CN112266423A - Method for optimizing yam polysaccharide extraction conditions by using response surface method - Google Patents
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Abstract
The invention relates to a method for optimizing yam polysaccharide extraction conditions by using a response surface method, which comprises the following steps: cleaning rhizoma Dioscoreae, peeling, slicing, lyophilizing, pulverizing, and removing pigment and oligosaccharide; extracting with distilled water, vacuum concentrating, precipitating with ethanol, centrifuging, removing protein, salt and small molecular substances, and freeze drying; determining variable parameters of yam polysaccharide extraction by adopting a response surface method; finally, determining the rate taking condition as follows: the temperature is 70.51 ℃, the time is 2.06h, the material-liquid ratio is 1:10.48, and the polysaccharide extraction rate is 12.04%. The method adopts a response surface method to determine the variable parameters of yam polysaccharide extraction, selects three factors which have more obvious influence on the yam polysaccharide extraction rate, namely extraction time, temperature and material-liquid ratio, designs three-factor three-level orthogonal test on the basis of single-factor test, further optimizes the optimal extraction process conditions of yam polysaccharide, and provides reference basis for comprehensive development and utilization of yam.
Description
Technical Field
The invention belongs to the technical field of food, and particularly relates to a method for optimizing yam polysaccharide extraction conditions by using a response surface method.
Background
The rhizoma Dioscoreae is a traditional food with homology of medicine and food, is tuber of Dioscorea (Dioscorea opposita Thunb) of Dioscoreaceae, and has effects of invigorating kidney-qi, nourishing spleen and stomach, relieving diarrhea and dysentery, and resolving phlegm and saliva. Yam polysaccharide (yam polysaccharide) is a main functional component in yam (Dioscorea opposita Thunb), and research shows that the yam polysaccharide has biological activities of anti-tumor activity, immunoregulation, antioxidation, diabetes resistance, mutation resistance and the like.
The extraction method of the plant polysaccharide is generally water bath extraction, has long time and high production cost, and is not beneficial to industrial production. The response surface method is an effective method for optimizing process conditions, a continuous variable curved surface model is established, the relation between a plurality of response variables and a series of variables is fitted through a regression equation, and the optimal combination condition of a plurality of test factors can be rapidly determined.
Through searching, the following two patent publications relevant to the present application are found:
1. a method for extracting rhizoma Dioscoreae polysaccharide (CN106957373A) comprises the following steps: 1) peeling fresh Chinese yam as raw material, and cutting into blocks; 2) adding the extracting solution, and pulping to obtain a yam homogenate; 3) centrifuging the yam homogenate to obtain yam homogenate centrifugation supernatant; 4) filtering the supernatant to remove starch to obtain filtrate; 5) adjusting the pH value of the filtrate, and carrying out magnetic stirring to obtain a suspension; 6) centrifuging the suspension, and precipitating rhizoma Dioscoreae protein; 7) taking the supernatant, adjusting the pH value, the ultrafiltration temperature and the ultrafiltration pressure, and performing ultrafiltration; 8) and (4) carrying out vacuum freeze drying on the trapped part to obtain the yam polysaccharide. The method for extracting the Chinese yam polysaccharide by using the ultrafiltration concentration method has the advantages of high polysaccharide extraction rate of 88.69%, simple operation and easy realization. The purity of the obtained yam polysaccharide is 43.84% as shown by component determination, infrared spectrum analysis, scanning electron microscope analysis and antioxidant activity determination, and the yam polysaccharide has high antioxidant activity.
2. The separation and purification method of Chinese yam polysaccharide (CN103910809A) comprises the steps of pretreatment, hot water bath leaching, protein removal by a Sevage method, crude polysaccharide separation by adopting high-speed counter-current chromatography, further purification by a SephadexG-100 chromatographic column and the like. The invention determines the extraction process flow and process parameters of the yam polysaccharide, determines the chromatographic conditions of high-speed countercurrent chromatography and SephadexG-100 purified yam polysaccharide, identifies the structure of the yam polysaccharide and determines the antioxidant and bacteriostatic activity of the yam polysaccharide.
