RU2461384C1 - Method for preparing biologically active concentrate of preserved antlers - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely a method for preparing a biologically active concentrate of preserved antlers. The method for preparing the biologically active concentrate of preserved antlers consisting in grinding and two-stage enzymatic hydrolysis of antlers to be followed by extraction; at the first stage hydrolysis in the presence of the pepsin enzyme is followed by centrifugation and filtration of the hydrolysate, while the second stage includes enzymatic hydrolysis of cake prepared by the first stage of fermentation in the presence of the papain enzyme; further the cake prepared by is extracted centrifugation and filtration of the hydrolysate after the second stage of fermentation that is followed by centrifugation and filtration; thereafter all the filtrates are dried under certain conditions.

EFFECT: method provides higher end product yield and highest possible extraction of the biologically active substances.

2 cl, 5 tbl

Description

The invention relates to medicine and veterinary medicine, more specifically to the technology of processing antlers to obtain a biologically active concentrate in the form of a powder.

A known method of obtaining a biologically active concentrate from raw deer antlers, including grinding (thawed and dried at room temperature) antlers to particles with a size of 100 μm, mixing the crushed antlers with water at a ratio of 1: 5, homogenizing the mixture and extraction for 4 hours at a temperature of 90 -93 ° C followed by enzymatic hydrolysis at a temperature of 38-40 ° C and pH 4.5 in the presence of papain with an activity of 30 thousand units based on 1-2% of the weight of antlers for 5 hours. After hydrolysis is complete, the hydrolyzate is centrifuged, filtered and dried at a temperature of 45-50 ° C and a vacuum of 0.9 atm (see patent RU No. 2386444, IPC A61K 35/32).

However, the developed method relates to the processing of raw antlers, which initially have a large (compared to canned antlers) amount of moisture. In addition, high-temperature extraction at the beginning of the process can cause irreversible denaturation of proteins and amino acids.

A known method of producing a biologically active concentrate from antlers of deer, including enzymatic hydrolysis of antlers with a degree of grinding of not more than 100 microns in the presence of water and homogenate of the animal’s pancreas at the rate of 100 g of gland per 1 kg of antler flour, followed by aqueous extraction of the hydrolyzate (mixture of liquid fraction and gland ) and its centrifugation and filtration. As an enzyme, take the pancreas of antler deer when breeding antler meal: water 1: 1.5 and the fermentation time 3-4 hours at a temperature of not more than 50 ° C, and the hydrolyzate is extracted at a weight ratio of hydrolyzate: water 1: 1.5 v for 3 hours and after centrifugation and filtration, drying is performed at a temperature not exceeding 50 ° C and a pressure of 0.9 atm (see patent RU 2386443, IPC A61K 35/32) (prototype).

However, the use of antler deer as a pancreatic enzyme is limited due to the current lack of industrial production of this enzymatic complex. The shelf life of fresh pancreas is quite limited, and high-temperature extraction of the hydrolyzate (mixture of liquid fraction and oilcake) causes the destruction of newly formed useful substances. It should also be noted that the yield of the finished concentrate according to both known methods does not exceed 12.5-13.0% (see article: V.G. Lunitsyn, V.A. Chelakh Method for producing antler concentrates, their biochemical composition // Mat. international scientific and practical conference, Altai State Agrarian University, Barnaul. - 2009. - T.3. - S.131-134).

It is necessary to develop a method for producing a biologically active concentrate from canned antlers, which ensures the maximum possible extraction of biologically active substances from canned antlers with a high yield of the finished product.

The specified technical result is achieved in that the method for producing a biologically active concentrate from canned antlers is characterized in that the antlers are crushed to particles of 100 μm and subjected to enzymatic hydrolysis followed by extraction. In this case, enzymatic hydrolysis is carried out in two stages, in the first stage in the presence of the pepsin enzyme at the rate of 2% of the antlers weight with the antler: water ratio of 1: 3 for 4 hours and a temperature of 40 ° C, followed by centrifugation and filtration of the hydrolyzate. In the second stage, the enzymatic hydrolysis of oilcake obtained after the first stage of fermentation in the presence of the papain enzyme with an activity of 30 thousand units is carried out at a rate of 2% of the initial weight of the antlers at a ratio of oil: water of 1: 1.5 for 4 hours at a temperature of 40 ° C, this extraction is subjected to cake obtained after centrifugation and filtration of the hydrolyzate after the second stage of fermentation at a ratio of cake: water 1: 5 for 3 hours and a temperature of 95-98 ° C. Drying of all filtrates is carried out at a temperature not exceeding 50 ° C and a vacuum of 0.9 atm. After each mixing of grinded antlers and oilcakes with water, the mixture is homogenized, and during fermentation, mixing is performed.

