CN109609472A - A kind of utilization method of soya whey wastewater - Google Patents
A kind of utilization method of soya whey wastewater Download PDFInfo
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- CN109609472A CN109609472A CN201910050792.6A CN201910050792A CN109609472A CN 109609472 A CN109609472 A CN 109609472A CN 201910050792 A CN201910050792 A CN 201910050792A CN 109609472 A CN109609472 A CN 109609472A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
- C12Y113/11012—Linoleate 13S-lipoxygenase (1.13.11.12)
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Abstract
The invention discloses a kind of utilization methods of soya whey wastewater, belong to food processing wastewater and utilize technical field.The present invention first pre-processes soya whey wastewater；[Bmim] Br ionic liquid is mixed with sylvite, soya whey wastewater pretreatment fluid is then added, and distilled water is added, the mass fraction for adjusting [Bmim] Br ionic liquid is 20% ~ 50%, and the mass fraction of sylvite is 17% ~ 35%；Adjusting solution ph is 5.4 ~ 6.4, after standing 30 ~ 180min, is centrifuged at 2 ~ 6 DEG C, and liquid is divided into two-phase up and down, and upper phase is lipoxygenase crude enzyme liquid.The present invention provides a kind of utilization methods of new soya whey wastewater, i.e., obtain the high lipoxygenase of industrial value using soya whey wastewater, and lipoxygenase recovery rate is high, with high purity, enzymatic activity is good.The method of the present invention is convenient for operation, and safe and efficient, raw material is Ke Xunhuanliyong, at low cost.
Description
Technical field
The present invention relates to food processing wastewaters to utilize technical field, in particular to a kind of utilization side of soya whey wastewater
Method.
Background technique
Soybean is a kind of protein content important crops more abundant, using it as the soybean protein isolate of Raw material processing
It is that a kind of purity of protein is high, the Refined Soybean protein product with deep shaping property, can be used as intermediate raw material, to be added to food raw
It is widely used in production.
Soybean protein isolate can use alkali extraction-acid precipitation, ultrafiltration concentration method, inverse micelle abstraction method, reversed-phased high performace liquid chromatographic
The methods of processed, wherein alkali extraction-acid precipitation is that current production soybean protein isolate is more mature, widely applied technique side
Method.But a large amount of soya whey wastewater can be generated sinking while technique produces soybean protein isolate using alkali soluble acid, and is given up
The substances such as albumen rich in, carbohydrate in water, easily result in waste of resources and environmental pollution.
Soybean protein isolate is widely used in food industry production, and annual demand is but every at 600,000 tons or so
The waste water that 1 ton of SPI will generate 40 tons or so is produced, the yield of waste water is very big.Currently, for soybean protein isolate processing waste water
Main methods be directly to be carried out multi-stage biological processing, using anaerobic and aerobic method reduce waste water in COD and
BOD, to reach national wastewater discharge standard, the processing cost is high, and a large amount of active material contained in waste water is not also closed
Reason utilizes.Therefore, benefit can be recycled by recycling the equal high value added products in soya whey wastewater using modern separation technology
With efficient resource, sewage is purified, preferable economic benefit and social benefit are generated.
In recent years, in order to reduce discharge of wastewater and environmental pollution, many scholars start to be dedicated to function in soya whey wastewater
The separation and Extraction, such as Jiang etc. of energy property substance recycle soybean using two-part foam separating technology from soya whey wastewater
Lactalbumin；Ling Xiuju etc. extracts the oligosaccharide in soya whey wastewater using membrane technology, and forms soy oligosaccharides sugar product.
However, the research about lipoxygenase separation and Extraction in soya whey wastewater has no research report.
Lipoxygenase (LOX), also known as lipoxidase, lipoxygenase belong to oxidoreducing enzyme, are a kind of containing non-heme
The protein of iron, single-minded can be catalyzed has suitable, the polyunsaturated fatty acid of suitable 4- alkene structure, passes through intramolecular oxygenation, formation tool
There is the hydro-oxidation derivative of conjugated double bond.Lipoxygenase (LOX) can lead to garden stuff processing product generate bad flavor, grease and
Deterioration etc. occurs for oil-containing food color during storage and processing.But lipoxygenase (LOX) adds as green food
Add agent, in wheat flour, the pigment in oxidable flour to be allowed to fade, brightens Flour product.Lipoxygenase (LOX) can
To apply to plant disease-resistant, in pest-resistant and shock reaction, the relationship of lipoxygenase (LOX) and plant disease-resistant wheat, tobacco,
It is all had been reported that on the crops such as rice, cotton.Lipoxygenase (LOX) industrially can be used for the work of dyestuff, coating, detergent etc.
