CN115353545A - Method for preparing lamb abomasum glycoprotein part by response surface method and application - Google Patents
Method for preparing lamb abomasum glycoprotein part by response surface method and application Download PDFInfo
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- CN115353545A CN115353545A CN202211169619.6A CN202211169619A CN115353545A CN 115353545 A CN115353545 A CN 115353545A CN 202211169619 A CN202211169619 A CN 202211169619A CN 115353545 A CN115353545 A CN 115353545A
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- glycoprotein
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- abomasum
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
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- Analytical Chemistry (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Water Supply & Treatment (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nutrition Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for preparing lamb abomasum glycoprotein part by a response surface method and application thereof, wherein the method takes liquid-material ratio, salt concentration and extraction temperature as independent variables, takes extraction rate and OH activity as response values, optimizes the extraction method of glycoprotein by using a response surface Box-Behnken experimental design, and has the yield of 24.6 +/-2.1%. The protein content measured by BCA method is 23.45 + -0.34%, the neutral sugar content measured by phenol-sulfuric acid method is 6.4 + -0.5%, and the monosaccharide components are arabinose, xylose, mannose, glucose and galactose. The antioxidant activity result shows that the glycoprotein part has stronger total reductionProductivity, DPPH and OH ‑ Free radical scavenging ability, and IC of the latter two 50 The values were 2.91. + -. 0.16mg/mL, 3.9. + -. 0.3mg/mL, respectively. The method for obtaining the lamb abomasum glycoprotein part is mature, simple and not complicated to operate, and easy to amplify and prepare in large quantity; the obtained lamb abomasums have good antioxidant activity and can be applied to the fields of medicine, health care and food.
Description
Technical Field
The invention relates to a method for preparing lamb abomasum glycoprotein parts by a response surface method and application thereof.
Background
Glycoproteins (glycoprotens) are the products of post-translational modifications of proteins. The proteins are modified by glycosylation to perform specific biological functions such as molecular biological recognition, intercellular and intracellular signaling, embryonic development, fertilization, immune defense, and inflammation, and most of membrane proteins and secreted proteins are glycoproteins. Animals, plants, microorganisms and the like contain abundant glycoproteins, and are important raw materials of active glycoproteins which are natural sources. In addition to natural sources, chemical semisynthesis is another route to active glycoproteins. However, because of the diversity of the linkage between the monosaccharides in the oligosaccharide chain, the oligosaccharide chain and the polypeptide chain, the extent of glycosylation reaction cannot be precisely controlled, resulting in more by-products than the desired product. Therefore, the active glycoprotein cannot be efficiently obtained by the chemical semi-synthesis method due to difficulties in separation and purification, low yield of the target glycoprotein compound and the like. Therefore, obtaining active glycoprotein from animal and plant and microorganism as raw materials is still the hot of natural drug research and new drug development.
Efficient extraction of active glycoproteins is an important prerequisite for obtaining glycoproteins with strong biological activity.
Huang Riming et al optimized the extraction process of glycoprotein from green squama fish using an orthogonal experimental method, and the extraction rate of glycoprotein under the optimal extraction conditions was only 0.052%.
The method for extracting glycoprotein from the muscle of cuttlefish by ultrasonic wave assisted extraction by Dai Hongjie et al by using a response surface optimization method has the extraction rate of 7.9 percent under the optimized condition.
Zhou Yuhui et al extracted active glycoprotein from tartary buckwheat by a water extraction method, and the extraction rate of the active glycoprotein is 2.31%.
The extraction rate of glycoprotein from acidic European nuts by water extraction of yellow cedar et al is 2.0-2.5%.
The lamb abomasums are used as traditional Chinese medicine raw materials for both medicine and food, and gastrointestinal dyspepsia and chronic gastritis are used for treating in traditional medicines. Over 10 kinds of secretory cells which are determined to be in the abomasum, such as G cells secreting gastrin, I cells secreting cholecystokinin-secretin, D cells secreting somatostatin, N cells secreting neurotensin and the like, contain abundant active glycoproteins.
The protein is easy to lose high-order structure and lose activity partially or completely under the conditions of ultrasound, high temperature, organic solvent and the like, and the glycoprotein as a derivative of the protein has similar characteristics; products for treating diseases or improving the physiological health of human bodies cannot be effectively obtained when the activity is reduced or completely lost, so that the utilization rate of raw materials is reduced, and the application of the products is limited; the preparation method of the patent glycoprotein has the defects of violent extraction conditions, low glycoprotein yield, no investigation on optimal extraction conditions and the like; obviously, the optimized extraction process and other active applications of the active ingredients of the lamb abomasum glycoprotein are still in an unprecedented state. Therefore, it is necessary to research the optimized extraction process and antioxidant activity of lamb abomasum glycoprotein to obtain a more effective production technology capable of improving the utilization rate of lamb abomasum, so as to expand the application and improve the economic benefit.
The reports related to glycoprotein from 2006 to 2021 were reviewed by using glycoprotein as a search keyword, wherein 88 patents for preparing active glycoprotein from natural animals and plants as raw materials are provided, 52 patents are screened from the raw materials by comparing the extraction method of glycoprotein with the selection of raw materials, and the treatment of raw materials of glycoprotein, the extraction of glycoprotein and the method for preparing crude glycoprotein or glycoprotein fraction are summarized.
Selecting raw materials; the patent application No. 202110922724.1 extracts probiotic active glycoprotein by taking lamb abomasums as raw materials, the application No. 202111360384.4 extracts hyaluronidase inhibition active glycoprotein by taking the abomasums of Tianshan red deer as raw materials, the two applications are both early researches of the subject, the application No. 202110922724.1 obtains the probiotic active glycoprotein by taking the lamb abomasums as raw materials, the extraction is carried out by an ultrasonic auxiliary extraction method at the temperature of 60 ℃, glycoprotein is obtained after ethanol precipitation and savage reagent deproteinization, and the yield is 13.8%. The application number of 202111360384.4 is that the glycoprotein is extracted by an ultrasonic-assisted extraction method at the temperature of 60 ℃ by using sodium chloride as an extracting agent; the present invention is further developed based on the previous work of the present subject, and the difference between them is that: the extraction temperature, the salt concentration and the feed-liquid ratio are obtained by single-factor experiments and response surface experiments, and the method has the advantages of reasonability, high efficiency, mild conditions and the like; thirdly, the phosphate buffer solution with the pH value closest to the normal physiological pH value of 7 is used as the extracting agent in the extraction process of the invention, so that the abomasum is the stomach of the ruminant with the real digestion significance and has the digestion function similar to the ordinary stomach of other animals, therefore, the abomasum has abundant secretory cells, the surface of the abomasum is protected by gastric mucus and is not damaged by gastric acid under the normal physiological condition, when the physiological condition is stripped, the gastric acid released by the rupture of the cells secreting gastric acid can instantaneously change the pH value of the extraction system so as to damage the pH value of the extraction system and further damage active substances released by other secretory cells, and the phosphate buffer added during the extraction can avoid the damage; glycoproteins, however, have similar characteristics as protein derivatives. The product with low activity or loss of biological activity has reduced application value.