By contrast, the present patent application is substantially different from the above patent publications.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for optimizing the extraction conditions of Chinese yam polysaccharide by using a response surface method.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for optimizing yam polysaccharide extraction conditions by using a response surface method comprises the following steps:
the preparation method comprises the steps of cleaning, peeling and slicing the Chinese yam, freeze-drying the Chinese yam in a freeze-drying machine overnight, and crushing the Chinese yam by a crusher and sieving the Chinese yam by a 30-50-mesh sieve; quantitatively weighing the sieved yam powder, adding 3-4 times of volume of anhydrous petroleum ether for degreasing, and then adding 4-5 times of volume of ethanol solution for removing 80-100% of pigment and oligosaccharide; dissolving the degreased and decolored powder in distilled water according to the feed-liquid ratio of 1:10-1:11, and leaching for three times at 70 ℃, wherein each time is 2-2.5 hours; mixing the extractive solutions, and vacuum concentrating to 1/5-1/6 volume of original volume; adding 3-4 times volume of ethanol, precipitating with ethanol at 4 deg.C overnight, centrifuging the precipitated solution, collecting precipitate, dissolving in water, removing protein in the solution by enzyme + sevage method, removing salt and small molecular substances by dialysis method, and freeze drying to obtain rhizoma Dioscoreae crude polysaccharide;
secondly, adopt the response surface method to confirm the variable parameter that chinese yam polysaccharide extracted, set up three-factor three-level's test mode and carry out the repeated test and obtain the experimental result, calculate chinese yam polysaccharide's extraction rate, adopt software to carry out multiple regression analysis again, it is to the regression equation of response value influence to reachd the experimental factor:
R(％)＝12.00+0.24A+0.34B+0.34C-0.65AB-0.064AC-0.26BC-1.55A2-1.18B2-1.60C2
in the formula, R is the extraction rate of the yam polysaccharide, a variable parameter A is the extraction temperature, a variable parameter B is the extraction time, and a variable parameter C is the ratio of material to liquid;
finally, determining the rate taking condition as follows: the temperature is 70.51 ℃, the time is 2.06h, the material-liquid ratio is 1:10.48, and the polysaccharide extraction rate is 12.04%.
In the first step, the using amount of petroleum ether is 3-4mL per gram of Chinese yam powder, the using amount of an ethanol solution for removing pigments and oligosaccharides is 4-5mL per gram of Chinese yam powder, and the using amount of the ethanol solution for alcohol precipitation is 3-4 mL.
In the first step, the rotating speed of the centrifuge is 6000r/min, and the time is 10 min.
In the step, the enzyme used for deproteinization in the step is papain, and the sevage reagent is chloroform: n-butanol is 4:1, and the protein removal times are 8; dialyzing with distilled water for three days by using a dialysis bag with the molecular weight cutoff of 8000-14000Da, and changing water twice every day.
And, the calculation formula of the polysaccharide extraction rate in the step II is as follows:
furthermore, the method comprises the steps of:
the polysaccharide extraction rate is measured by a phenol-sulfuric acid method.
The method comprises the following specific steps:
weighing an anhydrous glucose standard substance, and drying in a drying oven at 105 ℃ until the weight is constant; accurately weighing the dried anhydrous glucose, and performing constant volume to obtain a glucose standard solution of 0.1mg/mL for later use;
accurately measuring 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9mL of glucose standard solution in a test tube, and respectively supplementing 1mL of glucose standard solution with distilled water; adding 1.0mL of phenol solution with the mass concentration of 6% into each tube, then slowly adding 5mL of sulfuric acid, shaking and uniformly mixing, carrying out water bath at 40 ℃ for 15min, taking out and cooling to room temperature, and measuring the absorbance value at 490 nm;
(iii) taking the concentration C (mg/mL) of glucose as the abscissa, A490And drawing a standard curve by taking the ordinate.