Industry metaplasia produces, and is alternatively arranged as the intermediate of pharmaceutical synthesis.
Although LOX has in the fields such as food, chemical industry and has been widely used, directly mentioned from the raw material such as soybean
It takes, cost is too high, and there are no works to become the enzyme preparation that industry metaplasia produces；And LOX is recombinated by genetic engineering bacterium high efficient expression and is deposited
In problems such as safeties, so that it be made to be difficult to biggish breakthrough in terms of large-scale production and application.As it can be seen that finding suitable
The method for closing the acquisition lipoxygenase of industrial application is the problem of current urgent need to resolve.
It is found in researcher's early-stage study, a large amount of lipoxygenase is contained in SPI processing waste water, China is in the world
The maximum every annual meeting of SPI producing country generates a large amount of SPI processing waste water, in consideration of it, to rouge in soybean protein isolate processing waste water
SPI processing waste water is carried out resource utilization by fat oxygenase separation and Extraction, will generate huge economic and social value, tool
There is highly important realistic meaning.
Summary of the invention
In order to make up for the deficiencies of the prior art, the present invention provides a kind of utilization methods of soya whey wastewater.
The technical solution of the present invention is as follows:
A kind of utilization method of soya whey wastewater, comprising steps of
1) soya whey wastewater pre-processes
Soya whey wastewater is subjected to refrigerated centrifuge at 2 ~ 6 DEG C, retains supernatant, and adjust supernatant pH to 7 ~ 8, in 2 ~ 6
At DEG C, 2 ~ 5h is stood；Refrigerated centrifuge at 2 ~ 6 DEG C, supernatant is as soya whey wastewater pretreatment fluid；
2) lipoxygenase is extracted in soya whey wastewater pretreatment fluid
[Bmim] Br ionic liquid is mixed with sylvite, soya whey wastewater pretreatment fluid obtained by step 1) is added, and steaming is added
Distilled water, the mass fraction for adjusting [Bmim] Br ionic liquid is 20% ~ 50%, and the mass fraction of sylvite is 17% ~ 35%；Adjust solution
PH value is 5.4 ~ 6.4, after standing 30 ~ 180min, is centrifuged at 2 ~ 6 DEG C, and liquid is divided into two-phase up and down, and upper phase is lipoxidase
Enzyme crude enzyme liquid.
It preferably, further include step 3), lipoxygenase crude enzyme liquid utilizes the method preliminary purification being concentrated by ultrafiltration.
It preferably, further include step 4), enzyme solution obtained by step 3) is further purified using gel filtration chromatography,
Obtain pure lipoxygenase.
Preferably, it is concentrated by ultrafiltration in step 3) using the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size.
Further, using the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size, in 2 ~ 6 DEG C of temperature, 200 ~ 300 KPa of pressure
8 ~ 10 times of ultrafiltration concentrations are carried out to lipoxygenase crude enzyme liquid.
Preferably, gel filtration chromatography separation is carried out using SephadexG-75 in step 4).
Further, gel filtration chromatography utilizes the Sephadex G-75 gel column of 2-70KDa, with pH7.8,
The phosphate buffer of 0.02mol/L balances 2 ~ 3 column volumes, and loading after balance, with pH7.8, the phosphoric acid of 0.02mol/L is slow
Fliud flushing is elution；Eluent flow rate is 0.5mL/min.
Preferably, in step 2, the sylvite is potassium citrate, K2HPO4Or K2CO3。
Further, in step 2, the sylvite is K2HPO4。
Preferably, in step 2, borate buffer solution and the pH of sodium hydroxide solution regulation system are utilized.
Preferably, in step 2, the mass fraction of [Bmim] Br ionic liquid is 30% ~ 33%, the quality of sylvite
Score is 23% ~ 28%；Adjusting solution ph is 5.7 ~ 5.9.
