CN105481942A - Method for extracting protein in grape seeds - Google Patents
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- CN105481942A CN105481942A CN201510959637.8A CN201510959637A CN105481942A CN 105481942 A CN105481942 A CN 105481942A CN 201510959637 A CN201510959637 A CN 201510959637A CN 105481942 A CN105481942 A CN 105481942A
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- 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
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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
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- C07K1/30—Extraction; Separation; Purification by precipitation
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Abstract
The invention discloses a method for extracting protein in grape seeds, the extraction method includes the following steps: 1) degreased grape seed meal is subjected to smashing, sieving, extraction and suction filtration to obtain filtrate and filtration residues for standby application; 2, the filtrate obtained in step 1 is subjected to reduced-pressure concentration, and the distillate obtained through reduced-pressure concentration is for standby application; 3, the filtration residues obtained in step 1 and the distillate obtained in the step 2 are subjected to extraction, suction filtration, centrifugation, acid adding for isoelectric point adjustment, centrifugation, precipitation and vacuum drying to obtain crude protein. According to the method, the conditions of a process for extracting the protein in the grape seeds are optimized and researched by adopting an alkali-solution and acid-isolation method, the influences of raw materials, the material-liquid ratio, extraction time and extraction temperature on the extraction rate of the protein in the grape seeds are investigated, and by the adoption of the analysis on the results of single factor experiments and response surface optimization experiments, the best extraction conditions of the protein in the grape seeds are obtained as follows: the extraction time is 48.01 minutes, the extraction temperature is 60.89 DEG C, the material-liquid ratio is 1:32, and the protein extraction rate under the conditions is as high as 3.25%.
Description
Technical field
The present invention relates to method for extracting protein in a kind of Semen Vitis viniferae.
Background technology
Grape (grapes) is the defoliation liana of Vitaceae (Vitaceae) Vitis (Vitis).Fruit can be made into the products such as Sucus Vitis viniferae, raisin and grape wine, along with the turnout of grape product is increasing, waste such as Pericarpium Vitis viniferae, Semen Vitis viniferae, the grape dregs etc. produced in the course of processing is urgently fully utilized.
About research shows in grape waste containing the abundant material such as raisin seed oil, protein, tannin, trans-resveratrol, its protein content is high, amino acid classes is very complete, mineral substance enriches, all very important to the needs meeting human body, and these materials are also widely used in industries such as medicine, healthcare products, makeup, so how by these Semen Vitis viniferae wastes comprehensive development and utilization in addition, turn waste into wealth of far-reaching significance.If properly handled, not only can alleviate the pollution that brings to environment but also can create huge economic benefit, thus can reach the effect of doulbe-sides' victory.
Protein in Semen Vitis viniferae is mainly made up of 16 seed amino acids, and have 7 kinds to be the necessary amino acid of human body (8 seed amino acids of needed by human are α-amino-isovaleric acid, Isoleucine, phenylalanine, methionine(Met), Threonine, tryptophane, leucine, Methionin), so Semen Vitis viniferae protein has higher nutritive value and researching value in amino acid contained by Semen Vitis viniferae protein.The polypeptide that protein obtains after hydrolysis has better nutritive value, these low peptide materials can either as energy matter, the physiological function of human body can be regulated again, some special sequences that some research shows in Semen Vitis viniferae protein also have anti-oxidant, reduce blood pressure, promote the effect such as mineral absorption, adjusting blood lipid.So probing into the optimum process condition extracting protein from Semen Vitis viniferae has great using value.
The application's application Responds Surface Methodology is optimized research to protein extracting process in Semen Vitis viniferae, adopt polynary quadratic regression equation as the instrument of Function Estimation, level line and surface model is set up in conjunction with mathematics and statistics, make the extraction rate reached of protein in Semen Vitis viniferae to the highest, the comprehensive development and utilization for Semen Vitis viniferae provides certain scientific basis.
Summary of the invention
For Problems existing in background technology, the object of the present invention is to provide method for extracting protein in a kind of Semen Vitis viniferae, the application is optimized the condition of proteins extraction technique in Semen Vitis viniferae by alkali extraction-acid precipitation, investigate raw material, solid-liquid ratio, extraction time and Extracting temperature are on the impact of tannin extraction yield in Semen Vitis viniferae, by the interpretation of result that experiment of single factor and response surface optimization are tested, showing in best Semen Vitis viniferae that proteins extraction condition is extraction time is 48.01min, Extracting temperature is 60.89 DEG C, solid-liquid ratio is 1:32, with this understanding, the extraction yield of protein is up to 3.25%.
