Method for complex processing of fish raw material for obtaining hyaluronic acid and collagen

FIELD: biotechnologies.

SUBSTANCE: skins of pond fish are flushed with cold flushing water during 10-15 minutes. They are crushed to the size of 2-3 mm. Water extraction is performed at the temperature of 40-45°C during 40-50 minutes at the ratio of crushed skins to water, which is equal to 1:1 at periodic mixing. Then, they are filtered; liquid fraction is dried in a spraying drier at the drier outlet product temperature of 60-65°C during 15-25 minutes so that hyaluronic acid is obtained. Solid fraction is subject to bleaching during 12 hours with hydrogen peroxide-salt solution that is prepared by mixing of 1 l of 3% hydrogen peroxide and 20 g of sodium chloride. Treatment of bleached solid fraction is performed with 1.0-1.2% solution of sodium hydroxide during 24 hours at the temperature of 20-25°C with further neutralisation of the obtained mixture with 3% boric acid solution. Treatment of swollen solid fraction is performed with Pancreatin ferment preparation solution taken in the quantity of 0.5-0.6% to the weight of solid fraction during 1.5-2.0 hours at the temperature of 37-40°C. Flushing of solid fraction is performed with cold flushing water for removal of Pancreatin residues so that collagen is obtained. The obtained collagen, depending on the purpose, is supplied for drying in drying chambers with forced air circulation at the temperature of 18-20°C during 12 hours and storage in dry ventilated rooms at the temperature of not higher than 20°C during 24 months or frozen to the temperature of minus 18 - minus 20°C and stored at the temperature of minus 18 - minus 20°C during 24 months. The liquid fraction dried in the spraying chamber is stored at the temperature of 0-4°C during 12 months or dissolved in physiological buffer solution.

EFFECT: improvement of the method.

2 dwg, 1 tbl, 1 ex

 

The invention relates to fish processing and cosmetic industries, and in particular to methods of processing the skins of fish to obtain hyaluronic acid and collagen.

The closest in technical essence and the achieved effect is a method of processing raw material containing collagen, providing preliminary freezing of raw materials, grinding, degreasing, alkali-salt processing, washing, neutralization, grinding the obtained collagen, dissolving it in the organic acid and homogenization [Patent No. 2139937. The method of processing raw material containing collagen / Aotion; Opotow; Vggrtusahela; Nambouwalu; Geantrai No. 2139937; Claimed 07.12.1998; Publ. 20.10.1999].

The disadvantages of the method are mnogovershinnoe and duration of the process; the use of more aggressive reagents; the necessity of dissolving the collagen in the organic acid.

The technical problem of the invention is to develop a method for integrated processing of fish raw materials for the production of hyaluronic acid and collagen, which allows to obtain hyaluronic acid and collagen for one cycle, to implement waste-free technology for the processing of pond fish through the best use of secondary products cutting, to expand the raw material base of the cosmetic industry, and is also to reduce the time and simplify the process of obtaining the target product.

To solve the technical problem of the invention a method for complex processing of fish raw materials for the production of hyaluronic acid and collagen, characterized in that for obtaining hyaluronic acid and collagen using the skins pond fish, which are obtained from fish processing plants skins pond fish washed with cold running water for 10-15 minutes, reduce them to a size of 2-3 mm, conduct water extraction at a temperature of 40-45°C for 40-50 min at a ratio of shredded skin:water is 1:1 with periodic stirring, filtered, the liquid fraction is dried in a spray drier at the temperature of the product leaving the dryer 60-65°C for 15-25 min with a production of hyaluronic acid, the solid fraction is subjected to bleaching peroxide-salt solution is taken so that all the solid fraction was covered peroxide-salt solution, which is prepared by mixing 3%hydrogen peroxide with sodium chloride, from the calculation: 1 l of 3%hydrogen peroxide is introduced 20 g of sodium chloride, the process is conducted within 12 hours, after this time the peroxide-salt solution is drained, then blanched solid fraction is subjected to swelling in 1,0-1,2%solution of a hydroxide sodium taken in 1:1 ratio to the mass of the solid fraction, 24 h at 20-25°C is followed by neutralization of the mixture 3%solution of boric acid, next, handle the swollen solids solution of enzyme preparation "Pancreatin" (for example, "Pancreatin" Irbit himfarmzavod, Russia, according to the manufacturer, the solid amorphous powder with enzyme activity: proteolytic - 200 FIP units (IU enzyme activity), amylolytic - 3500 FIP units, lytic - 4300 FIP units), taken in an amount of 0.5-0.6% by weight solids, for 1.5-2.0 hours at a temperature of 37-40°C, and the enzyme preparation is prepared by mixing with water in the ratio 1:3, then hold washing the solids with cold running water to remove residual "Pancreatin"derived collagen, depending on destination, sent for drying in the drying chamber with forced air circulation at a temperature of 18-20°C for 12 h and stored in a dry ventilated room at a temperature not exceeding 20°C for 24 months or frozen to a temperature of minus 18 to minus 20°C and stored at a temperature of minus 18 to minus 20°C for 24 months, dried in a spray drier, the liquid fraction is stored at a temperature of 0-4°C for 12 months or dissolved in a physiological buffer solution.

