CN105038978A - Enzymatic degumming utilizing a mixture of pla and plc phospholipases - Google Patents

Abstract

A method for degumming an oil composition comprises the steps of (a) providing an oil composition containing a quantity of phospholipids, (b) contacting said oil composition simultaneously with one or more phospholipase A enzymes and one or more phospholipase C enzymes, under conditions sufficient for the enzymes to react with the phospholipids to create phospholipid reaction products, and (c) separating the phospholipids reaction products from the oil composition, the remaining oil composition after the separation being a degummed oil composition, whereby during step (b) the reaction of said one or more phospholipase A enzymes proceeds at a faster rate than it would in the absence of said one or more phospholipase C enzymes. In a preferred embodiment the reaction of step (b) continues for a duration of less than about one hour.

Description

Use the enzymatic degumming that PLA and PLC phospholipase cocktail carries out

The division that the application is the applying date is on 01 28th, 2008, application number is 200880009294.7, name is called the patent application of " using the enzymatic degumming that PLA and PLC phospholipase cocktail carries out ".

Technical field

The application relates to and from vegetables oil, to remove various phosphatide and Yelkin TTS (known be collectively referred to as " glue ") can be used for the degummed oil of foodstuffs production and/or non-food application or the enzyme method of fatty prod to manufacture.More specifically, the application relates to ferment treatment and removes the method for various phosphatide and Yelkin TTS, and the method can be applicable to raw oil or water degummed oil.In one embodiment, described enzyme reaction or treatment cycle can be less than about 1 hour.

Background technology

The crude vegetable oil obtained by squeezing or solvent-extraction process is the complex mixture of a kind of triglyceride level, phosphatide, sterol, tocopherol, free fatty acids, trace-metal and other minor compound.People wish to remove phosphatide, free fatty acids and trace-metal, thus produce that taste is light, the high-quality salad oil of lighter color and long quality-guarantee period.

Remove phosphatide can cause nearly allly refining loss together with vegetables oil.As shown in Figure 1, phosphatide is at one of two ends of its glycerol backbone with bound phosphate groups, and triglyceride level comprises at least one lipid acid.

This bound phosphate groups of phosphatide is " hydrophilic " or " happiness water ", and it represents that this functional group X is attracted by water.Fatty acid chain R1 and R2 of phosphatide is " lipophilic " or " happiness fat ", and it represents that they are attracted by lipid.Because phospholipid molecule has hydrophilic functional groups and lipophilic fatty acid chain simultaneously, therefore it is a kind of outstanding naturally occurring emulsifying agent.

The phosphoric acid functional group being expressed as the phosphatide of " X " in FIG determines its hydrophilicity.Functional group X in Fig. 1 can be any one in various known type, lists the sub-fraction in these types in table 2.

Phosphatide containing choline functional group and thanomin functional group has maximum affinity to glassware for drinking water, and has lower affinity containing the phosphatide of acid, acid-salt (calcium, magnesium and iron) and inositol functional group to glassware for drinking water.The salt of phosphatidic acid and phosphatidic acid is commonly called the phosphatide of hydration " can not " or NHP.Phosphatide measures in oil with " phosphorus content " of ppm usually.The Typical phospholipids amount existed in main oilseed crops is contained in table 1, and the percent profile in the phosphatide that exists in oil of various functional group.

Phosphatide forms

Table 1: the Typical phospholipids Content and distribution of common oilseeds.

Phosphatide is partially or completely removed from vegetables oil by multiple different any means known.Method the most frequently used in industry is that water comes unstuck, acid is come unstuck, alkali refining and enzymatic degumming.

Water comes unstuck

This technology is applied to usually can the raw oil of hydrated phospholipids containing high level.Due to the characteristic of its gentleness, the phosphatide of gained can be used as Yelkin TTS (a kind of naturally occurring emulsifying agent).The oil obtained from this technology is commonly called the industry " coming unstuck ", although just partly come unstuck.The oil come unstuck due to water still contains a large amount of phosphatide, particularly non-aqueous phosphatide, and other Technology (such as alkali refining or PLA1 enzyme come unstuck) may be needed to produce high quality oils that is final, that have high stability and low color.

In water degumming technology, at 60-75 DEG C, water (1 to 5%w/w) is added in raw oil, and vigorous stirring.Then oil is softly mixed 15 to 60 minutes, to help the hydration of the phosphatide existed in oil.Glue is expanded in the hydration of phosphatide or " glue " and aggegation is floss.This floss is emulsion or the mixture of hydrated gums and oil.This emulsion has the proportion higher than oil, and is separated by sedimentation, filtration or centrifugal industrial operation.Centrifugally produce two kinds of fluids, water degummed oil and wet glue.This water degumming technology is mainly only removed can the phosphatide of hydration.The residue phosphatide (50 to 250ppm) recorded as phosphotidats and/or PI can be removed in follow-up process operation.