In the prior art, the extraction method of glycoprotein from plants, marine products, insects and other raw materials; the method is suitable for the pretreatment and extraction of raw materials, but except the enzymolysis method, low acid or high alkali is mostly used, and ultrasonic or microwave-assisted extraction is carried out under the conditions of high temperature and the like; the pretreatment of raw materials is well known to depend on the characteristics and sources of the raw materials, so that the pretreatment of different raw materials is different, and the difference of the extraction method is excluded as follows: the 200910015667.8 patent uses acid water of pH 3 to extract pigment glycoprotein from Eucheuma muricatum; patent application No. 200910303102.X uses enzymolysis method to extract glycoprotein with antitumor activity from Eucheuma Gelatinosum; the brown algae double-oligo glycoprotein is extracted under the alkaline condition at the temperature of 40-100 ℃ in the patent with the application number of 202010934354.9; the precipitate extracted with sodium chloride at 70-100 deg.C under the condition of pH-8-9.5 with Tris-HCl buffer solution is subjected to extraction to obtain glycoprotein with application number of 201810172232.3.
After a large number of searches: almost all of the related patents and articles on glycoprotein of natural origin, such as 201110138032.4, 201110403339.2, 200910089580.5, 200910052066.4, 200710132671.3, 201610645106.6, 201910440149.4, 201910439568.6, 201811300319.0, 202111360384.4, etc., use one or two of the methods of trichloroacetic acid deproteinization, acetone precipitation, ethanol precipitation, savage reagent deproteinization, etc., to treat the deproteinized extract, and then the obtained product is defined as deproteinized.
The response surface method is a commonly used reliable, credible and efficient optimization method, and each raw material has different factors to be investigated according to the characteristics of the raw material, and has different obtained results according to different production target evaluation indexes, for example, the response surface method is used for optimizing the glycoprotein ratio extraction process, the yield of glycoprotein is 7.038%, but the actual activity and the application of the response surface method are not reflected, wherein the application number of 201410129587.6 adopts the sea horse as the raw material, the ultrasonic auxiliary extraction method is used, the salt concentration and the extraction time and the temperature are used as the investigation factors, and the yields of sugar and protein are used as indexes; the application number of 201610645106.6 is that the shiyuan is used as a raw material to extract the anti-aging active glycoprotein, the pH value and the temperature material-liquid ratio of an extracting solution are used as investigation factors, the protein content is used for replacing the extraction rate, the extraction process is optimized by a response surface method, and the average yield of the glycoprotein under the optimal extraction process condition is 5.96 percent.
The invention shows through the investigation of preliminary experiments that: the salt concentration, the temperature and the feed-liquid ratio are the largest factors influencing the extraction rate, so that the extraction rate obtained by adopting a response surface method can reach 24.6 +/-2.1 percent by taking the feed-liquid ratio, the temperature and the salt concentration as investigation factors and the extraction rate and the hydroxyl radical free radical clearance as evaluation indexes; therefore, the abomasum glycoprotein part of the lamb obtained by the method has strong antioxidant activity.
The invention solves the main problems that based on a lamb abomasum glycoprotein active ingredient system, the antioxidant active ingredient in the lamb abomasum is efficiently extracted, the blank of research on the lamb abomasum glycoprotein active ingredient is filled, and the application of abomasum raw materials in medicines, health products and foods is expanded based on the lamb abomasum glycoprotein antioxidant active ingredient.
Disclosure of Invention
The invention aims to provide a method for preparing lamb abomasum glycoprotein part by using a response surface method and application thereof, wherein the method takes liquid-material ratio, salt concentration and extraction temperature as independent variables, takes extraction rate and OH activity as response values, optimizes the extraction method of glycoprotein by using response surface Box-Behnken experimental design, obtains the lamb abomasum glycoprotein part with the extraction rate of 24.6 +/-2.1 percent, obtains the protein content of 23.45 +/-0.34 percent by using a BCA method, obtains the neutral sugar content of 6.4 +/-0.5 percent by using a phenol-sulfuric acid method, obtains the monosaccharide composition of 23.84 percent of arabinose, 11.60 percent of xylose, 28.24 percent of mannose, 8.68 percent of glucose and 27.63 percent of galactose by using a BCA method, and shows the antioxidant activity result that the extract has stronger total reducing capacity, DPPH and OH, and the antioxidant activity result shows that the extract has stronger total reducing capacity - Free radical scavenging ability, and IC of the latter two 50 The values were 2.91. + -. 0.16mg/mL and 3.9. + -. 0.3mg/mL, respectively. The method is simple and not tedious to operate, and easy to amplify and prepare in large quantities; the obtained lamb abomasum glycoprotein part has good antioxidant activity and can be applied to the fields of medicine, health care and food.
The invention relates to a method for preparing lamb abomasum glycoprotein part by a response surface method, which comprises the following steps:
a. cleaning feed in stomach cavity with tap water, removing fat and connective tissue, freezing at-80 deg.C for 3 hr, shearing, freeze drying, and pulverizing with liquid nitrogen to obtain lamb abomasums powder;
b. degreasing the lamb abomasum powder obtained in the step a for 3 times by using petroleum ether, naturally drying to obtain lamb abomasum degreased powder, and freezing and storing at the temperature of-20 ℃;
c. carrying out experiments on the feed-liquid ratio at 18mL/g,24mL/g,30mL/g,36mL/g, extraction temperature of 15 ℃,30 ℃,45 ℃,60 ℃, sodium salt concentration of 0.15M,0.3M,0.35M and 0.6M by using single factors to obtain the feed-liquid ratio of 24-36mL/g, temperature of 15-45 ℃ and sodium salt concentration of 0.2-0.6M;
d. taking the sodium salt concentration, the temperature and the liquid-material ratio obtained in the step C as independent variables, respectively naming the sodium salt concentration, the temperature and the liquid-material ratio as A, B and C, sequentially naming the extraction rate and the OH free radical scavenging capacity as response values as Y1 and Y2, utilizing Design Expert12 software to carry out Box-Behnken Design (BBD) Design, extracting the lamb abomasum defatted powder obtained in the step B, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at the temperature of 4 ℃ of 7000 for 10min, taking supernatant, and determining the extraction rate and the OH free radical scavenging activity;
e. respectively carrying out regression analysis on the extraction rate and the response surface experiment results of the OH free radical scavenging capacity, and carrying out regression fitting on the three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
extracting with extraction rate and OH free radical scavenging capacity as maximum values, wherein the concentration of sodium chloride salt is 0.4-0.6M, the temperature is 20-40 deg.C, and the liquid-material ratio is 25-35mL/g;
f. and e, extracting glycoprotein from the lamb abomasum defatted powder under the extraction condition obtained in the step e, centrifuging the extracting solution at the temperature of 4 ℃ at 7000 rpm for 10min, taking the supernatant, dialyzing and desalting for 60h by using deionized water as an exchange solution dialysis bag, freeze-drying the solution in the dialysis bag to obtain a product lamb abomasum glycoprotein part, weighing, calculating the extraction rate, and storing at the temperature of-20 ℃.
