CN116178482B

CN116178482B - Method for optimizing ultrasonic-assisted extraction of Tianshan red deer abomasum glycoprotein based on response surface method and application

Info

Publication number: CN116178482B
Application number: CN202211562019.6A
Authority: CN
Inventors: 阿布力米提·伊力; 阿吉艾克拜尔·艾萨; 杨子; 艾合米丁·外力
Original assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Current assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Filing date: 2022-12-07
Publication date: 2024-07-16
Anticipated expiration: 2042-12-07

Abstract

The invention relates to a method for optimizing ultrasonic auxiliary extraction of Tianshan red deer abomasum glycoprotein based on a response surface method and application thereof. Compared with the traditional method, the method has simple extraction steps, mature operation, easy mass production expansion, increased protein and total sugar content of the extract and higher antioxidant activity, and is one of ideal methods for preparing the total glycoprotein of the red deer.

Description

Method for optimizing ultrasonic-assisted extraction of Tianshan red deer abomasum glycoprotein based on response surface method and application

Technical Field

The invention relates to a response surface method-based ultrasonic-assisted extraction method for optimizing the siren abomasum glycoprotein of Tianshan deer and application thereof.

Background

The Tianshan red deer abomasum refers to the fourth stomach of Tianshan red deer, and has the functions of ruminant, such as cattle, sheep, deer, etc., and digestion and absorption as the function of mammal. Therefore, the gastric mucosa surface of abomasum contains abundant proteins, enzymes, probiotics, prebiotics and the like. Has immunity enhancing, digestion promoting, antiinflammatory, and antioxidant effects. The traditional Chinese medicine considers that deer stomach can warm cold and nourish stomach, and has good effects on gastritis, stomachache, gastric ulcer, gastrectasia, gastroparesis, gastroptosis, gastric mucosa prolapse, gastric hyperacidity, gastric acid deficiency and thermal gangrene in bones. Meanwhile, the deer stomach also has the effects of tonifying middle-jiao and Qi, strengthening stomach and strengthening body constitution, promoting digestion and absorption, treating dyspepsia, tonifying deficiency and enriching blood, has high medicinal value and is also a good health food. The "Ji Min" is related to the method of eating deer meat and viscera, and the "Ben Cao gang mu" is related to the medicinal value of the fourth stomach of other ruminants such as sheep stomach and cattle stomach; the habit of eating deer stomach is also available at home and abroad in the society at present, which plays a role in tonifying.

Glycoprotein (glycoprotein) is a complex macromolecule formed by covalent linkage of oligosaccharide chains and polypeptide chains, the sugar backbone is short, the number of sugar residues is generally not more than 15, and in most cases, the sugar content is less than the protein content. Sugar content also varies depending on the kind of protein. Sugar chains are typically branched and may include: monosaccharides (e.g., glucose, galactose, mannose, xylose), amino sugars (sugars with amino groups such as N-acetylglucosamine or N-acetylgalactosamine and acidic sugars (sugars with carboxyl groups such as sialic acid or N-acetylneuraminic acid). As a result of the presence of hydroxyl groups in the sugar, glycoproteins are more hydrophilic than simple proteins, meaning that the glycoproteins are more soluble in water than ordinary proteins, the hydrophilicity of the molecule also results in the presence of a characteristic fold in the tertiary structure of the glycoprotein.

Glycoproteins are widely found in animals, plants and microorganisms, and are of a wide variety and function. Many studies report that glycoproteins have significant pharmaceutical and health-care effects and that glycoproteins play a role in the structure, reproduction, immune system, hormonal regulation and protection of cells and organisms. The lipid bilayer surface glycoproteins present in the cell membrane, their hydrophilicity enables them to function in an aqueous environment where they function in intercellular recognition and binding of other molecules. Whereas cell surface glycoproteins can play an important role in crosslinking cells and proteins (e.g., collagen) to increase the strength and stability of tissue; in addition to this, some hormonal components in the human body also belong to glycoproteins such as Human Chorionic Gonadotrophin (HCG) and Erythropoietin (EPO), prothrombin, thrombin and fibrinogen. At present, most of the pharmaceutical protein preparations which are used clinically and have high-efficiency immunological activity are glycoproteins, and the functional diversity is the most active field of the current glycobiology research.

