CN102796613B - Demulsification method by soybean grease microbe extraction through aqueous enzymatic method - Google Patents
Demulsification method by soybean grease microbe extraction through aqueous enzymatic method Download PDFInfo
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Abstract
The invention relates to a demulsification method by soybean grease microbe extraction through an aqueous enzymatic method, which belongs to the plant grease extraction technology. The method comprises the following steps: 1) crushing soybean to obtain a puffed material by using an extruding puffed pretreatment, mixing the puffed material and water to obtain a mixed liquor, adding alkaline protease in the mixed liquor for enzymatic hydrolysis, centrifuging after enzymatic hydrolysis to obtain free oil, an emulsion, a hydrolysate and residues; 2) fermenting the mixed demulsifying bacteria with different complex formulation proportion through a MMSM inorganic salt liquid medium to obtain a mixed demulsifying bacteria whole liquid culture medium; 3) adjusting the pH value to the emulsion obtained in the step 1) to neutrality, fully and uniformly mixing with the mixed demulsifying bacteria whole liquid culture medium, and standing and performing constant temperature demulsification, centrifuging after demulsification to obtain soybean oil. The method of the invention has the advantages of simple technology device, low cost, easy degradation of waste liquid, low toxicity and no pollution generation, and the mixed demulsifying bacteria whole liquid culture medium has good thermostability and repeated usage.
Description
Technical field
The invention belongs to Vegetable oil lipoprotein and extract processing technology, relate generally to a kind of method of aqueous enzymatic extraction soybean oil microbial de-emulsification.
Background technology
In aqueous enzymatic extraction soybean oil method, breakdown of emulsion is very crucial step, and breakdown of emulsion is more thorough, and the rate of recovery of grease is higher.The quality of breakdown of emulsion directly has influence on the economic worth of whole technique.
The method of breakdown of emulsion is divided into chemical demulsification, physics breakdown of emulsion and biological demulsifying.Chemical demulsification method is to add chemical demulsifier; Centrifugal, change temperature, apply electrostatic field, adopt coalescing agent etc. to belong to physical method; Biological demulsifying method refers to utilizes the meta-bolites of microorganism cells itself or its metabolic process to realize emulsion breakdown.Due to the existence of the organic solvent residual of chemical method and the problem such as the demulsification efficiency of Physical is low, select biological method as breakdown of emulsion means.Hanmoungjai (2001) adopts proteolytic enzyme to act on rice bran, and to the method breakdown of emulsion boiling for the emulsion layer obtaining, oil is only 79% and 68% with the rate of recovery of protein.Aparna (2002) adopts 18000g ultra-high speed centrifugation breakdown of emulsion, and oily yield has improved 40% than contrast, but this centrifugation apparatus cannot be applied to scale operation.Moura etc. (2008) adopt proteolytic enzyme in conjunction with the method breakdown of emulsion of pH adjusting or Phospholipid hydrolase enzymolysis, the grease of 70%-80% in recyclable milk sap in soybean aqueous enzymatic method oil-producing technique.At soybean aqueous enzymatic method, carry in oil, Wu (2009) explores the mode breakdown of emulsion of enzymolysis and regulation system pH, Jung (2009) further reclaims free oil condition to endo-protease breakdown of emulsion and is optimized research, to the development for this environmentally friendly oil extracting process of propelling aqueous enzymatic method, contributes.In addition, foreign scholar adopts organic solvent directly to extract the oil in milk sap under study for action mostly, with the effect that after investigation enzymolysis, oil discharges from oil plant cell, and the research further breaking through from technological layer does not appear in the newspapers, and, existing demulsification technology exist demulsification efficiency low, to problems such as environments.
Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned the deficiencies in the prior art, and a kind of method of aqueous enzymatic extraction soybean oil microbial de-emulsification is provided, and reaches the object that reduces costs, improves demulsification efficiency, reduces pollution.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method of aqueous enzymatic extraction soybean oil microbial de-emulsification, the method comprises the following steps: (1) adopts expelling-expansion pretreatment to obtain expanded material the soybean after pulverizing, expanded material and water are mixed to get to mixed solution, in mixed solution, add Sumizyme MP to carry out enzymolysis, after enzymolysis, centrifugation obtains free oil, milk sap, hydrolyzed solution and residue; (2) Mo Haiwei genus bacillus, Bacillus subtillis and mud Nocardia bacteria are mixed with to mixing demulsifying bacteria with different compound proportions, mixing demulsifying bacteria is fermented through MMSM inorganic salt liquid substratum, described Mo Haiwei genus bacillus, Bacillus subtillis and mud Nocardia bacteria compound proportion are the 1-8% of MMSM inorganic salt liquid culture volume, fermentation condition is shaking speed 100-180 r/min, inoculum size is the 2-10% of culture volume, culture temperature 20-40 ℃, incubation time 10-30h, obtains mixing the full nutrient solution of demulsifying bacteria after fermentation; (3) milk sap step (1) being obtained adjusts pH neutral, fully mixes with mixing the full nutrient solution of demulsifying bacteria, carries out the reaction of constant temperature breakdown of emulsion after mixing, and breakdown of emulsion temperature is 20-60 ℃, and the breakdown of emulsion time is 0.5-2.5h, and after reaction, centrifugation obtains soybean oil.
The described preferred compound proportion of mixing demulsifying bacteria is: Mo Haiwei genus bacillus 2.19%, Bacillus subtillis 3.3%, mud Nocardia bacteria 2.51%.
The described preferred fermentation parameter of mixing demulsifying bacteria breakdown of emulsion is: shaking speed 142r/min, inoculum size is culture volume 5.9%, 29.6 ℃ of culture temperature, incubation time 20.65h.
Described breakdown of emulsion reaction conditions is: breakdown of emulsion temperature is 30 ℃, and the breakdown of emulsion time is 1.5h.
Present method adopts biological demulsifying bacteria to carry out breakdown of emulsion, biological demulsifying bacteria utilizes the lipopeptid class surfactant surfactin producing in somatic cells itself or its metabolic process to carry out breakdown of emulsion, thereby oil is separated out in milk sap, and then grease is released, have that required processing unit is simple, cost is low, demulsification efficiency is high, the degraded of waste liquid energy, hypotoxicity, feature that pollution is few, and mix the full nutrient solution Heat stability is good of demulsifying bacteria, reusable.
Accompanying drawing explanation
Fig. 1 the inventive method operational path block diagram;
The contour plots analysis of Fig. 2 mixing demulsifying bacteria interaction to demulsification efficiency;
The mixture experiment design optimum result of Fig. 3 mixing demulsifying bacteria proportioning to demulsification efficiency;
The response surface analysis figure of Fig. 4 mixing demulsifying bacteria proportioning to demulsification efficiency;
Fig. 5 shaking speed and inoculum size be the response surface to demulsification efficiency alternately;
Fig. 6 shaking speed and culture temperature be the response surface to demulsification efficiency alternately;
Fig. 7 inoculum size and culture temperature be the response surface to demulsification efficiency alternately;
Fig. 8 inoculum size and incubation time be the response surface to demulsification efficiency alternately;
Fig. 9 culture temperature and incubation time be the response surface to demulsification efficiency alternately;
The impact of Figure 10 breakdown of emulsion temperature on demulsification efficiency;
The impact of Figure 11 breakdown of emulsion time on demulsification efficiency.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the invention is described in detail,
A kind of method of aqueous enzymatic extraction soybean oil microbial de-emulsification, the method comprises the following steps: (1) adopts expelling-expansion pretreatment to obtain expanded material the soybean after pulverizing, expanded material and water are mixed to get to mixed solution, in mixed solution, add Sumizyme MP to carry out enzymolysis, after enzymolysis, centrifugation obtains free oil, milk sap, hydrolyzed solution and residue; (2) Mo Haiwei genus bacillus, Bacillus subtillis and mud Nocardia bacteria are mixed with to mixing demulsifying bacteria with different compound proportions, mixing demulsifying bacteria is fermented through MMSM inorganic salt liquid substratum, described Mo Haiwei genus bacillus, Bacillus subtillis and mud Nocardia bacteria compound proportion are the 1-8% of MMSM inorganic salt liquid culture volume, fermentation condition is shaking speed 100-180 r/min, inoculum size is the 2-10% of culture volume, culture temperature 20-40 ℃, incubation time 10-30h, obtains mixing the full nutrient solution of demulsifying bacteria after fermentation; (3) milk sap step (1) being obtained adjusts pH neutral, fully mixes with mixing the full nutrient solution of demulsifying bacteria, carries out the reaction of constant temperature breakdown of emulsion after mixing, and breakdown of emulsion temperature is 20-60 ℃, and the breakdown of emulsion time is 0.5-2.5h, and after reaction, centrifugation obtains soybean oil.
