CN112626041A - Process for extracting lipoxygenase from soybean whey protein wastewater by two aqueous phases - Google Patents
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Abstract
The invention belongs to the technical field of resource utilization of soybean whey wastewater, and relates to a process for extracting lipoxygenase from soybean whey protein wastewater by using aqueous two phases. With PEG6000- (NH)4)2SO4And (4) extracting by using a two-aqueous-phase system. The invention provides PEG6000- (NH)4)2SO4The invention adopts the double-water-phase extraction separation technology to separate the LOX in the soybean whey wastewater, can reduce COD and BOD in the wastewater, reduce resource waste and environmental pollution, and realize high-value utilization of the soybean whey wastewater.
Description
Technical Field
The invention belongs to the technical field of resource utilization of soybean whey wastewater, and relates to a process for extracting lipoxygenase from soybean whey protein wastewater by using aqueous two phases.
Background
The soybean whey wastewater is a byproduct generated by producing SPI from low-temperature defatted soybean meal by using an alkali-dissolving acid-precipitation process, statistics shows that 35-65t of soybean whey wastewater is generated by producing 1t of SPI, and the soybean whey wastewater contains rich nutrient substances, has an acidic pH value and is extremely easy to decay to cause serious pollution. At present, the main treatment methods of the soybean whey wastewater include an anaerobic method and an aerobic method to reduce the COD value and the BOD value in the wastewater so as to reach the national wastewater discharge standard, but the high treatment cost seriously reduces the production benefit and the economic benefit of enterprises, and a large amount of active substances in the wastewater are not fully utilized, so that the development and utilization of the active substances in the wastewater become one of research hotspots by taking the resource treatment of the soybean whey wastewater as a research target.
At present, most recent studies on soybean whey wastewater have focused on the recycling of active proteins in wastewater, such as Kunitz Trypsin Inhibitor (KTI), soybean whey protein, soybean isoflavone, and the like. Hydroperoxide derivatives generated by LOX oxidation of substrates are very important intermediates for drug synthesis and chemical synthesis, such as flavor substances or drug intermediates in catalytic synthesis, and can also be applied to the production of dyes and wash-coating agents, and LOX can be added in the processing of flour products to achieve the effect of whitening and strengthening tendons, so that LOX has wide application prospects in the fields of food, medicine, chemical industry and the like. However, there are few reports on the research on the extraction of LOX from soybean whey wastewater using modern separation techniques. Because LOX efficiently expressed and recombined by microbial fermentation or genetic engineering bacteria has many safety problems, it has not been applied to food processing and other industries, and therefore, it is very necessary to develop LOX with green safety, high purity and low cost.
Disclosure of Invention
The invention provides a novel process for extracting lipoxygenase from soybean whey protein wastewater, aiming at the problems of lipoxygenase extraction in the traditional soybean whey protein wastewater.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
a process for extracting lipoxygenase from soybean whey protein wastewater adopts PEG6000- (NH)4)2SO4And (4) extracting by using a two-aqueous-phase system.
Preferably, the extraction step is: freezing and centrifuging the soybean whey wastewater, and then reserving a supernatant; then reacting with PEG solution, (NH)4)2SO4Mixing the solution with distilled water to obtain an extraction mixed solution, adjusting the pH value, and standing; taking the lower phase (NH)4)2SO4The solution layer was centrifuged to obtain a precipitate containing lipoxygenase.
Preferably, the mass fraction of PEG6000 in the extraction mixed solution is 18-20% (NH)4)2SO410-12% of mass fraction, 18-25% of wastewater addition and 5-6 of pH.
The invention provides a process optimization model for extracting lipoxygenase from soybean whey protein wastewater, which has the following formula:
Y%=85.68+1.04A+0.76B+1.27C+0.40AB-1.03AC+0.57BC-1.78A2-2.95B2-1.45C2；
P=3.19+0.15A+0.11B+0.15C-0.030AB-0.16AC+0.040BC-0.27A2-0.47B2-0.17C2；
wherein Y% is extraction rate, P is purification multiple, A is PEG6000 mass fraction, and B is (NH)4)2SO4And C is PH in mass fraction.
