CN110196333B - Extraction method and application of green scale fish glycoprotein - Google Patents
Extraction method and application of green scale fish glycoprotein Download PDFInfo
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Abstract
The invention relates to the technical field of biochemistry, and discloses an extraction method and application of green scale fish glycoprotein, wherein the extraction method comprises the steps of pretreatment, ultrasonic treatment, hot water extraction, concentration alcohol precipitation, washing purification, dialysis freeze-drying and the like, and the extraction process of the green scale fish glycoprotein is contrastively optimized by combining a single-factor experiment with an orthogonal experiment method to obtain the optimal extraction process parameters of the green scale fish glycoprotein; the invention also proves that the green scale glycoprotein has a remarkable effect on the aspect of cell immunoregulation through cell experiment verification, and provides a reference basis for the application of the green scale glycoprotein in the research and development of medicines and foods.
Description
Technical Field
The invention relates to the technical field of biochemistry, and particularly relates to an extraction method and application of green scale fish glycoprotein.
Background
Glycoproteins are substances with biological activity in which oligosaccharide chains and polypeptides are covalently linked in various forms, and are one of important biological macromolecules in organisms, are widely found in animals, plants and microorganisms, even in unicellular organisms and viruses, and are distributed in various forms and types in intracellular and extracellular fluids and tissues of organisms to constitute various active substances in organisms. The natural glycoprotein has physiological activities of resisting oxidation, resisting tumor, improving immunity, etc. The glycoprotein plays an important role in growth and development, information transmission, immune system, nervous system and other various life activities, and is related to the occurrence and development of various diseases (such as cardiovascular diseases, liver and kidney diseases, diabetes, tumor and cancer and the like). With the development of science and technology and the importance of researchers at home and abroad on the development and utilization of marine resources, the research on glycoproteins from marine animals gradually becomes a hot spot, and attracts workers in many subjects such as chemistry, biology, biochemistry, immunology, cell biology, medicine and food science to conduct deep research in the field.
The green scale fish, also called green tangerine peel, green jade, green scale, willow leaf green and the like, is an animal of the herring family, is distributed in Bohai sea, yellow sea, east sea and south sea of China, contains rich protein, amino acid, vitamin, calcium, iron, phosphorus and other mineral elements essential to human bodies, and is a fish with rich nutrition and multiple physiological functions and development potentials. The green scale fish has wide application, can be used as a medicine, has the efficacy of detoxifying and reducing swelling, is mainly used for treating sea snake bite, and has special effects on relieving poisoning symptoms such as dyspnea, malnutrition, tooth obstruction, convulsion and the like. However, no research and report about the extraction method of the green scale fish glycoprotein and the regulation effect of the green scale fish glycoprotein on the immune system is found so far.
In summary, it is necessary to develop an extraction method and application of the green scale fish glycoprotein.
Disclosure of Invention
In order to overcome at least one defect (deficiency) in the prior art, the invention provides the extraction method and the application of the green scale fish glycoprotein.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for extracting green scale fish glycoprotein comprises the following steps:
① pretreating, removing scales of fresh Lepidogrammus pelagi, cleaning, draining, cutting fish, mincing in mincer to obtain paste, and weighing;
② ultrasonic treatment and hot water extraction, namely, uniformly mixing the minced green-scale fish meat obtained in the step ① and distilled water according to the material-liquid ratio of 1:20-40(g/m L), performing ultrasonic treatment for 20-40min to obtain a mixture of the green-scale fish, putting the mixture into a water bath, performing hot water extraction for 2-4h at 80-100 ℃, filtering, reserving the obtained filtrate, mixing the obtained filter residue and the distilled water according to the material-liquid ratio of 1:20-40(g/m L), performing ultrasonic treatment for 20-40min, performing hot water extraction for 1h at 80-100 ℃, filtering, and combining the two filtrates to obtain a mixed solution of the glycoprotein of the green-scale fish;
③ concentrating and precipitating with ethanol, adding the mixed solution obtained in step ② into a rotary evaporator, evaporating and concentrating at 50-70 deg.C to 1/5, adding 3 times of anhydrous ethanol, standing at 4 deg.C for 12-18h, precipitating with ethanol, centrifuging the precipitate, collecting precipitate, repeating the precipitation with ethanol to obtain supernatant with 3 times of anhydrous ethanol, and mixing the precipitates after precipitation with ethanol;
④ removing free protein, washing the precipitate obtained in step ③ with absolute ethyl alcohol and acetone in turn, wherein the amount of absolute ethyl alcohol and acetone is based on the amount of immersed precipitate, completely dissolving the precipitate with distilled water after washing, and removing free protein from the obtained solution by adopting a Sevage method to obtain a green scale fish glycoprotein solution;
⑤ lyophilizing by putting ④ the solution into dialysis bag, dialyzing in distilled water at 4 deg.C for 48 hr, lyophilizing the retentate to obtain glycoprotein powder, and weighing.