The described preferred compound proportion of mixing demulsifying bacteria is: Mo Haiwei genus bacillus 2.19%, Bacillus subtillis 3.3%, mud Nocardia bacteria 2.51%.
The described preferred fermentation parameter of mixing demulsifying bacteria breakdown of emulsion is: shaking speed 142r/min, inoculum size is culture volume 5.9%, 29.6 ℃ of culture temperature, incubation time 20.65h.
Described breakdown of emulsion reaction conditions is: breakdown of emulsion temperature is 30 ℃, and the breakdown of emulsion time is 1.5h.
Embodiment 1: mix the optimum compound proportion shaker test of demulsifying bacteria
1 materials and methods
1.1 test raw material
| decortication soybean sheet | harbin Jiu San grease group |
| protex-6L Alcalase | denmark novo company |
| mo Haiwei genus bacillus | key lab of Northeast Agricultural University |
| bacillus subtillis | key lab of Northeast Agricultural University |
| mud Nocardia bacteria | key lab of Northeast Agricultural University |
| mMSM inorganic salt liquid substratum | nH 4nO 34.0g, K 2hPO 44.0g, KH 2pO 46.0g, MgSO 4h 2o 0.2g, trace element solution lmL, whiteruss 4% (v/v), yeast extract paste 1.0g, glucose 10.0g, deionized water 1000mL.Sterilizing 15min at 121 ℃. |
1.2 test apparatuses and equipment
| Instrument | Manufacturer |
| CX-500 type Ultrasonic Cleaners | Beijing medical facilities two factories |
| High-pressure sterilizing pot | Tianjin Stettlen Instrument Ltd. |
| Bechtop | Shanghai Zhi Cheng analytical instrument Manufacturing Co., Ltd |
| Electronic analytical balance | Mettler-Toledo Instrument (Shanghai) Co., Ltd. |
| Beater disintegrating machine | China Tianjin Stettlen Instrument Ltd. |
| LDZ5-2 type table-type low-speed whizzer | Anting Scientific Instrument Factory, Shanghai |
| Electric-heated thermostatic water bath | Yuyao City east electric instrument factory |
| TGL-16G high speed tabletop centrifuge | Anting Scientific Instrument Factory, Shanghai |
| Electric precise stirrer | Jintan City, Jiangsu Province high honour instrument |
| Soxhlet extractor | Tianjin Glass Instrument Factory |
| Electric heating constant-temperature blowing drying box | Shanghai Yiheng Scientific Instruments Co., Ltd |
1.3 test method
1.3.1 the composition measurement of soybean
The mensuration of crude protein: GB6432-94 standard method is carried out
The mensuration of crude fat: in GB5512-85, Soxhlet extraction process is measured
The mensuration of moisture: GB304-87 are measured
Determination of ash: GB5009.4-85
1.3.2 technical process
Decortication soybean sheet → pulverizing → moisture adjusting → extruding puffing → pulverizing → adjusting pH value and the temperature → enzymolysis → enzyme that goes out → centrifugal → milk sap → adding in varing proportions composite demulsification bacterium carries out breakdown of emulsion → soybean oil
1.3.3 calculation formula
2 results and discussion
2.1 empirical factor level code table
On the basis of single factor research, choosing Mo Haiwei genus bacillus, Bacillus subtillis, 3 factors of mud Nocardia bacteria is independent variable(s), take demulsification efficiency as response value, according to mixture experimental designs principle, utilize simple form center of gravity (Simplex Centroid) design in Design-Expert software to carry out process optimization, its level of factor coding schedule is in Table table 2-1.