Ultrasonic wave is a mechanical wave, and ultrasonic wave extraction can apply the comprehensive effects of cavitation, vibration, crushing, stirring and the like generated by ultrasonic wave to the natural product component extraction process, and the process of extracting cell contents is achieved by damaging cell walls. The ultrasonic wave is mainly extracted by 3 mechanisms: cavitation effects, thermal effects and mechanical effects. Among them, cavitation is considered to be the most important, and when a large amount of ultrasonic waves act on the extraction medium, weak tension areas exist in the liquid of the system, and the liquid in these areas is torn into a plurality of small cavities, and these small cavities are rapidly swelled and closed, so that violent impact action occurs between liquid particles. Therefore, compared with the traditional mechanical stirring method, the ultrasonic auxiliary extraction method can shorten the extraction time and improve the extraction efficiency. Besides, the ultrasonic extraction does not influence the structure and activity of the extract, is not limited by the polarity and molecular mass of the components, and is suitable for extracting most of the effective components. Simple and easy operation, less impurity in the extracted liquid, and easy separation and purification of the effective components.

And searching 51 related files which are obtained by taking the response surface method optimization process as a keyword and optimize the macromolecular natural compound extraction process by using the response surface method, wherein 38 polysaccharide directions are in the protein polypeptide direction, 13 protein polypeptides are in the glycoprotein direction, and 0 glycoprotein is in the glycoprotein direction. 2 related patents are searched by taking ultrasonic extraction glycoprotein as a keyword, and the patent publication number is: CN101317622A is prepared from rhizoma Dioscoreae by ultrasonic-assisted water extraction at 15-50deg.C and ethanol precipitation to obtain rhizoma Dioscoreae glycoprotein with a yield of above 60%. The application number 202110922724.1 uses lamb abomasum as a raw material to obtain the probiotic active glycoprotein, which is extracted by an ultrasonic auxiliary extraction method at the high temperature of 60 ℃, and the glycoprotein is obtained after ethanol precipitation and savage reagent deproteinization, and the yield is 13.8%.

The invention is further researched on the basis of the extraction work of various glycoproteins in the early stage, and most of glycoprotein extraction related patents in the early stage are mechanically stirred and extracted by using buffer solution, water or NaCl solution; because of the limitation of experimental cost, the optimal condition can be screened only under the limited experimental conditions, so that the extraction rate is low, the activity of the extract is not strong, the optimal condition is not screened, and other obvious problems exist. The invention has the innovation points that modern ultrasonic auxiliary extraction is added on the extraction method, the ultrasonic auxiliary glycoprotein extraction process is subjected to condition optimization on three factors of ultrasonic power, extraction liquid salt concentration and extraction temperature by combining computer fitting software, the optimal extraction condition is calculated by computer assistance, and the glycoprotein optimal extraction process of animal products is screened out. In addition, the invention also uses three mechanisms as indexes to compare the antioxidant activity of the ultrasonic auxiliary method and the common mechanical stirring extraction method. The final result shows that the glycoprotein extract with larger extraction amount and stronger antioxidant activity can be obtained by the method on the premise of saving time and cost.

Through a large number of searches: the patent number CN110511293B,CN101317622A、CN113861304A,CN113861304A,CN113980088A,CN113861307A,CN112724274A,CN112812199A,CN102690321A,CN112697913A,CN112724276A,CN112724276A and other patents relate to ultrasound-assisted extraction of macromolecular compounds by means of ultrasound response, wherein the extraction conditions are different and the influence on the final product is different.

Disclosure of Invention

The invention aims to provide a response surface method-based method for optimizing ultrasonic-assisted extraction of antioxidant glycoprotein of the abomasum of Tianshan red deer and application thereof. The method washes the surface of the abomasum of Anshan red deer, removes the mucous membrane part, and removes the water in the raw materials. Pulverizing dry red deer, sieving to separate impurities, degreasing with petroleum ether, separating fat-soluble part and water-soluble solid, naturally air drying insoluble solid, extracting with 0.02M Phosphate Buffer Solution (PBS) with pH 7 as extraction solution under ultrasonic assistance, optimizing conditions with response surface method, centrifuging after extraction, desalting supernatant, and drying to obtain red deer glycoprotein. Compared with the traditional method, the method provided by the invention has the advantages that the required time is obviously shortened, the content of the extracted protein and the total sugar is increased, and the method is suitable for large-scale preparation, and is one of ideal methods for preparing the Tianshan red deer abomasum glycoprotein.

