RU2562581C1 - Method of producing biologically active agent from sea cucumber, having general tonic and immunomodulating properties - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing a biologically active agent from sea cucumber. A method of producing a biologically active agent from sea cucumber, having general tonic and immunomodulating properties, includes crushing the cleaned muscular sac, mixing the material with water, treating the obtained mixture with ultrasound, performing enzymatic hydrolysis of the mixture with proteolytic enzyme preparations, inactivating the enzyme, filtering and drying at certain conditions.

EFFECT: method widens the range of biologically active substances obtained from sea cucumber, having general tonic and immunomodulating properties.

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Description

The invention relates to the food industry and can be used to obtain a biologically active product from the Far Eastern commercial holothuria of the cucumaria cucumaria japonica and the Far Eastern trepang Apostichopus japonicus containing hydrolyzed collagen and triterpene glycosides.

It is known that the muscular tissue of holothuria contains a large amount of collagen, which is involved in the regeneration of cells and the maintenance of the structure and function of connective tissues [Popov A.M., Artyukov A.A., Glazunov V.P. et al. Antitumor and anticoagulant activity of collagen protein from the holothuria Apostichopus japonicus modified with proteolytic enzymes // Marine Biology. 2011.Vol. 37, No. 3. S.217-222].

Holteruria triterpene glycosides (saponins) exhibit a wide spectrum of biological activity. The results of studies of a complex of glycosides isolated from Cucumaria frondosa showed that these compounds exhibit antitumor and immunomodulating activity, the effectiveness of which depends on the dose. Data on the safety for the human body of low concentrations of these compounds make it possible to consider promising the development of biologically active food additives based on them [http://otherreferats.allbest.ru/medicine/00149623_0.html]. Low molecular weight collagen and hexosamines are the precursors of articular cartilage macromolecules and their administration causes a stimulating effect and facilitates the regeneration of cartilage tissue by using ready-made building material and the ability to accumulate in the foci of inflammation [Boonen S., Aerssens J., Mbuyi-Muamba JM, Dequeker J. General osteoarthritis protecting against osteoporetic fractures: review of the evidence. J Orthopedic Rheumatol. 1996. V.9. P.69-73 ;. Marshall V.J., Clinical Biochemistry / V.D. Marshall under the editorship of N. Novikova. - M .: BINOM; St. Petersburg: Nevsky dialect, 1999. - 367 p.].

A known method of producing adaptogen (RF Patent 2255747). The method consists in the destruction and hydrolysis of marine animal raw materials from the group including sea urchin, crab, trepang, mussels, squid, beluga whales, followed by separation of the cytoplasmic fraction and the addition of a salt solution of a polyvalent metal of food acid, and then acidity correction dried and converted into a form suitable for storage. The disadvantage of the claimed solution lies in the multi-stage process of obtaining the final product and the low content of collagen in it.

There is a method for the integrated processing of holothuria (Patent application of the Russian Federation No. 2002120596), in which holothurians are cut, the entrails, corollas and intraperitoneal film are removed and boiled in sea, fresh or salted water for 5-15 minutes at a ratio of 1: 1 or 1: 2 ( by weight). Cooking in the same cooking medium of new portions of raw materials is carried out from 3 to 5 times, obtaining a semi-finished product for the manufacture of food products, selecting the cooking frequency so that the amount of solids in the cooking water is from 4 to 10%, and the heat-treated cucumaria is dried at a temperature of 10 to 65 ° C to a water content in the product of 5-12% to obtain a semi-finished product for the production of a food product. Cooking media is collected and dried in a dryer of any type at a temperature of 40-120 ° C to a water content of 5-12% to obtain Akmar biologically active food additive. The resulting product is used as an adaptogenic agent in the form of powder, tablets, as well as in encapsulated form. Dietary supplement "Akmar" is a gray powder with various shades of brown, obtained from dried cooking water during the complex processing of holothuria, and consists of protein - 32.0-42.0%, water - 5.0-12.0% , minerals - 50.0-55.0%, lipids - 0.05-0.2%, the amount of triterpene glycosides is in the range of 5000-7000 μg / g, amino sugars - 0.5-2.0%.