Table 2-1 level of factor coding schedule
2.2 mixture experiment design experimental establishment and experimental results
This experimental applications mixture experiment design optimized method carries out process optimization.Take A, B, C is independent variable(s), take demulsification efficiency as response value R, mixture experiment design experimental program and the results are shown in Table 2-2.Experiment 1-10 is factorial experiment, and 11-15 is 5 center tests, in order to estimate experimental error.
Table 2-2 test arrangement and result
2.3 mixture experiment design interpretations
By statistical analysis software Design-Expert, carry out data analysis, set up quadratic regression model as follows:
Regression analysis and the results of analysis of variance are in Table 2-3.
Table 2-3 returns and the results of analysis of variance
From table 2-3, the linear relationship between equation dependent variable and independent variable(s) is obvious, and this model recurrence is (p<0.0001) significantly, and lose and intend item not remarkable (p>0.05), and this model R
2=98.85%, R
2 adj=98.16%, illustrate that this model and experimental fit are good, between independent variable(s) and response value, linear relationship is remarkable, can infer for the theory of this reaction.The p of AB, AC and BC is all less than 0.0001, and known three's interaction on the impact of response value demulsification efficiency significantly.By F check, can obtain factor contribution rate is: BC>AB>AC, i.e. the interaction > Mo Haiwei genus bacillus of Bacillus subtillis and mud nocardial interaction > Mo Haiwei genus bacillus and Bacillus subtillis and the nocardial interaction of mud.
Application responds face optimizing analytical procedure is analyzed regression model, finding optimal response result is that Mo Haiwei genus bacillus is 2.19%, Bacillus subtillis is 3.3%, and mud Nocardia bacteria is 2.51%, and response value demulsification efficiency has optimum value to be approximately 87.5462% left and right.
2.4 confirmatory experiments and simultaneous test
Under the top condition of trying to achieve in mixture experiment design analytical method, Mo Haiwei genus bacillus is 2.19%, and Bacillus subtillis is 3.3%, and mud Nocardia bacteria is 2.51%, carries out 3 parallel laboratory tests, and the mean value of 3 parallel laboratory test demulsification efficiency is 87.88%.Experimental value and regression equation predictor that response value is described are coincide good.
Embodiment 2: the screening experiment that mixes the fermentation optimum parameter of demulsifying bacteria breakdown of emulsion
Based on the optimum compound proportion of the determined compound bio demulsifying bacteria of embodiment 1, carry out the fermentation test of single factor compound bio demulsifying bacteria breakdown of emulsion, determine the scope of fermentation parameter (shaking speed, inoculum size, culture temperature, incubation time).Take demulsification efficiency as investigating index, carry out response surface and design 4 factor 5 hydraulic tests.