Based on the model, the extraction rate and the purification multiple are set to reach the maximum value, and the optimal extraction conditions are obtained by predicting through Design-expert.8.0.6.1 software: 19.17% of PEG6000 (NH)4)2SO410.52 percent of mass fraction, 20 percent of waste water addition and pH5.96. The predictive values for the extraction yield and the fold purification obtained are 86.10% and 4.23, respectively.
The regression equation formula for extraction (Y%), purification fold (P) is:
Y%=85.68+1.04A+0.76B+1.27C+0.40AB-1.03AC+0.57BC-1.78A2-2.95B2-1.45C2；
P=3.19+0.15A+0.11B+0.15C-0.030AB-0.16AC+0.040BC-0.27A2-0.47B2-0.17C2；
wherein, A is PEG6000 mass fraction, B is (NH)4)2SO4Mass fraction, C is pH value.
The optimization models of 3 variables of the extraction rate (Y%) and the purification multiple optimization are very obvious (P is less than 0.01), the simulation-missing items are not obvious (P is more than 0.05), and the determination coefficient R of the regression equation formula of the extraction rate (Y%) and the purification multiple (P)20.9951 and 0.9950, and 32.660 and 34.670, respectively, indicate that 99.5% of variability of experimental data can be explained by the model, the model has good fitting degree and high reliability, and therefore, experimental results can be analyzed and predicted by the regression equation formula of the extraction rate (Y%) and the purification multiple (P). In addition, primary item A, B, C, interactive item AC and secondary item A2、B2、C2The expression is very significant (P is less than 0.01), which indicates that A, B, C, AC and A2、B2、C2Has obvious influence on the extraction rate and the purification multiple. As can be seen from the analysis of variance, the influence of various factors on the LOX extraction rate of the soybean whey wastewater is as follows from big to small: c is more than A and more than B, and the influence of various factors on the LOX purification times in the soybean whey wastewater is in the following order: c is more than A and more than B.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the invention provides PEG6000- (NH)4)2SO4Separating and extracting LOX in the soybean whey wastewater by using a two-aqueous-phase extraction system, providing optimal extraction conditions, mutually verifying by combining an optimization model, and obtaining the optimal extraction conditions of PEG6000 mass fraction of 19.2%, (NH)4)2SO4The mass fraction was 10.5%, the amount of wastewater added was 20%, the pH was 6.0, and the LOX extraction rate and the purification fold were 85.45% and 4.06, respectively, under these conditions.
2. The invention adopts the double-aqueous phase extraction separation technology to separate LOX in the soybean whey wastewater, can reduce COD and BOD in the wastewater, reduce resource waste and environmental pollution, and realize high-value utilization of the soybean whey wastewater.
Drawings
FIG. 1 shows PEG- (NH)4)2SO4Double water phase system phase diagram.
FIG. 2 is a graph of a protein content standard.
FIG. 3 shows the effect of PEG6000 mass fraction and pH interaction on extraction yield.
Figure 4 is a graph of PEG6000 mass fraction and pH interaction on purification fold effect.
FIG. 5 is an SDS-PAGE analysis of the LOX purification process.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
Example 1 this example provides specific process steps for the aqueous two-phase extraction of lipoxygenase from soy whey protein wastewater.
500ml of soybean whey wastewater is taken at 8000r/min and 4 ℃ for freezing and centrifuging for 10min, and the supernatant is reserved and stored at 4 ℃ for standby. PEG6000 with mass fraction of 19.2% (NH) is prepared4)2SO410.5 percent of mixed solution (acidity) with the addition of 20.0 percent of waste water, adjusting the pH to 6.0 by using sodium hydroxide to construct a double-aqueous-phase system (the total amount is 10 g), fully and uniformly mixing, then standing for 3 hours, (NH)4)2SO4The layer was centrifuged at 12000r/min at 4 ℃ for 5min to obtain separated LOX in the precipitate.
Through detection, the LOX specific enzyme activity is 4027.68U/mg, and the recovery rate and the purity of the enzyme activity are 89.76 percent and 28.74 percent respectively.