The invention discloses a response surface method-based ultrasonic-assisted extraction method for optimizing the gastric glycoprotein of Tianshan red deer, which comprises the following steps:

a. washing the surface of the abomasum of the red deer, removing the mucous membrane part, dividing into squares with 5cm side length, freezing in a refrigerator with the temperature of-80 ℃ for 3 hours, removing the water in the raw materials by a freeze dryer after freezing, and weighing for later use;

b. Crushing the dry mucous membrane of the abomasum of the red deer in the step a in liquid nitrogen, sieving with a 40-mesh sieve, and storing in a refrigerator at the temperature of minus 40 ℃ by using a self-sealing bag;

c. Degreasing the Tianshan red deer abomasum mucosa powder obtained in the step b with petroleum ether for 3 times at normal temperature, performing magnetic stirring to assist degreasing each time, standing, and volatilizing a solvent under flowing air by using a lower layer precipitate to obtain Tianshan red deer abomasum mucosa degreasing powder;

d. Performing Box-Behnken Design by using Design Expert 11 software, extracting for 1 hour under the conditions of extracting temperature of 25-55 ℃ and extracting solution salt concentration of 0.3-0.9M, ultrasonic power of 300-700W and 6s off and 4s on for two times to obtain an extracting solution;

e. Centrifuging the Tianshan red deer abomasum glycoprotein extract obtained in the step d, removing residues, desalting with a dialysis bag with a cut-off of 3kKD, freezing the desalted extract, and freeze-drying at a temperature of-70 to-80 ℃ and a pressure of 10-20Pa to obtain the Tianshan red deer abomasum glycoprotein extract.

The application of the Tianshan red deer abomasum glycoprotein extract obtained by the method in preparing raw materials of antioxidant active products.

The invention relates to a response surface method-based ultrasonic-assisted extraction method for optimizing the glycoprotein of the abomasum of Tianshan deer and application thereof, wherein the method is characterized in that the surface of the abomasum of Tianshan deer is washed clean, the mucous membrane part is taken down, divided into squares, and the squares are put into a refrigerator at the temperature of minus 80 ℃ for freezing; removing water in the raw materials by a freeze dryer after freezing, and weighing for later use; crushing dry red deer, sieving to separate impurities, degreasing with petroleum ether, separating fat-soluble parts and water-soluble solids, naturally airing insoluble solids, using a buffer solution with the pH of 0.02M of 3-8 as an extraction solution, performing Box-Behnken Design by using Design Expert11 software, using the protein content of the extract as a response value, optimizing ultrasonic-assisted extraction of red deer abomasum glycoprotein by using a three-factor three-level response surface analysis method, and finally determining the extraction conditions as extraction temperature: after the extraction is finished, the extracting solution is centrifuged, dialysis and desalination are carried out by using a dialysis bag with the cutoff amount of 3kDa, and the dialysed extracting solution is subjected to vacuum freeze drying to prepare the Tianshan red deer glycoprotein. Finally, the extraction conditions are designed as follows: extraction temperature: 43 ℃, the salt concentration of the extracting solution is 0.57M/L, and the ultrasonic power is 445W. Under the condition, the sample extraction rate is 23.7%, the protein content is 33.4%, the total sugar content is 5.54%, and the uronic acid content is 2.53%. The extracted crude extract is subjected to infrared spectrum analysis and identified as glycoprotein substances through characteristic peaks.

Compared with the traditional method, the method for optimizing ultrasonic-assisted extraction of the red deer abomasum glycoprotein based on the response surface method has the advantages that the required time is obviously shortened, the content of the extracted protein and the total sugar is increased, the antioxidant activity is higher, meanwhile, the extraction steps are simple, the operation is mature, and the large-scale production is easy to realize, so that the method is one of ideal methods for preparing the red deer total glycoprotein. The invention can provide reference for the application and development of the Tianshan red deer glycoprotein in food and health products, and the obtained Tianshan red deer glycoprotein extract also provides basis for the subsequent separation and purification of glycoprotein monomer compounds.