1 materials and methods
1.1 materials, reagent
| decortication soybean sheet | harbin Jiu San grease group |
| protex-6L Alcalase | denmark novo company |
| mo Haiwei genus bacillus | key lab of Northeast Agricultural University |
| bacillus subtillis | key lab of Northeast Agricultural University |
| mud Nocardia bacteria | key lab of Northeast Agricultural University |
| mMSM inorganic salt liquid substratum | nH 4nO 34.0g, K 2hPO 44.0g, KH 2pO 46.0g, MgSO 4h 2o 0.2g, trace element solution lmL, whiteruss 4% (v/v), yeast extract paste 1.0g, glucose 10.0g, deionized water 1000mL.Sterilizing 15min at 121 ℃. |
1.2 key instrument equipment
| PHS-25 type acidometer | Shanghai great achievement instrument plant |
| Electronic analytical balance | Mei Lete-Tuo benefit instrument (Shanghai) Co., Ltd. |
| Whizzer | Beijing Medical Centrifugal Machine Factory |
| Electric precise stirrer | Jintan City, Jiangsu Province high honour instrument manufacturing company limited |
| Electric-heated thermostatic water bath | Yuyao City east electric instrument factory |
| Digestion instrument | Shanghai Qianjian Instrument Co., Ltd. |
| Planetary ball mill | Nanda Tianzun Electronics Co., Ltd., Nanjing |
| Soxhlet extractor | Tianjin Glass Instrument Factory |
1.3 experimental technique
1.3.1 the composition measurement of soybean
The mensuration of crude protein: GB6432-94 standard method is carried out
The mensuration of crude fat: in GB5512-85, Soxhlet extraction process is measured
The mensuration of moisture: GB304-87 are measured
Determination of ash: GB5009.4-85
1.3.2 technical process
Decortication soybean sheet → pulverizing → moisture adjusting → extruding puffing → pulverizing → adjusting pH value and the temperature → enzymolysis → enzyme that goes out → centrifugal → milk sap → adding in varing proportions compound bio demulsifying bacteria carries out breakdown of emulsion → soybean oil
2 results and discussion
2.1 empirical factor level code tables
On the basis of single factor research, choosing shaking speed, inoculum size, culture temperature and 4 factors of incubation time is independent variable(s), take demulsification efficiency as response value, according to center combination principle of design, the experiment of design response surface analysis, its level of factor coding schedule is in Table table 2-1.
Table 2-1 level of factor coding schedule
2.2 response surface experimental establishment and experimental results
This experimental applications response surface optimized method carries out process optimization.Take A, B, C, D, E is independent variable(s), take demulsification efficiency as response value Y, response surface experimental program and the results are shown in Table 2-3.Experiment 1-26 is factorial experiment, and the test of 27-36 Wei10Ge center, in order to estimate experimental error.
Table 2-2 test arrangement and result
2.3 response surface interpretations
By statistical analysis software SAS9.1, carry out data analysis, set up Quadratic response surface regression model as follows:
Regression analysis and the results of analysis of variance are in Table 2-3.
Table 2-3 returns and the results of analysis of variance
From table 2-3, the linear relationship between equation dependent variable and independent variable(s) is obvious, and this model recurrence is (p<0.0001) significantly, and lose and intend item not remarkable (p>0.05), and this model R
2=99.18%, R
2 adj=98.64%, illustrate that this model and experimental fit are good, between independent variable(s) and response value, linear relationship is remarkable, can infer for the theory of this reaction.By F check, can obtain factor contribution rate is: A>D>C>B, i.e. shaking speed > incubation time > culture temperature > inoculum size.
Application responds face optimizing analytical procedure is analyzed regression model, and finding optimal response result is that shaking speed is 142r/min, and inoculum size is 5.9%, and culture temperature is 29.6 ℃, and incubation time is 20.65h, and response surface has optimum value to be approximately 89.4583% left and right.
2.4 confirmatory experiments and simultaneous test
Under the top condition of trying to achieve in response surface analysis method, shaking speed is 142r/min, and inoculum size is 5.9%, and culture temperature is 29.6 ℃, and incubation time is 20.65h, carries out 3 parallel laboratory tests, and the mean value of 3 parallel laboratory test demulsification efficiency is 89.12%.Experimental value and regression equation predictor that response value is described are coincide good.