The lamb abomasum glycoprotein part obtained by the method is applied to the preparation of antioxidant active drugs.
The lamb abomasum glycoprotein part obtained by the method is applied to the preparation of an antioxidant activity health product.
The lamb abomasum glycoprotein part obtained by the method is applied to preparing antioxidant active food.
The invention relates to a method for preparing lamb abomasum glycoprotein part by using response surface method and application, wherein the method uses lamb abomasum as raw material, under the condition of magnetic stirring, the influence of factors such as extraction material-liquid ratio, extraction temperature and extraction salt concentration on extraction rate and antioxidant activity is inspected, and the optimal extraction condition is obtained by using response surface optimization method: the liquid-material ratio is 25-35mL/g, the salt concentration is 0.4-0.6M, the temperature is 20-40 ℃, the lamb abomasum glycoprotein is extracted by adopting a buffer system of a phosphate buffer solution with neutral pH 7 and under the conditions of non-mechanical cutting or ultrasonic crushing, and the highest extraction rate can reach 24.6 +/-2.1%. The method does not use violent extraction methods such as high temperature, ultrasonic and the like, and does not use organic solvent; and the antioxidant activity and the extraction rate are used as process evaluation indexes, so that the method has the advantages of mild preparation process, high yield, high activity, low pollution and the like.
One of the innovation points of the invention is that the preparation method of the lamb abomasum glycoprotein active ingredient is optimized by utilizing a response surface method, and the preparation method with stronger applicability, milder condition, higher extraction rate and less pollution is obtained; the lamb abomasum glycoprotein obtained by the method has antioxidant activity, so that the new activity application of lamb abomasum raw materials and glycoprotein components is expanded, and the application of the lamb abomasum glycoprotein is expanded.
The invention relates to an extraction method for optimizing lamb abomasum glycoprotein part based on a response surface method, which comprises the following steps of:
carrying out material-liquid ratio investigation on the obtained lamb abomasum defatted powder by taking the extraction rate as an index, carrying out single-factor experiments on four levels of 18mL/g,24mL/g,30mL/g and 36mL/g on the material-liquid ratio under the conditions of the extraction temperature of 37 ℃ and the sodium chloride concentration of 0.45M, adding deionized water into the obtained extracting solution for diluting by 4 times, centrifuging at the temperature of 4 ℃ at 7000 rotation speed for 10min, taking the supernatant, measuring the protein content by using a BCA method, calculating the extraction rate by using the following formula, drawing a curve graph of the extraction rate changing along with the material-liquid ratio, and obtaining the material-liquid ratio range of the optimal extraction rate;
extraction rate (%) = (C × N × V) × 100%/m yield of extract { in the formula C represents protein concentration (mg/mL); n represents a dilution factor; v represents volume (mL); m represents a raw material mass (g);
as shown in FIG. 1, the extraction rate slightly increased when the liquid-to-liquid ratio increased from 18 to 24, the extraction rate sharply increased from 24 to 30, and the extraction rate sharply decreased from 36, indicating that the liquid-to-liquid ratio has a certain effect on the extraction rate, and the optimum liquid-to-liquid ratio is in the range of 24 to 36.
C, taking the extraction rate as an index, carrying out single-factor experiments on four levels of 15 ℃,30 ℃,45 ℃ and 60 ℃ on the extraction temperature under the conditions that the feed-liquid ratio is 30mL/g and the concentration of sodium chloride is 0.45M, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at 7000 ℃ for 10min at the temperature of 4 ℃, taking the supernatant, calculating the extraction rate by using the same method in the step C, and drawing a curve of the extraction rate changing along with the extraction temperature to obtain the temperature range of the optimal extraction rate;
as shown in FIG. 2, the extraction rate tends to increase and decrease rapidly at an extraction temperature of 15 to 45 ℃ and the extraction rate at 60 ℃ is lower than that at 15 ℃, which indicates that the temperature has a certain influence on the extraction rate and that the optimum extraction temperature is in the range of 15 to 45 ℃.
C, taking the extraction rate as an index, carrying out single-factor experiments on the concentration of sodium chloride at four levels of 0.15M,0.3M,0.45M and 0.6M at a feed-liquid ratio of 30mL/g and an extraction temperature of 37 ℃, adding deionized water into the obtained extracting solution for diluting by 4 times, centrifuging at 7000 rotation speed at 4 ℃ for 10min, taking the supernatant, calculating the extraction rate by using the same method in the step C, drawing a curve of the extraction rate changing along with the concentration of the sodium chloride, and obtaining the concentration range of the sodium chloride with the optimal extraction rate;
the experimental results are shown in fig. 3: the extraction rate of the extract solution with the NaCl salt concentration of 0.2-0.6M tends to increase and decrease sharply, which shows that the NaCl salt concentration of the extract solution has certain influence on the extraction rate, and the optimal NaCl salt concentration of the extract solution is in the range of 0.2-0.6M.