The innovation point of the invention is that the extraction method of glycoprotein is researched from three dimensions of ultrasonic power, extraction temperature and salt concentration of an extracting solution, and simultaneously, the extracted compound is proved to be glycoprotein substances by combining various analysis methods such as infrared spectrum, monosaccharide analysis, amino acid analysis and the like, and the method is also laterally proved to be suitable for extracting glycoprotein substances, and the product has the characteristics of high extraction rate, higher protein content, higher sugar content and the like. In addition, the invention compares the antioxidant activity of the extract with that of the extract obtained by the traditional method through three indexes. The results show that glycoprotein compounds with higher activity can be obtained by ultrasound assistance. The extraction method is proved to be reasonable and effective. In addition, the invention is also a systematic study on the chemical composition level of the abomasum part of deer for the first time.

The ultrasonic-assisted extraction method and application of the Tianshan red deer abomasum glycoprotein based on the response surface method have the advantages that the obtained glycoprotein is higher in protein content than the traditional method, the extraction time is short, the method can be used for pilot scale production, the glycoprotein is subjected to ultrasonic-assisted extraction on the basis of retaining the original physical and chemical properties and nutritional ingredients of the glycoprotein, the extraction time is reduced, the obtained glycoprotein has higher antioxidant activity than the traditional method, and the defects that some compounds lose activity, the compounds decompose and the like caused by the traditional method are overcome. Active compounds are searched from natural products, and are new directions for development of modern medicine and food industries. The protein extraction rate of the natural active glycoprotein is improved, and the method has important practical significance for developing new natural product active products. The invention can provide reference for developing and pilot-scale industrial development of the Tianshan red deer abomasum glycoprotein active product.

The extraction method of the invention carries out ultrasonic auxiliary stirring extraction in a phosphate buffer solution containing a certain enzyme inhibitor and 0.3-0.9M sodium chloride pH7 at the temperature of 25-55 ℃ and with the power of 300-700W, the buffer solution can maintain the pH environment of the extraction solution, the glycoprotein denaturation caused by pH change in the extraction process is avoided, and the enzyme inhibitor is added, so that the degradation and inactivation of the active glycoprotein component in the extraction process can be avoided; the extraction temperature of 25-55 ℃ is close to the original normal physiological temperature of animal raw materials, which is more beneficial to extracting active ingredients and reducing degradation of the active ingredients; the extraction rate of the glycoprotein can be improved by properly increasing the salt concentration, and the salt-soluble glycoprotein can also be obtained; the proper ultrasonic assistance can quickly crush cells, and quicken the dissolution rate of active glycoprotein so as to save time cost; therefore, the preparation method of the Tianshan red deer abomasum glycoprotein active ingredient has the characteristics of easy amplification, high repeatability, high yield, low cost and no pollution.

Drawings

FIG. 1 is a graph of the response surface method of the present invention, 3-D plot, ultrasonic power and extraction temperature versus yield;

FIG. 2 is a graph of the response surface method of the present invention, 3-D plot, ultrasonic power and salt concentration versus yield;

FIG. 3 is a graph of the response surface method of the present invention showing the effect of salt concentration and extraction temperature on yield;

FIG. 4 is a graph of the effect of contour plot-ultrasonic power and extraction temperature on yield obtained by the response surface method of the present invention;

FIG. 5 is a graph of the effect of contour plot of ultrasonic power and salt concentration on yield obtained by the response surface method of the present invention;

FIG. 6 is a graph of contour plot obtained by the response surface method of the present invention versus salt concentration and extraction temperature versus yield;

FIG. 7 is an infrared spectrum of the extract obtained under the optimal conditions of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples and the accompanying drawings, but the purpose of these exemplary embodiments is to be construed as merely illustrative of the present invention and not limitative of the actual scope of the present invention in any way.

Example 1 (response surface)

d. Performing Box-Behnken Design by using Design Expert 11 software, taking the protein content of the extract as a response value, extracting for 1 hour under the conditions of extracting temperature of 25 ℃, extracting solution salt concentration of 0.9M, ultrasonic power of 300W,6s off and 4s on, and extracting twice to obtain extracting solution;

e. Centrifuging the Tianshan red deer abomasum glycoprotein extract obtained in the step d, removing residues, desalting with a dialysis bag with a cut-off of 3kKD, freezing the desalted extract, and freeze-drying under the condition that the temperature is-70 ℃ and the pressure is 10Pa to obtain the Tianshan red deer abomasum glycoprotein extract with the protein content of 22.61%.