Embodiment 3 mixes the screening experiment of the optimum parameter of demulsifying bacteria breakdown of emulsion
1.1 materials, reagent
| decortication soybean sheet | harbin Jiu San grease group |
| protex-6L Alcalase | denmark novo company |
| mo Haiwei genus bacillus | key lab of Northeast Agricultural University |
| bacillus subtillis | key lab of Northeast Agricultural University |
| mud Nocardia bacteria | key lab of Northeast Agricultural University |
| mMSM inorganic salt liquid substratum | nH 4nO 34.0g, K 2hPO 44.0g, KH 2pO 46.0g, MgSO 4h 2o 0.2g, trace element solution lmL, whiteruss 4% (v/v), yeast extract paste 1.0g, glucose 10.0g, deionized water 1000mL.Sterilizing 15min at 121 ℃. |
1.2 key instrument equipment
| PHS-25 type acidometer | Shanghai great achievement instrument plant |
| Electronic analytical balance | Mei Lete-Tuo benefit instrument (Shanghai) Co., Ltd. |
| Whizzer | Beijing Medical Centrifugal Machine Factory |
| Electric precise stirrer | Jintan City, Jiangsu Province high honour instrument manufacturing company limited |
| Electric-heated thermostatic water bath | Yuyao City east electric instrument factory |
| Digestion instrument | Shanghai Qianjian Instrument Co., Ltd. |
| Planetary ball mill | Nanda Tianzun Electronics Co., Ltd., Nanjing |
| Soxhlet extractor | Tianjin Glass Instrument Factory |
2 results and discussion
Determining of 2.1 the suitableeest breakdown of emulsion temperature
The breakdown of emulsion time is 1.5h, and the demulsification efficiency of take is 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and carries out breakdown of emulsion reaction as investigating index, choosing respectively breakdown of emulsion temperature, determines that the suitableeest breakdown of emulsion temperature is 30 ℃.
Determining of 2.2 the suitableeest breakdown of emulsion times
Breakdown of emulsion temperature is 30 ℃, and the demulsification efficiency of take is that 0.5h, 1h, 1.5h, 2h, 2.5h carry out breakdown of emulsion reaction as investigating index, choosing respectively the breakdown of emulsion time, determines that the suitableeest breakdown of emulsion time is 1.5h.
Claims (1)
1. the method for an aqueous enzymatic extraction soybean oil microbial de-emulsification, the method comprises the following steps: (1) adopts expelling-expansion pretreatment to obtain expanded material the soybean after pulverizing, expanded material and water are mixed to get to mixed solution, in mixed solution, add Sumizyme MP to carry out enzymolysis, after enzymolysis, centrifugation obtains free oil, milk sap, hydrolyzed solution and residue, it is characterized in that: (2) are by Mo Haiwei genus bacillus, Bacillus subtillis and mud Nocardia bacteria take compound proportion as: by the percentage calculation that accounts for MMSM inorganic salt liquid culture volume, Mo Haiwei genus bacillus 2.19%, Bacillus subtillis 3.3%, mud Nocardia bacteria 2.51% is mixed with mixing demulsifying bacteria, mixing demulsifying bacteria is fermented through MMSM inorganic salt liquid substratum, fermentation condition is shaking speed 142r/min, inoculum size is 5.9% of culture volume, 29.6 ℃ of culture temperature, incubation time 20.65h, after fermentation, obtain mixing the full nutrient solution of demulsifying bacteria, (3) milk sap step (1) being obtained adjusts pH neutral, fully mixes with mixing the full nutrient solution of demulsifying bacteria, carries out the reaction of constant temperature breakdown of emulsion after mixing, and breakdown of emulsion temperature is 30 ℃, and the breakdown of emulsion time is 1.5h, and after reaction, centrifugation obtains soybean oil.
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| CN108219915A (en) * | 2017-11-30 | 2018-06-29 | 潍坊友容实业有限公司 | A kind of Suaeda salsa seed oil preparation method |
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