CN102311867A - Method for extracting Cyperus esculentus oil by aqueous enzymatic extraction-freeze-thaw coupling technology - Google Patents
Method for extracting Cyperus esculentus oil by aqueous enzymatic extraction-freeze-thaw coupling technology Download PDFInfo
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Abstract
The invention discloses a method for extracting Cyperus esculentus oil by the combination of aqueous enzymatic extraction and freeze-thaw, selecting plump Cyperus esculentus without insect diseases, removing impurities on the surface by washing with the use of water, and drying water on the surface; crushing, weighing 500kg of a Cyperus esculentus powder, adding a mixed buffer solution of sodium carbonate and sodium bicarbonate at the solid-liquid ratio of 1:7 (m/v), adjusting pH value to 7, immersing for 1 hour at the temperature of 50 DEG C, killing enzyme for 10 min at the temperature of 90 DEG C; adopting a compound enzyme of alkali protease and cellulase mixed at the mass ratio of 2:1 (m/m) while the solid-liquid ratio is 1:6, the enzymatic hydrolysis time is 6 hours, the enzyme addition is 2.5% and the enzymatic hydrolysis temperature is 70 DEG C; placing the enzymatic hydrolysis solution into an ultralow temperature refrigerator at the temperature of minus 30 DEG C for 30 min after enzymatic hydrolysis, followed by thawing at room temperature; centrifuging for 20 min at the rotating speed of 4000r/min, carrying out secondary centrifugation, and separating crude oil to obtain the product. According to the invention, the oil extraction rate is raised, and good grease quality is also maintained.
Description
Invention field
The present invention relates to plant oil product extractive technique field, concrete, the present invention relates to a kind of cyperus esculentus oil extractive technique field.
Background technology
As everyone knows, in all compositions of human foods, grease is the highest as the value of the life energy, and its heat is 37.7kJ/g, exceeds about one times in protein and glucide.Oil prodution industry can be described as an eternal cause of human's demand, and the developing oil prodution industry prospect of China is very wide, it is predicted that the year two thousand twenty reaches the China of 1,500,000,000 populations, if press the pre-capita consumption 14kg/a estimation that FAO recommends.The grease aggregate consumption is 21Mt, promptly except that needs import 7Mt.Domestic still thunderbolt is produced the 14Mt edible oil, and national thus oil plant amount of finish also will increase by 72% on basis in 2000.
Cyperus esculentus has another name called tiger nuts (Cyperus esculentus L Var.Sativus Baeck.) and originates in African Mediterranean Sea bank, Cyperaceae Cyperus perennial plant.The a lot of local light and heat conditions in Xinjiang are similar with the country of origin (basin, the Nile, north African) of cyperus esculentus, and sand soil can satisfy growing of cyperus esculentus.Be a kind of important oil crops; Producing cyperus esculentus oil is one of main path that utilizes cyperus esculentus; Contain a large amount of cyperus esculentus albumen through lixiviate or in the cyperus esculentus grouts after squeezing, but, be difficult to eat because sex change or organic solvent pollute; Generally only be taken as feed, make the protein resource in the cyperus esculentus fail to be fully utilized.Therefore, exploring the extraction process of new cyperus esculentus oil, is very significant with the effect that reaches protein and the dual utilization of grease.
The traditional extraction technique of cyperus esculentus oil mainly is meant milling process and lixiviation process.The method that extracts of grease that milling process utilizes the squeezing action of mechanical external force will press in the material is called milling process, and the general technology flow process of milling process adopts: oil plant → cleaning → oven dry → press for extracting juice embryo → parch → steaming embryo → squeezing → crude oil; Lixiviation process utilizes organic solvent can " dissolve " method that greasy characteristic extracts the grease in the oil plant to be called lixiviation process or extraction process, and the lixiviation process typical process flow adopts oil plant → cleaning → oven dry → material embryo preparation → solvent extraction → crude oil.
At present, there are many technical problems in traditional oil plant reparation technology.In the milling process, supporting equipment is few, and technology is simple; Can make product keep original flavour, but this method residual oil content high (can reach 7～10%), cost is high, power consumption is big, labour intensity is big; And the protein denaturation degree in the grouts of squeezing back is high; Protein utilization rate is low, generally can only be used as fertilizer, thereby has wasted a large amount of quality plant albumen; In the lixiviation process, residual oil content is low, production efficiency is high, but this method equipment is many, the investment big; And the composition of crude oil is complicated, needs through further refining treatment, and is big to the flavor loss of oil product, and leaching oil colours pool is darker; With an organic solvent reduce the security of producing, increased the loaded down with trivial details property of technology, caused the pollution of environment; Meeting residual minim organic solvent in the while processed oil, this is unfavorable to human beings'health.