The technical result consists in a comprehensive receipt from the skins pond fish hyaluronic acid and collagen in one process cycle, EXT is of the functionality of secondary raw materials, the implementation of non-waste technology and deep processing of secondary products.

The method is as follows.

To obtain hyaluronic acid and collagen using the skins of the following species of fish: carp, carp, pike, carp, carp and others, the Proposed method can be used to produce hyaluronic acid and collagen from the skins of marine fish.

Adopted by the skin is subjected to washing with cold running water for 10-15 minutes to remove the mucus, dirt and retrieve globulin and albumen proteins. Next, the skin is crushed manual or automated way up to a size of 2-3 mm, because hyaluronic acid is soluble in warm water, then a water extraction of hyaluronic acid from the skin of pond fish. Extraction was carried out at a temperature of 40-45°C for 50 min with a ratio of the crushed skins:water is 1:1 with periodic stirring. The liquid is then filtered. The liquid fraction is used to produce GUK, and the solid fraction secrete collagen. The liquid fraction is dried in a spray drier at the temperature of the product leaving the dryer 60-65°C for 15-25 minutes Hyaluronic acid is stored in a dried form

at a temperature of 0-4°C for 12 months or dissolved in a physiological buffer solution.

Solid fraction of all Laut bleaching peroxide-salt solution, which is prepared by mixing 3%hydrogen peroxide and sodium chloride from the calculation: 1 l of 3%hydrogen peroxide is introduced 20 g of sodium chloride, peroxide-salt solution is taken in the same numbers, so that all the solid fraction was covered with it, the process is conducted within 12 hours, after this time the peroxide-salt solution is drained. Then bleached solid fraction is subjected to swelling in 1,0-1,2%sodium hydroxide solution taken in a 1:1 ratio to the mass of the solid fraction within 24 hours at 20-25°C, followed by neutralization of the mixture of 3%boric acid solution for 10 minutes Then swollen solid fraction is treated with a solution of enzyme preparation "Pancreatin" for the purpose of purification of collagen from the ballast globulin and albumen protein and fat components. The enzyme preparation contribute in the amount of 0.5 to 0.6% by weight solids, and the enzyme preparation is prepared by mixing with water in the ratio 1:3, the process is carried out for 1.5-2.0 hours at a temperature of 37-40°C, followed by washing with cold running water to remove residual "Pancreatin".

After washing in water, collagen or frozen to a temperature of minus 18 to minus 20°C or dried, and the drying is best conducted in the drying chambers with forced air circulation at a temperature which e 18-20°C during 12 hours

The dried collagen stored in dry, ventilated place at a temperature not higher than 20°C within 24 months, the frozen collagen stored at a temperature of minus 18 to minus 20°C for 24 months.

Method for integrated processing of fish raw materials for the production of hyaluronic acid and collagen is illustrated by the following example.

Example No. 1.

1 kg of skins carp subjected to washing in running cold water temperature of 18°C for 10 minutes to remove from the surface of the raw material mucus, dirt, extract ballast globulin and albumen proteins. Next skins carp crushed to a size of 2-3 mm manually, add 1 l of water, heat the mixture in thermostat to 40°C and incubated for 50 min at the same temperature and periodic stirring. After this time the liquid fraction is filtered off.

The liquid fraction is used to highlight the hyaluronic acid and the solid is used to obtain collagen.