Further, in the step ④, the specific step of removing the free protein by adopting the Sevage method is that Sevage reagent is added into the solution obtained by dissolving the precipitate, the solution is placed in a magnetic stirrer to oscillate for 1 hour at 4 ℃, then the solution is placed in a centrifuge to centrifuge for 15-20 minutes at the rotating speed of 12000r/min, the centrifuged solution sequentially comprises a water layer, a protein layer and an organic layer from top to bottom, the upper layer solution is sucked out by a pipette, the steps are repeated until the protein layer does not appear in the centrifuged solution, the protein in the solution is considered to be basically removed, the supernatant is collected, and the organic solvent is removed by rotary evaporation to obtain the green-scaled fish glycoprotein solution, wherein the Sevage reagent is used in 1/4 volume of the precipitate solution, and the Sevage reagent is formed by mixing n-butyl alcohol and chloroform according to the ratio of 1: 4.
Further, in the step ②, the ultrasonic treatment conditions include a temperature of 25-30 ℃, an ultrasonic power of 40W, an ultrasonic treatment time of 30min, and an ultrasonic cleaning machine as an apparatus used for ultrasonic treatment.
Further, in the step ②, in the hot water leaching process of the green scaled fish mixture and the filter residue, the material-liquid ratio of the extraction is 1:20(g/m L), and the temperature of the hot water leaching is 100 ℃.
Further, in step ③, when the alcohol precipitation operation is repeated on the supernatant, the amount of absolute ethanol added each time is equivalent to 3 times of the volume of the supernatant.
Further, in step ⑤, the dialysis bag used has a molecular weight cut-off of 5kDa, and the dialysate is changed every 6 hours during dialysis.
The extraction rate of the green scale fish glycoprotein of the present invention is 100% of the weight of the green scale fish glycoprotein powder obtained in step ⑤/the weight of the green scale fish meat mash obtained in step ①.
The invention also discloses application of the green scale fish glycoprotein prepared by the extraction method in cellular immunoregulation, and experiments prove that the green scale fish glycoprotein influences proliferation and phagocytic capacity of mouse-derived mononuclear macrophage leukemia cells (RAW264.7 cells) and the amount of NO, I L-6 and TNF- α secreted by the cells, so that the green scale fish glycoprotein has a cellular immunoregulation effect.
Compared with the prior art, the technical scheme of the invention has the beneficial effects that:
in the extraction method of the green scale fish glycoprotein, the ultrasonic treatment utilizes the characteristics of strong cavitation, strong vibration and high acceleration of ultrasonic waves to accelerate the dissolution of the green scale fish glycoprotein, thereby improving the extraction rate of the glycoprotein; the method for extracting the green scale fish glycoprotein by hot water extraction has the advantages of simple process, convenient operation, no need of toxic solvent and high safety; after concentration, ethanol is adopted to carry out alcohol precipitation on the glycoprotein solution, glycoprotein with different molecular weight stages can be collected and extracted, the integrity of the prepared glycoprotein variety is ensured, the preparation efficiency is improved, and the method is non-toxic and harmless; the Sevage method is adopted to effectively remove free protein, so that the interference of the free protein on the extraction rate and purity of the product is avoided, and the purity of the product is improved; and then salt and small molecular impurities in the product are removed by a dialysis method, the purity of the product is ensured, the dialysis retentate is subjected to freeze drying, the protein denaturation can be prevented, the inherent components in the protein can be maintained, and the finally obtained glycoprotein has the advantages of looseness, good solubility, stable structure and the like.
In a word, the extraction method of the green scale fish glycoprotein has the advantages of reasonable design, simple process, convenient and feasible operation, easy separation and purification of crude products, high product extraction rate and high purity; a large amount of toxic solvent is not needed in the extraction process, and the product has good safety; the preparation conditions are mild, the structure of the obtained green scale fish glycoprotein is stable, and the activity of the glycoprotein is ensured; the extraction method has the advantages that the used instrument reagent is simple and easy to obtain, so the preparation cost is low, and the extraction method has good repeatability and is easy to popularize and apply.
Drawings
FIG. 1 shows the effect of ultrasound time on the extraction rate of the glycoprotein from green scale fish;
FIG. 2 is a graph showing the effect of extraction time on the extraction rate of the glycoprotein from green fish;
FIG. 3 is a graph showing the effect of extraction temperature on the extraction rate of the glycoprotein from green scale fish;
FIG. 4 shows the effect of feed liquid ratio on the extraction rate of green scale fish glycoprotein;
FIG. 5 is a standard curve and a regression equation for measuring sugar content by phenol-sulfuric acid method;
FIG. 6 is a graph showing the effect of green scale glycoprotein on RAW264.7 cell proliferation;
FIG. 7 is a graph showing the effect of green scale glycoprotein on the phagocytic capacity of RAW264.7 cells;
FIG. 8 is a graph showing the effect of the green scale glycoprotein on NO secretion by RAW264.7 cells;
FIG. 9 shows the effect of the green scale glycoprotein on I L-6 secretion from RAW264.7 cells;
FIG. 10 is a graph showing the effect of the green fish glycoprotein on TNF- α secretion by RAW264.7 cells.