Along with the deep development of biotechnology, as far back as eighties of last century fifties just the someone propose, use zymin pre-treatment oil section, can improve oil-producing technique efficient, but economic factors has stoped its industrial applications.To the later stage seventies in last century, many new enzymes have dropped into suitability for industrialized production, and the production cost of enzyme constantly descends.Therefore, develop the interest that this technology has caused external many scholars again.1979, in enzyme Alcalase applied to VT 18 and soy proteinaceous water law is separated, the yield that makes oil was near 60% with the mikrobe egg for Olsen etc., and the protein yield is near 40%.1993, FulIBook found that in the proteolysis process, part oil is released out when from depleted watermelon seed, producing solubility X 1000.So he attempts will be by whole watermelon seed of the combinative enzyme hydrolysis that black mold produces making oil and X 1000, and achieve success.Subsequently, his use the same method stripping oil and protein from Semen Brassicae campestris and soybean has all been obtained expected effect.After the nineties, enzyme process separates oil and the extremely concern of Chinese scholars of proteinic research in the oil plant.1994, Tano-Debrah was a main body with proteolytic enzyme, is aided with cellulase and hemicellulase the butter seeds of trees are handled, and extraction yield improves 20%, and he thinks that adopting pair cell to have the enzyme of Degradation to handle the butter seeds of trees can improve extraction yield.2009; A.Moreau etc. have studied the technology of aqueous enzymatic extraction Semen Maydis oil; The oil extracting rate that he draws aqueous enzymatic extraction Semen Maydis oil reaches 57%; Kar Lin Nyam etc. then utilizes the response surface method to study the technology of aqueous enzymatic extraction OK a karaoke club Harry Semen Benincasae oil, adopts neutral protease oil extracting rate under optimal conditions to reach 71.55%.2010, researchs such as Shao Bing Zhang showed that peanut seed toasted before 20 minutes under (130～220 ℃) differing temps, did not have the output of remarkably influenced crude oil with the effect of Sumizyme MP and water, and crude oil extraction yield scope is 86～90%.
Aqueous enzymatic method technology is on the basis of Mechanical Crushing; Adopt the cell walls of enzyme (polygalacturonase, cellulase, glycase and proteolytic enzyme etc.) degrading plant oil plant; Or complex bodys such as degraded LPS, lipoprotein, discharge grease wherein, oil is separated from solids; Utilize non-oil component (protein and glucide) to the oil and the affine difference of water, and profit proportion difference and oil is separated with non-oil component.Operational path is following: fragmentation → enzyme → cooling → enzymolysis → spinning → crude oil → protein sieves → goes out in oil plant → cleaning
This technology is to be solvent with water, extracts at the same time to grow up on the basis of grease and oilseed protein technology, and be to study maximum enzyme process oil extracting process so far.After enzyme was handled, through centrifugal or press filtration solid-liquid separation, solid phase was oilseed protein and residue, after drying, can be used for albumen and fodder prodn, adopted method such as centrifugal to obtain oil and protein etc. behind the liquid phase breakdown of emulsion.
The aqueous enzymatic method oil extracting process is compared advantage and is mainly reflected in aspects such as economy, environment and safety and sanitation with traditional technology.With regard to greasy quality, in the aqueous enzymatic method oil-producing technique, phosphatide at first dissociates out from oil and need not degumming and refining.Most of experimental studies show that the oil quality that makes with aqueous enzymatic method and lixiviation process does not have too big difference.In sum, the aqueous enzymatic method oil extracting process has following advantage:
(1) operational condition is gentle, can nutritive substance be preserved as far as possible.The aqueous enzymatic method oil extracting process is compared with traditional milling process; Whole technology is all carried out under comparatively gentle condition; Even be the thermal treatment that the anti-nutrient substance in the destruction oil plant or the enzyme that goes out carry out raw material before enzymolysis; General employing boiling, rather than adopt the steaming in traditional oil-producing technique to fry, nutritive substance is destroyed few.
(2) in stripping oil, can non-oil ingredient such as soluble protein and glucide together be obtained.Adopt the aqueous enzymatic method oil extracting process from oil plant, to carry oil; The enzymolysis solution that oil plant obtains after enzymolysis is centrifugal is nutritious; Contain amounts of protein and soluble polysaccharide; Enzymolysis solution spraying drying behind ultrafiltration and concentration obtains the protein and the glucide powder of low fat, can be used as the batching of food and drink or animal-feed.
(3) compare with lixiviation process, aqueous enzymatic method adopts enzyme to handle oil plant, destroys cell walls, and grease is discharged from cell, has reduced facility investment and to the pollution of environment, has improved the security and the economy of technology.
(4) aqueous enzymatic method technology can remove some objectionable impurities that the oil plant raw material contains itself.Research shows; Sunflower seeds adopts aqueous enzymatic method technology to carry oil can remove chlorogenicacid and the coffic acid that wherein is prone to make the change of oilseeds cake generation look; Aqueous enzymatic method technology can successfully be removed goitrogenic sulfocompound in the vegetable seed; In addition, fishy smell material in bitter taste vegeto-alkali, the soybean and the pigment in the cottonseed can both be removed in aqueous enzymatic method technology to some extent in the plumage French beans.