In addition, after the aqueous two-phase extract is concentrated by 10 times through a 30KDa ultrafiltration membrane, the specific enzyme activity and the purification times of the LOX are 13255.45U/mg and 13.36 respectively, and the recovery rate of the enzyme activity reaches 86.27%, so that the process can basically keep the enzyme activity of the LOX.
1. Lipoxygenase enzyme activity assay
Definition of enzyme activity: at 25 ℃ and pH9.0, linoleic acid is taken as a substrate, and at 234nm, each increase of 0.001 of absorbance of a reaction system per minute corresponds to one enzyme activity unit.
Preparation of a substrate: 0.25mL of Tween 20 is dispersed in 10mL of 0.2mol/L borate buffer solution with the pH value of 9.0, 0.27mL of linoleic acid is continuously shaken and dropwise added to be uniformly mixed, then a NaOH solution with a certain concentration is dropwise added into the system to clarify the system, the pH value of the system is adjusted to 9.0, finally 0.2mol/L of borate buffer solution with the pH value of 9.0 is used for fixing the volume to 500mL to be used as a substrate solution, and the substrate solution is stored at the temperature of 4 ℃ for standby.
And (3) activity determination: dissolving the obtained precipitate containing the enzyme with deionized water (the volume of a sample solution prepared by deionized water is consistent with the volume of waste water for extraction) to obtain a sample solution, adding 0.2mL of the sample solution into 1.5mL of a substrate solution, uniformly mixing, reacting at a constant temperature of 25 ℃ for 3min, adding 5mL of absolute ethyl alcohol to terminate the reaction, adding 5mL of distilled water to clarify the reaction system, and measuring the absorbance at 234 nm. The blank sample is 1.5mL of substrate solution, 5mL of absolute ethanol is added, 0.2mL of sample solution is added, and 5mL of distilled water is added after the temperature is kept at 25 ℃ for 3 min.
2. Protein content determination
Measuring protein content by Coomassie brilliant blue method, collecting 6 clean test tubes, marking tube number, adding 1.0ml distilled water into 1 test tube as blank, adding 0.1mg/ml bovine serum albumin standard solution into the other 5 test tubes, and adding water to 1.0 ml. Then 5.0ml Coomassie brilliant blue G-250 reagent is added into each test tube, shaken evenly and placed for 5min, the light absorption value is measured at 595nm of an ultraviolet-visible spectrophotometer, and a protein standard curve is drawn. Using A595 absorbance as ordinateThe concentration of bovine serum albumin (mg/mL) was plotted on the abscissa as a standard curve, as shown in FIG. 1. The linear relation is better when the lambda =595nm and the protein concentration is in the range of 0-0.08mg/mL, and the regression equation is y =7.0379X +0.0033, R2=0.99898。
3. Preparation of aqueous two-phase diagram
The preparation of the two aqueous phase diagram adopts a cloud point titration method, 5g of 40 percent PEG solution is accurately weighed in a conical flask at 25 ℃, and the prepared 30 percent (NH) is slowly dripped by a burette4)2SO4The solution was observed for clarity until the solution in the tube began to appear cloudy. Standing for a period of time to separate the solution into distinct two-phase system, i.e. PEG solution as upper phase and (NH) as lower phase4)2SO4And (3) solution. Record (NH)4)2SO4The amount of (c) and the amount of (g) PEG added. Then deionized water was added to the flask to clarify it and the dropwise addition of (NH)4)2SO4And (5) making the solution turbid, and so on. Calculation of PEG and (NH) each time turbidity is reached4)2SO4Mass fraction in the system (wt%) and PEG mass fraction in wt% as ordinate, (NH)4)2SO4The mass fraction (wt%) is plotted on the abscissa, and a binodal phase diagram is obtained, as shown in fig. 1. As can be seen from FIG. 1, PEG- (NH)4)2SO4The two-phase region of the two-aqueous phase system increases with the average molecular weight of PEG, mainly because of the increase in PEG- (NH)4)2SO4In the aqueous two-phase system, the hydrophobicity of PEG is enhanced along with the increase of the average molecular weight of PEG, and the hydrophobicity of PEG and (NH) are increased4)2SO4Thereby reducing (NH)4)2SO4The phase forming concentration of (2). On the other hand, along with the increase of the average molecular weight of PEG, the electrostatic repulsion function between the hydrated ions and PEG molecules in the double-water phase system is enhanced, which is more beneficial to the formation of the double-water phase. Thus, 3 different molecular weights of PEG and (NH)4)2SO4The phase forming ability of (A) is as follows: PEG6000 > PEG4000 > PEG 2000.