Carrying out Box-Behnken Design (BBD) Design on the obtained lamb abomasum degreasing powder by using Design Expert12 software according to the single-factor range as the upper limit and the lower limit, wherein salt concentration, temperature and liquid-material ratio are used as independent variables and are respectively named as A, B and C, and the extraction rate and OH free radical scavenging capacity are used as response values and are sequentially named as Y1 and Y2; adding deionized water into the obtained extracting solution for diluting by 4 times, centrifuging at the temperature of 4 ℃ at 7000 rotation speed for 10min, taking supernatant, measuring OH free radical scavenging activity by using a conventional method, calculating the extraction rate by using the method in the step c, performing regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and performing regression fitting on three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A 2 -3.25B 2 -2.91C 2 +3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A 2 -4.55B 2 -5.78C 2 +4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
the obtained optimized extraction conditions were: the concentration of sodium chloride salt is 0.4-0.6M, the extraction temperature is 20-40 ℃, and the liquid-material ratio is 25-35mL/g;
lamb abomasum glycoprotein extracted response surface Box-Behnken experimental design
The upper and lower limits of the independent variable extracted from lamb abomasum glycoprotein are shown in Table 1:
TABLE 1 Box-Behnken experiment design independent variable upper and lower limit table extracted from lamb abomasum glycoprotein
According to the upper and lower limits of independent variables, the extraction rate and the OH free radical scavenging capacity are taken as corresponding values, and Design Expert12 software is used for carrying out Box-Behnken Design (BBD) Design to obtain 17 experimental conditions, wherein the experimental conditions are as follows:
table 2 Box-Behnken experimental condition table for lamb abomasum glycoprotein extraction process optimization
Diluting the obtained extract with deionized water 4 times, centrifuging at 4 deg.C 7000 rotation speed for 10min, collecting supernatant, and determining extraction rate and OH free radical scavenging activity, wherein the extraction rate is calculated by the following formula:
extraction rate (%) = (C × N × V) × 100%/m yield of extract { in the formula C represents protein concentration (mg/mL); n represents a dilution factor; v represents volume (mL); m represents a raw material mass (g) }
Carrying out regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and carrying out regression fitting on three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
the significance and fitness of the regression model to the extraction rate were evaluated by analysis of variance, the analysis of variance results are shown in table 3, and the corresponding surface graphs (fig. 4-6) show that: a model F value of 29.74 indicates that the model responds significantly to the extraction rate and the probability of noise occurrence is only 0.01%; the P value of the model is less than 0.0001, which shows that the model has obvious response to the extraction rate; the mismatching F value is 287.44, the P value is 0.1254, which shows that the mismatching has insubstantial property relative to pure error, and the effectiveness of the model is verified; in the regression model, the correlation coefficient R 2 0.9745, the adjusted determination coefficient Adj-R 2 0.9417, and a Coefficient of Variation (CV) of 5.43, which indicates that the experimental value has high correlation with the predicted value; analysis of variance showed that: variable A, B, C, A 2 、B 2 、C 2 The extraction rate is obviously influenced; the order of the P values of the three independent variables is A>B>C>Illustrating that the salt concentration (A) has the greatest effect on the yield and P<0.0001, indicating significant effect; the extraction temperature B and the liquid-to-material ratio C also have a significant influence P<0.05, and the effect of B is greater than C; AB, AC and BC had but not significant effect on extraction yield, P>0.05。
TABLE 3 regression analysis of variance table for extraction yield
A: salt concentration (M); b, the extraction temperature (DEG C); c: liquid-to-feed ratio (mL/g);
R 2 ＝0.9745；AdjR 2 =0.9417; note: * Indicates extreme significance; * And means significant, and significance is greater than;
evaluation of regression model for OH free radical scavenging by analysis of varianceThe analysis of variance results are shown in Table 4, except for the significance and fitness of the activity, and the corresponding surface plots (FIGS. 7-9) show: a model F value of 16.61 indicates that the model has a significant OH radical clearance and a noise occurrence probability of only 0.06%; the model P value is 0.0006, which shows that the model has obvious influence on the OH free radical clearance rate; the mismatching F value is 5.33, the P value is 0.0699, which shows that the mismatching has insubstantial property relative to the pure error, and the effectiveness of the model is verified; in the regression model, the correlation coefficient R2 is 0.9553, the adjusted determination coefficient Adj-R2 is 0.8977, and the Coefficient of Variation (CV) is 7.66, which shows that the experimental value has higher correlation with the predicted value; analysis of variance showed that: variable A, B, A 2 、B 2 、C 2 The extraction rate is obviously influenced; AB. The effect of AC on extraction rate was not significant; the P values of the three independent variables are in the order of B>A, indicates that the salt concentration (A) has the greatest effect on OH radical clearance and P is 0.0004, indicating a significant effect; a. The 2 、B 2 、C 2 The P value of (A) is less than 0.05, which shows that the extraction rate is significantly influenced;
TABLE 4 regression analysis of variance table for OH radical clearance
A: salt concentration (M); b, the extraction temperature (DEG C); c: liquid-to-feed ratio (mL/g);
R 2 ＝0.9553；AdjR 2 =0.8977; note: * Indicates significance, and significance is greater than;
according to the optimal scheme in the experimental design, in combination with the actual situation, the conditions set by the modified experimental operation are that the salt concentration is 0.4-0.6M, the extraction temperature is 20-40 ℃, and the liquid-material ratio is 25-35mL/g; the experiment is repeated for 3 times, the yield of the lamb abomasum glycoprotein is obtained by 24.6 +/-2.1 percent, the protein content is 23.45 +/-0.34 percent, the sugar content is 6.4 +/-0.5 percent, which is approximate to the theoretical value obtained by calculating by the response surface method, and the regression equation obtained by optimizing the response surface method has guiding significance.
According to the obtained optimal extraction conditions, glycoprotein part extraction is carried out on lamb abomasum defatted powder, the extract is centrifuged for 10min at 7000 ℃ and 4 ℃, supernatant is taken, dialysis and desalination are carried out for 60h by taking deionized water as exchange solution dialysis bag, the solution in the dialysis bag is freeze-dried, lamb abomasum glycoprotein part is obtained, weighing is carried out to calculate the extraction rate, the lamb abomasum glycoprotein part is stored at-20 ℃, and the extraction rate is calculated by the following formula: the extraction rate (%) = [ weight (g) of extract/weight (g) of degreased bulk drug) ] × 100%, namely the obtained product lamb abomasum glucoprotein position.
Carrying out protein content determination on the lamb abomasum glycoprotein part obtained by the method of the invention by using a BCA method; measuring the sugar content by a phenol-sulfuric acid method; analyzing monosaccharide composition; OH free radical scavenging capacity measurement; DPPH free radical scavenging ability and total reducing ability.