(5) aqueous enzymatic extraction oil, grease obtained output is high than milling process, and oil is as clear as crystal, and oil quality is good.
(6) aqueous enzymatic method is carried oil and can be reached and shorten the system oil time, improves the processing power of equipment, cuts down the consumption of energy.
(7) the relative energy consumption of aqueous enzymatic method technology is low, and the decline of its BOD of the waste water that is produced, the more non-enzyme process of COD value is about 35%～75%, is easy to carry out the anaerobic process wastewater treatment.
But aqueous enzymatic method is carried oil tech an inevitable shortcoming is arranged: oil extracting rate is lower, and this can cause the waste of raw material to a certain extent.
So far, enzyme process is carried oil and in multiple oilseeds that remove the above or oil fruit (Sweet Apricot seed, vegetable seed, walnut etc.), has all been obtained application.
In the prior art report; The relevant patent of declaring has: aqueous enzymatic method prepares the greasy method of shellfish internal organs; Number of patent application is 201010284737.2; Patent publication No. is CN101935584A, and the aqueous enzymatic method that this invention relates to a kind of environmental protection prepares the greasy methods of shellfish internal organs such as abalone, scallop.Mainly be to be raw material with fresh shellfish internal organs; At first utilize the vegetative cell in the broken shellfish internal organs of freeze-thaw technology; Utilize the intracellular enzymes such as proteolytic enzyme that discharge after the cytoclasis to carry out self-dissolving, exogenous enzyme is joined further degraded in solution, after the employing isoelectric point method combines the salting-out process breakdown of emulsion; High speed centrifugation is got the upper strata oil reservoir and is prepared shellfish internal organs grease, adopts the supercritical CO 2 extraction process that shellfish internal organs grease is made with extra care at last again.
But the oil extracting rate of aqueous enzymatic method is lower, it is reported, the oil extracting rate of aqueous enzymatic method reduces nearly 22% than traditional vegetables oil preparation method; Aqueous enzymatic method-freeze thawing coupling technique then can overcome this shortcoming; Improve the extraction yield of oil, therefore very far-reaching for the Research Significance of aqueous enzymatic method-freeze thawing coupling technique, with regard to cyperus esculentus; Severe cold, Qin Ye etc. once sent out the research of aqueous enzymatic extraction cyperus esculentus oil, did not see that relevant report is arranged but extract cyperus esculentus oil about aqueous enzymatic method-freeze thawing coupling technique.
Summary of the invention
The present invention is directed to prior art and do not see that the present invention provides a kind of method that adopts aqueous enzymatic method-freeze thawing coupling technique to extract cyperus esculentus oil about adopting aqueous enzymatic method-freeze thawing coupling technique to extract the state of the art of cyperus esculentus oil.
Particularly, the invention provides a kind of method that adopts aqueous enzymatic method-freeze thawing coupling technique to extract cyperus esculentus oil, concrete extraction step is following:
(1) cleaning: choose the surperficial impurity of cyperus esculentus water flush away full, no disease and pest and dry surface-moisture;
(2) pulverize: pulverize evenly with miniature Universalpulverizer;
(3) enzyme that goes out: take by weighing 500kg cyperus esculentus powder, yellow soda ash and sodium hydrogencarbonate are by the mixed solution damping fluid (mol/L) of 1: 7 (m/v) solid-liquid ratio, and adjust pH is 7,50 ℃ and soaks 1h, 90 ℃ of enzyme 10min that go out;
(4) enzymolysis: adopt Sumizyme MP and cellulase according to 2: 1 (m/m) blended prozymes, solid-liquid ratio is 1: 6 (m/v), and enzymolysis time is 6h, and the enzyme addition is 2.5%, and hydrolysis temperature is to carry out enzymolysis under 70 ℃ of conditions;
(5) freezing and melt: enzymolysis places Ultralow Temperature Freezer in-30 ℃ of held 30min enzymolysis solution after accomplishing, and at room temperature melts afterwards;
(6) centrifugal: centrifugal 20min under the 4000r/min rotating speed, and carry out secondary centrifuging, separate crude oil.
Among the present invention, greasy physical and chemical index is measured and is adopted this area domestic method, and average oleaginousness adopts the Suo Shi extraction process; Peroxide value is pressed GB/T5538-2005 Vegetable oil lipoprotein check peroxide value assay method and is measured; Acid value is pressed GB/T553O-2005 animal-plant oil acid value and acid test; Iodine value is pressed GB/T5532-1995 vegetables oil iodine value and is measured; Saponification value is pressed GB/T5534-1985 saponification value of lipids assay method.