Preferably, in step 2, time of repose is 100 ~ 130min.
The invention has the benefit that
1, the present invention provides a kind of utilization methods of new soya whey wastewater, that is, obtains industry using soya whey wastewater
Costly lipoxygenase, and lipoxygenase recovery rate is high, with high purity, enzymatic activity is good.
2, the method for the present invention is convenient for operation, and safe and efficient, raw material is Ke Xunhuanliyong, at low cost.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is reagent on the active influence situation map of LOX；
Fig. 2 is the influence situation map that [Bmim] Br mass fraction extracts LOX；
Fig. 3 is K2HPO4The influence situation map that concentration extracts LOX；
Fig. 4 is the influence situation map that system pH extracts LOX；
Fig. 5 is that SephadexG-75 chromatographs map；
Fig. 6 is 3 gained enzyme solution enzyme activity determination result of embodiment；
Fig. 7 is LOX purification process SDS-PAGE analysis chart；In Fig. 7,1-maker；2- soya whey wastewater；1 gained of 3- embodiment
Enzyme solution；2 gained enzyme solution of 4- embodiment；3 gained enzyme solution of 5- embodiment.
Specific embodiment
A kind of utilization method of soya whey wastewater, comprising steps of
1) soya whey wastewater pre-processes
Soya whey wastewater is subjected to refrigerated centrifuge at 2 ~ 6 DEG C, retains supernatant, and adjust supernatant pH to 7 ~ 8, in 2 ~ 6
At DEG C, 2 ~ 5h is stood；Refrigerated centrifuge at 2 ~ 6 DEG C, supernatant is as soya whey wastewater pretreatment fluid；
2) lipoxygenase is extracted in soya whey wastewater pretreatment fluid
[Bmim] Br ionic liquid is mixed with sylvite, soya whey wastewater pretreatment fluid obtained by step 1) is added, and steaming is added
Distilled water, the mass fraction for adjusting [Bmim] Br ionic liquid is 20% ~ 50%, and the mass fraction of sylvite is 17% ~ 35%；Adjust solution
PH value is 5.4 ~ 6.4, after standing 30 ~ 180min, is centrifuged at 2 ~ 6 DEG C, and liquid is divided into two-phase up and down, and upper phase is lipoxidase
Enzyme crude enzyme liquid.
It preferably, further include step 3), lipoxygenase crude enzyme liquid utilizes the method preliminary purification being concentrated by ultrafiltration.
It preferably, further include step 4), enzyme solution obtained by step 3) is further purified using gel filtration chromatography,
Obtain pure lipoxygenase.
Preferably, it is concentrated by ultrafiltration in step 3) using the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size.
Preferably, gel filtration chromatography separation is carried out using SephadexG-75 in step 4).
Preferably, in step 2, the sylvite is potassium citrate, K2HPO4Or K2CO3。
Further, in step 2, the sylvite is K2HPO4。
Preferably, in step 2, borate buffer solution and the pH of sodium hydroxide solution regulation system are utilized.
Preferably, in step 2, the mass fraction of [Bmim] Br ionic liquid is 30% ~ 33%, the quality of sylvite
Score is 23% ~ 28%；Adjusting solution ph is 5.7 ~ 5.9.
Preferably, in step 2, time of repose is 100 ~ 130min.
One, lipoxygenase enzyme activity determination method in present embodiment:
Enzyme activity definition: at 25 DEG C, under the conditions of pH9.0, using linoleic acid as substrate, at 234nm, reaction system 1min catalysis is generated
The enzyme concentration of 1umol product is defined as an enzyme-activity unit.
Substrate preparation: the polysorbas20 of 0.25mL is dispersed in the 0.2mol/L of 10mL, the borate buffer solution that pH is 9.0
In, the linoleic acid of 0.27mL is constantly shaken and be added dropwise, it is uniformly mixed, is then added dropwise into system certain density
NaOH solution clarifies system, and regulation system pH to 9.0, finally uses 0.2mol/L, and the borate buffer solution of pH9.0 is settled to
500mL is stored at 4 DEG C as substrate solution, spare.