Detailed Description
The present invention is further illustrated by the following specific examples, which are presently preferred embodiments of the invention, but are not intended to limit the scope of the invention, as claimed.
Example 1:
a method for extracting green scale fish glycoprotein comprises the following steps:
① pretreating, removing scales of fresh Lepidogrammus pelagi, cleaning, draining, cutting fish, mincing in mincer to obtain paste, and weighing;
②, ultrasonic treatment and hot water extraction, namely, uniformly mixing the minced green-scale fish meat obtained in the step ① and distilled water according to the material-liquid ratio of 1:20(g/m L), performing ultrasonic treatment for 30min to obtain a mixture of the green-scale fish, putting the mixture into a water bath, performing hot water extraction for 3h at 100 ℃, filtering, reserving the obtained filtrate for later use, mixing the obtained filter residue and the distilled water according to the material-liquid ratio of 1:20(g/m L), performing ultrasonic treatment for 30min, performing hot water extraction for 1h at 100 ℃, filtering, and combining the filtrates of the two times to obtain a mixed solution of the glycoprotein of the green-scale fish;
③ concentrating and precipitating with ethanol, adding the mixed solution obtained in step ② into a rotary evaporator, evaporating and concentrating at 50 deg.C to 1/5, adding 3 times of anhydrous ethanol into the concentrated solution, standing at 4 deg.C for 12h, precipitating with ethanol, centrifuging the precipitate, collecting precipitate, repeating the precipitation with ethanol to obtain supernatant with 3 times of anhydrous ethanol, and mixing the precipitates after precipitation with ethanol;
④ removing free protein, washing the precipitate obtained in step ③ with absolute ethyl alcohol and acetone in turn, wherein the amount of absolute ethyl alcohol and acetone is based on the amount of immersed precipitate, completely dissolving the precipitate with distilled water after washing, and removing free protein from the obtained solution by adopting a Sevage method to obtain a green scale fish glycoprotein solution;
⑤ lyophilizing by putting the solution of the glycoprotein of the scale fish obtained in step ④ into a dialysis bag with molecular weight cutoff of 5kDa, dialyzing in distilled water at 4 deg.C for 48h, changing the dialyzate every 6 hr during dialysis, lyophilizing the dialyzed retentate to obtain the glycoprotein powder of the scale fish, and weighing.
In the step ②, the ultrasonic treatment conditions are that the temperature is 28 ℃, the ultrasonic power is 40W, and the ultrasonic treatment equipment is an SB-4200DTD ultrasonic cleaning machine.
Adding a Sevage reagent into a solution obtained by dissolving a precipitate, placing the solution in a magnetic stirrer at 4 ℃ for oscillation for 1h, placing the solution in a centrifugal machine, centrifuging the solution at a rotating speed of 12000r/min for 15min, sequentially forming a water layer, a protein layer and an organic layer from top to bottom after centrifugation, sucking out the upper layer solution by using a pipette, repeating the steps until the protein layer does not appear in the solution after centrifugation, considering that the protein in the solution is basically removed, collecting a supernatant, and performing rotary evaporation to remove an organic solvent to obtain a green-scaled fish glycoprotein solution, wherein the dosage of the Sevage reagent is 1/4 volume of the precipitate solution, and the Sevage reagent is formed by mixing n-butyl alcohol and chloroform according to a ratio of 1: 4.
Example 2:
example 2 the procedure of example 1 is essentially the same, with the difference that:
in the step ②, ultrasonic treatment and hot water extraction, namely, uniformly mixing the green scale fish meat paste obtained in the step ① with distilled water according to the material-liquid ratio of 1:30(g/m L), carrying out ultrasonic treatment at 25 ℃ for 40min to obtain a green scale fish mixture, putting the green scale fish mixture into a water bath, carrying out hot water extraction at 90 ℃ for 2h, filtering, reserving the obtained filtrate, mixing the obtained filter residue with the distilled water according to the material-liquid ratio of 1:30(g/m L), carrying out ultrasonic treatment for 40min, carrying out hot water extraction at 90 ℃ for 1h, filtering, and combining the filtrates to obtain a green scale fish glycoprotein mixed solution;
in the step ③, in the concentration and alcohol precipitation, the mixed solution obtained in the step ② is added into a rotary evaporator, the mixed solution is evaporated and concentrated to 1/5 with the original volume at 60 ℃, 3 times of volume of absolute ethyl alcohol is added into the concentrated solution, the mixed solution is kept stand for 16 hours at 4 ℃, alcohol precipitation is carried out, the alcohol precipitate is centrifuged, precipitate is collected, the alcohol precipitation operation is repeated on the supernatant until no precipitate is separated out, the amount of the absolute ethyl alcohol added into the supernatant during each alcohol precipitation is 3 times of the volume, and the precipitate is combined after the alcohol precipitation;
in the step ④, free protein is removed, Sevage reagent is added into the solution obtained by dissolving the precipitate, the solution is shaken for 1h at the temperature of 4 ℃, and then the solution is centrifuged for 12min at the rotating speed of 12000r/min, and the other conditions and operations are the same.