Through the concrete summary of the invention of embodiment of the present invention, can reach following effect:
Prior art is known, and the aqueous enzymatic method oil extracting process is to add water raw material is worn into slurry earlier, and then enzyme-added, water is as disperse phase, and enzyme is hydrolyzed at this aqueous phase, and oil is separated from solids.Utilize the affine difference of non-oil component (protein and glucide) to oil and water; And profit proportion is different and oil is separated with non-oil component; Compare simple to operate, the safety of aqueous enzymatic method, solvent-free pollution with traditional oil extracting process; Both shorten operational path, guaranteed the quality of oil and fat product again.The present invention adopts aqueous enzymatic method-freeze thawing coupling technique extraction cyperus esculentus oil not only to improve oil extracting rate, and kept oil quality preferably.
Description of drawings:
Fig. 1 is shown as the process flow sheet that employing aqueous enzymatic method-freeze thawing coupling technique extracts cyperus esculentus oil.
Fig. 2 is shown as the influence figure of solid-liquid ratio to the cyperus esculentus oil extraction yield.
Fig. 3 is shown as the influence figure of temperature to the cyperus esculentus oil extraction yield.
Fig. 4 is shown as the influence figure of enzymolysis time to the cyperus esculentus oil extraction yield.
Fig. 5 is shown as the influence figure of enzyme addition to the cyperus esculentus oil extraction yield.
Fig. 6 is shown as the peroxide value comparison diagram of the oil of three kinds of process for extracting.
Fig. 7 is shown as the iodine number comparison diagram of the oil of three kinds of process for extracting.
Fig. 8 is shown as the acid number comparison diagram of the oil of three kinds of process for extracting.
Embodiment
Below, lift embodiment the present invention is described, still, the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, % all refers to the m/m mass percent.
Equipment and material have among the present invention:
Cyperus esculentus: Fu Hui agricultural industrialization development limited-liability company provides (Kelamayi City) by Xinjiang; Acidic cellulase, Sumizyme MP, neutral protease: outstanding promise enzyme ltd; Chloroform-glacial acetic acid mixed solution (getting chloroform 40mL ice acetic acid 60mL); Saturated solution of potassium iodide: analytical pure; Sulfothiorine: analytical pure; Neutral ether: analytical pure; 1% phenolphthalein ethanolic soln; 0.01mol/L hypo solution: analytical pure; 0.1mol/L sodium hydroxid standardized solution: analytical pure; Deionized water (laboratory self-control); Used other reagent are analytical pure in the experiment.
KQ-C glassware airflow drying device (magnificent instrument plant desired to give in Gong Yi city English); HH-2 digital display thermostat water bath (Jintan City's Medical Instruments factory); XMT-DA digital display adjusting apparatus (Jin Yao city AsiaSat instrument ltd); PHS-3C laboratory PH meter (going up marine rainbow benefit instrument ltd); TDL-5-A Series Centrifugal machine (Anting Scientific Instrument Factory, Shanghai); FW-100 high speed Universalpulverizer (the bright forever Medical Instruments in Beijing factory); Rotatory evaporator RE-52AA (Shanghai Yarong Biochemical Instrument Plant); Gas chromatograph-mass spectrometer (VARIAN CP-3800/Saturn2000) (VARIAN Oncology Systems).
All reagent selected for use among the present invention and instrument all are well known selecting for use, but do not limit enforcement of the present invention, and other reagent more well known in the art and equipment are all applicable to the enforcement of the following embodiment of the present invention.
Embodiment one: the method for extracting cyperus esculentus oil
Referring to accompanying drawing 1, adopt aqueous enzymatic method-freeze thawing coupling technique to extract the method for cyperus esculentus oil, concrete extraction step is following:
(1) cleaning: choose the surperficial impurity of cyperus esculentus water flush away full, no disease and pest and dry surface-moisture.
(2) pulverize: pulverize evenly with miniature Universalpulverizer.
(3) enzyme that goes out: take by weighing 500kg cyperus esculentus powder, yellow soda ash and sodium hydrogencarbonate are by the mixed solution damping fluid (mol/L) of 1: 7 (m/v) solid-liquid ratio, and adjust pH is 7,50 ℃ and soaks 1h, 90 ℃ of enzyme 10min that go out;
(4) enzymolysis: adopt Sumizyme MP and cellulase prozyme, proteolytic enzyme and cellulase were by 2: 1 (m/m).Solid-liquid ratio is 1: 6 (m/v), and enzymolysis time is 6h, and the enzyme addition is 2.5%, and hydrolysis temperature is to carry out enzymolysis under 70 ℃ of conditions;
(5) freezing and melt: enzymolysis places Ultralow Temperature Freezer in-30 ℃ of held 30min enzymolysis solution after accomplishing, and at room temperature melts afterwards.
(6) centrifugal: centrifugal 20min under the 4000r/min rotating speed, and carry out secondary centrifuging, separate crude oil.