4. Regression model and analysis of variance
In order to further optimize the extraction conditions of LOX, a response surface experiment of 3 factors 3 level is adopted to optimize and analyze the mass fraction of PEG6000, (NH4)2SO4 and pH, and the determination indexes are the extraction rate and the purification multiple of LOX.
TABLE 1 Box-Behnken Experimental design and results
Establishing a regression model and analyzing variance.
The experimental data of table 1 were subjected to regression analysis using Design-expert.8.0.6.1 software to obtain the extraction rate (Y%), and the regression equation for the multiple of purification (P) was:
formula (1): y% =85.68+1.04A +0.76B +1.27C +0.40AB-1.03AC +0.57BC-1.78A2-2.95B2-1.45C2；
Formula (2): p =3.19+0.15A +0.11B +0.15C-0.030AB-0.16AC +0.040BC-0.27A2-0.47B2-0.17C2。
And carrying out variance analysis on the two quadratic regression equations.
The optimization models of 3 variables of the extraction rate (Y%) and the purification multiple optimization are very obvious (P is less than 0.01), the simulation-missing items are not obvious (P is more than 0.05), and the determination coefficients R of the formula (1) and the formula (2)20.9951 and 0.9950, respectively, and 32.660 and 34.670, respectively, indicate that 99.5% of the variability of the experimental data can be explained by the model, the model has better fitting degree and higher reliability, and therefore, the experimental results can be analyzed and predicted by the formula (1) and the formula (1). In addition, primary item A, B, C, interactive item AC and secondary item A2、B2、C2The expression is very significant (P is less than 0.01), which indicates that A, B, C, AC and A2、B2、C2Has obvious influence on the extraction rate and the purification multiple. As can be seen from the analysis of variance, the influence of various factors on the LOX extraction rate of the soybean whey wastewater is as follows from big to small: c is more than A and more than B, and various factors purify LOX in soybean whey wastewaterThe influence of the numbers has a large to small order: c is more than A and more than B.
TABLE 2 regression model analysis of variance
Note: very significant, P < 0.01; is significant, P < 0.05; p > 0.05 is not significant.
4. Response surface analysis and optimization
The response surface graph is a three-dimensional space curved surface graph formed by the response values to each experimental factor, and the steeper the curved surface is, the more the factor influences the experimental result, namely, the change of the response value is shown. Quadratic regression fitting is carried out by using Design-expert.8.0.6.1 software, and the response surface graph and the contour line of the obtained quadratic regression equation are shown in fig. 2 and fig. 3.
As can be seen from fig. 2 and 3, the influence of ATPS on the LOX extraction rate and the purification multiple both tend to increase and decrease with the increase of PEG6000 mass fraction and pH, the response surface is steeper, and the contour line of the interaction between the two is elliptical, indicating that the influence of the interaction between the two on the response value is more significant, which is consistent with the result of variance analysis of each item of the regression equation, and the opening of the response surface is downward, indicating that the two response values can reach the maximum value. Therefore, on the basis of the established model, the extraction rate and the purification multiple are set to reach the maximum values, and the optimal conditions of the model obtained through Design-expert.8.0.6.1 software prediction are as follows: 19.17% of PEG6000 (NH)4)2SO410.52 percent of mass fraction, 20 percent of waste water addition and pH5.96. In this example, a single-factor experiment was also performed, and it was found that the model conclusion was similar to the extraction conditions obtained in the single-factor experiment, and the predicted values of the extraction rate and the purification multiple were 86.10% and 4.23, respectively. In order to verify the reliability of the regression model, the LOX of the soybean whey wastewater is extracted by adopting the optimized conditions, and the extraction process parameters are adjusted as follows: PEG6000 mass fraction 19.2%, (NH)4)2SO410.5 percent of mass fraction, 20.0 percent of wastewater addition amount and pH6.0, and the LOX extraction rate and the actual value of the purification multiple are obtained after 3 times of experimental verificationThe difference between the regression equation and the predicted value is respectively 85.45% and 4.06%, and the difference between the regression equation and the predicted value is respectively 0.65% and 0.17, so that the fitting degree of the model is better, and the regression equation can better predict the magnitude of the response value, so that the model has certain practical guiding significance.
