28-13; claimed 30.05.80; publ. 15.08.83, Bull. No. 30).

Enzymatic transformations of fatty foods into baking technologies are necessary to obtain high quality products.

The lipase enzyme hydrolyzes fats with the formation of fatty acids, mono- and diglycerides, which are surfactants - surfactants, enzymatic fat hydrolysates have a positive effect on the quality of bread. Its porosity, specific volume increase, stagnation slows down. The duration of the dough fermentation process is reduced.

The enzyme lipoxygenase plays an important role in the processes of “ripening” of flour after grinding. It oxidizes polyunsaturated fatty acids, and the resulting hydroperoxides oxidize SH groups in flour proteins, proteolytic enzymes, and their activator, glutathione. The formation of additional disulfide bonds in the tertiary and quaternary structure of flour proteins contributes to the creation of a stronger structure when they swell during the formation of the dough; the level of protein proteolysis is also reduced. The activity of wheat lipoxygenase is not very high, therefore sources of this enzyme with higher activity are used in bread making technology. These are soy flour and preparations of microbial origin.

Lipoxygenase does not oxidize esterified fatty acids. In this regard, its use in combination with lipase is necessary to increase the effect of its oxidative effect.

It is especially important to use a complex enzyme preparation of lipase and lipoxygenase when using flour with reduced baking characteristics.

The disadvantages of the known methods for producing lipase and lipoxygenase enzymes are that they are obtained separately, which is economically disadvantageous, and for the high efficiency of their influence on the quality of bakery products, a complex of these enzymes is necessary.

The disadvantages of the method of obtaining the enzyme preparation of lipoxygenase include the use of expensive components of a nutrient medium - soybean oil and soybean flour.

An object of the invention is to obtain a complex enzyme preparation of lipase and lipoxygenase and reducing the cost of the enzyme preparation and bakery products using it.

The technical problem is achieved by the fact that in the method for producing a complex enzyme preparation of lipase and lipoxygenase, including: preparation of a nutrient medium, deep cultivation of the producer strain and isolation of the enzyme, Rhizopus chinensis 1062 is used as the producer strain of the complex enzyme preparation, and in the preparation of the nutrient medium it is added (wt.%) soybean meal 2.5-3.5; sunflower oil 0.5-0.8, yeast extract 0.9-1.4; potassium hydrogen phosphate 0.32-0.39; sodium dihydrogen phosphate 0.25-0.29; the initial pH of the medium is 7.0.

The technical result of the invention is expressed in obtaining a complex of two enzymes - lipase and lipoxygenase with high activity; reducing the cost of bakery products while improving their quality.

The method is implemented as follows.

The producer strain Rhizopus chinensis 1062 was obtained from the All-Russian Collection of Microorganisms of the Russian Academy of Sciences, where it is stored under No. VKM F-1062.

A pure culture of Rhizopus chinensis 1062 is grown on an agarized medium composition (wt.%): Soy flour - 1.5; glucose - 1.0; sunflower oil - 0.5; ammonium dihydrogen phosphate - 0.6; initial pH value of 6.8-7.0 for 3-4 days at a temperature of (30 ± 1) ° C.

During the deep cultivation of the producer strain, the following components are used (wt.%): Soybean meal 2.5-3.5; sunflower oil 0.5-0.8; yeast extract 0.9-1.4; potassium hydrogen phosphate 0.32-0.39; sodium dihydrogen phosphate 0.25-0.29; the initial pH of the medium is 7.0.

Nutrient medium containing soybean meal, sunflower oil, yeast extract, potassium hydrogen phosphate, sodium dihydrogen phosphate and water are inoculated with Rhizopus chinensis 1062 strain and grown under optimal conditions (pH 7.0; temperature 30-32 ° С; duration 72 h) with aeration.

After the cultivation of the producer strain Rhizopus chinensis 1062, the culture fluid was separated from the biomass by filtration. A complex enzyme preparation is obtained from the culture fluid, precipitating it with acetone with a concentration of 70 vol.% At a temperature of minus 2 - minus 4 ° C.