The wet glue be separated is the oil in water emulsion mixtures of a kind of every two molecule phosphatide (or glue) containing at least one molecule triglyceride level (or oil).This oil in water emulsion can not physical sepn or recovery from emulsion, and is considered to a kind of process loss.This glue can be dried, and sell as food grade lecithin, but they are used to other lower application (such as animal-feed or commercial run) of economic worth usually used as by product.

The oily loss that emulsification causes is very large, and produces negative economic effects to the macroeconomy balance of treated oil process costs.

Acid is come unstuck

When target is overall removal phosphatide, this technology is applied to raw oil usually.The oil of gained is commonly referred to " unconventional glue " or " coming unstuck completely " in the industry.

At 60-90 DEG C, with the phosphoric acid of 250 to 2000ppm or citric acid treatment raw oil and vigorous stirring.Make the reactant salt 10 to 90 minutes of acid and NHP.Acid can improve the wetting ability of NHP, thus promotes their removal.Then, at 60-75 DEG C, water (1 to 5%w/w) to be added in acid-treated raw oil and vigorous stirring.Then oil is softly mixed 15 to 60 minutes, to help the hydration of phosphatide.Glue is expanded in the hydration of phosphatide or " glue " and aggegation is floss.This floss is emulsion or the mixture of hydrated gums and oil.The proportion of this emulsion higher than oil, and is separated by sedimentation, filtration or centrifugal industrial operation.Centrifugally obtain oil that acid comes unstuck and wet glue.This sour degumming technology can remove most phosphatide, but has still remained more phosphatide (25-100ppm) in this degummed oil, thus needs processing in addition.For food applications, this sour degummed oil will carry out bleaching and deodorizing usually, is called a kind of technique of " physical refining " in the industry.Thisly can not to use as food grade lecithin with acid-treated glue.

The same with water degumming technology, every two molecule phosphatide (or glue) triglyceride level containing at least one molecule (or oil) in the glue of the separation in this sour degumming technology and drying.The oil of this emulsification can not be physically separated or reclaim, and is considered to a kind of process loss, and its macroeconomy to treated oil process costs balance has negative economic effects.

Alkali refining

When target is for removing all phosphatide and free fatty acids, this technology is applied to rough or water degummed oil usually.

At 60-90 DEG C, with the phosphoric acid of 200 to 1000ppm or citric acid treatment is rough or water degummed oil, and vigorous stirring.Make the reactant salt 10 to 90 minutes of acid and NHP.Acid can improve the wetting ability of NHP, thus promotes their removal.In acid-treated oil, the sodium hydroxide solution (10-18%w/w) of dilution is added at 65-75 DEG C.The amount of sodium hydroxide (caustic alkali) is the amount based on the free fatty acids existed in oil, and on a dry weight basis excessive 0.05% to 0.20%.In this caustic solution and free fatty acids (generation soda soap), neutralize excessive acid, and help hydration and the emulsification of all residue phosphatide together with generated soda soap.

Mixed about 10 minutes of this sodium hydroxide/oil solution, then by sedimentation, filtration or be separated by industrial centrifugal.The oil of centrifugal acquisition caustic alkali process and soap stock.Then at 90-95 DEG C with the oil of softening water " washing " this caustic alkali process of 10 to 20%, and recentrifuge.The oil of centrifugal gained is called as " once by refining " (OnceRefined), and this water is commonly referred to " wash water " (WashWater).For food applications, bleaching and deodorizing usually should to be carried out to produce salad oil by " once by refining " oil.The alternative of washing is the oil with this caustic alkali process of absorptivity silica gel treatment, and the phosphatide that filter is walked the remaining soap in initial centrifugation and do not removed.

The same with sour degumming technology as water, every two molecule phosphatide (or glue) triglyceride level containing a part (or oil) in the glue of the separation in this alkali-refining process and drying.This oil in water emulsion can not be physically separated or reclaim, and is considered to a kind of process loss.In addition, this sodium hydroxide will react with neutral oil and generate soap, thus reduce overall oily productive rate further, and produce negative economic effects to the macroeconomy balance of treated oil process costs.

Ferment treatment

The another kind of refining techniques adopted in vegetables oil industry is " enzyme catalysis refining " or " enzymatic degumming ".When target is that when removing phosphatide completely, enzymatic degumming can be used.Generally speaking, enzymatic degumming process of the prior art has been used in by oil that the one (being generally water to come unstuck) in other method is come unstuck.For food applications, this enzymatic degumming oil carries out bleaching and deodorizing subsequently, is called a kind of technique of " physical refining " in the industry.Enzymatic degumming provides and to come unstuck better oily productive rate than water, acid or caustic alkali, and has better economic benefit.

This enzymic catalytic reaction changes the character of phosphatide, the cracking part of some phosphatide.Which reduce the emulsification property of phosphatide, the oil that when making separation gel from oil, loss is less, thus save oil.The activated enzyme of phosphatide tool is commonly called " Phospholipid hydrolase ".The position classification that Phospholipid hydrolase can react on phospholipid molecule according to this enzyme, and be called as PLA1, PLA2, PLC and PLD.The position display that phospholipid molecule reacts with dissimilar Phospholipid hydrolase is in Fig. 3.