The method for preparing the abomasum glycoprotein part of the lamb by the response surface method and the application have the advantages that:
the yield of the obtained glycoprotein part is higher than that of other methods, and the method is easy to amplify pilot scale tests. On the basis of keeping the original physical and chemical properties and nutritional ingredients of the glycoprotein, the yield is improved by extracting at a lower temperature, and the obtained glycoprotein has higher antioxidant activity. The search for new antioxidants from natural plants is a new direction for the development of modern medicine and food industries. The research on the antioxidation of the polysaccharide and the derivatives thereof and the development of new natural antioxidants have important practical significance. The research can provide reference for the industrial development of lamb abomasum glycoprotein in antioxidant and anti-inflammatory foods, health products and medicines.
Drawings
FIG. 1 is a graph showing the results of a single-factor experiment of the liquid-to-material ratio of the glycoprotein site of the present invention;
FIG. 2 is a graph showing the results of a single-factor temperature experiment on a glycoprotein site according to the present invention;
FIG. 3 is a graph showing the results of a single-factor experiment of the salt concentration at the glycoprotein site of the present invention;
FIG. 4 is a 3D surface plot of the effect of the salt concentration (A) and extraction temperature (B) on the extraction rate of the glycoprotein fraction of the present invention;
FIG. 5 is a 3D plot of the effect of salt concentration (A) and liquid-to-feed ratio (C) on extraction yield for a glycoprotein site of the present invention;
FIG. 6 is a 3D plot of the effect of glycoprotein site temperature (B) and liquid-to-feed ratio (C) on extraction yield according to the present invention;
FIG. 7 shows OH dependence on the salt concentration (A) and extraction temperature (B) of the glycoprotein site of the present invention - A 3D surface plot of the effect of free radical clearance;
FIG. 8 shows the ratio of salt concentration (A) and liquid-to-liquid ratio (C) to OH of the glycoprotein site of the present invention - A 3D surface plot of the effect of free radical clearance;
FIG. 9 shows the temperature (B) and liquid-to-liquid ratio (C) versus OH of the glycoprotein site of the present invention - A 3D surface plot of the effect of free radical clearance;
FIG. 10 shows OH groups at the glycoprotein site of the present invention - Experimental result chart of scavenging ability of free radical;
FIG. 11 is a graph showing the results of experiments on the scavenging ability of DPPH radicals at the glycoprotein site of the present invention;
FIG. 12 is a graph showing the results of experiments on the total reducing ability of the glycoprotein site of the present invention.
Detailed Description
Example 1
a. Cleaning feed in stomach cavity with tap water, removing fat and connective tissue, freezing at-80 deg.C for 3 hr, shearing, freeze drying, and pulverizing with liquid nitrogen to obtain lamb abomasums powder;
b. degreasing the lamb abomasum powder obtained in the step a for 3 times by using petroleum ether, naturally drying to obtain lamb abomasum degreased powder, and freezing and storing at the temperature of-20 ℃;
c. the material-liquid ratio is respectively tested by single factor at 18mL/g,24mL/g,30mL/g,36mL/g, extraction temperature of 15 ℃,30 ℃,45 ℃,60 ℃, sodium salt concentration of 0.15M,0.3M,0.35M and 0.6M, and the material-liquid ratio is 24mL/g, the temperature of 15 ℃ and the sodium salt concentration of 0.6M;
d. taking the sodium salt concentration, the temperature and the liquid-material ratio obtained in the step C as independent variables, respectively naming the sodium salt concentration, the temperature and the liquid-material ratio as A, B and C, sequentially naming the extraction rate and the OH free radical scavenging capacity as response values as Y1 and Y2, utilizing Design Expert12 software to carry out Box-Behnken Design (BBD) Design, extracting the lamb abomasum defatted powder obtained in the step B, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at the temperature of 4 ℃ of 7000 for 10min, taking supernatant, and determining the extraction rate and the OH free radical scavenging activity;
e. respectively carrying out regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and carrying out regression fitting on three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
extracting conditions are obtained by taking the extraction rate and the OH free radical scavenging capacity as maximum values, wherein the concentration of sodium salt is 0.4M, the temperature is 20 ℃, and the liquid-material ratio is 25mL/g;
f. and e, extracting glycoprotein from 10g of lamb abomasum defatted powder under the extraction condition obtained in the step e, centrifuging the extracting solution at the temperature of 4 ℃ at 7000 rpm for 10min, taking the supernatant, dialyzing and desalting the supernatant for 60h by using deionized water as exchange fluid in a dialysis bag, freeze-drying the dialysate in the dialysis bag to obtain a product lamb abomasum glycoprotein part, weighing, calculating the extraction rate, and storing at the temperature of-20 ℃.
The protein content of the extract is determined by BCA method, and the sugar content is determined by phenol-sulfuric acid method;
the extraction rate was calculated using the following formula: extraction ratio (%) = (weight of extract (g)/weight of defatted raw material (g)) × 100%
The extraction rate is 18.7%, the protein content is 17.2% +/-0.6%, and the sugar content is 5.3% +/-0.8%;
example 2
a. Cleaning feed in stomach cavity with tap water, removing fat and connective tissue, freezing at-80 deg.C for 3 hr, shearing, freeze drying, and pulverizing with liquid nitrogen to obtain powder;
b. degreasing the lamb abomasum powder obtained in the step a for 3 times by using petroleum ether, naturally drying to obtain lamb abomasum degreased powder, and freezing and storing at the temperature of-20 ℃;
c. the material-liquid ratio is respectively tested by single factor at 18mL/g,24mL/g,30mL/g,36mL/g, extraction temperature of 15 ℃,30 ℃,45 ℃,60 ℃, sodium salt concentration of 0.15M,0.3M,0.35M and 0.6M, and the material-liquid ratio is 24mL/g, the temperature of 30 ℃ and the sodium salt concentration of 0.4M;
d. taking the sodium salt concentration, the temperature and the liquid-material ratio obtained in the step C as independent variables, respectively naming the sodium salt concentration, the temperature and the liquid-material ratio as A, B and C, sequentially naming the extraction rate and the OH free radical scavenging capacity as response values as Y1 and Y2, utilizing Design Expert12 software to carry out Box-Behnken Design (BBD) Design, extracting the lamb abomasum defatted powder obtained in the step B, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at the temperature of 4 ℃ of 7000 for 10min, taking supernatant, and determining the extraction rate and the OH free radical scavenging activity;
e. respectively carrying out regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and carrying out regression fitting on three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
extracting conditions are obtained by taking the extraction rate and the OH free radical scavenging capacity as maximum values, wherein the concentration of sodium salt is 0.5M, the temperature is 30 ℃, and the liquid-material ratio is 30mL/g;
f. and e, extracting glycoprotein from 10g of lamb abomasum defatted powder under the extraction condition obtained in the step e, centrifuging the extracting solution at the temperature of 4 ℃ at 7000 rpm for 10min, taking the supernatant, dialyzing and desalting the supernatant for 60h by using deionized water as an exchange solution dialysis bag, freeze-drying the solution in the dialysis bag to obtain a lamb abomasum glycoprotein extract, weighing the extract to calculate the extraction rate, and storing the extract at the temperature of-20 ℃ to obtain a product lamb abomasum glycoprotein part.