Example 2

d. Performing Box-Behnken Design by using Design Expert 11 software, taking the protein content of the extract as a response value, extracting for 1 hour under the conditions of the extraction temperature of 55 ℃, the salt concentration of the extract of 0.3M, the ultrasonic power of 700W,6s off and 4s on, and extracting twice to obtain an extract;

e. Centrifuging the Tianshan red deer abomasum glycoprotein extract obtained in the step d, removing residues, desalting with a dialysis bag with a cut-off of 3kKD, freezing the desalted extract, and freeze-drying under the condition that the temperature is minus 75 ℃ and the pressure is 15Pa to obtain the Tianshan red deer abomasum glycoprotein extract with the protein content of 21.12%.

Example 3

d. Performing Box-Behnken Design by using Design Expert 11 software, taking the protein content of the extract as a response value, extracting for 1 hour under the conditions of extracting temperature of 40 ℃, extracting solution salt concentration of 0.6M, ultrasonic power of 500W,6s off and 4s on, and extracting twice to obtain extracting solution;

e. Centrifuging the Tianshan red deer abomasum glycoprotein extract obtained in the step d, removing residues, desalting with a dialysis bag with a cut-off of 3kKD, freezing the desalted extract, and freeze-drying under the condition that the temperature is minus 78 ℃ and the pressure is 20Pa to obtain the Tianshan red deer abomasum glycoprotein extract with the protein content of 31.97%.

Example 4

d. Performing Box-Behnken Design by using Design Expert 11 software, taking the protein content of the extract as a response value, extracting for 1 hour under the conditions of the extraction temperature of 43 ℃, the salt concentration of the extract of 0.57M, the ultrasonic power of 445W,6s off and 4s on, and extracting twice to obtain an extract;

e. Centrifuging the Tianshan red deer abomasum glycoprotein extract obtained in the step d, removing residues, desalting with a dialysis bag with a cut-off of 3kKD, freezing the desalted extract, and freeze-drying under the condition that the temperature is-80 ℃ and the pressure is 10-20Pa to obtain Tianshan red deer abomasum with glycoprotein extract content of 33.4%.

Example 5

Carrying out infrared spectrum analysis on the Tianshan red deer abomasum glycoprotein extract obtained in the examples 1-4, and identifying the Tianshan red deer abomasum glycoprotein extract as a glycoprotein compound through characteristic peaks; measuring the protein content, measuring the total sugar content by using a phenol sulfuric acid method, measuring the uronic acid content by using a m-hydroxybiphenyl method, and measuring the antioxidant activity by using DPPH free radical, ABTS free radical and hydroxyl free radical clearance as indexes;

the method comprises the steps of (1) extracting a red deer abomasum mucosa degreasing sample obtained in the step C under the condition of a table 1 by using Design-Expert11 software Design and using an ultrasonic cell crusher (Bian instruments limited company, shanghai, china), detecting the protein content of a red deer abomasum glycoprotein crude product extracted by using a BCA method, and finally performing multiple regression fitting on the data to determine a binary multiple regression equation of the red deer abomasum glycoprotein protein extraction rate (Y) and the extraction temperature (A), the extract salt concentration (B) and the ultrasonic power (C):

Y＝0.966568+0.666113*A+39.18895*B+0.027281*C+0.176731*A*B-0.000036*A*C+0.021998*B*C-0.008765*A^2-49.43639*B^2-0.000043*C^2;

Table 1 response surface conditions:

Evaluating the significance and the fitness of the regression model by using an analysis of variance, wherein the obtained analysis of variance results are shown in a table 2, and a curve graph (fig. 1-3) and a contour graph (fig. 4-6) of a response surface; the results show that: the P value (possibility) of the model is less than 0.0001, which indicates that the model has obvious response to the extraction rate; the model F value is 17.74, which shows that the test accuracy is higher; the P value of the mismatch is 0.1817, the F value is 2.38, which shows that the mismatch is not obvious relative to the pure error, and the validity of the model is verified; in the regression model, the correlation coefficient R2 is 0.9410, the adjusted determined coefficient adj-R2 is 0.8880, and the variation Coefficient (CV) is 4.76%, which indicates that the experimental value and the predicted value have certain correlation; analysis of variance shows that: variables A, B, C, AB, A2, B2, C2, all have an effect on the protein content of the extract; the order of the P values of the three independent variables was B > a > C, indicating that the effect of ultrasonic power (C) on yield was greatest, while the effect of salt concentration (B) of the extract on protein content was least. However, the interaction terms of salt concentration and ultrasonic power (BC) have a significant effect on yield (P < 0.05);