Determination of activity: 0.2mL sample solution is added in 1.5mL substrate solution, the isothermal reaction at 25 DEG C after mixing
After 3min, 5mL dehydrated alcohol is added and terminates reaction, adding 5mL distilled water clarifies reaction system, measures and inhales at 234nm
Luminosity.Blank sample is that 5mL dehydrated alcohol is first added in 1.5mL substrate solution, and 0.2mL sample solution is being added, permanent at 25 DEG C
After warm 3min, 5mL distilled water is added.
The above enzyme activity determination method refers to following two document:
1, influence [D] Southern Yangtze University of the Wang Ren super-pressure to soybean lipoxygenase, nutrition inhibiting factor and property of protein,
2008.
2、 Catod L, Halmos A L, Small D M. Measurement of lipoxygenase in
Australian white wheat flour: the effect of lipoxygenase on the quality
properties of white salted noodles[J]. Journal of the Science of Food and
Agriculture, 2006, 86(11): 1670-1678. DOI: 10.1002/jsfa.2539.
Two, SDS-PAGE analysis experiment in present embodiment
SDS-PAGE analysis is carried out to LOX sample solution with reference to Laemmli method, sample is with 2Loading buffer carries out molten
Solution, and 3min is heated in boiling water bath, 3min is centrifuged with 8000r/min.Resolving gel concentration is 15%, and concentration gum concentration is 5%, electricity
Pole buffer is pH8.3 Tris-Gly buffer, and separation gel buffer liquid is 1.5mol/L pH8.8 Tris-HCl buffer,
Concentration glue buffer is 1.0mol/L pH6.8 Tris-HCl buffer, at room temperature electrophoresis 120min.With examining after electrophoresis
Mas bright blue R-250 coloring agent carries out dyeing 8h, and decoloration for 24 hours, then carries out photographic analysis.And according to protein content in sample
Linearly relevant principle carries out protein precipitation rate and purity analysis to PAGE gel electrophoretic band gray value, using solidifying
Glue imaging system acquires electrophoretogram, gray value analysis is carried out to each electrophoretic band with Image J software, based on formula (1) and (2)
Calculate protein precipitation rate (PR) and purity (PU) in each sample.
Wherein, PGproRepresent testing protein gray value in precipitating；EGproRepresent testing protein gray scale in soya whey wastewater
Value；Np and Ne respectively represents precipitating and soya whey wastewater extension rate；SGproTesting protein gray value in representative sample；
SGallRepresent the total gray value of all proteins in precipitating.
Embodiment 1
1) soya whey wastewater pre-processes
It takes appropriate soya whey wastewater under the conditions of 4 DEG C, with 12000r/min refrigerated centrifuge 15min, retains supernatant and be adjusted to
PH7.5, after 4 DEG C of standing 3h, refrigerated centrifuge 15min, supernatant is saved backup as soya whey wastewater pretreatment fluid, 4 DEG C；
2) lipoxygenase is extracted in soya whey wastewater pretreatment fluid
By [Bmim] Br ionic liquid and sylvite (potassium citrate, K2CO3、K2HPO4) mixing, soy-bean whey obtained by step 1) is added
Wastewater Pretreatment liquid, and distilled water is added；It is adjusted with borate buffer solution and certain density NaOH solution regulation system pH molten
Liquid pH value is 5.4 ~ 6.4, after standing 30 ~ 180min, with 8000r/min, 10min is centrifuged at 4 DEG C, liquid is divided into upper and lower two
Phase, upper phase are lipoxygenase crude enzyme liquid.
Influence of 1.1 reagents to enzymatic activity
The reagent of various concentration has different degrees of influence to the enzymatic activity of LOX, and choosing concentration gradient respectively is 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40% potassium citrate, K2CO3、K2HPO4And [Bmim] Br solution, probe into each of various concentration
At phase reagent on the active influence of LOX, as a result as shown in Figure 1.
As shown in Figure 1, within the scope of 5%-40%, activity ratio's [Bmim] Br concentration shows a increasing trend, and has to LOX activity
Significant activation, and K2CO3Concentration significantly inhibits LOX activity within the scope of 5%-40%, and enzymatic activity
Than between 32.4%-50.7%.Potassium citrate is active on LOX to be influenced smaller, i.e. activity ratio is 100% or so, and K2HPO4
For concentration within the scope of 5%-40%, activity ratio is in first to increase the trend dropped afterwards, and activity ratio is kept between 100%-122%,
Illustrate K2HPO4There is apparent facilitation to LOX activity.In contrast, [Bmim] Br-K2HPO4The ATPS of composition is more advantageous
LOX in extracting soybean whey wastewater.