As seen from Figure 3, different Phospholipid hydrolases can obtain different compounds when reacting from phosphatide.In addition, the Phospholipid hydrolase of each type has speed of reaction and the optimum reaction condition (pH, water % and temperature) of himself.PLA needs the reaction times at least about 4 hours usually when being used alone, and usually needs the reaction times of about 1 hour when PLC is used alone.Known enzyme catalytic treatment should be less than or equal to 8 times at pH and carry out, thus reduces undesirable oily saponification as far as possible, but PLA optimum response pH is 4.5, and the optimum response pH of PLC is 7.0.Often kind of enzyme also has different thermotolerances.PLA enzyme will sex change at about 50 DEG C, and PLC enzyme will sex change at about 65 DEG C.

There is the aminoacid sequence wide coverage in the literature of phospholipase activity, and be disclosed in patent, and it is reported that the part in them has activity to the phosphatide existed in vegetables oil.All these are known in the art.

A kind of business-like PLA1 enzyme product with phospholipase activity is the phospholipase A1 of Novozymes ultra.According to application table oil and the description in fat (ApplicationSheetOils & Fats) #2002-185255-01 and 2002-05894-03 of Novozymes, when this product can mix with the degummed oil with 1-1.5% water citric acid-NaOH being buffered to 4.5<pH<7.0 at 40 DEG C <T<55 DEG C, can polarization lysophospholipid and polarity lipid acid.This PLA1 selective hydrolysis glycerol backbone rolls into a ball relative lipid acid with phosphate functional, as shown in Figure 4.

The reaction formed obtains lysophospholipid and lipid acid.This lysophospholipid molecule lost a hydrophilic functional group, and is hydrophilic in the residue alcohol groups of reaction site.After having two hydrophilic sites, this lysophospholipid molecule is water-soluble, and loses its emulsification property.This PLA1 degumming technology by no longer removing any neutral oil with glue, and only loses original phospholipid molecule, thus decreases refining loss.

Although enzymatic degumming provides significant advantage relative to oil treater, it also has certain disadvantages.Shortcoming is that the reaction of enzyme and phosphatide may be slow, comparatively consuming time.Especially, the reaction of phospholipase A and phosphatide may need a few hours, and its time depends on response variable, as pH, temperature, relative concentration and mixing condition.The reaction times of this prolongation is worth the macroeconomy of enzymatic degumming technique and has serious negative impact.Due to PLA sluggish, enzymatic degumming carries out usually in the oil compositions come unstuck through water.So, oil must be come unstuck twice, is low to moderate the product that can realize its intended applications to obtain phosphorus concentration.

PLC enzyme known in the art is reacted with phosphatide by selective hydrolysis phosphate functional group, as shown in Figure 5.The reaction formed generates triglyceride (" DAG ") and phosphatidic acid group.This triglyceride molecule no longer has phosphate functional group, and does not need to be removed.This PLC degumming technology only removes phosphate functional group by retaining original phospholipid molecule, thus decreases refining loss.But PLC does not react with all phosphatide of existing in oil.Generally speaking, PLC does not react with phosphatidic acid (PA) or phospholipid inositol (PI), as shown in Figure 2.But PA and PI is the non-aqueous phosphatide be retained in after water comes unstuck in oil.Therefore the oil of PLC process must use caustic alkali process further, thus removes residual glue.

Some PLC known only with specific phosphatidic acid radical reaction.Such as, the known PI specificity PLC being accredited as PI-PLC.

Therefore, one aspect of the present invention provides the enzymatic degumming method of oil, and wherein said enzymic catalytic reaction speed is faster than enzymatic degumming technique of the prior art.

Another aspect provides the method for the speed of reaction improving phospholipase A used in enzymatic degumming technique.

Another aspect provides the method that oil compositions is come unstuck, wherein can process hydratability phosphatide and non-aqueous phosphatide in same process simultaneously.

Another aspect provides enzymatic degumming and treatment process, the time length of wherein said enzymic catalytic reaction is less than about 1 hour.

Following reference relates to the Study on enzymatic degumming of oil.

