Method to produce chitosan-nucleic hydrolysate

FIELD: biotechnologies.

SUBSTANCE: fish roe is homogenised. Fish roe hydrolysis is carried out with a ferment preparation "Collagenase" in presence of an inhibitor for 10-12 hours. Chitosan is added to the produced hydrolysate at the ratio of 0.5-1.0:1.0. Components are mixed.

EFFECT: invention makes it possible to accelerate process of chitosan-nucleic complex production.

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The invention relates to biotechnology, in particular to methods for nucleic acid-protein hydrolysates from fish raw, and can be used in fish, meat and other food industries, as well as in medicine, biochemistry and agricultural production.

Analysis of scientific-technical and patent documentation showed that there are different ways to obtain a complex of biologically active products. Basically, it is obtaining protein hydrolysates containing biologically valuable substances, such as amino acids, deoxynucleoside, oligonucleotides and other

However, the main disadvantage of this method is that the resulting nucleic acid products have a low potential for use. Due to the fact that they have high molecular weight, transfection and delivery of these substances directly into the cell of the organism is limited. The required compaction of such molecules. This is often used cationic polymers that contribute to this process. However, use of these systems is limited due to the poor biocompatibility. Therefore, the creation of biocompatible complex polymer systems is an urgent task in the field of Biomedicine, cosmetics and other industries.

A method of obtaining peptides/amino acids from raw materials containing protein. The way which engages the grinding raw material and heating it, add water and hydrolysis using endogenous enzymes. The precondition for this hydrolysis is the use of alkaline enzymes. Optimal fermentation is carried out at pH of 7.6 to 8.2 at a temperature of 44-45°C. For termination of hydrolysis using elevated temperature above 70°C. the resulting hydrolyzate is subjected to membrane filtration for the separation of fractions of peptides/amino acids of the desired molecular weight. Hydrolizates the filtrate is concentrated to the desired level of concentration of peptides and amino acids (p. the Russian Federation No. 2333663, A23J 3/04, 2006).

The main disadvantage of this method is a multistage process and inefficient use of raw materials, because in the process of obtaining peptides/amino acids diverted valuable products of hydrolysis, in particular, DNA (deoxyribonucleic acid).

In addition, the use of alkaline environment for hydrolysis is also a negative factor, since an alkaline environment promotes the development of microflora, and you have to use UV-irradiation or the other, which complicates the production process.

A method of obtaining a product enriched in free amino acids by enzymatic hydrolysis of protein-containing raw materials. The method is carried out by enzymatic hydrolysis for 1.5 to 6.0 hours at a temperature of 35-40°C. To obtain Leche is BNO-preventive product of protein raw material is crushed and poured 5-20%water-alcohol solution at a ratio of 1:1-1:5 and alkalinized with NaOH solution to pH 8.0 to 8.5. The hydrolysis is carried out with the use of enzyme preparations in an amount of 0.1 to 1.0% by weight of raw materials. Inactivation of enzymes is carried out by heating a mixture of up to 70-80°C for 15-20 minutes. Target product produce by filtering (p. the Russian Federation No. 2171066, A23L 1/325, A23L 1/33, 2001).

The disadvantage of this method is the use of an alkaline environment, which is favorable for the development of pathogenic microflora.

In addition, continuous monitoring of the pH of the medium.

In addition, the use of high temperature for inactivation of the enzyme contributes to the further disintegration of the obtained product.

Closest to the claimed method is a method of obtaining a nucleic acid-protein hydrolysate from marine animal products, namely milk salmon and sturgeon, by enzymatic hydrolysis of raw materials by collagenase. The method involves homogenization milk and autolysis at pH 4.5 to 5.0, a temperature of 37-39°C for 46-50 hours. This gomogenizirovannom ROE is subjected to degreasing flocculation with chitosan. The autolysate is separated into a liquid fraction, which is heated up to 80°C to inactivate the enzymes, and the precipitate, which is subjected to enzymatic hydrolysis. Then the hydrolysate and liquid fraction are combined and the mixture is used as the finished product (p. the Russian Federation No. 2055482, A23J 3/04, A23J 3/00, A23J 3/34, AK 35/60, 1996).

The disadvantage JV is soba is the length of the process to obtain the hydrolysate, more than 60 hours. According to the authors of the claimed invention stage of autolysis - excessive stage of the process, because the future use of enzymatic hydrolysis, which, in principle, allows to obtain the same cleavage products in a shorter time.

The objective of the invention is to develop a method of producing chitosan-nucleic acid hydrolysate with high physiological value, but also increase the yield of low molecular weight nucleic acid product.

The problem is solved in that in the method for production of chitosan-nucleic gidrolizata the enzymatic hydrolysis is carried out in the presence of inhibitor for 10-12 hours, and the chitosan is added to the hydrolysate after hydrolysis at a ratio of 0.5 to 1.0:1.0, followed by mixing of the components.

The technical result is to accelerate the process of obtaining a nucleic acid hydrolysate.

The claimed technical result is achieved due to the fact that the hydrolysis of the raw materials are enzyme preparation "Collagenase" in the presence of inhibitor for 10-12 hours, and the chitosan is added in a ratio of 0.5 to 1.0:1.0 in the hydrolysate after hydrolysis.

To achieve the required result of hydrolysis are within 10-12 hours. To determine the optimum time of hydrolysis experiments were carried out and the results are presented in table 1.