The liquid fraction is dried in a spray drier at a temperature of product at the outlet of the apparatus 61°C for 15 minutes After drying, the product is a white amorphous powder, soluble in water. The output of biologic - 12% in terms of SV. Hyaluronic acid is stored in dried form at a temperature of 2°C for 12 months.

The solid fraction is subjected to bleaching,to do this, prepare a solution of 3%hydrogen peroxide and sodium chloride, taken from the calculation: 1 l of 3%hydrogen peroxide is introduced 20 g of sodium chloride and pour this solution on the solid fraction for 12 hours, so that all the solid fraction was covered peroxide-salt solution. After this time the peroxide-salt solution is drained.

Then bleached pulp for swelling pour 1 liter of a 1.0%aqueous solution of sodium hydroxide for 24 hours at 20°C. After swelling the mass of the solid fraction is 1.5 kg

Then hold the neutralization of sodium hydroxide 3%boric acid solution for 10 minutes

For purification of the solid fraction from the ballast globulin and albumen protein and fat components are treated with a solution of enzyme preparation "Pancreatin". The solid fraction add 3 l of water, heat the mixture to 37°C, contribute to 7.5 g of Pancreatin and maintain a 2.0 h at the same temperature and periodic stirring. After this time the solid fraction was washed with cold running water to remove residual enzyme preparation.

After rinsing in water the resulting collagen frozen at minus 18°C or dried, and the drying is best conducted in the drying chambers with forced circulation of air and at a temperature of 18°C for 12 hours

The dried collagen stored in dry ventilated by which edenia at a temperature not exceeding 20°C for 24 months.

As can be seen from table 1, the proposed method allows to obtain hyaluronic acid and collagen in one process cycle, hyaluronic acid, after drying, is a white amorphous powder, soluble in water, the yield of hyaluronic acid is 10-12% in terms of MW, the output of collagen - 45-50% by weight of the crushed skins. In the process of water extraction a significant impact on the yield of hyaluronic acid has a temperature. When the temperature reaches 50°C maximum observed output of the biopolymer, the use of 3%hydrogen peroxide leads to a significant increase in interstitial pressure in the connective tissue, pushing its structural elements, causes the formation of multiple ties radical type between the active groups of the side chains of collagen molecules and allows you to achieve in the future better absorption of sodium hydroxide. Analysis of experimental data shows that upon receipt of collagen after 3 h after the beginning of the peroxide-alkaline hydrolysis the degree of swelling of the skins of fish is about 50%. The maximum value of the degree of swelling of the sample is observed after 24 h after the beginning of the hydrolysis. The mechanism of action of sodium hydroxide is in violation and the weakening of some of the hydrogen bridges, partial rupture of protein-carbohydrate member is, surrounding the bundles of collagen fibrils, the impact of sodium hydroxide and hydrogen peroxide promotes better penetration of alkali in the structure of collagen fibrils.

The impact of "Pancreatin" leads to a weakening and partial rupture of relations between protein, protein-lipid complexes, which leads to the extraction from the skins of mucopolysaccharides, leaching protein formations structureless substance of the dermis.

In the method for integrated processing of fish raw materials for the production of hyaluronic acid and collagen possible use of the enzyme preparation "Pancreatin" different manufacturers (Bovines, Russia; Mikos, Russia; OOO Indukern-Rus, Russia; Samson-Med, Russia; Hubei Maxpharm industries Co., China; Biozym Gesellshaft fur Enzymtechnologie GmbH, Germany, and others)that have similar enzymatic activity with the enzyme preparation "Pancreatin" Irbit himfarmzavod, Russia.

Drying of collagen best conducted in the drying chambers with forced air circulation at a temperature of 18-20°C for 12 H. the Advantage of this method is that the dried collagen in appearance corresponds to the given type of product, drying does not require additional power costs.

A water extraction for isolation of hyaluronic acid at a temperature above 50°C does not lead to substantially the mu-change output GUK, but creates conditions for the development of denaturation and coagulation processes, reducing the purity and quality of the drug. Temperatures below 50°C reduces the rate of extraction, and therefore slows down the whole technological cycle.

When using hydrogen peroxide at a concentration below 3%, does not achieve the desired effect, and the use of hydrogen peroxide in concentrations above 3%, results in excessive oxidant.