Embodiment two: the mensuration of average oleaginousness
Sample with the 3g left and right sides porphyrize of packing in the filter paper packet of having weighed; Seal bag mouthful, put into 105 ± 2 ℃ the dry 3h of baking oven, move to and put into the extracting tube with long forceps after being cooled to room temperature in the moisture eliminator; Inject 1.67 times anhydrous diethyl ether of a siphon amount, the sample bag is immersed in the ether fully.Connect the extractor each several part; Connect the water of condensation current, in water bath with thermostatic control, carry out extracting, regulate water temperature between 70～80 ℃; Make the ether of following of condensation become beaded (reflux 7 times/more than the h), extracting (approximately need 6～12h) till with filter paper drop inspection oil stains-less to extracting ether in the tube.After extracting finishes, take out filter paper packet, make ether volatilization (the extracting room temperature is advisable with 12～25 ℃) in the ventilation with long forceps.Ether in the extraction flask reclaims separately.Treat after the ether volatilization filter paper packet to be placed 105 ± 2 ℃ of dry 2h of baking oven, put into moisture eliminator and be cooled to till the constant weight.
Crude fat content W (%)=(b-c)/(b-a) * 100%
In the formula: a: weighing bottle adds filter paper packet heavy (g)
B: weighing bottle adds filter paper packet and oven dry appearance heavy (g)
C: solid residue weighed (g) after weighing bottle added filter paper packet and extracting
The oleaginousness that is recorded cyperus esculentus by traditional Suo Shi extraction process is 30.85%.
Embodiment three: the selection of enzyme and the mensuration of enzyme activity
1. the mensuration of enzyme activity
(1) mensuration of proteinase activity
1. reagent
0.1mol/L yellow soda ash---sodium bicarbonate buffer liquid (pH10);
5% Tricholroacetic Acid;
1% casein solution:
Taking by weighing 5 gram caseins and place 500ml 0.1mol/L yellow soda ash---sodium bicarbonate buffer liquid (pH10), heating makes its dissolving.
0.4mol/L sodium carbonate solution;
100 μ g/ml tyrosine solutions;
Folin reagent:
In 2000ml ground reflux, add sodium wolframate (Na2WO4.2H20) 100 grams, Sodium orthomolybdate (Na2MO2.2H20) 25 grams add 700 milliliters in water, 50 milliliters of 85% phosphoric acid, and 100 milliliters of concentrated hydrochloric acids, slow fire refluxed 10 hours.Take off reflux exchanger, add Lithium Sulphate 50 grams, several of 50 milliliters in water and dense bromine waters (99%) were boiled 15 minutes again; To remove unnecessary bromine (still having green need add bromine water again after cold), cooling adds water and is settled to 1000 milliliters, mixing; Filter, it is golden yellow that the reagent that makes should show, and stores in the brown bottle.
2. step
The extraction of a, enzyme liquid
Take by weighing 1 gram crude zyme preparation and place mortar, add less water and grind together, be settled to 100ml then, from volumetric flask, take out 1ml enzyme liquid and be settled to 100ml again, the enzyme liquid after the dilution obtains clarifying enzyme liquid behind filter paper filtering.
The mensuration of b, enzyme activity
Sample hose: the enzyme liquid (0.1～1.0ml) and use that 0.1mol/L yellow soda ash---sodium bicarbonate buffer liquid (pH10) is supplied volume to 1.0ml of getting different amounts; In each sample hose, add 1ml then more in advance in 1% casein solution of 40 ℃ of insulations; Mixing 40 ℃ of insulations 10 minutes, respectively adds the 2ml5% Tricholroacetic Acid immediately; Shake up rapidly, place half a hour in room temperature.
Blank pipe: in every test tube, add 1.0ml 1% casein solution and 2.0ml5% Tricholroacetic Acid earlier, after shaking up, add the enzyme liquid (enzyme liquid concentration is identical with sample hose respectively) of 1.0ml again,, below operate same sample hose 40 ℃ of insulations 10 minutes.With enzyme reaction mixture filtering and collecting filter liquid; In 1ml filtrating, add 5ml0.4mol/L sodium carbonate solution and 1ml Folin reagent; Shaking up the back and in 40 ℃ of waters bath with thermostatic control, developed the color 20 minutes, is contrast with blank tube reaction liquid, the absorbancy of reaction solution in the working sample pipe under 680nm.
3. the calculating of enzyme activity:
Enzyme activity (gram zymin units)=4/10 * K * OD * n
In the formula 4---the reaction mixture TV is 4ml
10---the enzyme reaction time is 10 minutes
K---the OD value is 1 o'clock suitable tyrosine micrograms in the typical curve
N---the extension rate of enzyme liquid
(2) mensuration of cellulase activity is pressed the measuring method mensuration of GB/T23881-2009 feeding cellulase vigor.