The invention has the advantages and positive effects that:
1. the method optimizes the yam polysaccharide extraction process by using a response surface analysis method on the basis of a single-factor test. The method adopts a response surface method to determine the variable parameters of yam polysaccharide extraction, selects three factors which have more obvious influence on the yam polysaccharide extraction rate, namely extraction time, temperature and material-liquid ratio, designs three-factor three-level orthogonal test on the basis of single-factor test, further optimizes the optimal extraction process conditions of yam polysaccharide, and provides reference basis for comprehensive development and utilization of yam.
2. The method adopts a response surface method to determine the variable parameters of the yam polysaccharide extraction, selects three factors which have more obvious influence on the yam polysaccharide extraction rate, namely temperature, time and material-liquid ratio, and designs a response surface analysis test with three factors and three levels on the basis of a single-factor test, thereby optimizing the optimal extraction process conditions of the yam polysaccharide.
3. The method provided by the invention has the advantages that the statistical software Design expert8.0 is used for simply, conveniently and reliably carrying out optimization test and data analysis, the process parameters for extracting the Chinese yam polysaccharide are optimized, a better effect is obtained, and a foundation is laid for the popularization and application of the new Chinese yam polysaccharide variety. Saving the cost for industrial production.
Drawings
FIG. 1 is a graph showing the effect of extraction temperature on the extraction rate of yam polysaccharide in the present invention;
FIG. 2 is a graph showing the effect of extraction time on the extraction rate of yam polysaccharide in the present invention;
FIG. 3 is a graph showing the effect of feed liquid ratio on the extraction rate of yam polysaccharide in the present invention;
FIG. 4 is a graph showing the response surface and contour plot of temperature and time to the extraction rate of yam polysaccharide in the present invention; wherein, the upper graph is a response surface and a contour map of the time to the extraction rate of the yam polysaccharide, and the lower graph is a response surface and a contour map of the temperature to the extraction rate of the yam polysaccharide;
FIG. 5 is a response surface and contour plot of the temperature versus feed liquid ratio for the yam polysaccharide extraction rate of the present invention; wherein, the upper graph is a response surface and a contour map of the extraction rate of the yam polysaccharide compared with the feed liquid, and the lower graph is a response surface and a contour map of the extraction rate of the yam polysaccharide compared with the temperature;
FIG. 6 is a response surface and contour plot of the time to feed liquid ratio versus yam polysaccharide extraction rate in accordance with the present invention; wherein, the upper graph is a response surface and a contour map of the extraction rate of the yam polysaccharide compared with the feed liquid, and the lower graph is a response surface and a contour map of the extraction rate of the yam polysaccharide compared with time.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products if no special description is provided, the method used in the invention is all conventional methods in the field if no special description is provided, and the mass of all the materials used in the invention is the conventional use mass.
A method for optimizing yam polysaccharide extraction conditions by using a response surface method comprises the following steps:
the preparation method comprises the steps of cleaning, peeling and slicing the Chinese yam, freeze-drying the Chinese yam in a freeze-drying machine overnight, and crushing the Chinese yam by a crusher and sieving the Chinese yam by a 30-50-mesh sieve; quantitatively weighing the sieved yam powder by using a balance, adding 3-4 times of volume of anhydrous petroleum ether for degreasing, and then adding 4-5 times of volume of ethanol solution for removing 80% -100% of pigment and oligosaccharide; dissolving the degreased and decolored powder in distilled water according to the feed-liquid ratio of 1:10-1:11, and leaching for three times at 70 ℃, wherein each time is 2-2.5 hours; mixing the extractive solutions, and vacuum concentrating to 1/5-1/6 volume of original volume; adding 3-4 times volume of ethanol, precipitating with ethanol at 4 deg.C overnight, centrifuging the precipitated solution, collecting precipitate, dissolving in water, removing protein in the solution by enzyme + sevage method, removing salt and small molecular substances by dialysis method, and freeze drying to obtain rhizoma Dioscoreae crude polysaccharide;
secondly, adopt the response surface method to confirm the variable parameter that chinese yam polysaccharide extracted, set up three-factor three-level's test mode and carry out the repeated test and obtain the experimental result, calculate chinese yam polysaccharide's extraction rate, adopt software to carry out multiple regression analysis again, it is to the regression equation of response value influence to reachd the experimental factor:
R(％)＝12.00+0.24A+0.34B+0.34C-0.65AB-0.064AC-0.26BC-1.55A2-1.18B2-1.60C2
in the formula, R is the extraction rate of the yam polysaccharide, a variable parameter A is the extraction temperature, a variable parameter B is the extraction time, and a variable parameter C is the ratio of material to liquid;
finally, determining the rate taking condition as follows: the temperature is 70.51 ℃, the time is 2.06h, the material-liquid ratio is 1:10.48, and the polysaccharide extraction rate is 12.04%.