The influence that 1.2 [Bmim] Br mass fractions extract LOX
In K2HPO4In the case of mass fraction, pH, waste water additive amount and extraction time remain unchanged, [Bmim] Br mass fraction pair
LOX extraction yield, purification and the influence compared be as shown in Figure 2 in soya whey wastewater.
As shown in Figure 2, increase compared to the increase with [Bmim] Br mass fraction, extraction yield and purification are with [Bmim]
Br mass fraction increase and in first increasing the trend subtracted afterwards, when [Bmim] Br mass fraction is 32%, the extraction yield and purifying of LOX
Multiple reaches maximum, and extraction yield and the maximum value of purification are respectively 86.84% and 2.05.
This is because with the increase of [Bmim] Br mass fraction, enzyme molecule to its compatibility also with increase, increase from
Electrostatic interaction between sub- liquid and enzyme molecule, and with [Bmim] Br mass fraction increase ATPS compared to also with
Increase, this be conducive to improve LOX extraction yield.When [Bmim] Br mass fraction is higher than it is best when, yin present in sample from
Son can prevent LOX from combining with [Bmim] Br.Comprehensively consider, when [Bmim] Br mass fraction is 32%, most beneficial for extracting soybean
LOX in whey wastewater.
1.3 K2HPO4The influence that concentration extracts LOX
Fig. 3 is K2HPO4Mass fraction is to LOX extraction yield, purification and the influence compared.From the figure 3, it may be seen that working as K2HPO4Quality
Score is within the scope of 17%-35%, and the extraction yield and purification of LOX is with K2HPO4The increase of mass fraction is in first increasing becoming of adding afterwards
Gesture, and work as K2HPO4When mass fraction is 26%, extraction yield and purification reach maximum, and respectively 88.91% and 2.37.And it is
System is compared to K2HPO4The increase of concentration tapers off trend.
The main reason for this trend formation is then with K2HPO4The increase of mass fraction, phase is structural under ILATPS
Reinforce, and the upper phase structure rich in ionic liquid is poor, then LOX is easier to be present in phase, and works as K2HPO4Mass fraction is high
When 26%, the salting out of phase is reinforced under ILATPS, leads to the extraction yield and purification decline of LOX.Therefore, K2HPO4Concentration
It is best to LOX effect of extracting when being 26%.
The influence that 1.4 system pH extract LOX
PH to compare, the influence of extraction yield and purification it is as shown in Figure 4.As shown in Figure 4, between pH5.4-6.4, compared to not
Become, extraction yield and purification are in first to increase to subtract trend afterwards, and when pH is 5.8, extraction yield and purification are reached with the increase of pH
To maximum value, respectively 90.83% and 2.79.
Data show that the isoelectric point of LOX is 5.6-5.6, and there are 3 kinds of isodynamic enzymes.When utilization borate buffer solution tune
When section system pH, enzyme molecule institute is electrically charged and how much is affected, and then causes enzyme molecule and imitate at the hydrogen bond between phase reagent
It answers and charge effect, and different pH can also have an impact the degree of dissociation of salt in aqueous phase system.Therefore, system pH and enzyme point
The isoelectric point difference of son is bigger, and distribution of the enzyme molecule in two-phase is more uneven.
The influence that 1.5 extraction times extract LOX
Extraction agent to compare, the influence of extraction yield and purification it is as shown in Figure 5.
As shown in Figure 5, extraction time within the scope of 30min-180min to compare, the influence of extraction yield and purification
It is more significant.With the increase of extraction time, compared to constant, extraction yield and purification are also in first to increase to subtract trend afterwards, maximum value
Respectively up to 91.12% and 2.83, and extraction time is 120min.
1.6 response surface experimental analyses
According to experiment of single factor result it is found that due to [Bmim] Br mass fraction, K2HPO4Mass fraction and pH are to soy-bean whey
The extraction yield of LOX and purification have more significant influence in waste water.Therefore, Box-Behnken center combination design is selected, it is right
It carries out further optimization analysis, and is worth in response with extraction yield and purification.Experimental result is as shown in table 1.