Table 1
The content of nucleic acids during hydrolysis
The duration of hydrolysis, hWithnucleic acidsmg/ml (concentration)
01,12
27,15
4EUR 7.57
67,79
88,15
108,58
128,95
248,95

These tables indicate the increase of the number of nucleic acids (nucleic acid products) with increasing duration of hydrolysis up to 12 hours At a subsequent increase in the time of hydrolysis up to 24 h, the concentration of nucleic acid products remains unchanged. Thus, practically, it was found that for the complete hydrolysis of fish ROE and allocation of the maximum number of nucleic acid products of hydrolysis enough 10-12 hours.

It was also investigated the qualitative composition of nucleic what products the resulting hydrolysis. At the end of hydrolysis were obtained following nucleic acid products: oligonucleotides, mononucleotides, oligonucleotides. Compared with the prototype already after 10-12 hours were obtained low molecular weight nucleic acid products that are better absorbed by the body, i.e. they are more physiologically valuable. To determine the qualitative composition of the hydrolysate was applied standard method of thin-layer chromatography.

The results are presented in figure 1.

The findings also suggest that the optimal duration of hydrolysis is 12 h, since the subsequent increase in the time of hydrolysis does not change the qualitative composition of the nucleic material in the hydrolysate.

Thus, it was experimentally established that the hydrolysis is carried out in 10-12 hours for maximum isolation of physiologically valuable products of hydrolysis, which are well absorbed by the body. In addition, they are easy to contact with chitosan, which provides access them directly into the cell body. We thus solve the set task of obtaining chitosan-nucleic acid complex.

To reduce the activity of the enzyme in the hydrolysis of use inhibitor, such as sorbic acid or anhydrous calcium chloride. In addition, they contribute to the suppression Mick is Flory of the hydrolysate, it allows to increase the shelf life of the product.

The claimed result is also achieved when introducing chitosan hydrolysate, i.e. after the hydrolysis step, unlike the prototype, where chitosan is used to hydrolysis, for degreasing gomogenizirovannykh Molok and after flocculation is removed from the homogenate.

In the claimed invention the chitosan contribute in the hydrolysate, which contains a complex of nucleic acid products with a negative potential, causing the molecules of chitosan bearing positively charged amino group, can form a biopolymer complex. It is this complex has potential advantages in comparison with known nucleic acid products. It can transfection of nucleic material directly into the cell of an organism that is fast enough to affect the response of the organism. Moreover, this complex protects the nucleic acid material from biodegradation during transport in the flow of biological fluid, because it is in a bound state. Since chitosan has low toxicity and good biocompatibility, the use of such a complex has great possibilities. In addition, chitosan inhibits the growth of bacteria, which helps to preserve the product.

All this leads to the conclusion that the obtained complex Hito is an-nucleic acid product can have a wide practical application.

On the basis of experiments it was established that in the hydrolysate add the chitosan in the ratio of 0,5-1,0:1,0.

Thus, if the ratio of chitosan hydrolysate is less than 0.5:1.0 then decreases its physiological value, the product loses the ability to be functional.

If the ratio of chitosan hydrolysate is more than 1.0:1.0 then deteriorate the organoleptic properties of the product, you receive astringent taste, resulting in a decrease in consumer power.

The method is as follows.

Take fish ROE, add to them the water and acetate buffer, put everything in a blender and homogenize. In the homogenate make an enzyme inhibitor and mix. The mixture is incubated for 10-12 hours. After hydrolysis, the hydrolysate contribute chitosan in the ratio of 0.5 to 1.0:1.0 and intensively stirred. If necessary, the hydrolysate may be subjected to concentration and drying.

Example 1. Take 1.0 kg of frozen milk pink, add to them 8,0 litres of water and 1.0 liter of acetate buffer to obtain a pH of 5.0. All this is placed in a blender and homogenize. In the homogenate make 0,007 kg enzyme preparation "Collagenase and 0.01 kg of sorbic acid and mixed. The mixture is incubated at a temperature of 38°C for 12 hours. Then take a 10.0 l of hydrolysate, make 1%solution of chitosan in the amount of 5.0 liters (the rate of 0.5:1.0) and intensively stirred. The product is evaporated to a volume of 1.0 liter.

Example 2. Take 1,0 kg ROE herring, add to them 8,0 litres of water and 1.0 liter of acetate buffer to pH of 5.0. The mixture is homogenized. In the homogenate make 0,007 kg enzyme preparation "Collagenase" and 0.001 kg sorbic acid and mixed. The mixture is incubated for 10 hours at a temperature of 37°C. and then hydrolyzed make a 1%solution of chitosan in the amount of 10.0 liters (ratio 1:1) and intensively stirred. The product is evaporated to a volume of 1.0 liter.

Example 3. Perform in example 1, but as raw material take chum salmon ROE. As an inhibitor take 0,02 kg of calcium chloride, and the mixture incubated for 11 hours.

The method of obtaining chitosan-nucleic acid hydrolysate, involving the hydrolysis of fish ROE enzyme preparation "Collagenase" and the use of chitosan, wherein the hydrolysis is carried out in the presence of inhibitor for 10-12 h, and chitosan is added to the hydrolysate after hydrolysis at a ratio of 0.5 to 1.0:1.0, followed by mixing of the components.



 

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