Preferably, in the step of performing the alcohol precipitation, the using amount of petroleum ether is 3-4mL per gram of Chinese yam powder, the using amount of an alcohol solution for removing pigments and oligosaccharides is 4-5mL per gram of Chinese yam powder, and the using amount of the alcohol solution for alcohol precipitation is 3-4 mL.
Preferably, the rotating speed of the centrifuge in the step of the process is 6000r/min, and the time is 10 min.
Preferably, the enzyme used for deproteinization in the step of the process is papain, and the sevage reagent is chloroform: n-butanol is 4:1, and the protein removal times are 8; dialyzing with distilled water for three days by using a dialysis bag with the molecular weight cutoff of 8000-14000Da, and changing water twice every day.
Preferably, the calculation formula of the polysaccharide extraction rate in the step II is as follows:
preferably, the method further comprises the steps of:
the polysaccharide extraction rate is measured by a phenol-sulfuric acid method.
Preferably, the specific steps are as follows:
weighing an anhydrous glucose standard substance, and drying in a drying oven at 105 ℃ until the weight is constant; accurately weighing the dried anhydrous glucose, and performing constant volume to obtain a glucose standard solution of 0.1mg/mL for later use;
accurately measuring 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9mL of glucose standard solution in a test tube, and respectively supplementing 1mL of glucose standard solution with distilled water; adding 1.0mL of phenol solution with the mass concentration of 6% into each tube, then slowly adding 5mL of sulfuric acid, shaking and uniformly mixing, carrying out water bath at 40 ℃ for 15min, taking out and cooling to room temperature, and measuring the absorbance value at 490 nm;
(iii) taking the concentration C (mg/mL) of glucose as the abscissa, A490And drawing a standard curve by taking the ordinate.
Specifically, the preparation and detection are as follows:
first, sample preparation
Cleaning rhizoma Dioscoreae, peeling, slicing, lyophilizing overnight in a lyophilizer, pulverizing with a pulverizer, and sieving with 30 mesh sieve. Quantitatively weighing the sieved yam powder by using a balance, adding anhydrous petroleum ether for degreasing, and then adding an ethanol solution for removing part of pigments and oligosaccharides; the powder of the degreasing and the decoloration is leached for three times at 70 ℃ with a feed-liquor ratio of 1:10, and each time is leached for 2.5 hours. Mixing extractive solutions, and vacuum concentrating to a certain volume. Adding 4 times volume of ethanol, precipitating with ethanol at 4 deg.C overnight, centrifuging the precipitated solution, collecting precipitate, dissolving in water, removing protein in the solution by enzyme + sevage method, removing salt and small molecular substances by dialysis method, and freeze drying to obtain rhizoma Dioscoreae crude polysaccharide.
Second, experimental design and statistical analysis
1. The single factor experiment finally determines the rate taking conditions as follows: the temperature is 70.51 deg.C, the time is 2.06h, the material-liquid ratio is 1:10.48, and the polysaccharide extraction rate is 12.04%
(1) Leaching temperature: determining the extraction time to be 2h, and the material-liquid ratio is 1:20, extracting for 3 times in water bath at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C and 90 deg.C, collecting the leaching solution, and repeating for three times. The polysaccharide content is quantitatively determined by adopting a phenol-sulfuric acid method, the extraction rate of the yam polysaccharide is calculated, and each experiment is carried out in parallel for three times.