The United States Patent (USP) 5,264,367 of the people such as Aalrust describes the purposes of phospholipase A1, A2 or B process oil, this oil by refining first to 50-250ppm phosphorus.The technology described in this patent is commercially called as aalrust points out to act on Yelkin TTS due to these enzymes, " being used for method of the present invention having the oil (as crude soybean oil) of high density Yelkin TTS by nonsensical ".This reaction, at the temperature of 20-80 DEG C, is carried out in the citric acid of pH scope 3-7 and salt thereof.This patent points out that described enzyme should thoroughly be dispersed in oil, and the drop that its enzyme-aqueous solution is less than 10 μm as diameter exists.The measurements and calculations mode of weighed average is not disclosed.Emulsifying agent is used to dissolve the Phospholipid hydrolase obtained from Pancreatinum or pancreas product, and it comprises fat.Aalrust points out that the oil owing to reclaiming comprises the phosphorus being less than 5ppm, and it is applicable to being become edible oil by physical refining.Subsequently, the details of technology described in Aalrust is disclosed in multiple publication (Dahlke, K. and Eichelsbacher, M., and -Lurgi'sroutetoPhysicalRefininginProceedingoftheWorldConf erenceonOilseedandEdibleOilsProcessing, Istanbul, Turkey, 1996, ed.Kaseoglu, Rhee and Wilson; The people such as Dalke, K., FirstExperienceswithEnzymaticOilRefining, Inform, vol.6, No.12, Dec1995).The commerical test Data support disclosed in these publications purposes of this technology to oil, its P content is 40-180ppm, but not higher.According to another disclosure, " this technique is without any need for special equipment.All pumps, agitator, mixing tank, heat exchanger and whizzer are standard design, and can obtain from Duo Jia supplier." Dahlke, K. and Eichelsbacher, M., and -Lurgi'sroutetoPhysicalRefininginProceedingoftheWorldConf erenceonOilseedandEdibleOilsProcessing, Istanbul, Turkey, 1996, ed.Kaseoglu.RheeandWilson, the 56th page.

The United States Patent (USP) 5 of the people such as Yagi, 532,163 disclose the oil of every 100 parts by weight or the fatty enzymatic process using the water (water of a preferred 50-200 parts by weight) of at least 30 parts by weight to carry out reacting to carry out phospholipase A1, A2 or B and the oil containing 100-10,000ppm phosphorus.Described oil is washed subsequently with the water of the oil of every 100 parts by weight or fatty 30%-200% parts by weight or acidic aqueous solution.Total Water l oad scope required in this technique is 60% to the 400%w/w of handled oil.The generation of a large amount of elutriant makes this kind of method not too economical in the factory.

The United States Patent (USP) 6 of the people such as Loeffler, 001.640 discloses a kind of technique, wherein one or more vegetables oil with phosphorus containing components adopt the phospholipase cocktail process obtained by aspergillus (Aspergillus), this mixture comprise have A1 activity, A2 active, there are two kinds of active enzymes simultaneously, and there is the enzyme of lysophospholipase activity.This patent is pointed out because Phospholipid hydrolase can attack Yelkin TTS, uses the method to be unpractiaca to the oil (such as crude soybean oil) with high lecithin content.

The people such as Loeffler disclose and should be less than 4 times at pH and carry out enzymic catalytic reaction, and emulsion droplet is less than 20 μm.The mode of measurements and calculations emulsion droplet size weighed average is not disclosed.This patent points out that the product of gained is less than 15ppm or less residual P by having.When pH by oil known in the art is down to 4 or lower, will the glue existed in oil be hydrated, and be separated from reaction medium.The glue of this hydration will play the effect of emulsifying agent, thus will carry oil when they are separated, cause the loss of oil.Oily lossy data in glue is not provided.

The United States Patent (USP) 6,103,505 of the people such as Clausen discloses discovery and the activity of removing the specific Phospholipid hydrolase (A1, A2 or B) of phosphatide for enzyme catalysis, and produces the method for these enzymes.This enzymatic degumming technique have employed US5,264, the method described in 367, does not adopt any additional processing step.

The United States Patent (USP) 6 of the people such as Hasida; 127; 137 discovery and the activity disclosing some Phospholipid hydrolase, these enzymes 0.5-5% water, pH be 1.5-3, temperature mixes 1 to 12 hour with degummed oil (50-250ppm phosphorus) under being the condition of 30 – 45 DEG C can remove the aliphatic acyl groups that phospholipid molecule exists simultaneously.

The United States Patent (USP) 6,143.545 of the people such as Clausen discloses discovery and the activity of removing the specific Phospholipid hydrolase (A1, A2 or B) of phosphatide for enzyme catalysis, and produces the method for these enzymes.This enzymatic degumming technique have employed US5,264, the method described in 367, does not adopt any additional processing step.

The United States Patent (USP) 6,548,633 of the people such as Edwards discloses the sequence of the cDNA of encoding secreted proteins.On the 44th hurdle, this patent points out that the albumen of this invention can be used for above-cited United States Patent (USP) 6,001, the vegetable oil enzymatic degumming disclosed in 640.This patent points out that in same paragraph the albumen of this invention can combine other enzymes in " cocktail " further, thus improves the efficiency of feed utilization of animal.

The U.S. Patent Application No. 10/556 of the people such as Dayton, the 816 enzymatic degumming techniques disclosing a kind of improvement, the pH of the enzymic catalytic reaction wherein cushioned is reduced to less than 4.5 after enzymic catalytic reaction completes, thus removing the dirt of equipment (particularly heat exchanger and separation centrifuges), this dirt can be formed by the precipitation of calcium and magnesium salts under the Optimal pH needed for enzymic activity.