The protein content of the extract is determined by BCA method, and the sugar content is determined by phenol-sulfuric acid method;
the extraction rate was calculated using the following formula: extraction ratio (%) = (weight of extract (g)/weight of defatted raw material (g)) × 100%
The extraction rate is 23.6%, the protein content is 22.06% + -1.6%, and the sugar content is 6.15% + -0.8%.
Example 3
a. Cleaning feed in stomach cavity with tap water, removing fat and connective tissue, freezing at-80 deg.C for 3 hr, shearing, freeze drying, and pulverizing with liquid nitrogen to obtain powder;
b. degreasing the lamb abomasum powder obtained in the step a for 3 times by using petroleum ether, naturally drying to obtain lamb abomasum degreased powder, and freezing and storing at the temperature of-20 ℃;
c. the material-liquid ratio is respectively tested by single factor at 18mL/g,24mL/g,30mL/g,36mL/g, extraction temperature of 15 ℃,30 ℃,45 ℃,60 ℃, sodium salt concentration of 0.15M,0.3M,0.35M and 0.6M, and the material-liquid ratio of 36mL/g, temperature of 45 ℃ and sodium salt concentration of 0.2M is obtained;
d. taking the sodium salt concentration, the temperature and the liquid-material ratio obtained in the step C as independent variables, respectively naming the sodium salt concentration, the temperature and the liquid-material ratio as A, B and C, sequentially naming the extraction rate and the OH free radical scavenging capacity as response values as Y1 and Y2, utilizing Design Expert12 software to carry out Box-Behnken Design (BBD) Design, extracting the lamb abomasum defatted powder obtained in the step B, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at the temperature of 4 ℃ of 7000 for 10min, taking supernatant, and determining the extraction rate and the OH free radical scavenging activity;
e. respectively carrying out regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and carrying out regression fitting on three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
obtaining extraction conditions by taking the extraction rate and the OH free radical scavenging capacity as maximum values, wherein the concentration of sodium chloride is 0.4M, the temperature is 30 ℃, and the liquid-material ratio is 31mL/g;
f. and e, extracting glycoprotein from 10g of lamb abomasum defatted powder under the extraction condition obtained in the step e, centrifuging the extracting solution at the temperature of 4 ℃ at 7000 rpm for 10min, taking the supernatant, dialyzing and desalting the supernatant for 60h by using deionized water as exchange solution through a dialysis bag, freeze-drying the solution in the dialysis bag to obtain a product lamb abomasum glycoprotein part, weighing, calculating the extraction rate, and storing at the temperature of-20 ℃.
Measuring protein content by using a BCA method, and measuring sugar content by using a phenol-sulfuric acid method;
the extraction rate was calculated using the following formula: extraction ratio (%) = (extract (g)/weight of defatted raw material (g)) × 100%
The extraction rate is 24.2%, the protein content is 23.564% + -1.2%, and the sugar content is 7.18% + -0.4.
Example 4
a. Cleaning feed in stomach cavity with tap water, removing fat and connective tissue, freezing at-80 deg.C for 3 hr, shearing, freeze drying, and pulverizing with liquid nitrogen to obtain lamb abomasums powder;
b. degreasing the lamb abomasum powder obtained in the step a for 3 times by using petroleum ether, naturally drying to obtain lamb abomasum degreased powder, and freezing and storing at the temperature of-20 ℃;
c. the material-liquid ratio is respectively tested by single factor at 18mL/g,24mL/g,30mL/g,36mL/g, extraction temperature of 15 ℃,30 ℃,45 ℃,60 ℃, sodium salt concentration of 0.15M,0.3M,0.35M and 0.6M, and the material-liquid ratio is 30mL/g, the temperature of 30 ℃ and the sodium salt concentration of 0.6M;
d. taking the sodium salt concentration, the temperature and the liquid-material ratio obtained in the step C as independent variables, respectively naming the sodium salt concentration, the temperature and the liquid-material ratio as A, B and C, sequentially naming the extraction rate and the OH free radical scavenging capacity as response values as Y1 and Y2, utilizing Design Expert12 software to carry out Box-Behnken Design (BBD) Design, extracting the lamb abomasum defatted powder obtained in the step B, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at the temperature of 4 ℃ of 7000 for 10min, taking supernatant, and determining the extraction rate and the OH free radical scavenging activity;
e. respectively carrying out regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and carrying out regression fitting on three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
obtaining extraction conditions by taking the extraction rate and the OH free radical scavenging capacity as maximum values, wherein the concentration of sodium chloride is 0.5M, the temperature is 36 ℃, and the liquid-material ratio is 29mL/g;
f. and e, extracting glycoprotein from 10g of lamb abomasum defatted powder under the extraction condition obtained in the step e, centrifuging the extracting solution at the temperature of 4 ℃ at 7000 rpm for 10min, taking the supernatant, dialyzing and desalting the supernatant for 60h by using deionized water as an exchange solution dialysis bag, freeze-drying the solution in the dialysis bag to obtain a lamb abomasum glycoprotein extract, weighing the extract to calculate the extraction rate, and storing the extract at the temperature of-20 ℃ to obtain a product lamb abomasum glycoprotein part.
Measuring protein content by using a BCA method and measuring sugar content by using a phenol-sulfuric acid method;
the extraction rate was calculated using the following formula: extraction ratio (%) = (extract (g)/weight of defatted raw material (g)) × 100%
The extraction rate is 24.05%, the protein content is 22.3% +/-1.2%, and the sugar content is 6.5% +/-0.4%.