In the present invention, a titrimetric method based on determining the amount of fatty acids released during the hydrolysis of olive oil is used to determine the lipase activity of an enzyme preparation. The amount of enzyme that catalyzes the formation of 1 μmol of oleic acid in 1 hour of hydrolysis is taken as a unit of lipolytic activity. (Laboratory workshop on the technology of enzyme preparations / I.M. Gracheva, Yu.P. Grachev, M.R. Mosichev, etc. - M .: Light and food industry, 1978. - 271 p.).

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 solution of the substrate; 0.5 cm ³ enzyme solution, 5 cm ³ 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% ethanol. To prepare a control sample, ethyl alcohol is added to 1 cm ^{3 of the} reaction mixture after 5-10 s from the start of the reaction.

Linoleic acid is used as a substrate.

For a unit of lipoxygenase activity, the change in extinction is taken to be 0.001 in 1 min, referred to 1 g of the drug. (Oganesyan N.A., Borisova I.G., Solomatin D.A., Budnitskaya E.V. Isoenzyme composition and activity of LOs of some wheat varieties of the species Triticum aestivum // Dokl. AN SSSR. 1983. T. 269. No. 4 S.1002-1005).

The composition of the nutrient medium ensures the biosynthesis of two enzymes — lipase and lipoxygenase, and thus obtaining a complex enzyme preparation from the culture fluid.

The method is illustrated by examples.

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

Soybean meal 2,3

Sunflower oil 0.3

Yeast extract 0.8

K ₂ HPO ₄ 0.30

NaH ₂ PO ₄ 0.22

Water Else

The initial pH is adjusted to 7.0.

In Erlenmeyer flasks with a capacity of 750 cm ³ contribute 100 cm ^{3 of} nutrient medium. The medium is sterilized for 1 h at 0.1 MPa, then it is cooled and inoculated with an aqueous-spore suspension of the producer strain Rhizopus chinensis 1062 in an amount of 1% by volume of the medium. An aqueous-spore suspension of the producer strain Rhizopus chinensis 1062 is prepared as follows: a bevel of a 4-day culture in 18 × 180 mm test tubes is filled with 10 cm ^{3 of} sterile water and the spores are washed off. The producer strain Rhizopus chinensis 1062 is incubated on a rocking chair at a speed of 1.7-1.8 s ^-1 , temperature (30 ± 2) ° C for 72 hours. After the cultivation of the producer strain, the mycelium and suspended particles of the medium are separated from the culture fluid filtering.

The enzyme preparation is obtained by precipitation from the culture fluid with acetone in the cold (minus 2 - minus 4 ° C) at a concentration of 70 vol.% And dried under vacuum.

The activity of lipase is 260800 units / g, lipoxygenase - 221300 units / g.

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

Soybean meal 2.5

Sunflower oil 0.5

Yeast Extract 0.9

K ₂ HPO ₄ 0.32

NaH ₂ PO ₄ 0.25

Water Else

The initial pH is adjusted to 7.0.

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

The resulting enzyme preparation has a lipase activity of 295200 u / g, lipoxygenase - 245600 u / g.

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

Soybean meal 3,5

Sunflower oil 0.8

Yeast Extract 1.4

K ₂ HPO ₄ 0.37

NaH ₂ PO ₄ 0.29

Water Else

The initial pH is adjusted to 7.0.

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

The resulting enzyme preparation has a lipase activity of 325700 units / g, lipoxygenase - 252400 units / g.

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

Soybean meal 3.7

Sunflower oil 0,9

Yeast extract 1.5

K ₂ HPO ₄ 0.40

NaH ₂ PO ₄ 0.30

Water Else

The initial pH is adjusted to 7.0.

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

The resulting enzyme preparation has a lipase activity of 280300 units / g, lipoxygenase - 232700 units / g.