(3) enzyme of different enzymes is lived
By the enzyme activity determination experimental result, drawing standard curve, the regression equation of cellulase activity are Y=0.0088X-0.0109 (R
2=0.9962), good in 10～50mg/mL scope internal linear relation.At wavelength 540nm place, by experiment gained absorbance, the substitution formula records cellulosic enzyme activity.Regression equation Y=0.0086X-0.0073 (the R of proteolytic enzyme
2=0.9975), good in 10～60ug/mL scope internal linear relation.At wavelength 660nm place, by experiment gained absorbance, the substitution formula records the enzyme activity of two kinds of proteolytic enzyme.
Through enzyme activity determination, the enzyme activity of three kinds of different enzymes such as following table:
The enzyme of the different enzymes of table 1 is lived
| The enzyme title | Sumizyme MP | Neutral protease | Cellulase |
| Enzyme (U/g) alive | 12.9×10 4 | 29×10 4 | 15×10 4 |
2. the righttest single enzyme is confirmed
The selection of enzyme is 1: 7 with fixing solid-liquid ratio, and three kinds of enzymes are lived addition/substrate than identical time of effect down at the righttest separately pH, temperature and identical enzyme respectively, and relatively the oil extracting rate of three kinds of enzymes draws best enzyme.Under the constant prerequisite of best enzyme addition, it is compound to add other enzymes, draws best enzyme combination.
The enzymatic hydrolysis condition of the different enzymes of table 2 and oil yield
| Enzyme | React initial pH | Hydrolysis temperature (℃) | Enzymolysis time (h) | Oil yield (%) |
| |
60 | 4 | 25.48 | |
| Neutral protease | 7.5 | 60 | 4 | 50.59 |
| Sumizyme MP | 9.0 | 60 | 4 | 59.16 |
| Cellulase | 5.5 | 55 | 4 | 45.04 |
Can know that by table 2 without the control group of any enzyme effect, the cyperus esculentus oil yield is very low, has only 25.48%.Add different enzymes, the cyperus esculentus oil yield is significantly improved.This possibly be because: protein is hydrolyzed into peptide or ammonia under the proteolytic enzyme effect, thereby has caused in the cotyledon cell collapse of the tenuigenin structure relevant with protein and the destruction that surrounds greasy protein film, and discharges grease.The ability of three kinds of different enzyme raising cyperus esculentus oil yield is followed successively by: Sumizyme MP＞neutral protease＞cellulase; Wherein best with the Sumizyme MP action effect; The cyperus esculentus oil yield is 59.16%, and the cyperus esculentus oil yield that cellulase degradation obtains is minimum, is 45.04%.
3. prozyme is confirmed
Fixedly solid-liquid ratio is 1: 7, and add other enzymes at the righttest pH of best enzyme, temperature and under the constant prerequisite of Sumizyme MP addition compound, is index with the oil extracting rate, confirms optimum prozyme.
The different enzyme combinations of table 3 are to the influence of cyperus esculentus oil yield
Annotate: [1] adds cellulase again for adding Sumizyme MP earlier;
[2] add Sumizyme MP again for adding cellulase earlier
Can know that by table 3 Sumizyme MP and other enzyme recombination energy significantly improve the cyperus esculentus oil yield.But add Sumizyme MP earlier and add cellulase again; The cyperus esculentus oil yield slightly descends on the contrary, this possibly be because, the action pH slant acidity of cellulase; Under the study condition of this oil extracting process; Part cyperus esculentus protein molecule sex change sedimentation has been wrapped up a part of cyperus esculentus oil simultaneously, thereby has been reduced oily yield.Finally confirm the processing condition of selecting for use Sumizyme MP and cellulase to add simultaneously.
Prozyme is to be mixed by Sumizyme MP and cellulase, and market is seen various Sumizyme MPs usually and all is fit to technical scheme provided by the invention, the also suitable technical scheme provided by the invention of various cellulases.
Embodiment four: aqueous enzymatic method-freeze thawing coupling technique is carried oily experiment of single factor
(1) solid-liquid ratio is to the influence of cyperus esculentus oil extraction yield: at 60 ℃; The ph optimum scope of prozyme, the addition of enzyme are 2%, enzymolysis time 4h; Solid-liquid ratio is respectively under 1: 5,1: 6,1: 7,1: 8,1: 9 the condition, the influence of research different feed liquid comparison extraction yield.
The result is referring to accompanying drawing 2.Can be known that by accompanying drawing 2 along with the increase of water consumption, the yield of cyperus esculentus oil rose before this then and to descend, be to reach maximum at 1: 7 o'clock at solid-liquid ratio, is 61.82%.This be because: water consumption more after a little while, the flowability of solute is poor, enzyme-to-substrate contact is insufficient; Limited the carrying out of enzyme reaction, along with the increase of water consumption, the flowability of solute is also good more; Yet; The concentration of enzyme and substrate also descends thereupon, and reaction also can change slow gradually. but after solid-liquid ratio surpassed 1: 7, increasing degree was slowed down.For this reason, select for use solid-liquid ratio be 1: 7 for the Comprehensive Experiment upper limit.