Example 5
a. Cleaning feed in stomach cavity with tap water, removing fat and connective tissue, freezing at-80 deg.C for 3 hr, shearing, freeze drying, and pulverizing with liquid nitrogen to obtain lamb abomasums powder;
b. degreasing the lamb abomasum powder obtained in the step a for 3 times by using petroleum ether, naturally drying to obtain lamb abomasum degreased powder, and freezing and storing at the temperature of-20 ℃;
c. the material-liquid ratio is respectively tested by single factor at 18mL/g,24mL/g,30mL/g,36mL/g, extraction temperature of 15 ℃,30 ℃,45 ℃,60 ℃, sodium salt concentration of 0.15M,0.3M,0.35M and 0.6M, and the material-liquid ratio is 30mL/g, the temperature of 36 ℃ and the sodium salt concentration of 0.6M;
d. taking the sodium salt concentration, the temperature and the liquid-material ratio obtained in the step C as independent variables, respectively naming the sodium salt concentration, the temperature and the liquid-material ratio as A, B and C, sequentially naming the extraction rate and the OH free radical scavenging capacity as response values as Y1 and Y2, utilizing Design Expert12 software to carry out Box-Behnken Design (BBD) Design, extracting the lamb abomasum defatted powder obtained in the step B, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at the temperature of 4 ℃ of 7000 for 10min, taking supernatant, and determining the extraction rate and the OH free radical scavenging activity;
e. respectively carrying out regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and carrying out regression fitting on three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
obtaining extraction conditions by taking the extraction rate and the OH free radical scavenging capacity as maximum values, wherein the concentration of sodium chloride is 0.5M, the temperature is 36 ℃, and the liquid-material ratio is 30mL/g;
f. and e, extracting glycoprotein from 10g of lamb abomasum defatted powder under the extraction condition obtained in the step e, centrifuging the extracting solution at the temperature of 4 ℃ at 7000 rpm for 10min, taking the supernatant, dialyzing and desalting the supernatant for 60h by using deionized water as exchange solution through a dialysis bag, freeze-drying the solution in the dialysis bag to obtain a product lamb abomasum glycoprotein part, weighing, calculating the extraction rate, and storing at the temperature of-20 ℃.
Measuring protein content by using a BCA method, and measuring sugar content by using a phenol-sulfuric acid method;
the extraction rate was calculated using the following formula: extraction ratio (%) = (extract (g)/weight of defatted raw material (g)) × 100%
The extraction rate is 23.9%, the protein content is 23.3% +/-1.2%, and the sugar content is 5.8% +/-0.4%.
Example 6
a. Cleaning feed in stomach cavity with tap water, removing fat and connective tissue, freezing at-80 deg.C for 3 hr, shearing, freeze drying, and pulverizing with liquid nitrogen to obtain powder;
b. degreasing the lamb abomasum powder obtained in the step a for 3 times by using petroleum ether, naturally drying to obtain lamb abomasum degreased powder, and freezing and storing at the temperature of-20 ℃;
c. the material-liquid ratio is respectively tested by single factor at 18mL/g,24mL/g,30mL/g,36mL/g, extraction temperature of 15 ℃,30 ℃,45 ℃,60 ℃, sodium salt concentration of 0.15M,0.3M,0.35M and 0.6M, and the material-liquid ratio is 29mL/g, temperature of 35 ℃ and sodium salt concentration of 0.5M;
d. taking the sodium salt concentration, the temperature and the liquid-material ratio obtained in the step C as independent variables, respectively naming the sodium salt concentration, the temperature and the liquid-material ratio as A, B and C, sequentially naming the extraction rate and the OH free radical scavenging capacity as response values as Y1 and Y2, utilizing Design Expert12 software to carry out Box-Behnken Design (BBD) Design, extracting the lamb abomasum defatted powder obtained in the step B, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at the temperature of 4 ℃ of 7000 for 10min, taking supernatant, and determining the extraction rate and the OH free radical scavenging activity;
e. respectively carrying out regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and carrying out regression fitting on three factors to obtain a regression equation:
Y1(％)＝23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2(％)＝36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
obtaining extraction conditions by taking the extraction rate and the OH free radical scavenging capacity as maximum values, wherein the concentration of sodium chloride is 0.4M, the temperature is 30 ℃, and the liquid-material ratio is 32mL/g;
f. and e, extracting glycoprotein from 10g of lamb abomasum defatted powder under the extraction condition obtained in the step e, centrifuging the extracting solution at the temperature of 4 ℃ at 7000 rpm for 10min, taking the supernatant, dialyzing and desalting the supernatant for 60h by using deionized water as exchange solution through a dialysis bag, freeze-drying the solution in the dialysis bag to obtain a product lamb abomasum glycoprotein part, weighing, calculating the extraction rate, and storing at the temperature of-20 ℃.
Measuring protein content by using a BCA method and measuring sugar content by using a phenol-sulfuric acid method;
the extraction rate was calculated using the following formula: extraction ratio (%) = (extract (g)/weight of defatted raw material (g)) × 100%
The extraction rate is 24.6%, the protein content is 23.35% +/-1.2%, and the sugar content is 6.45% +/-0.4%.
Example 7
Analysis of monosaccharide composition of lamb abomasum glycoprotein:
hydrolysis: adding 4mL of 2mol/L trifluoroacetic acid into a headspace bottle containing 10mg of glycoprotein sample, sealing, hydrolyzing at 110 ℃ for 6h at constant temperature, adding an appropriate amount of methanol, reducing pressure, evaporating to dryness, and repeating for 3 times; acetylated derivatives: adding glycolic acid 8mg, pyridine 0.5mL, heating at 90 deg.C for 30min, cooling, adding acetic anhydride 0.5mL, heating at 90 deg.C30min, after cooling N 2 Blow-drying, redissolving the acetylated monosaccharide with chloroform and then analyzing by gas chromatography (GS), the results of which are shown in table 7:
TABLE 5 analysis table of polar molar ratio of monosaccharide composition of lamb abomasum glycoprotein extract
As can be seen from the table: the monosaccharide composition of the glycoprotein part obtained by the invention has the mole percentage size of mannose, galactose, arabinose, xylose and glucose.