5. Concentrating and purifying
The two-aqueous phase extraction LOX experiment was performed according to the two-aqueous phase extraction conditions proposed in this example, and the two-aqueous phase extraction solution was concentrated and purified by using a 30KDa modified polyethersulfone ultrafiltration membrane and sephadex g-75, and the purification results are shown in table 3. And SDS-PAGE analysis was performed on the samples at each purification stage, and the results are shown in FIG. 4.
TABLE 3 LOX isolation and purification results
As can be seen from Table 3, the LOX specific enzyme activity in the extract obtained by aqueous two-phase extraction was 4027.68U/mg, the enzyme activity recovery rate was 89.76%, and the purification fold was 4.06. After further concentration and purification by a 30KDa ultrafiltration membrane and SephadexG-75, the specific enzyme activity of the obtained LOX chromatography liquid is 21223.75U/mg, the purification multiple is improved by 21.39 times, and the recovery rate and the purity of the enzyme activity are 38.30% and 70.03% respectively. It can be seen from fig. 5 that there are many protein bands in the soybean whey wastewater (lane 2), the molecular weight is distributed between 10 KDa and 100KDa, lane 3 is aqueous phase extract, and it can be seen from the figure that the protein bands and protein content are significantly reduced, indicating that the purification process has a good purification effect on LOX in the soybean whey wastewater, lanes 4 and 5 are 30KDa ultrafiltration concentrate and sephadex g-75 chromatography liquid, respectively, and it can be seen from the figure that the protein below 35KDa is substantially removed by concentration and purification with 30KDa ultrafiltration membrane, and after gel filtration chromatography, the sample purity is high, indicating that the purification process has a good purification effect on LOX in the soybean whey wastewater.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.
Claims (5)
1. A process for extracting lipoxygenase from soybean whey protein wastewater is characterized in that PEG6000- (NH) is adopted4)2SO4And (4) extracting by using a two-aqueous-phase system.
2. The process for extracting lipoxygenase from soybean whey protein waste water as claimed in claim 1, wherein the supernatant of the soybean whey waste water is retained after refrigerated centrifugation; then reacting with PEG solution, (NH)4)2SO4Mixing the solution with distilled water to obtain an extraction mixed solution, adjusting the pH value, and standing; to obtain (NH)4)2SO4The layer was centrifuged to obtain a precipitate containing lipoxygenase.
3. The process for extracting lipoxygenase from soybean whey protein wastewater as claimed in claim 2, wherein the mass fraction of PEG6000 in said extraction mixture is 18-20% (NH)4)2SO410-12% of mass fraction, 18-25% of wastewater addition and 5-6 of pH.
4. The process optimization model for lipoxygenase in wastewater from soybean whey protein extraction as claimed in any of claims 1-3, wherein the formula is as follows:
Y%=85.68+1.04A+0.76B+1.27C+0.40AB-1.03AC+0.57BC-1.78A2-2.95B2-1.45C2；
P=3.19+0.15A+0.11B+0.15C-0.030AB-0.16AC+0.040BC-0.27A2-0.47B2-0.17C2；
wherein Y% is extraction rate, P is purification multiple, A is PEG6000 mass fraction, and B is (NH)4)2SO4And C is PH in mass fraction.
5. The process optimization model for extracting lipoxygenase from soybean whey protein wastewater as claimed in claim 4, wherein the extraction rate and the purification multiple are set to be maximum values, and the optimal conditions for extraction are predicted by Design-expert.8.0.6.1 software: 19.17% of PEG6000 (NH)4)2SO410.52 percent of mass fraction, 20 percent of waste water addition and pH5.96.
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