1 response surface experimental design of table and result
Regression analysis is carried out using experimental data of the Design-Expert.8.0.6.1 software to table 1, obtains extraction yield (Y%),
The regression equation of purification (P).
Variance analysis is carried out to two above quadratic regression, the results are shown in Table 2.
2 two quadratic regressions of table carry out the results of analysis of variance
By Tables 1 and 2 it is found that the extremely significant (P of the Optimized model of extraction yield (Y%) and 3 variables of purification (P) optimization
< 0.0001), lose quasi- Xiang Jun not significantly (P > 0.05), and the coefficient of determination R of two quadratic regression equations2Respectively 0.9988
With 0.9990, signal-to-noise ratio (Adeq Precision) is respectively 64.271 and 69.321, illustrates the variability of 99% experimental data
It can be explained with this model, model-fitting degree is preferable, and confidence level is higher, therefore available regression equation divides experimental result
Analysis and prediction.
In addition, first order A, B, C and quadratic term A2、B2、C2It shows as extremely significant (P < 0.0001), interaction item AB and BC table
It is now significant (P < 0.001), shows A, B, C, A2、B2、C2, AB, BC have a significant impact extraction yield and purification.From side
Difference analysis it is found that in each factors on soybean whey wastewater lipoxygenase extraction yield the descending sequence of influence are as follows: A
([Bmim] Br mass fraction) > C(pH) > B(K2HPO4Mass fraction), and lipoxidase in each factors on soybean whey wastewater
The influence of the enzyme rate of recovery, which has, arrives greatly small sequence are as follows: A([Bmim] Br mass fraction) > B(K2HPO4Mass fraction) > C(pH).
The optimal conditions of model is obtained by Design-Expert.8.0.6.1 software prediction are as follows: [Bmin] Br mass point
Number is 32.58%, K2HPO4Mass fraction is 25.75%, pH 5.74, extraction time 120min, gained extraction yield and purifying times
Several predicted values is respectively 91.54% and 2.82.And the LOX of soya whey wastewater is extracted using above-mentioned optimal conditions, institute
The actual value for obtaining LOX extraction yield and purification is respectively 91.21% and 2.57, differs 0.33% and 0.25 respectively with predicted value,
Illustrate that the model has preferable degree of fitting.
Embodiment 2
1) soya whey wastewater pre-processes
It takes appropriate soya whey wastewater under the conditions of 4 DEG C, with 12000r/min refrigerated centrifuge 15min, retains supernatant and be adjusted to
PH7.5, after 4 DEG C of standing 3h, refrigerated centrifuge 15min, supernatant is saved backup as soya whey wastewater pretreatment fluid, 4 DEG C；
2) lipoxygenase is extracted in soya whey wastewater pretreatment fluid
By [Bmim] Br ionic liquid and K2HPO4Mixing is added soya whey wastewater pretreatment fluid obtained by step 1), and is added
Distilled water, [Bmin] Br mass fraction are 32.58%, K2HPO4Mass fraction is 25.75%；With borate buffer solution and certain dense
It is 5.74 that the NaOH solution regulation system pH of degree, which adjusts solution ph, after standing 120min, with 8000r/min, is centrifuged at 4 DEG C
10min, liquid are divided into two-phase up and down, and upper phase is lipoxygenase crude enzyme liquid；
3) lipoxygenase crude enzyme liquid utilizes the Methods For Purification being concentrated by ultrafiltration
Using ultrafiltration system and the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size, two-stage ultrafiltration mode is selected, in 200 KPa of pressure
10 times of ultrafiltration concentrations are carried out to lipoxygenase crude enzyme liquid.