The object of the invention is to be achieved through the following technical solutions:
Method for extracting protein in a kind of Semen Vitis viniferae, described extracting method comprises the steps:
1) the defatted grape seed dregs of rice carried out pulverize, sieve, lixiviate, suction filtration obtains filtrate and filter residue is for subsequent use;
2) filtrate in step 1) is carried out concentrating under reduced pressure, the distillate obtained by concentrating under reduced pressure is for subsequent use;
3) by the filter residue in step 1) and step 2) in the distillate that obtains carries out lixiviate, suction filtration, centrifugal, acid adding adjust iso-electric point, centrifugal, precipitation, vacuum-drying obtain crude protein.
Further, the pulverizing in described step 1), sieving is specially: get the appropriate defatted grape seed dregs of rice, pulverize with high-speed multifunctional pulverizer, then use 60 object sieve for subsequent use.
Further, the extracting solution of the lixiviate in described step 1) is ethanol, and the volume fraction of described ethanol is 30-70%, and the Semen Vitis viniferae dregs of rice after sieving and the solid-liquid ratio of described ethanol are: 1:40-1:80, and extraction time is 2-4h, and Extracting temperature is 40-80 DEG C.
Further, the extracting solution of the lixiviate in described step 1) is ethanol, and the volume fraction of described ethanol is 51.70%, and the Semen Vitis viniferae dregs of rice after sieving and the solid-liquid ratio of described ethanol are: 1:60, and extraction time is 3.08h, and Extracting temperature is 61.87 DEG C.
Further, the extracting solution of lixiviate in described step 3) is sodium hydroxide solution, and the concentration of described sodium hydroxide solution is 0.02mol/L, and solid-liquid ratio is: 1:20-1:40, and extraction time is 30-70min, and Extracting temperature is 40-80 DEG C.
Further, the extracting solution of lixiviate in described step 3) is sodium hydroxide solution, and the concentration of described sodium hydroxide solution is 0.02mol/L, and solid-liquid ratio is: 1:32, and extraction time is 48.01min, and Extracting temperature is 60.89 DEG C.
The present invention has following positive technique effect:
The application's application Responds Surface Methodology is optimized research to protein extracting process in Semen Vitis viniferae, adopt polynary quadratic regression equation as the instrument of Function Estimation, level line and surface model is set up in conjunction with mathematics and statistics, make the extraction rate reached of protein in Semen Vitis viniferae to the highest, the comprehensive development and utilization for Semen Vitis viniferae provides certain scientific basis.
Accompanying drawing explanation
Fig. 1 is the influence curve figure of different pH to extracting solution of protein light absorption value;
Fig. 2 is the impact of different feed liquid aligned protein extraction yield;
Fig. 3 is the impact of different Extracting temperature on extraction rate of protein;
Fig. 4 is that different extraction time is on the impact of extraction rate of protein;
Fig. 5 is the impact (isogram) of interaction on extraction rate of protein of extraction time and Extracting temperature;
Fig. 6 is the impact (3D surface chart) of interaction on extraction rate of protein of extraction time and Extracting temperature;
Fig. 7 is the impact (isogram) of interaction on extraction rate of protein of extraction time and solid-liquid ratio;
Fig. 8 is the impact (3D surface chart) of interaction on extraction rate of protein of extraction time and solid-liquid ratio;
Fig. 9 is the impact (isogram) of interaction on extraction rate of protein of Extracting temperature and solid-liquid ratio;
Figure 10 is the impact (3D surface chart) of interaction on extraction rate of protein of Extracting temperature and solid-liquid ratio.
Embodiment
Below, with reference to accompanying drawing, more fully illustrate the present invention, accompanying drawing acceptance of the bid shows exemplary embodiment of the present invention.But the present invention can be presented as multiple multi-form, and should not be construed as the exemplary embodiment being confined to describe here.But, these embodiments are provided, thus make the present invention comprehensively with complete, and scope of the present invention is fully conveyed to those of ordinary skill in the art.