Example 8
OH - Radical scavenging ability:
reagent: 6mM FeSO 4 Solution: 166.8mg of FeSO are weighed 4 Dissolving with deionized water, and making the volume to be 100mL.6mM salicylic acid: 138.12mg of salicylic acid was weighed and dissolved in ethanol to 100mL.0.1% hydrogen peroxide: diluting 100 mu L of hydrogen peroxide with 3% concentration to 3mL by using deionized water;
sample preparation: dissolving 6mg of lamb abomasum glycoprotein extract with 2mL of deionized water, centrifuging for later use, and diluting the extract into a series of concentrations;
activity assay was performed by adding 300. Mu.L of 6mM salicylic acid followed by 300. Mu.L of 6mM FeSO 4 The solution, 500. Mu.l of sample solution and 150. Mu.L of 0.1% hydrogen peroxide were mixed in a 4.5mL EP tube to give a sample group; 3 replacing the sample solution with a sample solvent to serve as a control group; using deionized water to replace a hydrogen peroxide solution as a blank control group, wherein the absorbance is as follows; adding the reagent, mixing uniformly, placing at 37 ℃, incubating for 30min, transferring to a 96-well plate, and measuring the absorbance at lambda =510 nm; vc is used as a positive control, and the determination method is the same as that of the sample; each set of experiments was repeated 3 times;
OH radical clearance (%) = (a) 0 -A i- A)×100％/A 0
In the formula A 0 Represents the absorbance of the control group; a. The i Representing the absorbance of the reagent blank; a represents the absorbance of the sample group；
As can be seen from FIG. 10, OH of glycoprotein extract - Radical scavenging ability increases with increasing concentration, is concentration dependent, and IC 50 2.91±0.16mg/mL。
Example 9
DPPH radical scavenging activity:
sample preparation: dissolving 6mg of lamb abomasum glycoprotein extract with 2mL of deionized water, centrifuging, and diluting into a series of concentrations for later use;
and (3) activity determination: samples of 400. Mu.L series concentration were combined with 400. Mu.L of 0.02% DPPH-methanol in 1.5mL EP tubes as a sample set; mixing 400 μ L deionized water with 400 μ L0.02% DPPH-methanol solution to obtain control group; 400 μ L of Vc of serial concentrations was mixed with 400 μ L of 2mM DPPH-methanol solution as a positive control; after mixing well, incubation at 37 ℃ for 30min, transfer to 96-well plates and measure absorbance at λ =517 nm;
DPPH radical clearance (%) = (a) 0- A-A i )×100％/A 0
A 0 Absorbance of control group; a is the absorbance of the sample; a. The i Blank sample; the calculation method of the Vc free radical clearance is the same as that of the sample;
as can be seen from FIG. 11, the DPPH radical scavenging ability of the glycoprotein extract is increased with the increase of the concentration, and the glycoprotein extract has concentration dependence and IC 50 3.9±0.3mg/mL。
Example 10
Total reducing power:
reagent preparation: 1% potassium ferricyanide solution: 1g of potassium ferricyanide is weighed and dissolved in deionized water, and the volume is determined to be 100mL.0.1% ferric chloride solution: 0.1g of ferric chloride is weighed and dissolved in deionized water, the volume is 100mL, and 10% trichloroacetic acid solution: weighing 5g of trichloroacetic acid, dissolving in deionized water, and fixing the volume to 50mL;
sequentially adding 300 mu L of sample solution, 150 mu L of 50Mm phosphate buffer solution with pH of 7.2 and 200 mu L of 1% potassium ferricyanide solution, uniformly mixing, incubating at 50 ℃ for 20min, rapidly cooling, adding 200 mu L of 10% trichloroacetic acid solution, 200 mu L of deionized water and 200 mu L of 0.1% ferric chloride solution, uniformly mixing, standing at room temperature for 5min, and measuring absorbance at 700nm of ultraviolet; the positive control Vc group changes the sample solution into the Vc solution, and the rest operations are the same as the sample; blank group: deionized water is used for replacing a sample solution, the rest operations are the same as those of the sample, and finally the strength of the total reducing capacity is evaluated according to the change of absorbance;
as can be seen from fig. 12, the DPPH radical scavenging ability of the glycoprotein extract was increased with the increase in concentration, and was concentration-dependent.
Claims (4)
1. A method for preparing lamb abomasum glycoprotein part by a response surface method and application thereof are characterized by comprising the following steps:
a. cleaning feed in stomach cavity with tap water, removing fat and connective tissue, freezing at-80 deg.C for 3h, cutting, freeze drying, and pulverizing with liquid nitrogen to obtain lamb abomasums powder;
b. b, degreasing the lamb abomasums powder obtained in the step a for 3 times by using petroleum ether, naturally drying the defatted powder to obtain lamb abomasums defatted powder, and freezing and storing the defatted powder at the temperature of-20 ℃;
c. the material-liquid ratio is respectively tested by single factors at 18mL/g,24mL/g,30mL/g,36mL/g, the extraction temperature is 15 ℃,30 ℃,45 ℃,60 ℃, the sodium salt concentration is 0.15M,0.3M,0.35M and 0.6M, and the material-liquid ratio is 24-36mL/g, the temperature is 15-45 ℃, and the sodium salt concentration is 0.2-0.6M;
d. taking the sodium salt concentration, the temperature and the liquid-material ratio obtained in the step C as independent variables, respectively naming the sodium salt concentration, the temperature and the liquid-material ratio as A, B and C, sequentially naming the extraction rate and the OH free radical scavenging capacity as response values as Y1 and Y2, utilizing Design Expert12 software to carry out Box-Behnken Design (BBD) Design, extracting the lamb abomasum defatted powder obtained in the step B, adding deionized water into the obtained extracting solution to dilute by 4 times, centrifuging at the temperature of 4 ℃ of 7000 for 10min, taking supernatant, and determining the extraction rate and the OH free radical scavenging activity;
e. respectively carrying out regression analysis on the response surface experiment results of the extraction rate and the OH free radical scavenging capacity, and carrying out regression fitting on three factors to obtain a regression equation:
Y1 (%) =23.34-3.68A2-3.25B2-2.91C2+3.53A+1.45B+0.8288C+0.7825AB-0.0875AC-0.84BC
Y2 (%) =36.43-5.99A2-4.55B2-5.78C2+4.97A+2.54B+0.31C+2.4AB-0.8AC-2.4BC
extracting with extraction rate and OH free radical scavenging capacity as maximum values, wherein the concentration of sodium chloride salt is 0.4-0.6M, the temperature is 20-40 deg.C, and the liquid-to-material ratio is 25-35 (mL/g);
f. and e, extracting glycoprotein from the lamb abomasum defatted powder under the extraction condition obtained in the step e, centrifuging the extracting solution at the temperature of 4 ℃ at 7000 for 10min, taking the supernatant, dialyzing and desalting the supernatant by using deionized water as an exchange solution through a dialysis bag at 60h, freeze-drying the solution in the dialysis bag to obtain the lamb abomasum glycoprotein part, weighing, calculating the extraction rate, and storing at the temperature of-20 ℃.
2. Use of the abomasum glycoprotein fraction of lamb obtained according to the method of claim 1 for the preparation of a medicament with antioxidant activity.
3. Use of the abomasum glycoprotein fraction of lamb obtained according to the method of claim 1 for the preparation of a health product with antioxidant activity.
4. Use of the abomasum glycoprotein fraction of lamb obtained according to the method of claim 1 in the preparation of a food product with antioxidant activity.
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