The disadvantage of this method is that collagen does not reach the cooking water, and in muscle tissue it is contained in a non-hydrolyzed high molecular weight state and is therefore insoluble.

A known method of obtaining funds of animal origin with anticoagulant action (RF Patent 2302250). The invention discloses an anticoagulant agent representing a collagen fragment with a molecular weight of about 12 kDa, additionally containing 10.0-12.0% carbohydrates, 6.0-7.5% sulfate groups, micro and macro elements. The method of obtaining funds consists in homogenization, extraction with distilled water at a pH of 8.0-8.5, then processing the extract with an enzyme preparation at a temperature of 35-37 ° C at a pH of 7.5-8.5 for 3.5-4, 5 hours, then adding ethanol to a mixture cooled to room temperature until it is 40% in the extract and then separating the formed filamentary precipitate by pressing on the filter, washing in succession with 40% and 96% ethanol and drying, then the product is dissolved in 0.05-0 , 1 M sodium bicarbonate solution, then fermentolysis is repeated in the same Slovenia; ethanol is added to the resulting solution to a final concentration concentration of 50%; the precipitate is squeezed out on a fabric filter, washed with a 50% solution of ethyl alcohol in distilled water, and then 96% ethyl alcohol and dried in air; then the product is dissolved in water and precipitated with acid, the precipitate is separated by centrifugation, dissolved in sodium bicarbonate, then the protein-containing solution is dialyzed or ultrafiltered on a membrane that passes substances with a molecular weight of 15 kDa, after which the desalted protein solution is separated by passing through a sterilizing membrane with a pore size of 0.2 μm, then the protein fraction passing through the membrane is collected, and the target product is dried.

The disadvantages of this method should be recognized as the complexity, multi-stage process, as well as the use of organic solvents, during the extraction of which the loss of triterpene glycosides occurs.

Closest to the technical solution is a method for producing hydrolysates from cucumaria (RF Patent No. 2095000 “Method for producing hydrolyzate from hydrobionts, a method for preparing a fermented milk product and a method for determining the amount of triterpene glycosides”), including grinding the purified muscle bag, mixing with water, adding to the crushed raw materials a proteolytic enzyme from the crab viscera in the amount of 1000-3500 PE per 1 kg of cucumaria, 3% sodium chloride and 0.1% sodium benzoate, hydrolysis at a temperature round 30-45 ° C for 14-24 hours, followed by inactivation of the enzyme by heating and separation of the liquid part by filtration.

The disadvantages of this method is the long duration of the fermentolysis process, the addition of preservatives, as well as the lack of information about the content in the liquid part of the product, separated from the hydrolyzate, collagen, including low molecular weight.

The objective of the proposed solution is the development of a new effective way to obtain a biologically active agent from holothuria, with tonic and immunomodulating properties, containing both triterpene glycosides and low molecular weight collagen, while significantly reducing the preparation time of the finished product and expanding the spectrum of biologically active substances obtained from commercial holothuria

To solve the problem, in a method for producing a biologically active agent from holothuria, having general strengthening and immunomodulating properties, including grinding raw materials — a cleaned muscle bag, mixing raw materials with water, processing with a proteolytic enzyme, inactivating the enzyme, filtering and drying, according to the invention, the crushed raw materials are mixed with water in a ratio of 1: 3-1: 5, before enzymatic hydrolysis is treated with ultrasound with a power of 150-200 W / cm ³ for 5-15 minutes, and enzymatic hydrolysis is carried out infused with proteolytic enzyme preparations: either protamex, or megaterin, or esperase, or savinase in an amount of 1.5-3 P / E per 1 g of raw material, at a pH of 7.9-8.1 for 2.5-3.5 hours

The method is as follows:

Freshly caught or frozen raw materials - commercial holothurians are exempted from internal organs, which are sent to receive preparations containing carotenoids. The cleaned muscle bag is crushed to a homogeneous state on a spinning top or meat grinder with a diameter of 2.0 mm of the grill holes. The crushed raw materials are mixed with water in the ratio of raw materials: water 1: 3-1: 5, sonication is performed at a power of 150-200 W / cm ³ for 3-15 minutes to suppress microbiological processes, increase the degree of grinding of raw materials and destruction of tissue level structures . The pH of the extract was adjusted to 7.9-8.1 with a 0.1 M sodium hydroxide solution. Then carry out enzymatic hydrolysis using proteolytic enzyme preparations or protamex, or esperase, or savinase in a ratio of 1.5-3 P / E per 1 g of raw material. Fermentolysis conditions: temperature 35-42 ° С, time 2.5-3.5 h. After fermentolysis, the extract is kept at 70-80 ° C for 15-25 min to inactivate the enzyme and filtered through calico, after which the filtrate is sent for freeze-drying or drying of a different type at a temperature of the final product not higher than 45 ° C and residual moisture of the dry product not more than 10%. The dry product is ground to a powder state and encapsulated.

The final product obtained by the proposed method is a powder from light to dark gray in color, containing low molecular weight collagen, triterpene glycosides as the main biologically active components, which determines its general strengthening and immunomodulating properties.

A biologically active product is obtained that has general strengthening and immunomodulating properties, in the form of a dry powder, which contains:

Protein, including collagen, wt.%, Not less than 70/42 Low molecular weight collagen, in% of the total content collagen, not less 60 Triterpene glycosides, mg / g, not less than 5 Water, wt.%, No more 10

In the study of the composition of the obtained product, the following methods were used. The amount of protein was determined by Kjeldahl (Lurie I.S., Skokan L.E., Tsitovich A.P. Technological and microbiological control in the confectionery industry. Reference book. M .: KolosS. - 2003. - 416 p.), Fractionation of collagen was carried out using gel electrophoresis in 7% SDS page (L. Osterman. Methods for the study of proteins and nucleic acids. Electrophoresis and ultracentrifugation. - M .: Nauka, 1981, 287 p.), determination of triterpene glycosides (Chumak AD, Pavel K.G., Timchishina G.N. Determination of triterpene glycosides in holoturia // News of the TINRO Center. - 199 5. - T. 118. -P.36-40).

The product can be used to prepare a biologically active food supplement, which is a source of low molecular weight collagen, triterpene glycosides, used as a general strengthening agent for increased physical exertion, for recovery from injuries and surgeries [Shulgina L.V., Blinov Yu.G., Zagorodnaya G.I. et al. New products for surgical patients based on a hydrolyzate from cucumaria. // Sat materials of the Russian scientific. conf. "New biomedical technologies using dietary supplements." - Vladivostok, 1998. - P.168 - 171; Goncharenko O.G., Grossman N.V. Medical and preventive nutrition from cucumaria. // Nutrition issues. - 1994. - No. 4. - S. 38 - 39; Prokopenko D.V., Glotova I.A. Prospects for the creation of functional beverages for athletes // Journal "Modern high technology" No. 3, 2010, pp. 66-67; Melnikov V.I. Sports Pharmacology: Dietary Supplements Derived from Seafood // Theory and Practice of Physical Culture - 2007 - No. 9, p. 44-45].

The technical result provided by the invention is to develop a new method for producing a biologically active agent from holothuria, which has general strengthening and immunomodulating properties, while significantly reducing the preparation time of the finished product (BAS) due to the use of ultrasonic treatment and new conditions for the enzymatic hydrolysis of raw materials - the muscular sac of holothuria. In addition, the range of biologically active substances obtained from commercial holothurians is expanding.

Ultrasonic disintegration can be used as the main method of destruction of the cell membrane. The destruction of cell structures (lysis) using ultrasound is used to extract intracellular compounds. With intense ultrasonic action, enzymes and proteins can be released from cells or subcellular organelles as a result of destruction of the cell structure. In this case, a substance that must be dissolved in a liquid is in an insoluble state. To extract it, it is necessary to open the cell membrane. Ultrasound allows you to achieve greater penetration of the solvent into the tissue and improve mass transfer. Ultrasonic waves cause cavitation in the liquid, destroy the cell walls and promote the release of components of the cell matrix, as well as provide lysis of the cell walls of microorganisms (http://bio-x.ru/articles/ultrazvukovaya-dezintegraciya-metod-razrusheniya-kletochnoy-obolochki-drozhzhey )

The need for processing parameters arises from studies performed by the authors of this application regarding the identification of the degree of disintegration of raw materials under the influence of ultrasonic treatment, the degree of protein hydrolysis during enzymatic hydrolysis, the transition of biologically active components to a soluble state, shown in tables 1, 2 and 3.