The U.S. Patent Publication 2004/0005399 of the people such as Chakrabarti discloses enzymatic process, it uses and adds separately enzyme and buffering system and shortly continue/the reaction times, then bleach with 2-4% Fuller's earth and 0-1% gac, dewaxing subsequently obtains the oil that phosphorus content is 5ppm.Bleaching process and dewaxing process all can remove residual phosphorus from oil.In addition, the people such as Chakrabarti point out that the oily loss that caused by glue is within the scope of the 30-40% of the glue be separated, and points out this enzymic catalytic reaction not carry out completely, cause due to the oily emulsification in removed phosphatide formed the oily loss of height extremely.

The U.S. Patent Publication 2005/0059130A1 of the people such as Bojsen discloses discovery and the activity of removing the specific Phospholipid hydrolase of phosphatide for enzyme catalysis, and produces the method for these enzymes.This announcement relates to United States Patent (USP) 5, and 264, what disclose in 367 passes through process vegetables oil to reduce phospholipids content.

The U.S. Patent Publication 2005/0108789A1 of the people such as Gramatikova (is now United States Patent (USP) 7,226,771) claim disclose can Phosphoric acid glycerol esters ester bond effectively in cracked oil (such as vegetables oil) can the phosphoric acid choline of water extraction and the Phospholipid hydrolase (such as, phospholipase A, B, C, D stem tuber specific proteases) of triglyceride to generate.At the 108th section, this application points out that these Phospholipid hydrolases can be used for the enzymatic degumming of vegetables oil further, and the PLC of this invention can be used for associating or replace Commercial Oil coming unstuck (such as technique, wherein phosphatide is hydrolyzed by PLA1 and PLA2) in PLA1 and PLA2.At the 474th section, this application is pointed out that PLC can be used alone or combines PLA and is used, to remove non-aqueous phosphatide from the oil once come unstuck by water, but the reaction conditions that this application does not provide above-mentioned two kinds of enzymes to share.Because PLA enzyme is different with the optimum reaction condition of PLC enzyme, do not provide operation embodiment in this application, how fail art in light technician uses PLA and PLC enzyme simultaneously.This application points out Phospholipase C further, D1 and D2 can be used for carrying out enzymatic degumming to the oil of once come unstuck and do not come unstuck (rough), and contribute to alkali refining.

Summary of the invention

The present invention relates to the Degumming method of oil compositions, the method comprises:

A () provides the oil compositions containing a certain amount of phosphatide,

(b) by described oil compositions simultaneously with one or more phospholipase As and one or more Phospholipase Cs being enough to contact under the condition making described enzyme and described phosphatide react, to generate phosphatide reaction product, and

C () is separated described phosphatide reaction product from described oil compositions, after being separated, remaining oil becomes degummed oil composition,

Wherein in step (b), the speed of reaction of one or more phospholipase As described is faster than the speed of reaction of this phospholipase A when lacking one or more Phospholipase Cs described.In preferred embodiments, the duration of the reaction of step (b) is less than about one hour.

The amount of the water needed for present invention process can be less than about 5%, advantageously can be reduced by least in about 3%, preferably about 1.5-2.0%.

The pH of described system can be regulated before or after adding a kind of or all enzymes to described oil compositions.Oily productive rate is maximized based on the composition of phosphatide contained in crude product.

Particularly, the present invention relates to Phospholipase C (PLC) and phospholipase A (PLA) are used from enzyme reaction to remove the method for the phosphatide existed in oil.More specifically, the present invention relates to combination and add Phospholipase C (PLC) and/or phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A1 (PLA1) and/or Phospholipase A2 (PLA2), with maximum oil productive rate, and reduce the amount of the waste product generated.Surprisingly, find when two kinds of enzymes use together, the situation when kinetics of enzyme reaction is far used alone faster than any one enzyme.In addition, even if when finding not to be optimized the reaction conditions of at least one enzyme, this reaction is still more rapid than expection.In addition, find that this reaction can be carried out within the time being less than one hour, and speed of response can reach about 30 minutes soon.

Advantageously, handled oil both can be raw oil, also can be water degummed oil.Described enzyme can be added into oil separately or together, but two kinds of enzymes will contact with oil simultaneously.According to the present invention, enzymic catalytic reaction parameter (such as water concentration, temperature, pH, churning time and enzyme concn) can be controlled, thus optimize the reaction of the certain enzyme combination in specific oil system.

Accompanying drawing explanation

Fig. 1 shows the chemical structure of general phosphatide and general triglyceride level.

Fig. 2 shows functional group and the structure of common phosphatide.

Fig. 3 shows the position of Phospholipid hydrolase reaction on phospholipid molecule with dissimilar.

Fig. 4 shows the reaction of phosphatide and PLA1 enzyme and the product of gained.

Fig. 5 shows the reaction of phosphatide and PLC enzyme and the product of gained.

Fig. 6 summarizes the result of embodiment 13-30, wherein each level of each evaluated empirical factor is depicted to the average residual phosphorus come unstuck in sample.

Fig. 7 summarizes the result of embodiment 31-38, wherein each level of each evaluated empirical factor is depicted to the average residual phosphorus come unstuck in sample.

