Method for preparing collagen

FIELD: biotechnology.

SUBSTANCE: method involves purification of protein raw, addition of water and ice, milling a mixture followed by hydrolysis. After hydrolysis collagen-containing solution is homogenized and collagen is separated. Hydrolysis is carried out for two stages. The first stage is carried out by treatment of the reaction mixture with lipase from fungus Rhizopus oryzae, and at the second stage a proteolytic enzyme as neutral protease is used. Invention provides preparing collagen approaching to natural collagen by physicochemical and structural-mechanical properties. Invention can be used in food processing industry, cosmetic, medicinal and other branched of industry.

EFFECT: improved preparing method.

4 cl, 3 ex

 

The invention relates to biotechnology and can be used in food, cosmetic, medical and other industries.

It is well known that collagen is the main structural protein of all existing beings. Any changes to this structural protein lead to diseases of the skin, internal organs and, ultimately, to the aging of the whole organism ("Trends Glycosci. Glucotetechnol.", 1992, V.4, No. 19, str-498; "Protein" / Ed. Neurath, and Bailey, K.M.: Foreign literature, 1959, s.400-438, Antipova, VA, Glotova IA / "foundations of rational use of raw material containing collagen in the meat industry". Voronezh.: Voronezh state technology. Academy, 227 S., 1998).

From the literature it is also known that collagen is partially changes its conformation and its unique properties at low (below 5) and high (above 10) pH values. Therefore, in most cases, researchers are not natural collagen, which is especially valuable due to their physico-chemical properties that enable its use in various industries, even in such as electronic industry, as one of the best dielectrics, and its high molecular weight fraction (L. Jesperson, Tompson P. Eur. 1992. Eur. 14254. Composting compact Quel Assur. Criter., str-203).

Currently, numerous well-known methods of obtaining the collagen is from various raw material sources (Neklyudov A.D., Ivankin A.N. "Biologically active compounds from natural objects. Properties, structure-function relationship. M: at MSFU, 2003, str-412). However, the above methods give the possibility of obtaining the collagen and its fractions, which, at best, close in its properties to the precursor of collagen, upon which he builds his chain - tropocollagen.

A known method of producing collagen, in which raw materials containing collagen, it is proposed to first soaked in water, cooled by ice at pH 5.5, and then increasing the pH to 10.5, to process the resulting conglomerate with alcalase and then again to reduce the solution pH to 5.5. Filtration and subsequent sterilization of the solution ensures collagen (see RF patent №2094999, class. And 22 With 13/00, From 07 To 1/36, 1997 - prototype).

A disadvantage of the known method is used as the proteolytic enzyme alkaline alcalase for 3 hours at pH values above 10, which, as is known from the above sources, can lead to partial hydrolysis links of collagen between oxidizing and glutamic acid, the destruction of essential amino acids and partial deglycosylated protein molecule, resulting in a broken collagen structure and, therefore, all its valuable physical and chemical properties. In addition, homogenization of collagen OS is p at pH 3.0. At these pH values is the hydrolysis of peptide bonds between Gli-Pro and Gli-Respondents, and between Gli ili and Gli-Lei and other amino acids. Apply the enzyme after coagulation of collagen in isoelectric point (pH 5.5) is inefficient process, since it involves simultaneous capture other related proteins, and therefore becomes ineffective subsequent processing alcalase. In addition, the protein retains its configuration, and hence their chemical-physical and structural-mechanical properties of neutral conditions at pH of 6.5-7.5, i.e. those conditions that are most acceptable for the existence of most mammals.

The aim of the invention is to obtain collagen, close to the natural protein in their physico-chemical and structural-mechanical properties using known and available proteoliticheskikh enzymes.

This goal is achieved by the fact that in the method of producing collagen, which includes cleaning of raw protein Supplement water and ice mass ratio of 1:1, grinding the mixture, the hydrolysis of the crushed mixture for 1.5-3 hours proteoliticeski enzyme technology pH values less than 11 and a temperature in the range 37-40°With, the homogenization of the collagen solution by centrifugation, then the internal medicine collagen by washing with water at pH equal to 5.5, and then obtaining the desired product in pure form, a pre-protein raw material is crushed in the cutter at a temperature of 0-5°and soaked in cold water for one hour, then spend two-stage hydrolysis of the resulting reaction mixture in the presence of 5% ethanol by volume with intermediate centrifugation and washing with water, providing the technological values of pH in the range of 7.5-8.0 and ionic strength of the reaction mixture in the range of 0.05-0.1 n through supplements of sodium carbonate and sodium chloride, respectively, and the first stage of hydrolysis is performed by processing the reaction mixture by lipase from the fungus Rhizopus oryzae, and the second stage by processing proteoliticeski enzyme, for example a neutral protease, trypsin or chymotrypsin.