As can be seen from the examples, during the cultivation of Rhizopus chinensis 1062, the following composition of the medium provides the greatest effect, wt.%: Soybean meal 2.5-3.5; sunflower oil 0.5-0.8; yeast extract 0.9-1.4; K ₂ HPO ₄ 0.32-0.39; NaH ₂ PO ₄ 0.25-0.29 (Examples 2 and 3). Compared with known methods, it becomes possible to obtain an enzyme preparation with the highest activity of lipase and lipoxygenase.

Thus, the proposed method allows to obtain a complex enzyme preparation of lipase and lipoxygenase and to reduce the production costs of these two enzymes used in the baking industry and to simultaneously reduce the cost of bakery products when using them, increase their quality, reduce the duration of the dough preparation process and slow down the staling process .

In baking, it is more efficient to use not each enzyme separately, but both at the same time to improve the quality of bakery products. A complex enzyme preparation is introduced at the stage of preparation of the dough. Lipase Rhizopus chinensis 1062 hydrolyzes oils to fatty acids, mono- and diglycerides, which improve the physical properties of the dough and the quality of products. The time of fermentation of the dough is reduced, the staling of products is slowed down. Lipoxygenase Rhizopus chinensis 1062 oxidizes polyunsaturated fatty acids formed during the hydrolysis of fats and oils to hydroperoxides. The latter give an additional positive effect in the technology for the preparation of bakery products.

This is confirmed by example 5.

Example 5. Prepare the dough for a bar for tea in a sparse manner. Flour was used with reduced baking properties - with weak gluten. The formulation and mode of preparation of the dough are given in table. 1.

The recipe for the test for control and prototypes does not differ - flour is added up to 100 kg, water - by calculation, salt - 1.5 kg, sugar - 5 kg and margarine - 8 kg. The duration of the fermentation of the test in the control case is 160-180 minutes, and in the experimental - 110-120 minutes; final acidity is the same - 3.4-3.6 degrees.

Due to the partial hydrolysis of fat by lipase of a complex enzyme preparation, the fermentation process of the test is intensified (the inhibitory effect of fat on yeast is reduced).

Tea bar quality indicators are presented in Table 2.

Prototypes of products have better porosity, volume, and form stability even when using flour with reduced baking properties.

Thus, the essence of the processes is reduced to the intensification of the oxidative effects on the protein-proteinase complex of flour. The colloidal test system is more elastic, characterized by high mechanical properties. As a result, the dough more steadily maintains the optimum of its physical properties, is characterized by better shape stability and significant gas-holding ability. This is especially important when processing flour with reduced baking qualities.

Products with the addition of a complex enzyme preparation, lipase and lipoxygenase, have a more elastic crumb and retain freshness longer, since the total crumb deformation determined on the penetrometer is higher in comparison with control samples - in prototypes, the strain values decrease more slowly during storage (Table 3 )

The proposed method allows to obtain a complex enzyme preparation consisting of lipase and lipoxygenase. This not only reduces the cost of production of the drug compared to receiving them separately, but also reduces the cost of bakery products, since the dosage of the complex drug is reduced by at least 1.5 times and amounts to 0.02-0.03% of flour. A dosage of lipase or lipoxygenase alone is given 0.02-0.05%. The use of such a preparation in bread baking makes it possible to shorten the duration of the dough fermentation process, improve the quality of finished products, and slow down their staleness. The use of such a preparation is especially important in the processing of flour with weak gluten or an insufficient amount of it in flour.

No analogues of the invention were found.

Claims (1)

A method of obtaining a complex enzyme preparation of lipase and lipoxygenase, involving deep cultivation of the producer strain Rhizopus chinensis VKM F-1062 on a nutrient medium of the following composition, wt.%: soybean meal 2.5-3.5 sunflower oil 0.5-0.8 yeast extract 0.9-1.4 sodium dihydrogen phosphate 0.25-0.29 potassium hydrogen phosphate 0.32-0.39 distilled water the rest at an initial pH of 7.0, followed by isolation.