Ultrasonic processing of the extracts was carried out using an IKASONIC U 50 control device, with an exposure intensity (power) of 100-250 W / cm ³ for 3-15 minutes. The high power of ultrasonic exposure causes an increase in the temperature of the processed raw materials, which affects the native structure of proteins and can cause their denaturation. As is known from literature, denatured collagen is less susceptible to hydrolysis. Therefore, when studying the effect of ultrasonic exposure on the temperature of the processed raw materials, power and duration were selected that did not cause a significant increase in temperature. As can be seen from the table, a decrease in the power of ultrasonic exposure and an increase in intensity does not allow us to obtain the desired degree of destruction of biopolymers and an increase in the content of solids in the liquid part, an increase in power above predetermined limits leads to an increase in the amount of liquid fraction, but at the same time, some protein components denature goes into an insoluble state, and the amount of dissolved solids decreases. The best results of the disintegration of raw materials, under which there is no strong heating, are achieved under the following conditions: with a power of 150 W / cm ³ and a time of 10-15 minutes, a power of 200 W / cm ³ and a time of 5 minutes

In enzymatic hydrolysis, collagen molecules are destroyed to free amino acids and di-, tri-, tetra- and polypeptides with a molecular weight of less than 5-10 kDa. The degree of collagen hydrolysis was evaluated by the accumulation of free amino groups by the method of formol titration and low molecular weight protein fragments by gel electrophoresis in the final product using various enzyme preparations. The degree of hydrolysis should not be lower than 35%, and the content of low molecular weight collagen should be at least 60% of the total collagen content.

Initially, enzymatic hydrolysis was carried out on raw materials without preliminary ultrasonic treatment (Table 2).

Table 3 presents the results on the content of free amino groups and low molecular weight fractions of collagen after processing the raw materials with ultrasound and subsequent enzymatic hydrolysis. Exposure to ultrasound with a power of 200 W / cm ³ for 5 min allows obtaining fermentolizates from holothuria tissue with a higher degree of protein hydrolysis and increasing the amount of hydrolyzed collagen (with a molecular weight of less than 10 kDa).

The results allow us to conclude that the destruction of connective tissue proteins (collagen) and the hydrolysis of an interstitial substance containing proteoglycans under the influence of ultrasound, which contributes to a deeper degree of hydrolysis during processing with enzyme preparations.

Thus, the objective of the invention was solved by using the processing of raw materials by ultrasound before enzymatic hydrolysis, which contributed to the destruction of cell membranes, protein extraction and a change in their structure for greater protein availability as a substrate for enzyme preparations (http://selo-delo.ru/12 -vliyanie-ultrazvukovogo-oblucheniya).

Case Studies

Example 1

1 kg of freshly frozen purified muscle bag of sea cucumber is ground in a spinning top or meat grinder, add 3 l of water. The mixture is stirred and sonicated at 25 Hz at 100 W / cm ³ for 10 minutes. The pH of the extract was adjusted with a 1 M sodium hydroxide solution to 8.0. Then enzymatic hydrolysis is carried out by adding to the mixture 100 ml of a protamex solution with a proteolytic activity of 300 U / g at a concentration of 0.1 g / ml. The mixture was kept at 37 ° C for 3 hours. Then the mixture was heated to a temperature of 80 ° C and held for 20 minutes to inactivate the enzyme. The mixture is filtered through calico and sent to freeze-drying or drying of a different type at the temperature of the final product not higher than 45 ° C and the residual moisture content of the dry product not more than 10%. The dry product is ground to a powder state and encapsulated. The final product contains protein, including collagen - 67/40%, low molecular weight collagen with a molecular weight of less than 5 kDa - 60% of the total collagen content, triterpene glycosides - 7 mg / g.