Example 3:
example 3 the procedure of example 1 is essentially the same, except that:
in the step ②, ultrasonic treatment and hot water extraction, namely, uniformly mixing the green scale fish meat paste obtained in the step ① with distilled water according to the material-liquid ratio of 1:40(g/m L), performing ultrasonic treatment at 30 ℃ for 20min to obtain a green scale fish mixture, putting the green scale fish mixture into a water bath, performing hot water extraction at 80 ℃ for 4h, filtering, reserving the obtained filtrate, mixing the obtained filter residue with the distilled water according to the material-liquid ratio of 1:40(g/m L), performing ultrasonic treatment for 20min, performing hot water extraction at 80 ℃ for 1h, filtering, and combining the filtrates to obtain a green scale fish glycoprotein mixed solution;
in the step ③, in the concentration and alcohol precipitation, the mixed solution obtained in the step ② is added into a rotary evaporator, the mixed solution is evaporated and concentrated to 1/5 with the original volume at 70 ℃, 3 times of volume of absolute ethyl alcohol is added into the concentrated solution, the mixed solution is kept stand for 18 hours at 4 ℃, alcohol precipitation is carried out, the alcohol precipitate is centrifuged, precipitate is collected, the alcohol precipitation operation is repeated on the supernatant until no precipitate is separated out, the amount of the absolute ethyl alcohol added into the supernatant during each alcohol precipitation is 3 times of the volume, and the precipitate is combined after the alcohol precipitation;
in the step ④, free protein is removed, Sevage reagent is added into the solution obtained by dissolving the precipitate, the solution is shaken for 1h at the temperature of 4 ℃, and then the solution is centrifuged for 20min at the rotating speed of 12000r/min, and the other conditions and operations are the same.
First, comparative experiment:
the optimal extraction conditions of the extraction method of the green scale fish glycoprotein are contrastingly optimized through the combination of a single-factor experiment and an orthogonal experiment, and the main factors influencing the extraction rate of the green scale fish glycoprotein are determined as follows: ultrasonic treatment time, extraction temperature and material-liquid ratio.
The ultrasonic treatment time refers to the time for carrying out ultrasonic treatment on the green scale fish flesh paste (or the filter residue obtained by the first extraction).
The extraction time and the extraction temperature refer to the time and the temperature of hot water leaching.
The feed-liquid ratio refers to the mass-volume ratio of the green scale fish meat paste (or filter residue obtained by first extraction) to the distilled water in the processes of ultrasonic treatment and hot water extraction, and is expressed by g/m L.
1. Single factor experiment
And carrying out single-factor experiments by using the ultrasonic time, the extraction temperature and the feed-liquid ratio in the extraction process of the green scale fish glycoprotein.
① influence of ultrasonic time on extraction rate of green scale fish glycoprotein
100g of green scale fish meat paste is weighed each time, the extraction temperature is 90 ℃, the extraction time is 3h, the material-liquid ratio is 1:30(g/m L), and the influence of different ultrasonic time (20 min, 30min and 40min respectively) on the extraction rate of the green scale fish glycoprotein is researched under the condition that the other extraction steps are kept consistent, wherein the experimental results are shown in a table 1 and an attached drawing 1:
TABLE 1 influence of ultrasound time on the extraction yield of the glycoprotein from green scale fish
As can be seen from Table 1 and attached figure 1, when the extraction time is 3h, the extraction temperature is 90 ℃, and the material-liquid ratio is 1:30(g/m L), the extraction rate of the green scale fish glycoprotein shows a tendency of increasing and then decreasing along with the extension of the ultrasonic time, and when the ultrasonic time is 30min, the extraction rate is 0.052% at most.
② influence of extraction time on extraction rate of green scale fish glycoprotein
100g of green scale fish meat paste is weighed each time, the influence of different extraction times (2 h, 3h and 4h respectively) on the extraction rate of the green scale fish glycoprotein is researched under the condition that the ultrasonic time is 30min, the extraction temperature is 90 ℃, the material-liquid ratio is 1:30(g/m L) and the other extraction steps are kept consistent, and the experimental results are shown in a table 2 and an attached figure 2:
TABLE 2 Effect of extraction time on the extraction yield of the glycoprotein from green-scaled fish
As can be seen from Table 2 and attached figure 2, when the ultrasonic time is 30min, the extraction temperature is 90 ℃, and the feed-to-liquid ratio is 1:30(g/m L), the extraction rate of the green scale fish glycoprotein increases and then decreases with the extension of the extraction time, and when the extraction time is 3 hours, the extraction rate of the glycoprotein is 0.052% at the maximum.