Embodiment 3
1) soya whey wastewater pre-processes
It takes appropriate soya whey wastewater under the conditions of 4 DEG C, with 12000r/min refrigerated centrifuge 15min, retains supernatant and be adjusted to
PH7.5, after 4 DEG C of standing 3h, refrigerated centrifuge 15min, supernatant is saved backup as soya whey wastewater pretreatment fluid, 4 DEG C；
2) lipoxygenase is extracted in soya whey wastewater pretreatment fluid
By [Bmim] Br ionic liquid and K2HPO4Mixing is added soya whey wastewater pretreatment fluid obtained by step 1), and is added
Distilled water, [Bmin] Br mass fraction are 32.58%, K2HPO4Mass fraction is 25.75%；With borate buffer solution and certain dense
It is 5.74 that the NaOH solution regulation system pH of degree, which adjusts solution ph, after standing 120min, with 8000r/min, is centrifuged at 4 DEG C
10min, liquid are divided into two-phase up and down, and upper phase is lipoxygenase crude enzyme liquid；
3) lipoxygenase crude enzyme liquid utilizes the method preliminary purification being concentrated by ultrafiltration
Using ultrafiltration system and the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size, two-stage ultrafiltration mode is selected, in 200 KPa of pressure
10 times of ultrafiltration concentrations are carried out to lipoxygenase crude enzyme liquid.
4) enzyme solution obtained by step 3) is further purified using gel filtration chromatography, obtains pure lipoxygenase；Gel
Filtration Chromatography utilizes the Sephadex G-75 gel column of 2-70KDa, and with pH7.8, the phosphate buffer of 0.02mol/L is balanced
2 ~ 3 column volumes, loading after balance, with pH7.8, the phosphate buffer of 0.02mol/L is elution；Elute liquid stream
Speed is 0.5mL/min；Automatic collection sample merges appearance part.Sephadex G-75 chromatographs map as shown in figure 5, enzyme activity
As a result as shown in Figure 6
Soya whey wastewater, 1 gained enzyme solution of embodiment, 2 gained enzyme solution of embodiment, 3 gained enzyme solution of embodiment LOX parameter list such as
Shown in table 3, SDS-PAGE analysis, as a result as shown in Figure 7.
3 soya whey wastewater of table, 1 gained enzyme solution of embodiment, 2 gained enzyme solution of embodiment, 3 gained enzyme solution of embodiment LOX
Parameter list
As shown in Table 3, as can be seen from the table, the specific enzyme activity of LOX is 2823.94U/ in liquid in upper phase liquid obtained by embodiment 1
Mg, the enzyme activity rate of recovery and purity are respectively 56.79% and 31.46%, and purification 2.82 has reached industrial production mark
It is quasi-.After 30KDa ultrafiltration membrane and SephadexG-75 are further concentrated and purified, final gained concentrate specific enzyme activity is
18055.24U/mg, purification improve 17.03 times, and the enzyme activity rate of recovery is 18.05%, and the purity of final LOX extract liquor is
68.55%.And SDS-PAGE analysis is carried out to the sample of each purification phase, acquired results are as shown in Figure 4.Swimming lane 2 is in figure
Soya whey wastewater, and protein band molecular weight distribution, between 10-100KDa, swimming lane 3 is 1 gained enzyme solution of embodiment, swimming lane 4
It is respectively 3 gained enzyme solution of 2 gained enzyme solution of embodiment and embodiment, as can be seen from the figure protein band and albumen with swimming lane 5
Matter content is gradually reduced reduction, illustrates that this purification process has preferable purification effect to LOX in soya whey wastewater, can from figure
Concentrated and purified with finding out by 30KDa ultrafiltration membrane, foreign protein removes substantially, and by after gel permeation chromatography, sample purity compared with
Height illustrates that this purification process has preferable purification effect to the LOX in soya whey wastewater.
It can be obtained by embodiment 1, embodiment 2, embodiment 3:
1. obtaining [Bmim] Br-K by experiment of single factor and Box-Behnken central combination design2HPO4Extract LOX
Optimum condition are as follows: [Bmin] Br mass fraction 32.58%, K2HPO4Mass fraction 25.75%, pH 5.74, extraction time are
120min, and LOX extraction yield and purification are respectively 91.21% and 2.57 with this condition, distinguish phase with response surface predicted value
Poor 0.33% and 0.25, and LOX specific enzyme activity is 2823.94U/mg in extract liquor, the enzyme activity rate of recovery and purity are respectively 56.79% He
31.46%。
2. influence [Bmim] Br-K has been determined by experiment of single factor2HPO4The major influence factors of system are [Bmim] Br
Mass fraction, K2HPO4Mass fraction and pH, face variance analysis is it is found that LOX extracts in each factors on soybean whey wastewater according to response
The sequence for taking the influence of rate descending are as follows: A([Bmim] Br mass fraction) > C(pH) > B(K2HPO4Mass fraction), and it is each
There is the influence of the LOX rate of recovery in factors on soybean whey wastewater arrives greatly small sequence are as follows: A([Bmim] Br mass fraction) > B
(K2HPO4Mass fraction) > C(pH).