The proposed method allows to obtain hyaluronic acid and collagen for one cycle, to implement waste-free technology for the processing of secondary products cutting pond fish, rational use of Malostranske raw materials of the fishing industry to reduce the duration of the technological process, to use the skins of all fish species, including marine.

Table 1
Criteria comparisonThe placeholderA new technical solution
Name stages
The reception of the skin of pond fishNo dataSkins take lots
Flushing Ethyl alcohol with the purpose of bleeding cocks ' combs (raw material:ethyl alcohol 1:2)Rinse with cold running water (τ=10-15 min, t=18-20°C)
GrindingGrinding skins to a size of 2-3 mmGrinding skins to a size of 2-3 mm
ExtractionA solution of the enzyme preparation "Collagenase" content 0,035-0,040% by weight of raw materials for 45-50 minutes at a temperature of 45-50°C and a pH of 6.8 to 7.0.Water (τ=40-50 min, t=40-45°C)
FilteringVacuumMechanical filter
Deposition GUKThe deposition of 96%ethyl alcohol (liquid:ethanol 1:3)Not required
DryingFreeze-dryingDrying in a spray dryer (τ=15-25 min, t=60-65°C)
WhiteningNo data3%hydrogen peroxide solution, 2% sodium chloride, τ=12 h
SwellingMolochnosokovoj bath, containing 4-5% NaOH and 6-7% Na2SO4, LCD=3.1,0-1,2%solution of sodium hydroxide, τ=24 h, t=20-25°C
Neutralization90 minutes at a temperature of 18-20°C ammonium sulfate in an amount of 5% by weight of the feedstock under stirring3%boric acid solution, τ=10 min
Enzymatic processingNo dataProcessing enzyme preparation "Pancreatin" (t=37-40°C; τ=1.5 to 2.0 hours; dosage of 0.5-0.6% by weight of raw material, LCD=1-3)
The total time of the process (without drying or freezing)
There is insufficient dataLess than 2 days

Method for integrated processing of fish raw materials for the production of hyaluronic acid and collagen, which wash with cold running water for 10-15 minutes skins pond fish, grinding skins to a size of 2-3 mm, water extraction at a temperature of 40-45°C for 40-50 min at a ratio of crushed raw material:water 1:1 with periodic stirring, filtering, drying of liquid fraction in the spray dryer at a temperature of the product leaving Sushil is 60-65°C for 15-25 min with a production of hyaluronic acid, the solid fraction is subjected to bleaching within 12 h of peroxide-salt solution prepared by mixing 1 l of 3%hydrogen peroxide with 20 g of sodium chloride, processing bleached solid fraction 1,0-1,2%sodium hydroxide solution for 24 h at 20-25°C, followed by neutralization of the mixture 3%solution of boric acid, solid fraction enzyme preparation "Pancreatin", taken in an amount of 0.5-0.6% by weight solids, for 1.5-2.0 hours at a temperature of 37-40°C, washing the solid faction cold running water to remove residual "Pancreatin" with the receipt of collagen derived collagen, depending on destination, sent for drying in the drying chamber with forced air circulation at a temperature of 18-20°C for 12 hours or freeze up to a temperature of minus 18 to minus 20°C.



 

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FIELD: chemistry.

SUBSTANCE: disclosed are versions of a method of producing cross-linked polysaccharides, involving reaction of at least one polysaccharide selected from amino-polysaccharide, amino-functionalised polysaccharide containing one or more amino groups which can be cross-linked by reducing sugar, and combinations thereof, with at least one reducing sugar. The invention also discloses polysaccharides obtained using the disclosed method, a method of producing cross-linked matrices based on polysaccharides and matrices obtained using this method. The obtained matrices may include polysaccharide matrices and composite cross-linked matrices, including polysaccharides cross-linked with proteins and/or polypeptides.

EFFECT: obtained polysaccharides have satisfactory resistance to enzymatic degradation coupled with rheological properties of the preparation for injection, obtained matrices exhibit various physical, chemical and biological properties.

29 cl, 12 dwg, 6 tbl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: peptides consist of four amino-acid residues that are used for stimulation of collagen production with fibroblast.

EFFECT: invention allows effective stimulation of collagenoses in fibroblast cells.

11 cl, 3 dwg, 7 tbl, 6 ex

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