③ influence of extraction temperature on extraction rate of green scale fish glycoprotein
100g of green scale fish meat paste is weighed each time, the ultrasonic time is 30min, the extraction time is 3h, the material-liquid ratio is 1:30(g/m L), and the influence of different extraction temperatures (80 ℃, 90 ℃ and 100 ℃) on the extraction rate of the green scale fish glycoprotein is researched under the condition that the other extraction steps are kept consistent, wherein the experimental results are shown in a table 3 and an attached drawing 3:
TABLE 3 influence of extraction temperature on the extraction yield of the glycoprotein from green-scaled fish
As can be seen from Table 3 and attached figure 3, when the ultrasonic time is 30min, the extraction time is 3h, and the feed-liquid ratio is 1:30(g/m L), the extraction rate of the green scale fish glycoprotein is continuously increased along with the increase of the extraction temperature, and the glycoprotein yield is 0.068% at the highest when the extraction temperature reaches 100 ℃.
④ influence of feed liquid ratio on extraction rate of green scale fish glycoprotein
100g of the green scale fish meat paste is weighed each time, the influence of different feed-liquid ratios (1: 20, 1:30 and 1:40 respectively) on the extraction rate of the green scale fish glycoprotein is researched under the conditions that the ultrasonic time is 30min, the extraction time is 3h, the extraction temperature is 100 ℃ and the other extraction steps are kept consistent. The results are shown in table 4 and figure 4:
TABLE 4 influence of feed liquid ratio on the extraction rate of the glycoprotein from green scale fish
As can be seen from Table 4 and attached figure 4, when the ultrasonic time is 30min, the extraction time is 3h, and the extraction temperature is 90 ℃, the extraction rate of the green scale fish glycoprotein is firstly increased and then decreased along with the increase of the feed-liquid ratio, and when the feed-liquid ratio is 30m L/g, the yield of the glycoprotein is 0.052% at most.
2. Orthogonal experiment
Based on a single-factor experiment result, the optimal extraction process parameters are verified through an orthogonal experiment, 4 factors including ultrasonic time (A), extraction time (B), extraction temperature (C) and material-liquid ratio (D) are selected as experiment factors, and L is carried out by taking the extraction rate of the green scale fish glycoprotein as an index9(34) Orthogonal experimental design is adopted to study the influence of each factor level on the extraction rate and content of the green scale fish glycoprotein and the optimal extraction process conditions, and the factor level design is shown in table 5:
TABLE 5 orthogonal experiment factor horizon
Orthogonal experiments were performed according to the design of orthogonal experimental factor level table 5, and the results of the green scale glycoprotein orthogonal experiments are shown in table 6:
TABLE 6 orthogonal experimental results table
Note: in Table 6, K1Represents the sum of the extraction rates of each experiment at a level of 1 for each factor; k2Represents the sum of the extraction rates of each experiment at a level of 2 for each factor; k3Represents the sum of the extraction rates of each experiment at a level of 3 for each factor; k is a radical of1Represents the average value of the extraction rate of each experiment at a level of 1 for each factor; k is a radical of2Mean values representing the extraction yield per experiment at 2 levels for each factor; k is a radical of3Represents the average of the extraction rate of each experiment at a level of 3 for each factor; the range R refers to k corresponding to the same factor1、k2、k3The difference between the median maximum and minimum.
As can be seen from Table 6, RC>RB>RA>RDTherefore, the significant sequence of the influence of the four factors on the extraction rate of the green scale fish glycoprotein is the extraction temperature>Extraction time>Time of ultrasound>The material-liquid ratio, wherein the influence degree of the extraction temperature and the extraction time is larger, and the influence degree of the ultrasonic time and the material-liquid ratio is smaller. It follows that the optimal combination of influence factors for the extraction of the green scale fish glycoprotein is A2B2C3D1The ultrasonic time is 30min, the extraction time is 3h, the extraction temperature is 100 ℃, the material-liquid ratio is 1:20(g/m L), the optimal process condition for extracting the green scale fish glycoprotein is provided, and the extraction rate of the green scale fish glycoprotein under the condition is 0.057%.
II, a green scale glycoprotein purity verification experiment:
the purity of the green scale fish glycoprotein prepared in the embodiment 1 of the invention is verified through experimental determination. The glycoprotein is a macromolecular compound, the purity standard of which cannot be measured by the common purity standard of a small molecular compound, because even pure glycoprotein is not uniform in a microscopic scale, and therefore, the purity of the green scale fish glycoprotein prepared by the method is characterized by the following modes: the green fish skin glycoprotein has a purity of sugar content + protein content.
① determination of sugar content
The determination method comprises the following steps: determining the absorbance of glucose with different concentrations by a phenol-sulfuric acid method to formulate a concentration-absorbance standard curve, preparing a solution with a certain concentration from the green-scale fish glycoprotein sample, measuring the absorbance of the solution according to the operation under the standard curve term, substituting the solution into a concentration-absorbance standard curve equation, and calculating to obtain the sugar content in the glycoprotein.
The preparation of the standard curve comprises accurately weighing 1mg of anhydrous glucose standard substance, adding appropriate amount of distilled water for dissolving, fixing the volume to the scale mark with a 10m L volumetric flask, shaking up, and preparing into 0.1mg/m L standard solution.