(2) temperature was to the influence of cyperus esculentus oil extraction yield: solid-liquid ratio 1: 7; The ph optimum scope of prozyme, the addition of enzyme are 2%, enzymolysis time 4h; Temperature is respectively under 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ the condition, and the research differing temps is to the influence of extraction yield.
The result is referring to accompanying drawing 3.Can know that by accompanying drawing 3 along with the rising of temperature, cyperus esculentus oil must take the lead in raising and then descend.The highest when enzymolysis reaches 60 ℃, be 55.85%.After hydrolysis temperature surpassed 60 ℃, protein was prone to sex change takes place under the influence of long-time heat, and along with the exposure and the expansion of hydrophobic grouping, protein molecule is assembled, and solubleness descends, thereby causes the cyperus esculentus oil yield to descend.Thereby, confirm that hydrolysis temperature is 60 ℃.
(3) enzymolysis time is to the influence of cyperus esculentus oil extraction yield: at 60 ℃, and the ph optimum scope of prozyme, the addition of enzyme is 2%, solid-liquid ratio 1: 7, enzymolysis time are respectively under the condition of 2h, 4h, 6h, 8h, study the influence of different enzymolysis times to extraction yield.
The result is referring to accompanying drawing 4.Can be known that by accompanying drawing 4 yield of cyperus esculentus oil increases along with the prolongation of enzymolysis time, above behind the 4h, increase trend is mild; Enzymolysis time is long more, oil plant cell degradation abundant more, and the effect of enzyme is complete more; Consider production cost, so the selection enzymolysis time is 4h.
(4) the enzyme addition is to the influence of cyperus esculentus oil extraction yield: at 60 ℃; The ph optimum scope of prozyme, solid-liquid ratio 1: 7, enzymolysis time 4h; The enzyme addition is respectively under 1%, 1.5%, 2%, 2.5% the condition, studies the influence of different enzyme additions to extraction yield.
The result is referring to accompanying drawing 5.Can be known that by accompanying drawing 5 the cyperus esculentus oil extraction yield rises along with the increase of enzyme dosage, especially between 1.5～2.O%, ascensional range is bigger.When enzyme concentration surpassed 2%, cyperus esculentus oil extraction yield ascendant trend tended towards stability, and at this moment, substrate is saturated by enzyme institute, and therefore selecting enzyme concentration is 2% (w/w).
Embodiment five: aqueous enzymatic method-freeze thawing coupling technique is proposed oily orthogonal optimization experiment
With selected prozyme serves as to extract enzyme; Investigate the influence of single factor solid-liquid ratio, hydrolysis temperature, enzymolysis time and enzyme addition respectively to the cyperus esculentus oil extraction yield; To select suitable solid-liquid ratio, hydrolysis temperature, enzymolysis time and enzyme addition scope; Carry out 4 factors, 3 levels again and carry out the orthogonal optimization test,, select optimum extracting technology parameter to investigate the influence of composite factor to the cyperus esculentus oil extraction yield.
The factor of table 6 cyperus esculentus oil enzymolysis and design levels table
Carry out interpretation of result with orthogonal design assistant II v3.1.1 statistical software.
Table 7 Orthogonal experiment results table
The variance analysis of table 8 orthogonal experiment
Can know that by table 6,7,8 order that influences cyperus esculentus oil yield factor is:
Enzyme addition＞enzymolysis time＞solid-liquid ratio＞temperature.
Optimum combination A1B3C3D3, promptly solid-liquid ratio is 1: 6, and enzymolysis time is 6h, and the enzyme addition is 2.5%, and hydrolysis temperature is 70 ℃.Carry out confirmatory experiment with this understanding, obtain the extraction rate reached 75.24% that aqueous enzymatic method-freeze thawing coupling technique extracts cyperus esculentus oil.
Embodiment six: Different Extraction Method is to the influence of the extraction yield of cyperus esculentus oil
The present invention has compared the influence of solvent method, aqueous enzymatic method and aqueous enzymatic method-freeze thawing coupling technique to the rate of proposing of cyperus esculentus oil, result such as following table:
Table 9 different methods is to the influence of the extraction yield of cyperus esculentus oil
Embodiment seven: the physico-chemical property of cyperus esculentus oil relatively
For estimating the physico-chemical property that aqueous enzymatic method-freeze thawing coupling technique extracts cyperus esculentus oil, the cyperus esculentus oil that cyperus esculentus oil and solvent method and aqueous enzymatic method extracted that this method is extracted has carried out physical and chemical index mensuration respectively, measures result such as following table:
The comparison of table 10 cyperus esculentus oil physico-chemical property
Can know that by table 10 all minimum through cyperus esculentus oil acid number, saponification value and the peroxide value of the integrated processing gained of aqueous enzymatic method-freeze-thaw technology, but iodine number is the highest, the cyperus esculentus oil that acid number is low to show this method gained degree of becoming sour is lower, more helps the preservation of cyperus esculentus oil; The reason that saponification value is lower possibly be to contain due to the less free fatty acids in this method gained cyperus esculentus oil; Peroxide value is low, and then to have reacted the cyperus esculentus oil freshness of this method gained higher, has good edibleness; The iodine number height explains that then the degree of unsaturation of this method gained cyperus esculentus oil is higher.