The essence of the claimed method lies in the fact that the optimization of the method for producing a biologically active concentrate is carried out by intensifying the process of enzymatic hydrolysis, based on the use of two enzymes (pepsin and papain) sequentially one after the other, acting on crushed antlers, while the priority of exposure to pepsin, and high-temperature only cake is subjected to extraction (solid fraction after centrifugation and filtration).

To identify the optimal parameters of the technological process for the production of biologically active concentrate, a series of experimental experiments was carried out. The first two experiments were aimed at identifying the optimal parameters of the fermentation process of canned antlers ground to particles with a size of 100 μm using pepsin (1 experiment) at pH -2 and papain (2 experiment) at pH 4.5 as an enzyme. After fermentation, the centrifuged cake was subjected to extraction with identical, for both experiments, parameters of the extraction process, namely, the ratio of cake: water 1: 5, for 3 hours at a temperature of 95-98 ° C, followed by drying of the hydrolyzate and extract (after centrifugation and filtration ) at a temperature not exceeding 50 ° C and a vacuum of 0.9 atm. After each mixing of antlers and oilcakes, the mixture was homogenized, and during fermentation, mixing was performed.

Table 1 shows the results of the first experiment, namely the yield of the concentrate and its biological activity with the ratio of antlers: water 1: 1.5; 1: 3; 1: 4.5, fermentation time 3, 4, 5 hours and a pepsin content of 1.5, 2.0, 2.5% of the amount of milled antlers. The fermentation process was carried out at 40 ° C.

Table 1 The yield of concentrate and biological activity,% Experience number Enzyme The amount of enzyme,% The yield of concentrate,% and its biological activity,% with the ratio of antlers: water and fermentation time, hour 1: 1,5 1: 3.0 1: 4,5 3 four 5 3 four 5 3 four 5 1,5

one pepsin 2.0

2.5

Note: numerator - concentrate yield,%; denominator - biological activity,%

As can be seen from table 1, the maximum yield and biological activity of the finished concentrate was observed at the following process parameters: the amount of pepsin - 2% by weight of the antlers, with the ratio of preserved antlers: water 1: 3 and the duration of the fermentation process 4 hours followed by (after centrifugation and filtration) by extraction of oilcake at a ratio of oilcake: water 1: 5 for 3 hours at a temperature of 95-98 ° C and drying the filtrates at a temperature not exceeding 50 ° C and a vacuum of 0.9 atm.

Table 2 shows the similar results of the second experiment, where papain was used as the enzyme, and due to the specificity of its properties and according to preliminary studies, the fermentation time was 4, 5, and 6 hours.

table 2 The yield of concentrate and biological activity,% Experience number Enzyme The amount of enzyme,% The yield of concentrate,% and its biological activity,% with the ratio of antlers: water and fermentation time, hour 1: 1,5 1: 3.0 1: 4,5 four 5 6 four 5 6 four 5 6 1,5

one papain 2.0

2.5

Note: numerator - concentrate yield,%; denominator - biological activity,%

The optimal technological parameters of the process, as can be seen from the results of table 2, when using papain as an enzyme are: the amount of papain is 2%, the ratio of antlers: water is 1: 1.5, and the fermentation process takes 6 hours.

Pepsin is a proteolytic enzyme of the hydrolase class, which breaks down central peptide bonds in protein and peptide molecules to form simpler peptides and free amino acids, such as tyrosine and phenylalanine, but, unlike other proteolytic enzymes, it does not have strict specificity. The action of pepsin is carried out in an environment with a pH of 1-2.

Papain - is able to hydrolyze almost any peptide bonds and break down proteins deeper than proteases of animal origin, with the exception of bonds formed by proline residues. Moreover, it contains 212 amino acid residues in its composition, thereby deliberately enriching the finished product. The action of papain is carried out in an environment with a pH of 4.5.

The third and fourth experiments were aimed at identifying the possibility of enzymatic hydrolysis by two types of enzymes to intensify the process, as well as at identifying the optimal sequence for introducing enzymes into the process.

Table 3 shows the experimental (experiment 3) data of the process for producing a biologically active concentrate, upon receipt of which the canned antlers were treated first with pepsin at optimal process parameters (according to the first experiment), and then, after centrifugation and filtration of the hydrolyzate, the cake was exposed papain (in the technological mode of the second experiment).

Table 3 The yield of concentrate and biological activity,% Experience number enzyme The amount of enzyme,% Antler ratio: water Enzyme time, hour Exit
concentrate,% Biological activity,% 3 pepsin 2.0 1: 3 four - - 2.0 1: 1,5 four 43.5 38,4 papain 2.0 1: 1,5 5 40.9 35.6 2.0 1: 1,5 6 39.1 34.1

As can be seen from table 3, the optimal result for the yield of concentrate and its biological activity when using two enzymes (with the priority of introducing pepsin into the process) was 43.5% - the yield of concentrate and 38.4% - biological activity. The optimal mode is characterized by the following parameters: at the first stage - exposure to pepsin at a dose of 2% of the weight of the ground antlers with the ratio of antlers: water 1: 3 for 4 hours at a temperature of 40 ° C, at the second stage - the effect on the cake centrifuged and filtered from the hydrolyzate after the first stage with papain in the ratio of cake: water 1: 1.5 for 4 hours at a temperature of 40 ° C. The cake was extracted after the second stage according to the previously described technological regime.