This application provides method for extracting protein in a kind of Semen Vitis viniferae, this extracting method comprises the steps: 1) the defatted grape seed dregs of rice carried out pulverize, sieve, lixiviate, suction filtration obtains filtrate and filter residue is for subsequent use; 2) filtrate in step 1) is carried out concentrating under reduced pressure, the distillate obtained by concentrating under reduced pressure is for subsequent use; 3) by the filter residue in step 1) and step 2) in the distillate that obtains carries out lixiviate, suction filtration, centrifugal, acid adding adjust iso-electric point, centrifugal, precipitation, vacuum-drying obtain crude protein.Get the appropriate defatted grape seed dregs of rice, pulverize with high-speed multifunctional pulverizer, sieve (60 order) is for subsequent use.
As shown in Figure 1, each group of Semen Vitis viniferae extracting solution of protein acid adding is transferred to different pH value, and after pelleting centrifugation, the light absorption value of mensuration; As shown in Figure 1, as pH=3.8, light absorption value is minimum.So pH=3.8 is Semen Vitis viniferae isoelectric point of protein, under this pH condition, the solubleness of protein is minimum, and precipitation capacity is maximum, and in Semen Vitis viniferae, the extraction yield of protein is the highest.
As shown in Figure 2, get the alkali lye that 1g raw material correspondence adds different volumes, the protein of different solid-liquid ratios to Semen Vitis viniferae is set and extracts, calculate the extraction rate of protein of different feed liquid ratio.Typical curve according to drawing searches protein concn corresponding to measured light absorption value, calculates protein content and extraction yield; Can obviously be found out by Fig. 2, the increase extraction rate of protein with solid-liquid ratio increases gradually, is because the protein increasing stripping with alkali lye gets more and more.When solid-liquid ratio is about 1:30, the extraction rate reached of protein is to maximum, and increases solid-liquid ratio extraction rate of protein again and slightly decline, and change is mild.Too much extraction agent not only can cause the wasting of resources but also can bring difficulty to follow-up work, so give up the level that solid-liquid ratio is 1:40, and selects solid-liquid ratio to be that three levels of 1:25,1:30,1:35 are optimized experiment, chooses more excellent level further.
As shown in Figure 3, set according to the Suitable ranges of proteins extraction the comparison that 6 graded levels carry out extraction rate of protein in Semen Vitis viniferae, calculate the extraction yield of protein under different Extracting temperature.Typical curve according to drawing searches protein concn corresponding to measured light absorption value, calculates protein content and extraction yield; Can obviously be found out by Fig. 3, along with the increase of Extracting temperature, in Semen Vitis viniferae, the extraction yield of protein also increases to some extent, and when about 60 DEG C, extraction rate of protein is the highest, and when temperature continues to increase, extraction rate of protein reduces gradually.This be due to temperature lower time, increasing of temperature will promote the dissolving of protein in alkali lye, and the protein content of extraction increases gradually.But chemical reaction structural changes easily occurs protein under excessive temperature condition, extraction rate of protein is caused to decline.
As shown in Figure 4, the comparison of extraction rate of protein in Semen Vitis viniferae is carried out according to suitable time range set 5 levels of proteins extraction in related data, the light absorption value of sample liquid under more different extraction time, search protein concn corresponding to measured light absorption value according to the typical curve drawn again, calculate protein content and extraction yield; Can obviously be found out by Fig. 4, within the specific limits, extraction rate of protein increases with the lengthening of extraction time, for extraction rate of protein during about 50min reaches maximum upon extracting, at this moment the stripping substantially of the protein in raw material, the extraction yield lengthening extraction time protein again declines to some extent, may be because overlong time causes the structure of protein to there occurs a little change in alkali lye, its solubleness is declined.
On the basis of experiment of single factor, application Design-Expert.8.05b software, adopt Box-BehnkenDesign founding mathematical models, to choose extraction time (X1), extraction temperature (X2), solid-liquid ratio (X3) three factors for investigating object, with the mean value of extraction rate of protein (Y) for response value, the response surface of Three factors-levels is adopted to carry out experimental design, by the test-results of Box-BehnkenDesign design gained in table 1.