Respectively sucking glucose standard solutions of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 and 4.0m L into a 10m L volumetric flask, adding distilled water to a constant volume to be marked, shaking up to prepare a series of glucose standard solutions.
Respectively sucking 200 mu L series standard solutions into a 2m L centrifuge tube, respectively adding 100 mu L of the prepared 5% phenol reagent, shaking up, then adding 500 mu L of concentrated sulfuric acid, shaking up the mixed solution, standing for 20min, measuring the light absorption value of the solution at 490nm, drawing a standard curve and establishing a regression equation, wherein the standard curve and the regression equation are shown in the attached figure 5.
Accurately weighing 10mg of green scale fish glycoprotein, adding distilled water to dissolve, fixing the volume to 10m L, wherein the final concentration of the sample is 1mg/m L, accurately absorbing 0.1m L of each sample solution, measuring the light absorption value according to a standard curve manufacturing method, and calculating the sugar content of the glycoprotein sample to be 15.2% according to the measured light absorption value and an established regression equation.
② determination of protein content
Weighing 20mg of green scale fish glycoprotein in a hydrolysis tube, adding hydrochloric acid solution with the concentration of 18m L being 6 mol/L, filling nitrogen for 2min, vacuumizing and sealing the tube, placing the tube in an oven for hydrolysis at 110 ℃ for 24h, placing the hydrolyzed solution in a water bath kettle at 100 ℃ to fully volatilize the hydrochloric acid so as to remove the acid in the hydrolyzed solution, washing the solution for 3 times by using double distilled water, continuously evaporating the residual hydrochloric acid, fixing the volume to 5m L by using distilled water, filtering the solution by using a 0.45 mu m microporous filter membrane, and measuring and analyzing the amino acid content of the filtrate by using an amino acid analyzer, wherein the measurement result is shown in table 7:
TABLE 7 amino acid content test results
As can be seen from Table 7, the amino acid content of the present invention-produced capelin is 59.74%, i.e., the protein content of the capelin is 59.74%,
in summary, the purity of the present invention, i.e. sugar content + protein content, is 74.94%.
Thirdly, immunoregulation experiment of green scale fish glycoprotein:
experiments prove that the cell immunoregulation effect of the green scale fish glycoprotein prepared in the embodiment 1 of the invention is realized, and the experimental method and the results are as follows:
1. subject: mice derived mononuclear macrophage leukemia cells (RAW264.7 cells).
2. The main reagents are as follows:
3. culture and passaging of cells
Culture of RAW264.7 cells: complete medium containing 10% fetal bovine serum and 1% double-resistant DEME in 5% (V/V) CO2Culturing in a constant-temperature incubator at 37 ℃;
when the cells were attached to the bottom of the flask for passaging, the cells were digested with pancreatin digest containing 0.25% EDTA. The cell adheres loosely, the digestion time of the membrane enzyme is not suitable to be too long, and the cell should be handled lightly in the experiment.
4. Effect of Lepidoptera glycoprotein on RAW264.7 cell proliferation
Determining influence of green scale fish glycoprotein on proliferation capacity of RAW264.7 macrophage by MTT method, collecting RAW264.7 cell in logarithmic growth phase, counting with blood count plate, and adjusting cell suspension concentration to 3 × 104Cells/m L, inoculating to a 96-well plate, culturing at 37 ℃ for 24h at 100 mu L per well, discarding the supernatant, adding sample solutions of the green squama fish glycoprotein with different concentrations (10-120 mu g/m L), setting 3 multiple wells per concentration, culturing the culture solution at 37 ℃ for 24h as a blank control, adding 10 mu L of MTT solution per well after 24h, continuously culturing in an incubator for 4h, then sucking off the supernatant, adding 100 mu L of dimethyl sulfoxide per well, shaking and mixing uniformly, and detecting the absorbance value at 570nm by an enzyme reader, wherein the experimental result is shown in figure 6.
As shown in figure 6, in the concentration range of 10-120 mug/m L, the alepidote glycoprotein can promote the proliferation of RAW264.7 macrophages, in the concentration range of 10-80 mug/m L, the promotion effect is more obvious along with the increase of the concentration of the alepidote glycoprotein, and in 80-120 mug/m L, although the cell proliferation promotion effect is realized, the effect is lower than 80 mug/m L.
5. Effect of Lepidoptera glycoprotein on phagocytic Capacity of RAW264.7 cells
The influence of the green scale glycoprotein on the phagocytosis of RAW264.7 macrophages is detected by adopting a neutral red phagocytosis experiment. Taking cells in logarithmic growth phase, counting by using a blood counting chamber,adjusting the cell suspension concentration to 3 × 104Cells/m L, inoculating to a 96-well plate, culturing at 37 ℃ for 24h at 100 mu L per well, discarding the supernatant, adding sample solutions of green squama fish glycoprotein with different concentrations (10-120 mu g/m L), setting 6 multiple wells per concentration, using the culture solution as a blank control, using L PS solution with 2 mu g/m L as a positive control, culturing at 37 ℃ for 24h, discarding the supernatant after 24h, adding 100 mu L neutral red solution (0.5mg/m L) per well, continuing to culture for 1h, discarding the supernatant after 1h, washing 3 times with PBS solution, adding 100 mu L cell lysate (glacial acetic acid; ethanol ═ 1:1(v/v)) per well, incubating overnight at room temperature, shaking, and detecting the light absorption value at 540nm by a microplate reader, wherein the experimental results are shown in FIG. 7.