Detailed Description Of The Invention

The present invention relates to the improvement to oil compositions enzymatic degumming technique.The present inventor finds, surprisingly, uses the combination of enzyme can improve the reaction kinetics of phosphatide cracking.Specifically, compared with the technique of alone phospholipase A, the enzymatic degumming technique adopting the combination of Phospholipase C and phospholipase A to carry out provides the lower degummed oil product of phosphorus content within the shorter reaction times.In addition, be surprisingly found out that this reaction can be carried out within the time being less than one hour, and speed of response can reach 30 minutes soon.This point is unexpected by it, because PLA needs the reaction times at least about 4 hours usually when being used alone, and usually needs the reaction times of about 1 hour when PLC is used alone.In addition, the optimum response pH of PLA is 4.5, and the optimum response pH of PLC is 7.0.Often kind of enzyme also has different thermotolerances.PLA enzyme will sex change at about 50 DEG C, and PLC enzyme will sex change at about 65 DEG C.In addition, the thermostability of enzyme known in the art is improved by rite-directed mutagenesis.The enzyme of this clone can be thermally-stabilised at up to the temperature of 80 DEG C, and the use of this clone's enzyme within the scope of the present invention.

The minimizing in reaction times obtains confirmation by PLA.When combinationally using with PLC, its reaction times is sharply shortened, and can be less than about 1 hour in some embodiments, or even for PLC and under the acid-reaction condition of non-optimal.The present inventor finds under suitable conditions further, likely will be reduced to 30 minutes low the reaction times.

Find that adjustable water concentration is to meet the demand of particular process environment.Therefore, when wanting the wastewater flow rate reducing this Process Production, water concentration can be reduced to about 1-2%, is reduced to about 1.5% especially.Alternatively, when wanting the efficiency improving this degumming technology, water concentration can be increased to about 4-5%, is increased to about 4.5% especially.

An advantage of the present invention is that oil to be come unstuck both can be raw oil, also can be once to carry out by one of existing method the oil that comes unstuck.Can realize degumming of oil in a single step to the particular advantages of oil treater.The oil that can carry out processing according to the present invention can include but not limited to: canola oil, Viscotrol C, Oleum Cocois, Fructus Coriandri oil, Semen Maydis oil, oleum gossypii seminis, hazelnut oil, hempseed oil, linseed oil, awns nut oil, white awns caul-fat, neat's foot oil, sweet oil, plam oil, palm-kernel oil, palm olein, peanut oil, rapeseed oil, Rice pollard oil, Thistle oil, camellia caul-fat, soybean oil, sunflower seed oil, Yatall MA, Chinese toon oil and vegetables oil.

Phospholipase A used in method of the present invention both can be phospholipase A1, also can be Phospholipase A2.Phospholipase C used in method of the present invention both can be Phospholipase C, also can be inositol specific phospholipase C.Many kinds of enzymes in phospholipase A and Phospholipase C family can business obtain; And expect that such enzyme and equivalent thereof will be suitable for using in the present invention.

In the method for the invention, the different phosphate lipase used together in enzymatic degumming technique of the present invention can be added into pending oily forward slip value together.Alternatively, they can in turn or be added into separately in oil simultaneously.No matter be add in turn or simultaneously, described enzymic catalytic reaction will carry out when two kinds of enzymes are present in reaction mixture simultaneously.

Degumming technology of the present invention can at pH lower than about 8, preferably between about 3-7, most preferably carry out under about 4-5.The pH of described enzyme degumming technology realizes by adding known damping fluid.Known citric acid and sodium hydroxide are suitable for this object.Other buffer reagent can use as required, to regulate pH under specific reaction conditions.

The temperature of enzymatic degumming technique of the present invention can in the scope of about 40-80 DEG C, preferably in the scope of about 40-60 DEG C, more preferably in the scope of about 45-55 DEG C.Be surprisingly found out that, in the method for the invention, PLA comes unstuck and in the optimum temps more than 45 DEG C of himself, and can carry out closer at the temperature of the optimum operating temperature of PLC, and can not excessively sex change.

The inventive process provides single step degumming technology, wherein the phospholipids content of oil (even raw oil) can be reduced to and be less than 50ppmP, is preferably less than 20ppmP, is more preferably less than 10ppmP, is most preferably less than 5ppmP.

After enzymatic degumming completes, the oil come unstuck is separated with glue, the requirement of the target terminal purposes as required or according to this degummed oil product, and this oil come unstuck can carry out other procedure of processing known in the art, such as bleaching or deodorizing.