The experimental design of table 1 response surface optimization and response value
| Test number | x 1 | x 2 | x 3 | Y extraction yield/% |
| 1 | 50 | 60 | 30 | 3.19 |
| 2 | 50 | 70 | 35 | 2.69 |
| 3 | 50 | 60 | 30 | 3.13 |
| 4 | 60 | 50 | 30 | 1.84 |
| 5 | 50 | 60 | 30 | 3.24 |
| 6 | 40 | 60 | 35 | 2.83 |
| 7 | 50 | 70 | 25 | 2.13 3 --> |
| 8 | 50 | 60 | 30 | 3.02 |
| 9 | 50 | 50 | 35 | 2.57 |
| 10 | 50 | 50 | 25 | 1.58 |
| 11 | 40 | 70 | 30 | 2.52 |
| 12 | 60 | 60 | 35 | 2.59 |
| 13 | 40 | 60 | 25 | 2.09 |
| 14 | 60 | 70 | 30 | 2.28 |
| 15 | 40 | 50 | 30 | 2.49 |
| 16 | 60 | 60 | 25 | 1.46 |
| 17 | 50 | 60 | 30 | 3.09 |
After experimental result regression fit, obtain light absorption value to solid-liquid ratio, extraction time and the polynary quadratic regression equation of extraction time:
Y=3.13-0.22A+0.14B+0.43C+0.10AB+0.097AC-0.11BC-0.43A2-0. in 43B2-0.47C2 formula: Y---extraction rate of protein
A---extraction time
B---Extracting temperature
C---solid-liquid ratio
In order to check the reliability of regression equation, carried out variance analysis, analytical results is in table 2.
Table 2 analysis of variance table
| Source of variation | Sum of squares | Degree of freedom | All square | F value | P value |
| Regression model | 4.86 | 9 | 0.54 | 80.2 | < 0.0001** |
| A | 0.39 | 1 | 0.39 | 57.46 | 0.0001** |
| B | 0.16 | 1 | 0.16 | 24.11 | 0.0017** |
| C | 1.46 | 1 | 1.46 | 216.97 | < 0.0001** |
| AB | 0.042 | 1 | 0.042 | 6.24 | 0.0412* |
| AC | 0.038 | 1 | 0.038 | 5.64 | 0.0492* |
| BC | 0.046 | 1 | 0.046 | 6.86 | 0.0345* |
| A 2 | 0.76 | 1 | 0.76 | 113.26 | < 0.0001** |
| B 2 | 0.76 | 1 | 0.76 | 113.26 | < 0.0001** |
| C 2 | 0.91 | 1 | 0.91 | 135.54 | < 0.0001** |
| Residual error | 0.047 | 7 | 6.74E-03 | ||
| Lose and intend item | 0.018 | 3 | 5.95E-03 | 0.81 | 0.5502 |
| Net error | 0.029 | 4 | 7.33E-03 | ||
| Total value | 4.91 | 16 |
Note: " * " is that significantly (p<0.05) " * * " is highly significant (p<0.01).
Lose as can be seen from the above table and intend item P=0.5502>0.05, show to lose plan not remarkable; The P<0.01 of regression model, shows model highly significant; The correction coefficient of determination (relation conefficient) R2Adj of model is 0.9780, illustrates that this model can reflect the change of 97.80% response value.Above 3 fitting degree all indicating this model are fine, and therefore this regression model can be used for analysis design mothod data and predicts optimum extraction condition more exactly.
Once the F value size of item reflects the influence degree of each factor to response value, and F value shows that more greatly the influence degree of this factor to response value is larger.Can find out thus, the influence degree order of each factor to tannin extraction yield is: solid-liquid ratio > extraction time > Extracting temperature.
As shown in Figure 5-10, response surface isogram and 3D figure can reflect the impact of each factor on extraction rate of protein intuitively, and extraction time, interaction between Extracting temperature and solid-liquid ratio Three factors, optimal processing parameter can be found out more accurately.So, on the basis of regression model the results of analysis of variance, utilize software Design-Expert8.05b to do extraction time, Extracting temperature and solid-liquid ratio to the response surface of extraction rate of protein impact in Semen Vitis viniferae and isogram; Above isogram and 3D surface chart reflect the impact of the interaction between each factor on extraction rate of protein intuitively, namely when a factor is fixed wherein, two other factor interaction is on the impact of simple protein extraction yield, and can find out optimal processing parameter.
3 isogram ovalizes of the application, illustrate that the extraction yield of the interaction of each factor on protein has impact.Obviously can find out that isocontour density degree has larger difference from the graph, 3D surface chart obliquity is had nothing in common with each other, show that the interaction of each factor on extraction rate of protein impact significantly, the influence power size sequence of interaction to extraction rate of protein of each factor is: BC>AB>AC.
The determination of optimum process condition:
(1) optimization of optimum parameter
Solve according to the regression equation of response phase method computing gained and seek its maximum of points.