The papain fermentation time (with an optimal result) was reduced by 2 hours compared to the second experiment, most likely due to the preliminary effect on antlers and pepsin, which actively cleaves almost all peptide bonds.

A similar experience, with the only difference being that papain was used as the first enzyme introduced into the process, is shown in Table 4.

Table 4 The yield of concentrate and biological activity,% Experience number enzyme The amount of enzyme,% Antler ratio: water Enzyme time, hour The yield of concentrate,% Biological activity,% four papain 2.0 1: 3 6 - - pepsin 2.0 1: 1,5 3 35,4 30.5 2.0 1: 1,5 four 32.6 28.8 2.0 1: 1,5 5 32,0 28.0

According to the results of the fourth experiment, where the yield of the concentrate and its biological activity were significantly lower than in the third experiment, it follows that the optimal sequence for introducing enzymes during joint enzymatic hydrolysis is: at the first stage of fermentation - pepsin, at the second - papain.

Based on four experiments, it should be noted that the optimal regime for obtaining a biologically active concentrate is the first stage of exposure to milled antlers with the enzyme pepsin in the amount of 2% of the antlers weight when the antlers are diluted (ratio): water 1: 3 at a fermentation time of 4 hours and after centrifugation and filtration of the hydrolyzate, the effect on the cake with pepsin (stage 2) in the amount of 2% of the initial weight of the antlers when diluting (ratio) the cake: water 1: 1.5 for 4 hours, followed by the extraction of centrifuged and about filtered (after the end of the second stage of fermentation) meal with a cake: water ratio of 1: 5 for 3 hours at a temperature of 95-98 ° C and drying of collected filtrates at a temperature not exceeding 50 ° C and a vacuum of 0.9 atm, table 5.

Example. One kilogram of canned antlers was crushed in a crusher, and then in a mill to particles with a size of 100 microns. Next, to 1 kg of antler flour was added 3 l of distilled water (1: 3 ratio) and 20 g of pepsin and the mixture was homogenized for 5 minutes, fermentation was carried out at 40 ° C for 4 hours after homogenization and stirring constantly, every 30 minutes, stirring , at the end of the process, the hydrolyzate was subjected to centrifugation and filtration. 20 g of papain with an activity of 30 thousand units were added to the resulting cake, mixed with water in a ratio of 1: 1.5, and fermented at 40 ° C for 4 hours with constant stirring, every 30 minutes, then the hydrolyzate was centrifuged and filtered. Water was added to the resulting cake in a ratio of 1: 5 and the mixture was extracted for 3 hours at a temperature of 95-98 ° C, followed by centrifugation and filtration. All the obtained filtrates were dried in a vacuum dryer at a temperature not exceeding 50 ° C and a pressure of 0.9 atm to a moisture content of 4-5%. The concentrate was packaged in a sealed vacuum packaging.

Thus, the technological methods of the claimed method for producing concentrate from canned antlers provide a high 43.5% yield of the concentrate (with its high biological activity - 38.4%), which is 30.8% higher than by known technologies.

Claims (2)

1. A method of obtaining a biologically active concentrate from canned antlers, characterized in that the antlers are crushed to particles with a size of 100 μm and subjected to enzymatic hydrolysis followed by extraction, while enzymatic hydrolysis is carried out in two stages, in the first stage in the presence of pepsin enzyme at a rate of 2% from the weight of antlers with the ratio of antlers: water 1: 3 for 4 hours and a temperature of 40 ° C, followed by centrifugation and filtration of the hydrolyzate, and in the second stage, enzymatic hydrolysis of the meal is carried out, after the first stage of fermentation, in the presence of the papain enzyme with an activity of 30 thousand units, at the rate of 2% of the initial weight of the antlers with the ratio of cake: water 1: 1.5 for 4 hours at a temperature of 40 ° C, then the cake obtained after centrifugation and filtration of the hydrolyzate after the second stage of fermentation at a ratio of cake: water 1: 5 for 3 h and a temperature of 95-98 ° C, followed by centrifugation and filtration, and all filtrates are dried at a temperature not exceeding 50 ° C and a vacuum of 0.9 atm to a moisture content of 4-5%. 2. The method according to claim 1, characterized in that after each mixing of the milled antlers and oilcakes with water, the mixture is homogenized, and in the fermentation processes, mixing is performed.