In FIG. 7, Black represents a blank control group containing culture medium alone, CK represents a negative control group containing culture medium + cells, and L PS represents a positive control group containing culture medium + cells + 2. mu.g/m L in L PS solution.
As shown in figure 7, the capelin glycoprotein can promote the phagocytosis ability of RAW264.7 macrophages within the concentration range of 10-120 mu g/m L, and the promotion effect is more obvious along with the increase of the concentration of the capelin glycoprotein within the concentration range of 10-80 mu g/m L, while the capelin glycoprotein has the promotion effect within the concentration range of 80-120 mu g/m L, but the effect is not obviously different from that of the capelin glycoprotein at 80 mu g/m L.
6. Effect of Pilenmex glycoprotein on NO, I L-6 and TNF- α secretion from RAW264.7 cells
When acting on RAW264.7 cells, glycoprotein can induce the generation of various cellular immune response reactions, and the immune regulation effect is realized by promoting the proliferation of cells and producing cytokines, wherein NO is an active substance in organisms, the release amount of NO is an important index for judging whether the immune regulation activity of macrophages is enhanced, RAW264.7 cells are stimulated by glycoprotein and can promote the secretion of cytokines, such as Tumor Necrosis Factor (TNF) and interleukin (I L), and the like, which can regulate the immune function of organisms to a certain degree and play an important role in immune response reactions, and lipopolysaccharide (L PS) is a main component in cell walls of gram-negative bacteria, has toxicity to hosts, has the effect of inducing cells to generate immune response reactions and is commonly used as a positive control for evaluating the immune regulation activity.
Taking cells in logarithmic growth phase, counting, diluting cell suspension with culture medium, and adjusting cell suspension concentration to 5x105Cells/m L, inoculated into 24-well plates, cultured at 37 ℃ for 24h at 100 mu L per well, and discarded supernatant, the experimental group added with various concentrations (10-120 mu g/m L) of sample solution of the green fish glycoprotein, each concentration set 4 multiple wells, negative control group (CK) ═ culture solution + Cells, positive control group (L PS) ═ L PS solution (2 mu g/m L) + culture solution + Cells, each group of Cells cultured at 37 ℃ for 24 h.
① detecting influence of alepidote glycoprotein on NO content secreted by RAW264.7 cells by Griess reagent, sucking cell culture supernatant 50 mu L, adding 50 mu L Griess reagent I, adding 50 mu L Griess reagent II solution, shaking and mixing uniformly, detecting light absorption value at 540nm by enzyme labeling instrument, and simultaneously detecting NaNO of serial concentrations (1, 2, 5, 10, 40, 60, 100 nmol/L) diluted by DMEM culture solution2The solution is used as a standard substance, a standard curve is drawn, a regression equation is established, the content of NO in the cell supernatant is calculated according to the regression equation, and the experimental result is shown in figure 8.
As can be seen from FIG. 8, the green scale glycoprotein can significantly promote NO secretion of RAW264.7 cells, and is dose-dependent, and when the concentration of the green scale glycoprotein reaches 60 μ g/m L, the amount of NO released is about 35 times that of the blank control group.
② the influence of the alepidote glycoprotein on the I L-6 content secreted by RAW264.7 cells is detected by adopting an E L ISA reagent method, the content of I L-6 in cell supernatant is detected by absorbing the cell culture supernatant with 100 mu L and adopting an I L-6E L ISA reagent according to the detection method described in the kit specification, and the experimental result of the influence of the alepidote glycoprotein on the I L-6 content secreted by RAW264.7 cells is shown in figure 9.
As can be seen from the attached figure 9, compared with the positive control group, the effect of the green scale fish glycoprotein on promoting the secretion of I L-6 by RAW264.7 cells is not obvious, but the secretion of I L-6 is increased along with the increase of the dosage, and the promotion effect is gradually obvious, when the concentration of the green scale fish glycoprotein reaches 60 mu g/m L, the secretion of I L-6 is improved by 4.75 times compared with the blank control group.
③, detecting the influence of the green scale glycoprotein on the content of TNF- α secreted by RAW264.7 cells by adopting an E L ISA reagent method, sucking cell culture supernatant fluid of 100 mu L, detecting the content of TNF- α in the cell supernatant fluid by adopting a TNF- α E L ISA reagent according to a detection method described by a kit instruction, and the experimental result of the influence of the green scale glycoprotein on the TNF- α secreted by the RAW264.7 cells is shown in figure 10.