To implement the present invention as raw materials are washed out of the colon, kaniga or skin of mammals and birds, as well as swim bladders of animals and fish, separated from the associated proteins. The resulting mass is pulverized in the bowl cutter at 0÷5°, soaked in cold water for one hour. After that, the water is separated and the weight of the newly added water containing NaCl and 5% ethanol by volume of the mixture was adjusted pH to 7.5 by means of sodium carbonate, the resulting mass is heated to 40°and to cosiderable the lipase activity is not below 40000, specific hydrolysis of olive oil. The mixture is stirred for 1-3 hours at the above temperature, monitoring the change in pH and keeping it in the range of 7.5 to 8.0. At the end of this procedure, the aqueous solution is drained, the remaining mass is washed thoroughly with cold water. After washing it, water is added and NaCl, keeping the ionic strength of the solution in the range of 0.05-0.1 N. the resulting suspension is again heated up to 37-40°To add to the reaction mixture 5% ethanol and neutral protease of any origin for the hydrolysis of proteins associated with collagen, in the complete absence of collagenase. The hydrolysis is carried out under stirring for 1-3 hours, depending on the activity of a selected enzyme, maintaining the temperature of the reaction mass 37-40°and monitoring the process to increase the nitrogen of the amino groups liberated during the hydrolysis, using a method formolite titration. The process is finished after the ending increase in amino nitrogen. After the process is finished, bring the pH value to 5.5 (isoelectric point of collagen), the reaction mass is centrifuged, washed several times with water, dried in a vacuum at a temperature not exceeding 40°and the resulting dry product is ground. The result is a powder collagen containing 5-6% moisture, about 1% ash and having a molecular mA is su defined by electrophoresis in 12% polyacrylamide gel, equal 1000-1100 kDa. In addition to collagen with a molecular weight of 1000 kDa, the product contains about 5-7% of its high molecular weight impurities with molecular weight of 300-500 kDa. Protein also contains more than 10% carbohydrates, determined after acid hydrolysis, indicating the preservation of the collagen of its glycosidic shell. The following example illustrates the invention.

Example 1. 10 kg of kanagi after machining crushed in the cutter with the addition of 10 kg of ice so that the temperature of the reaction mass does not exceed 5°S, and then transferred into the reactor where add 10 l of cold water. The mixture is stirred at this temperature for one hour, followed by centrifugation to separate the water from the biomass, washed several times with cold water and again transferred into the reactor where add 100 l of water, 365 g of NaCl and 5 l of ethanol. The reaction mass is slowly heated up to 37-40°C, after which it add 45-50 g, for example, fungal lipase from the fungus Rhizopus oryzae activity 50000 units, for hydrolysis of the lipids present in the reaction mass, maintaining the pH of the solution in the range of 7.5 to 8.0 with sodium carbonate. After completion of the hydrolysis process (about 1 hour), the reaction mass is centrifuged. Washed with water and again placed in the reactor. Add Aut 250-300 g NaCl, so that the ionic strength of the reaction medium was below 0.05-0.1 g, and 10-15 g of the enzyme, depending on its initial activity. As enzyme use a neutral protease G-10x from Bas. Subtilis. The reaction mixture was stirred at 37-40°C for 1.5 to 3 hours before termination of the increase in amino nitrogen, after which there was added a solution of Na2CO3to obtain the pH of the reaction mixture of 5.5. The reaction mixture is cooled to 7-10°C, then centrifuged, washed several times demineralized water and dried in a vacuum evaporation apparatus at 40°C to a residual moisture content of 6-8%. The obtained dry product is ground and determine its physico-chemical characteristics. The yield of dry collagen from 10 kg of kanagi with a humidity of 40% is 5 kg (83%), counting the protein in raw materials. The product has a moisture content of 5%, the carbohydrate content, with certain astronomy reagent, 10%, ash 0.7 percent, the molecular weight 970-1120 kDa. As proteoliticheskogo enzyme can be used trypsin, chymotrypsin, fungal protease.

Example 2. Is carried out analogously to example 1, but instead of kanagi taken fish bubble. The output of the dry collagen powder 90%, ash content of 0.5%, the carbohydrate content of 12%, a molecular weight of 1,000 kDa. The content of other proteins is virtually absent.

Example 3. Is carried out analogously to example 1, but instead of kanagi ispolzuetsyaprintsip intestine of ruminants. The yield of dry powder collagen 89%, ash 0.7%, carbohydrate content of 15%, a molecular weight of 1200 kDa.

Thus, it is possible to obtain collagen, similar in its physical-chemical and structural-mechanical properties to the natural, from belaustegui waste of food raw materials.

1. A method of producing collagen, which includes cleaning of raw protein Supplement water and ice mass ratio of 1:1, grinding the mixture, the hydrolysis of the crushed mixture for 1.5-3 h with a proteolytic enzyme in technological pH values less than 11 and a temperature in the range 37-40°With, the homogenization of the collagen solution by centrifugation, then the Department of collagen by washing with water at pH 5.5 and the subsequent obtaining of the desired product in pure form, wherein the pre-protein raw material is crushed in the cutter at a temperature of 0-5°and soaked in cold water within one hour, then spend two-stage hydrolysis of the resulting reaction mixture in the presence of 5% ethanol by volume with intermediate centrifugation and washing with water, providing the technological values of pH in the range of 7.5-8.0 and ionic strength of the reaction mixture in the range of 0.05-0.1 n through supplements of sodium carbonate and sodium chloride, respectively, and the first stage of hydrolysis is performed by which the processing of the reaction mixture by lipase from the fungus Rhizopus oryzae, and in the second stage using proteolytic enzyme in the form of neutral protease.

2. The method according to claim 1, characterized in that the use of the proteolytic enzyme in the form of trypsin.

3. The method according to claim 1, characterized in that the use of the proteolytic enzyme in the form of chymotrypsin.

4. The method according to claim 1, characterized in that the use of the proteolytic enzyme in the form of a fungal protease.



 

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