TABLE 2 regression model analysis of variance

A, extraction temperature (DEG C); b, salt concentration (M/L) of the extracting solution; ultrasonic power (W); r2= 0.9410; adj-r2= 0.8880;

Verification experiment:

According to design expert11 fitting calculation, the theoretical optimal extraction scheme is as follows: extracting temperature 42.841 ℃, salt concentration 0.572M/L, ultrasonic power 444.638W, and modifying conditions set by experimental operation to be extracting temperature 43 ℃, salt concentration 0.57M/L and ultrasonic power 445W; the experiment is repeated for 3 times, the content of the Tianshan red deer abomasum glycoprotein is 33.4%, and the theoretical value obtained by calculation of the response surface method is approximated, so that the regression equation obtained by optimization of the response surface method has guiding significance.

Example 6 (magnetic stirring extraction method, comparative)

A. Washing the surface of the abomasum of Anshan red deer, removing the mucous membrane, dividing into squares with 5cm side length, and freezing in a refrigerator at-80deg.C for 3 hr. Removing water in the raw materials by a freeze dryer after freezing, and weighing for standby.

B. c, crushing the Anshan red deer abomasum anhydrous mucous membrane obtained in the step a in liquid nitrogen, sieving with a 40-mesh sieve, and storing in a refrigerator at the temperature of minus 40 ℃ by using a self-sealing bag;

d. c, extracting the sample obtained in the step c twice under the conditions that the extraction temperature is 43 ℃ and the salt concentration of an extracting solution is 0.57M/L by using a magnetic stirrer for 60min each time;

e. centrifuging the Tianshan red deer abomasum extract obtained in the step d, removing residues, desalting with a dialysis bag with a cut-off of 3kKD, freezing the desalted extract, and freeze-drying at a temperature of-70 to-80 ℃ and a pressure of 10-20Pa to obtain Tianshan red deer abomasum glycoprotein extract.

Carrying out infrared spectrum analysis on the crude extract extracted under the optimal condition, and identifying the crude extract as glycoprotein compounds through characteristic peaks;

The protein content of the obtained glycoprotein crude extract was measured by BCA method, the total sugar content was measured by phenol sulfuric acid method, the uronic acid content was measured by m-hydroxybiphenyl method, and the antioxidant activity was measured by DPPH radical scavenging rate as an index.

Example 7

Infrared spectroscopic analysis of crude extracts of the abomasum of Tianshan deer obtained in examples 1-4 and example 6 was carried out to determine the type of compound (FIG. 7)

The experimental method comprises the following steps: and adopting a potassium bromide tabletting method to carry out infrared spectrum detection. Mixing the extract with dry potassium bromide, grinding into fine powder particles, and scanning with infrared spectrophotometer in the range of 400-4000cm ^-1.

Example 8

Content measurement and comparison of the Tianshan red deer abomasum glycoprotein extracts obtained in examples 1 to 4 and example 6: the results are shown in Table 3;

The experimental method comprises the following steps:

Protein content detection: standard bovine serum albumin was used as a standard curve using BCA kit method, protein samples: mixing the working solutions in a ratio of 1:8, adding the mixture into a gold 96-well plate, incubating the mixture for 30min at 37 ℃, detecting absorbance at the wavelength of 562nm by using an enzyme-labeled instrument, and carrying the absorbance of the sample into a standard curve to obtain the concentration of the sample;

Sugar content detection: using the phenol sulfuric acid method, glucose standard was used as a standard curve according to 4:6:3, adding 5% phenol solution and concentrated sulfuric acid into a 96-well plate, incubating at 95 ℃ for 10min, detecting absorbance at 490nm by using an enzyme-labeled instrument, and bringing the absorbance of the sample into a standard curve to obtain the concentration of the sample;

Determination of uronic acid content: using m-hydroxybiphenyl method, taking galacturonic acid as standard substance as standard curve, adding 200 μl sample into 1.5ml centrifuge tube, adding 1ml sodium tetraborate-sulfuric acid solution under ice bath condition, shaking, reacting in boiling water bath for 15min, cooling to room temperature, adding 15 μl m-hydroxybiphenyl solution, mixing, reacting at room temperature for 40min, transferring 250 μl into 96-well plate, detecting absorbance at wavelength 525nmc with enzyme-labeled instrument, and substituting sample absorbance into standard curve to obtain sample concentration.