Embodiment eight: Different Extraction Method gained cyperus esculentus oil GC-MS analyzes
The cyperus esculentus oil that the solvent method that employing is known, traditional water enzyme process, aqueous enzymatic method-freeze thawing coupling technique extract, GC-MS analytical results lipid acid composition is seen table 11.
Table 11 cyperus esculentus oil lipid acid moity table
Can know that by table 11 unsaturated fatty acids is isolated 4 peaks altogether in the cyperus esculentus oil, is respectively tetradecenoic acid, Zoomeric acid, oleic acid, linolic acid; The unsaturated fatty acids that identifies solvent method processing cyperus esculentus oil accounts for 70.09% of grease total amount, and wherein oleic acid content reaches 61.23%, is linolic acid 6.20% secondly; The unsaturated fatty acids that aqueous enzymatic method is handled cyperus esculentus oil accounts for 70.17% of grease total amount, and wherein oleic acid content reaches 62.29%, is linolic acid 6.67% secondly; And the unsaturated fatty acids of process aqueous enzymatic method-cyperus esculentus oil that the freeze thawing coupling technique is handled accounts for 75.61% of grease total amount; Wherein oleic acid content reaches 67.07%; Secondly be linolic acid 6.80%, variant with people's such as Yan Xiaoxin analytical results, reason is a used experimental raw cyperus esculentus place of production difference.Can know with the contrast of the cyperus esculentus oil unsaturated fatty acid content that aqueous enzymatic method-freeze thawing coupling technique different methods is handled through solvent method, traditional water enzyme process through table 10 pair, aqueous enzymatic method-freeze thawing coupling technique to lipid acid form influence not remarkable.But the total content of the grease unsaturated fatty acids after the freeze thawing treatment exceeds 5.52% than solvent method, exceeds 5.44% than traditional water enzyme process, and reason is the autoxidation that low temperature can be avoided unsaturated fatty acids preferably.It is thus clear that the cyperus esculentus oil researching value after the freeze thawing treatment is very high.
Embodiment nine: Different Extraction Method gained cyperus esculentus oil oil quality relatively
For estimating the physico-chemical property that aqueous enzymatic method-freeze thawing coupling technique extracts cyperus esculentus oil, the cyperus esculentus oil that cyperus esculentus oil and solvent method, aqueous enzymatic method extracted that this method is extracted has carried out quality parameter mensuration respectively, measures the result referring to accompanying drawing 6,7,8.Can know that by accompanying drawing 6,7,8 cyperus esculentus oil acid number, the peroxide value of handling gained through aqueous enzymatic method-freeze thawing coupling technique are all minimum, but iodine number is the highest, and along with time lengthening oil quality index changes less; Acid number is low to show that the cyperus esculentus oil free fatty acid content of this method gained is few, more helps the follow-up refining of cyperus esculentus oil; Peroxide value is low, and then to have reacted the cyperus esculentus oil freshness of this method gained higher, has good edibleness; The iodine number height explains that then the degree of unsaturation of this method gained cyperus esculentus oil is higher.Aqueous enzymatic method-freeze thawing coupling technique is described at the extraction yield that improves the aqueous enzymatic extraction cyperus esculentus oil simultaneously, oil quality is good, and promptly storage property is good.
Claims (1)
1. one kind is adopted aqueous enzymatic method-freeze thawing coupling to extract the method for cyperus esculentus oil, it is characterized in that described concrete extraction step is following:
(1) cleaning: choose the surperficial impurity of cyperus esculentus water flush away full, no disease and pest and dry surface-moisture;
(2) pulverize: pulverize evenly with miniature Universalpulverizer;
(3) enzyme that goes out: take by weighing 500kg cyperus esculentus powder, yellow soda ash and sodium hydrogencarbonate are by the mixed solution damping fluid (mol/L) of 1: 7 (m/v) solid-liquid ratio, and adjust pH is 7,50 ℃ and soaks 1h, 90 ℃ of enzyme 10min that go out;
(4) enzymolysis: adopt Sumizyme MP and cellulase prozyme, proteolytic enzyme and cellulase were by 2: 1 (m/m), and solid-liquid ratio is 1: 6 (m/v), and enzymolysis time is 6h, and the enzyme addition is 2.5%, and hydrolysis temperature is to carry out enzymolysis under 70 ℃ of conditions;
(5) freezing and melt: enzymolysis places Ultralow Temperature Freezer in-30 ℃ of held 30min enzymolysis solution after accomplishing, and at room temperature melts afterwards;
(6) centrifugal: centrifugal 20min under the 4000r/min rotating speed, and carry out secondary centrifuging, separate crude oil.
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