Various preferred embodiment of the present invention is listed in embodiment hereafter, and what provide together also has the comparative examples adopting prior art.In each embodiment hereafter, separation mixing tank (overheadmixer) is the ElectorKG type Heidolph mixing tank with flat blade; Normal stirring adopts 90rpm, and vigorous stirring adopts 350rpm.Whizzer is installed the DeLavalGyro-Tester for " rotary drum unit (TheBowlUnit) " be continuously separated.This centrifuge drum is closed by the plug screw installed.Ultra-Turrax refiner SD-45 by having G450 rotor stator realizes shear-mixed under 10,000rpm.PLA1 enzyme is sold by the NovozymesA/S of Denmark ultra (lot number LYN050070), concentration is 11.2 units/mg.PLA2 enzyme is sold by the ABEnzymes of Germany mPL (lot number Ch:4738), concentration is 2000 units/mg.PLC enzyme is the Purifine of California DiversaCorporationofSanDiego Company ^tM.In embodiment 1-12, PLC's batch is BD16449, and concentration is 205 units/mg.In embodiment 13-38, PLC's batch is 90BU002A1, and concentration is 27.5 units/mg.In the oil of process, remain the amount of phosphatide by the official method Ca20-99 of ppmP with reference to American Oil Chemical Society, " by the analysis of inductively coupled plasma atomic emission to the phosphorus in oil " measures.

Embodiment

Embodiment 1

1965.4 grams of crude soybean oil containing 746ppm phosphorus are heated to 70-75 DEG C by contrast: water comes unstuck-uses and is separated mixing tank under normal stirring.39.4 grams of deionized waters are added, vigorous stirring 1 minute to this warm oil.Mixing tank is decelerated to usual dispatch (90rpm) to flocculate 30 minutes to allow glue.Then this oil centrifugal, collects the oil and wet glue that are separated.Residual phosphorus in water-degummed oil is 80.7ppm.

Embodiment 2

Contrast: coming unstuck with the single enzyme that phospholipase A1 (PLA1) carries out-use is separated mixing tank, and the crude soybean oil that 1997.9g contains 746ppm phosphorus is under agitation heated to 75-80 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, mixture is sheared 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40-45 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 40-45 DEG C, first adds the deionized water of 60.0 grams, then by mixture shear-mixed 1 minute, then add 0.1044 gram of Novozymes's ultraPLA1, by entire mixture shear-mixed 1 minute.At the temperature of 41-48 DEG C, with separation mixing tank, this oil mixt usual dispatch is stirred 4 hours.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.Residual phosphorus in PLA1-degummed oil is 31.7ppm.

Embodiment 3

2011.1 grams of crude soybean oil containing 746ppm phosphorus are heated to 55-60 DEG C by contrast: coming unstuck with the single enzyme that Phospholipase C (PLC) carries out-use is separated mixing tank under normal stirring.Add 60.3 grams of deionized waters, and by this mixture shear-mixed 1 minute.Add the Purifine of 0.1051 gram of Diversa ^tM(PLC lipase B D16449, containing 205U/mg), and mixture is sheared 1 minute.Stir normal at 50-63 DEG C for oil mixt 1 hour.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.Residual phosphorus in PLC degummed oil is 70.9ppm.

Embodiment 4

Contrast: use PLC-in this comparative examples after PLA comes unstuck, according to prior art, oil sample originally reacts separately with this kind of enzyme respectively under the optimum reaction condition of often kind of enzyme, and 2110.5 grams of crude soybean oil containing 560.1ppm phosphorus are heated to 60 DEG C under normal stirring.Add the Purifine of 63 grams of deionized waters and 0.1123 gram of Diversa ^tM(PLC lipase B D16449, containing 205U/mg), and mixture is sheared 1 minute.At 55-56 DEG C, oil mixt is stirred 1 hour with usual dispatch.Then this oil centrifugal, collects oil and wet glue.In order to form the damping fluid of pH4.5, first adding the citric acid solution of 2.0 grams of 50%w/w to this PLC-degummed oil, by solution shear 1 minute, then stirring one hour under usual dispatch with being separated mixing tank; Then add 4 molar sodium hydroxide solutions of 1.8 milliliters, and by this oil mixt shear-mixed 10 second, add 59 grams of deionized waters, by this mixture shear-mixed 1 minute.After forming damping fluid, add 0.1061 gram of Novozymes's ultraPLA1, then shears 1 minute by entire mixture.At the temperature of about 45 DEG C, oil is stirred 4 hours with usual dispatch.Then this oil centrifugal; Collect the oil and wet glue that are separated.In the oil that PLA1 order is come unstuck after first PLC, residual phosphorus is 3.2ppm.

Embodiment 5

Together with PLC with PLA1, neutral pH, 1 little the reaction time-2004.9 grams of crude soybean oil containing 560.1ppm phosphorus are heated to 45 DEG C under normal stirring at 45 DEG C.The Purifine of 60 grams of deionized waters, 0.1037 gram of Diversa is added in the oil of neutral pH ^tM(PLC enzyme) and 0.1076 gram of Novozymes's ultra (PLA1 enzyme), shears 1 minute by entire mixture.The mixture of oil and enzyme is stirred 1 hour with usual dispatch at the temperature of about 45 DEG C.Then this oil centrifugal, collects the oil and wet glue that are separated.The oil of this PLC and PLA1 combination enzyme mixture process reacts to generate for 1 hour and has the degummed oil that 13.2ppm remains phosphorus at neutral pH and 45 DEG C.

This residual phosphorus value is starkly lower than and is used alone PLA (embodiment 3) or be used alone the value of PLC (embodiment 3) gained under its top condition under its top condition.