Y=3.13-0.22A+0.14B+0.43C+0.10AB+0.097AC-0.11BC-0.43A2-0.43B2-0.47C2
As X1=-0.198, X2=0.089, X3=0.428, Y value is maximum, and namely to obtain best processing condition be thus extraction time is 48.01min, and Extracting temperature is 60.89 DEG C, solid-liquid ratio be 1:32 at this moment extraction rate of protein up to 3.25%.
(2) checking of top condition
The optimum process condition drawn is adopted to carry out proof test.Using the mean value of three parallel tests as experimental result, the extraction rate of protein obtained is 3.24% differ 0.01% with theoretical value, and relative error is 0.31%, and practical measurement value conforms to substantially with theoretical expectation values, therefore the extraction conditions reliable in parameters adopting response phase method to obtain, has practical value.
In sum, the application to be optimized the condition of proteins extraction technique in Semen Vitis viniferae by alkali extraction-acid precipitation and to probe into, investigate the impact on extraction rate of protein in Semen Vitis viniferae of raw material, solid-liquid ratio, extraction time and Extracting temperature, by the interpretation of result that experiment of single factor and response surface optimization are tested, showing in best Semen Vitis viniferae that proteins extraction condition is extraction time is 48.01min, Extracting temperature is 60.89 DEG C, solid-liquid ratio is 1:32, with this understanding, the extraction yield of protein is up to 3.25%.
Described just in order to the present invention is described above, be construed as the present invention and be not limited to above embodiment, meet the various variants of inventive concept all within protection scope of the present invention.
Claims (6)
1. a method for extracting protein in Semen Vitis viniferae, is characterized in that, described extracting method comprises the steps:
1) the defatted grape seed dregs of rice carried out pulverize, sieve, lixiviate, suction filtration obtains filtrate and filter residue is for subsequent use;
2) filtrate in step 1) is carried out concentrating under reduced pressure, the distillate obtained by concentrating under reduced pressure is for subsequent use;
3) by the filter residue in step 1) and step 2) in the distillate that obtains carries out lixiviate, suction filtration, centrifugal, acid adding adjust iso-electric point, centrifugal, precipitation, vacuum-drying obtain crude protein.
2. method for extracting protein in Semen Vitis viniferae according to claim 1, it is characterized in that, pulverizing in described step 1), sieving is specially: get the appropriate defatted grape seed dregs of rice, pulverize with high-speed multifunctional pulverizer, then use 60 object sieve for subsequent use.
3. method for extracting protein in Semen Vitis viniferae according to claim 1, it is characterized in that, the extracting solution of the lixiviate in described step 1) is ethanol, the volume fraction of described ethanol is 30-70%, the Semen Vitis viniferae dregs of rice after sieving and the solid-liquid ratio of described ethanol are: 1:40-1:80, extraction time is 2-4h, and Extracting temperature is 40-80 DEG C.
4. method for extracting protein in Semen Vitis viniferae according to claim 3, it is characterized in that, the extracting solution of the lixiviate in described step 1) is ethanol, the volume fraction of described ethanol is 51.70%, the Semen Vitis viniferae dregs of rice after sieving and the solid-liquid ratio of described ethanol are: 1:60, extraction time is 3.08h, and Extracting temperature is 61.87 DEG C.
5. method for extracting protein in Semen Vitis viniferae according to claim 1, it is characterized in that, the extracting solution of lixiviate in described step 3) is sodium hydroxide solution, the concentration of described sodium hydroxide solution is 0.02mol/L, solid-liquid ratio is: 1:20-1:40, extraction time is 30-70min, and Extracting temperature is 40-80 DEG C.
6. method for extracting protein in Semen Vitis viniferae according to claim 5, it is characterized in that, the extracting solution of lixiviate in described step 3) is sodium hydroxide solution, the concentration of described sodium hydroxide solution is 0.02mol/L, solid-liquid ratio is: 1:32, extraction time is 48.01min, and Extracting temperature is 60.89 DEG C.
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| CN107604034A (en) * | 2017-11-06 | 2018-01-19 | 石河子大学 | A kind of grape pip protein zymolyte and preparation method thereof |
| CN109380728A (en) * | 2018-10-17 | 2019-02-26 | 汤臣倍健股份有限公司 | A kind of composition and health food with auxiliary blood pressure reduction effect |
| CN110698535A (en) * | 2019-10-26 | 2020-01-17 | 陇东学院 | Preparation method of fructus cannabis protein extracting solution |
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| CN107604034A (en) * | 2017-11-06 | 2018-01-19 | 石河子大学 | A kind of grape pip protein zymolyte and preparation method thereof |
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