Example 2

Raw trepang is cut, removing peri-pharyngeal corollas and entrails.

2 kg of raw material - peeled and washed muscle bag of sea cucumber is ground in a meat grinder, add 8 l of water. The mixture is stirred and sonicated at 30 Hz at 150 W / cm ³ for 5 minutes. The pH of the extract was adjusted with a 1 M sodium hydroxide solution to 7.9. Then, enzymatic hydrolysis is carried out by adding to the mixture 100 ml of a solution of megaterina with proteolytic activity of 250 U / g with a concentration of 0.24 g / ml. The mixture was kept at 40 ° C for 3.5 hours. The mixture was then heated to 80 ° C and held for 20 minutes to inactivate the enzyme. The mixture is filtered through calico and sent to freeze-drying or drying of a different type at a temperature of the final product not higher than 45 ° C and a residual moisture content of the dry product of 10%. The dry product is ground to a powder state and encapsulated. The final product contains protein, including collagen - 69/41%, low molecular weight collagen with a molecular weight of less than 5 kDa - 64% of the total collagen content, triterpene glycosides - 6 mg / g.

Example 3

1 kg of freshly frozen raw materials - the cleaned muscle bag of cucumaria is ground in a meat grinder and 5 l of water are added. The mixture is stirred and sonicated at 30 Hz 300 W / cm ³ for 8 minutes. The pH of the extract was adjusted to pH 8.1 with 1 M sodium hydroxide solution. Then enzymatic hydrolysis is carried out by adding to the mixture 25 ml of esperase solution with a proteolytic activity of the enzyme of 60 U / ml. The mixture was kept at 42 ° C for 2.5 hours. The mixture was then heated to 80 ° C and held for 20 minutes to inactivate the enzyme. The mixture is filtered through calico and sent to a freeze-drying or other type of drying at a temperature of the final product of 45 ° C and a residual moisture content of the dry product of 10%. The dry product is ground to a powder state and encapsulated. The final product contains protein, including collagen - 72/43%, low molecular weight collagen with a molecular weight of less than 5 kDa - 65% of the total collagen content, triterpene glycosides - 13 mg / g.

Example 4

2 kg of raw cucumaria are cut, purifying the muscular bag from the viscera, and crushing it in a meat grinder. 6 l of water is added to the ground mass, mixed thoroughly and sonicated at 25 Hz 300 W / cm ³ for 10 minutes. The pH of the extract was adjusted to pH 8.0 with 1 M sodium hydroxide solution. Then enzymatic hydrolysis is carried out by adding to the mixture 25 ml of a solution of savinase with a proteolytic activity of 60 U / ml. The mixture was kept at 42 ° C for 3.5 hours. The mixture was then heated to 80 ° C and held for 20 minutes to inactivate the enzyme. The mixture is filtered through calico and sent to freeze-drying or drying of a different type at the temperature of the final product not higher than 45 ° C and the residual moisture content of the dry product not more than 10%. The dry product is ground to a powder state and encapsulated. The final product contains protein, including collagen - 74/44%, low molecular weight collagen with a molecular weight of less than 5 kDa - 68% of the total collagen content, triterpene glycosides 12 mg / g.

Claims (1)

A method of obtaining a biologically active agent from holothuria having general strengthening and immunomodulating properties, including grinding raw materials - a cleaned muscle bag, mixing raw materials with water, processing with a proteolytic enzyme, inactivating the enzyme, filtering and drying, characterized in that the crushed raw materials are mixed with water in a ratio of 1 : 3-1: 5, before enzymatic hydrolysis is treated with ultrasound with a power of 150-200 W / cm ³ for 5-15 minutes, and enzymatic hydrolysis is carried out by enzyme preparations pr oteolytic effect: either protamex, or megaterin, or esperase, or savinase in an amount of 1.5-3 P / E per 1 g of raw material, at a pH of 7.9-8.1 for 2.5-3.5 hours.