3. during enzyme purification, ILATPS extract liquor after 30KDa ultrafiltration membrane is concentrated 10 times, the specific enzyme activity of LOX and
Purification is respectively 9222.89U/mg and 9.21, and the enzyme activity rate of recovery is up to 49.91%, and after gel permeation chromatography, LOX compares enzyme
Living finally to reach 18055.24U/mL, the purification of LOX improves 17.03 times, and the enzyme activity rate of recovery is 18.05%, LOX extract liquor
Final purity up to 68.55%.
4. being analyzed using SDS-PAGE LOX purification process, show based on the analysis results, by SephadexG-
The sample of 75 purifying is essentially single protein band, illustrates to have obtained the higher LOX solution of purity by purification process.
Claims (10)
1. a kind of utilization method of soya whey wastewater, which is characterized in that comprising steps of
1) soya whey wastewater pre-processes
Soya whey wastewater is subjected to refrigerated centrifuge at 2 ~ 6 DEG C, retains supernatant, and adjust supernatant pH to 7 ~ 8, in 2 ~ 6
At DEG C, 2 ~ 5h is stood；Refrigerated centrifuge at 2 ~ 6 DEG C, supernatant is as soya whey wastewater pretreatment fluid；
2) lipoxygenase is extracted in soya whey wastewater pretreatment fluid
[Bmim] Br ionic liquid is mixed with sylvite, soya whey wastewater pretreatment fluid obtained by step 1) is added, and steaming is added
Distilled water, the mass fraction for adjusting [Bmim] Br ionic liquid is 20% ~ 50%, and the mass fraction of sylvite is 17% ~ 35%；Adjust solution
PH value is 5.4 ~ 6.4, after standing 30 ~ 180min, is centrifuged at 2 ~ 6 DEG C, and liquid is divided into two-phase up and down, and upper phase is lipoxidase
Enzyme crude enzyme liquid.
2. the utilization method of soya whey wastewater as described in claim 1, it is characterised in that: further include step 3), lipoxidase
Enzyme crude enzyme liquid utilizes the method preliminary purification being concentrated by ultrafiltration.
3. the utilization method of soya whey wastewater as claimed in claim 2, it is characterised in that: further include step 4), step 3) institute
It obtains enzyme solution further to purify using gel filtration chromatography, obtains pure lipoxygenase.
4. the utilization method of soya whey wastewater as claimed in claim 2, it is characterised in that: utilize 30KDa size in step 3)
Modified poly (ether-sulfone) ultrafiltration membrane be concentrated by ultrafiltration.
5. the utilization method of soya whey wastewater as claimed in claim 3, it is characterised in that: utilize SephadexG- in step 4)
75 carry out gel filtration chromatography separation.
6. the utilization method of soya whey wastewater as described in claim 1, it is characterised in that: in step 2, the sylvite is lemon
Lemon acid potassium, K2HPO4Or K2CO3。
7. the utilization method of soya whey wastewater as claimed in claim 6, it is characterised in that: in step 2, the sylvite is
K2HPO4。
8. the utilization method of soya whey wastewater as described in claim 1, it is characterised in that: slow using borate in step 2
The pH of fliud flushing and sodium hydroxide solution regulation system.
9. the utilization method of soya whey wastewater as described in claim 1, it is characterised in that: in step 2, [Bmim] Br ion
The mass fraction of liquid is 30% ~ 33%, and the mass fraction of sylvite is 23% ~ 28%；Adjusting solution ph is 5.7 ~ 5.9.
10. the utilization method of soya whey wastewater as described in claim 1, it is characterised in that: in step 2, time of repose is
100~130min。
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| CN112626041A (en) * | 2020-12-24 | 2021-04-09 | 齐鲁工业大学 | Process for extracting lipoxygenase from soybean whey protein wastewater by two aqueous phases |
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