Example 9

The DPPH radical scavenging rate comparison results of the Tianshan red deer abomasum glycoprotein extracts obtained in examples 1 to 4 and example 6 are shown in Table 3;

The experimental method comprises the following steps:

Scavenging DPPH radical activity: placing 100 μl of DPPH ethanol solution into a 96-well plate, adding the same amount of sample solution, mixing, performing parallel experiments on each sample for 3 times, performing light-shielding reaction at 37 ℃ for 30min, measuring absorbance at 517nm by using an enzyme-labeled instrument, calculating DPPH free radical clearance according to the following formula, and measuring sample concentration at 5mg/ml;

Wherein A ₀ is absorbance of a blank group system without adding a sample; a _i is the absorbance of the system added to the sample experimental group; a _j is the absorbance of the control group containing only the sample;

Example 10

The results of comparing the ABTS radical clearance rates of the Tianshan red deer abomasum glycoprotein extracts obtained in examples 1-4 and example 6 are shown in Table 3;

The experimental method comprises the following steps:

Scavenging DPPH radical activity: placing 100 μl of ABTS free radical aqueous solution (absorbance is 0.78 at 734nm of ultraviolet) into a 96-well plate, adding the same amount of sample solution, mixing, performing parallel experiments on each sample for 3 times, performing light-proof reaction at room temperature for 10min, measuring absorbance at 734nm with an enzyme-labeled instrument, calculating the ABTS free radical clearance according to the following formula, and measuring sample concentration at 5mg/ml;

example 11

Results of comparing the hydroxy radical clearance of the Tianshan red deer abomasum glycoprotein extracts obtained in examples 1-4 and example 6 are shown in Table 3;

The experimental method comprises the following steps:

Scavenging hydroxyl radical activity: taking 70 mu l of salicylic acid-ethanol solution and 70 mu l of 0.1% hydrogen peroxide solution, placing the solution into a 96-well plate, adding 70 mu l of sample solution, uniformly mixing, performing 3 times of parallel experiments on each sample, reacting for 30min at room temperature, measuring the absorbance at 510nm by using an enzyme-labeling instrument, and calculating the hydroxyl radical clearance according to the following formula, wherein the concentration of the sample is 5mg/ml;

TABLE 3 ultrasound assisted extraction and comparison of chemical Properties of traditional extraction methods

Note that: 1. the extraction time is 1 hour, the extraction is twice, and the feed-liquid ratio is 1:30.

Example 12

The glycoprotein extracts obtained in examples 1 to 4 and example 6 were subjected to monosaccharide analysis, and the analysis results are shown in Table 4;

The experimental method comprises the following steps:

Sample acid hydrolysis: placing 5mg of the sample into a 20ml screw cap (with a built-in spacer) fine mouth bottle, adding 4ml 2mol/l trifluoroacetic acid, screwing the bottle cap, shaking uniformly, reacting at 110 ℃ for 6 hours, evaporating to dryness, dissolving with methanol, repeating for 3-4 times, and removing the trifluoroacetic acid.

PMP pre-column derivatization: dissolving the spin-dried sample and the mixed standard substance in water, taking 150 mu L of the mixture and adding the mixture into a 4ml centrifuge tube, adding 150 mu L of 0.6mol/L sodium hydroxide and 300 mu L of 0.5 mol/L1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution, uniformly mixing, reacting at 70 ℃ for 100min, and adding 150 mu L of 0.6mol/L hydrochloric acid for neutralization after the reaction is finished. Adding chloroform with equal volume, extracting, collecting water layer, repeating for three times, removing upper layer, and standing.

HPLC monosaccharide composition analysis: and (3) performing gradient elution on the derivatized sample and the mixed standard substance by setting a time program on a PBS solution with 17% acetonitrile in the A phase and a 10% acetonitrile in the B phase. And comparing the peak-out time of the mixed standard substance to judge the types of the monosaccharides in the sample, and comparing the relative peak areas in the same sample to judge the content of the monosaccharides.