(2) Leaching time: adding distilled water according to the material-liquid ratio of 1:20, determining the optimal extraction temperature, extracting for 3 times for 1h, 1.5h, 2h, 2.5h and 3h respectively, collecting the extractive solution, and repeating for three times in parallel.
(3) The material-liquid ratio: extracting with distilled water at the ratio of 5, 10, 15, 20, 25 for 3 times according to the determined optimal extraction temperature and time, collecting the extract, and repeating for three times.
2. Response surface method optimization design
According to the single-factor experiment result, three factors of extraction temperature, extraction time and feed-liquid ratio are selected, a response surface test is designed according to three levels of the three factors, the yam polysaccharide extraction rate is taken as a response value, the optimal extraction condition is obtained, and the test is verified. The experimental design factors and levels are shown in table 1.
TABLE 1 test factors and level settings
The extraction temperature (A), the extraction time (B) and the feed-liquid ratio (C) are taken as the extraction rate of the polysaccharide, and the test scheme and the results are shown in Table 2.
Table 2 experimental protocol and extraction rate for response surface analysis
3. Model building and statistical analysis
And performing multiple regression analysis according to the obtained data to obtain a multiple quadratic regression equation between the corresponding variable extraction temperature, the extraction time and the ratio of the material to the liquid and the response value polysaccharide extraction rate.
R(％)＝12.00+0.24A+0.34B+0.34C-0.65AB-0.064AC-0.26BC-1.55A2-1.18B2-1.60C2
The results of the anova analysis in Table 3 show that the model has a high significance due to the modeling term P < 0.0001. The simulation loss term 0.5400 is more than 0.05, which indicates that the model has high reliability; regression correlation coefficient R20.9994 and coefficient of determination R2 Adj0.9986, this indicates that the test value of the test has a relatively high fit to the predicted value, and can be used relatively well to predict the test result.
TABLE 3 analysis of variance of test data
Note: r2＝0.9994 R2 Adj＝0.9986
Thirdly, analyzing and optimizing test results
The influence of extraction temperature on the extraction rate of yam polysaccharide is shown in fig. 1, the influence of extraction time on the extraction rate of yam polysaccharide is shown in fig. 2, and the influence of feed liquid ratio on the extraction rate of yam polysaccharide is shown in fig. 3.
The software is used to perform plot analysis according to the regression equation, and the response surface of the regression equation and the contour diagram 4 thereof are obtained, as shown in fig. 5 and 6.
The optimal extraction conditions calculated by software are that the extraction temperature is 70.51 ℃, the time is 2.06h, the material-liquid ratio is 1:10.48, and the polysaccharide extraction rate is 12.04%. According to actual operation, the optimal conditions are adjusted to be that the ratio of material to liquid is 1: 10.5, time 2h and leaching temperature 71 ℃. The average value of the actual extraction rate of the Chinese yam polysaccharide measured by a phenol-sulfuric acid method is 11.92%, and compared with a predicted value, the relative deviation is 0.99%, which shows that the optimal extraction process obtained by using response surface analysis has higher reliability.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (7)
1. A method for optimizing yam polysaccharide extraction conditions by using a response surface method is characterized by comprising the following steps: the method comprises the following steps:
the preparation method comprises the steps of cleaning, peeling and slicing the Chinese yam, freeze-drying the Chinese yam in a freeze-drying machine overnight, and crushing the Chinese yam by a crusher and sieving the Chinese yam by a 30-50-mesh sieve; quantitatively weighing the sieved yam powder, adding 3-4 times of volume of anhydrous petroleum ether for degreasing, and then adding 4-5 times of volume of ethanol solution for removing 80-100% of pigment and oligosaccharide; dissolving the degreased and decolored powder in distilled water according to the feed-liquid ratio of 1:10-1:11, and leaching for three times at 70 ℃, wherein each time is 2-2.5 hours; mixing the extractive solutions, and vacuum concentrating to 1/5-1/6 volume of original volume; adding 3-4 times volume of ethanol, precipitating with ethanol at 4 deg.C overnight, centrifuging the precipitated solution, collecting precipitate, dissolving in water, removing protein in the solution by enzyme + sevage method, removing salt and small molecular substances by dialysis method, and freeze drying to obtain rhizoma Dioscoreae crude polysaccharide;