As can be seen from FIG. 10, the green fish scale glycoprotein can significantly promote TNF- α secretion of RAW264.7 macrophage in the concentration range of 5-60 μ g/m L, and is dose-dependent, compared with the blank control group, when the concentration of the green fish scale glycoprotein reaches 60 μ g/m L, the secretion of TNF- α is 21 times that of the blank control group.
In conclusion, the low-dose green scale fish glycoprotein can remarkably improve the proliferation and phagocytosis capacity of the RAW264.7 cells and remarkably promote the RAW264.7 cells to secrete NO, I L-6 and TNF- α, so that the green scale fish glycoprotein has an immunoregulation effect, and provides a reference basis for the research and development of green scale fish applied to immunoregulation medicaments and health-care foods and the maximum development and utilization of the green scale fish.
The above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. It will be apparent to those skilled in the art that other variations and modifications may be made in the foregoing description, and it is not necessary or necessary to exhaustively enumerate all embodiments herein. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (9)
1. The application of the extracted green scale fish glycoprotein in cellular immune regulation is characterized in that the extracted green scale fish glycoprotein is used for promoting NO, I L-6 and TNF- α secreted by RAW264.7 cells in the cellular immune regulation;
the extracted green scale fish glycoprotein is extracted by an extraction method comprising the following steps:
① pretreating, removing scales of fresh Lepidoporus japonicus, cleaning, draining, cutting fish meat, and mincing to obtain meat paste;
②, ultrasonic treatment and hot water extraction, namely uniformly mixing the minced green-scale fish meat obtained in the step ① with distilled water according to the material-liquid ratio of 1:20g/m L-1: 40g/m L, performing ultrasonic treatment for 20-40min to obtain a mixture of the green-scale fish, putting the mixture into a water bath pot, performing hot water extraction for 2-4h at the temperature of 80-100 ℃, filtering, keeping the obtained filtrate for later use, performing repeated ultrasonic treatment and hot water extraction on the obtained filter residue, filtering, and combining the two filtrates to obtain a mixed solution of the green-scale fish glycoprotein;
③ concentrating and precipitating with ethanol, adding the mixed solution obtained in step ② into a rotary evaporator, evaporating and concentrating at 50-70 deg.C to 1/5, adding 3 times of anhydrous ethanol, standing at 4 deg.C for 12-18h, precipitating with ethanol, centrifuging the precipitate, collecting precipitate, precipitating the supernatant with ethanol until no precipitate is separated out, and mixing the precipitates;
④ removing free protein, washing the precipitate obtained in step ③ with anhydrous ethanol and acetone in sequence, dissolving the precipitate with distilled water, and removing free protein from the obtained solution by Sevage method to obtain the green scale fish glycoprotein solution;
⑤ lyophilizing by dialysis, namely filling the above-mentioned ④ solution into dialysis bag, placing in distilled water, dialyzing for 48h, and lyophilizing the retentate to obtain the final product.
2. The application of the extracted capelin glycoprotein in cellular immunoregulation as claimed in claim 1, wherein in the step ④, the specific steps of removing free protein by adopting the Sevage method are that Sevage reagent is added into the solution obtained by dissolving the precipitate, the solution is shaken for 1h at 4 ℃, then the solution is centrifuged for 15-20min at 12000r/min, the supernatant is collected, and the steps are repeated until no protein layer is separated out.
3. The use of extracted capelin glycoprotein in cellular immunomodulation according to claim 2, wherein in step ④, the Sevage method is used to remove free protein under the condition that the Sevage reagent is used in an amount corresponding to 1/4 volume of the precipitate solution, and the Sevage reagent is a mixture of n-butanol and chloroform at a ratio of 1: 4.
4. The use of extracted lepidopteran glycoprotein according to claim 1, wherein said sonication in step ② is performed for 30 min.
5. The use of the extracted capelin glycoprotein in cellular immune modulation according to claim 1, wherein the ratio of the capelin puree to the distilled water in the extraction step ② is 1:20g/m L, the hot water extraction temperature is 100 ℃, and the extraction time is 3 h.
6. The application of the extracted capelin glycoprotein in cell immunoregulation according to claim 1, wherein in the step ②, when the obtained filter residue is repeatedly extracted, the filter residue is mixed with distilled water according to a material-liquid ratio of 1:20g/m L-1: 40g/m L, is subjected to ultrasonic treatment for 20-40min, and is subjected to hot water extraction at 80-100 ℃ for 1 h.
7. The use of extracted lepidopteran glycoprotein in cellular immunomodulation according to claim 1, wherein in step ②, the sonication is performed under conditions of a temperature of 25-30 ℃ and a sonication power of 40W.
8. The use of the extracted capelin glycoprotein of claim 1, wherein the amount of absolute ethanol added in each time the alcohol precipitation operation is repeated on the supernatant in step ③ is equal to 3 times the volume of the supernatant.
9. The use of extracted lepidopteran glycoprotein of claim 1, wherein in step ⑤, dialysis is performed at 4 ℃, the molecular weight cut-off of the dialysis bag is 5kDa, and the dialysate is changed every 6 hours during dialysis.
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