TABLE 4 composition of the major monosaccharides of the extract by ultrasound-assisted extraction

Example 13

The glycoprotein extracts obtained in examples 1 to 4 and example 6 were subjected to amino acid composition analysis, and the analysis results are shown in Table 5;

The experimental method comprises the following steps:

sample acid hydrolysis: placing 8mg of sample in a 20ml screw cap (with an internal spacer) fine mouth bottle, adding 10ml 0.1% phenol and 6mol/l hydrochloric acid aqueous solution, screwing the cap, vibrating and mixing uniformly, reacting for 24 hours at the temperature of 110 ℃, completely transferring the reaction solution into a 100ml distillation bottle, decompressing and steaming until the reaction solution is nearly dry at the temperature of 75 ℃ to remove residual hydrochloric acid in the reaction solution, dissolving residues in 12ml0.2mol/l hydrochloric acid aqueous solution for three times, transferring to a 20ml glass test tube with a plug scale, and then fixing the volume to 20ml by pure water until the reaction solution is derived;

Phenyl isothiocyanate derivatization: placing 200 mu l of acid hydrolyzed sample solution into a 1.5ml centrifuge tube, adding 100 mu l of 1mol/l triethylamine acetonitrile solution and 100 mu l of 0.2mol/l phenyl isothiocyanate acetonitrile solution, uniformly mixing, reacting for 1 hour at room temperature, then adding 400 mu l of normal hexane, tightly screwing a cover, vigorously shaking for 5-10 seconds, standing for layering, mixing 200 mu l of lower solution with 800 mu l of water, and filtering by a 0.22 mu m needle filter to obtain the sample solution to be analyzed;

HPLC amino acid composition analysis: according to Diamonsil AAA amino acid analysis and use instructions, respectively carrying out HPLC analysis on the derived amino acid mixed standard substance and the sample, comparing the peak time of a spectrogram obtained by the sample with the peak time of different amino acids in the mixed standard substance, and judging the types of the amino acids contained in the sample; comparing the area ratio of each peak in the spectrogram of the sample to judge the proportion of amino acid contained in the sample;

TABLE 5 composition of main amino acids of extract by ultrasound-assisted extraction

The invention discloses a response surface method-based ultrasonic-assisted extraction method for optimizing the gastric glycoprotein of red deer in Tianshan and application thereof. The optimal extraction conditions of the mutual correlation of the salt concentration of the extracting solution and the ultrasonic power, namely the extraction temperature 42.841 ℃, the salt concentration of 0.572M/L and the ultrasonic power of 444.638W are obtained through the experimental design of related software and the fitting of experimental results. The extraction conditions were modified to an extraction temperature of 43℃and an extract salt concentration of 0.57M/L, ultrasonic power of 445W, depending on the actual conditions. By comparing the protein content, sugar content and DPPH free radical clearance rate (Table 3) of glycoprotein obtained by ultrasonic assisted extraction and traditional extraction method, the indexes of ultrasonic assisted extraction are superior to those of traditional method on the premise of the same extraction time, the protein content reaches 33.4%, the sugar content reaches 5.54%, the uronic acid content reaches 2.53%, which is higher than the protein content of 23.82%, the sugar content of 3.19% and the uronic acid content of 1.06% in the traditional extraction method. In terms of antioxidant activity, the clearance rate of ultrasonic-assisted extraction of three indexes (DPPH free radical, ABTS free radical and hydroxyl free radical clearance rate) under the same experimental concentration (2.5 mg/ml) is higher. This is because the ultrasonic energy breaks up the cells better, thus more efficient extraction, reducing the extraction time and also reducing the disadvantages of macromolecular decomposition due to the extraction temperature. The absorption of the relevant wave band of the infrared spectrum proves that the crude extract is Tang-protein substances. The results show that: the response surface method is effective in optimizing ultrasonic auxiliary extraction, is more effective, is more environment-friendly, has higher activity than the traditional method, is easy to scale-up production, and is one of ideal methods for preparing the red deer glycoprotein.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. The ultrasonic-assisted extraction method for optimizing the Tianshan red deer abomasum glycoprotein based on the response surface method is characterized by comprising the following steps of:

d. performing Box-Behnken Design by using Design Expert 11 software, extracting for 1 hour under the conditions of extracting temperature of 25-55 ℃ and extracting solution salt concentration of 0.3-0.9M, ultrasonic power of 300-700W,6 s off and 4 s on for two times to obtain an extracting solution;

2. Use of a Tianshan red deer abomasum glycoprotein extract obtained according to the method of claim 1 for the preparation of a raw material for an antioxidant active product.