secondly, adopt the response surface method to confirm the variable parameter that chinese yam polysaccharide extracted, set up three-factor three-level's test mode and carry out the repeated test and obtain the experimental result, calculate chinese yam polysaccharide's extraction rate, adopt software to carry out multiple regression analysis again, it is to the regression equation of response value influence to reachd the experimental factor:
R(％)＝12.00+0.24A+0.34B+0.34C-0.65AB-0.064AC-0.26BC-1.55A2-1.18B2-1.60C2
in the formula, R is the extraction rate of the yam polysaccharide, a variable parameter A is the extraction temperature, a variable parameter B is the extraction time, and a variable parameter C is the ratio of material to liquid;
finally, determining the rate taking condition as follows: the temperature is 70.51 ℃, the time is 2.06h, the material-liquid ratio is 1:10.48, and the polysaccharide extraction rate is 12.04%.
2. The method for optimizing yam polysaccharide extraction conditions by using a response surface method as claimed in claim 1, wherein the method comprises the following steps: the method comprises the steps of using 3-4mL of petroleum ether per gram of Chinese yam powder, using 4-5mL of ethanol solution per gram of Chinese yam powder when removing pigments and oligosaccharides, and using 3-4mL of ethanol solution during alcohol precipitation.
3. The method for optimizing yam polysaccharide extraction conditions by using a response surface method as claimed in claim 1, wherein the method comprises the following steps: the rotating speed of the centrifuge in the step is 6000r/min, and the time is 10 min.
4. The method for optimizing yam polysaccharide extraction conditions by using a response surface method as claimed in claim 1, wherein the method comprises the following steps: the enzyme used for deproteinizing in the step is papain, and the sevage reagent is chloroform: n-butanol is 4:1, and the protein removal times are 8; dialyzing with distilled water for three days by using a dialysis bag with the molecular weight cutoff of 8000-14000Da, and changing water twice every day.
5. The method for optimizing yam polysaccharide extraction conditions by using a response surface method as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following steps of:
6. the method for optimizing the extraction conditions of yam polysaccharide by using the response surface method as claimed in any one of claims 1 to 5, wherein: the method further comprises the steps of:
the polysaccharide extraction rate is measured by a phenol-sulfuric acid method.
7. The method for optimizing yam polysaccharide extraction conditions by using a response surface method as claimed in claim 6, wherein the method comprises the following steps: the method comprises the following specific steps:
weighing an anhydrous glucose standard substance, and drying in a drying oven at 105 ℃ until the weight is constant; accurately weighing the dried anhydrous glucose, and performing constant volume to obtain a glucose standard solution of 0.1mg/mL for later use;
accurately measuring 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9mL of glucose standard solution in a test tube, and respectively supplementing 1mL of glucose standard solution with distilled water; adding 1.0mL of phenol solution with the mass concentration of 6% into each tube, then slowly adding 5mL of sulfuric acid, shaking and uniformly mixing, carrying out water bath at 40 ℃ for 15min, taking out and cooling to room temperature, and measuring the absorbance value at 490 nm;
(iii) taking the concentration C (mg/mL) of glucose as the abscissa, A490And drawing a standard curve by taking the ordinate.
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| CN114957500A (en) * | 2021-12-24 | 2022-08-30 | 济宁医学院 | Selenylation modification method for improving immunocompetence of Chinese yam polysaccharide and application thereof |
| CN115852947A (en) * | 2022-12-22 | 2023-03-28 | 成都理工大学 | Construction method of prestressed artificial anchor cable of artificial digging pile |
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