Embodiment 6

Together with PLC with PLA1, neutral pH, 4 little the reaction times-2003.7 grams of crude soybean oil containing 560.1ppm phosphorus are heated to 45 DEG C under normal stirring at 45 DEG C.Add the Purifine of 60 grams of deionized waters, 0.1040 gram of Diversa ^tM(PLC enzyme) and 0.1085 gram of Novozymes's ultra (PLA1 enzyme), shears 1 minute by entire mixture.At the temperature of about 45 DEG C, oil mixt is stirred 4 hours with usual dispatch.Then this oil centrifugal, collects the oil and wet glue that are separated.The technique of this PLC and PLA1 combination enzyme mixture is reacted under neutral ph to generate for 4 hours and is had the degummed oil that 10.5ppm remains phosphorus.

Compared with embodiment 5, this residual phosphorus value was only slightly improved, and the display reaction times was increased to 4 hours and does not produce significantly impact to the effect of this degumming technology from 1 hour.

Embodiment 7

Together with PLC with PLA1, pH4.5,1 little the reaction time-2021.4 grams of crude soybean oil containing 547.9ppm phosphorus are heated to 75-80 DEG C under normal stirring with being separated mixing tank at 45 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.This oil mixt is stirred 1 hour under usual dispatch.By oil cooling to 40-45 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Add the Purifine of 61.0 grams of deionized waters, 0.1184 gram of Diversa ^tM(PLC enzyme) and 0.1038 gram of Novozymes's ultra (PLA1 enzyme), shears 1 minute by entire mixture.At the temperature of about 45 DEG C, oil mixt is stirred 1 hour with usual dispatch.Then this oil centrifugal, collects the oil and wet glue that are separated.The technique of this PLC and PLA1 combination enzyme mixture is reacted to generate for 1 hour and is had the degummed oil that 2.4ppm remains phosphorus at the temperature of pH4.5 and 45 DEG C.

This residual phosphorus value is roughly equivalent to, even a little less than the value (in embodiment 4, various enzyme reacts separately under the top condition of himself) of embodiment 4.Surprisingly, when two kinds of enzymes not to reaction times of PLA the best and not to the pH of PLC the best and temperature under react time come unstuck effect and two kinds of enzymes top condition respectively at himself under react time effect of coming unstuck suitable.

Embodiment 8

Together with PLC with PLA1, pH4.5,4 little the reaction times-2069.3 grams of crude soybean oil containing 547.9ppm phosphorus are heated to 75-80 DEG C under normal stirring at 45 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, this mixture is sheared 1 minute, then stir 1 hour under usual dispatch.Mixture is cooled to 40-45 DEG C, then adds 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Add the Purifine of 63 grams of deionized waters, 0.1112 gram of Diversa ^tM(PLC enzyme) and 0.1258 gram of Novozymes's ultra (PLA1 enzyme), shears 1 minute by entire mixture.At the temperature of about 45 DEG C, oil mixt is stirred 4 hours with usual dispatch.Then this oil mixt centrifugal, collects the oil and wet glue that are separated.The technique of this PLC and PLA1 combination enzyme mixture is reacted to generate for 4 hours and is had the degummed oil that 2.5ppm remains phosphorus under pH4.5.

This residual phosphorus value was roughly equal to the value of embodiment 7, and the display reaction times was increased to 4 hours and does not produce significantly impact to the effect of this degumming technology from 1 hour.

Embodiment 9

Together with PLC with PLA1, pH4.5,1 little the reaction time-1985.2 grams of crude soybean oil containing 547.9ppm phosphorus are heated to 75-80 DEG C under normal stirring at 55 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, this mixture is sheared 1 minute, then stir 1 hour under usual dispatch.Mixture is cooled to 40-45 DEG C, then adds 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Add the Purifine of 63.0 grams of deionized waters, 0.1085 gram of Diversa ^tM(PLC enzyme) and 0.1045 gram of Novozymes's ultra (PLA1 enzyme), shears 1 minute by entire mixture.At the temperature of 55 DEG C, oil mixt is stirred 1 hour with usual dispatch.Then this oil centrifugal; Collect the oil and wet glue that are separated.The technique of this PLC and PLA1 combination enzyme mixture is reacted to generate for 1 hour and is had the degummed oil that 2.3ppm remains phosphorus at the temperature of pH4.5 and 55 DEG C.

This residual phosphorus value is roughly equal to the value of embodiment 7 and 8, shows temperature of reaction is about increased to about 55 DEG C from 45 DEG C does not make the effect of degumming technology form obvious difference, although can sex change under being generally expected to the temperature of PLA1 more than 50 DEG C.

Embodiment 10

PLC together with 2 times of PLA1 concentration, pH4.5,1 little the reaction time-1992.2 grams of crude soybean oil containing 547.9ppm phosphorus are heated to 75-80 DEG C under normal stirring at 45 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, this mixture is sheared 1 minute, then stir 1 hour.Mixture is cooled to 40-45 DEG C, then adds 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.The Purifine of 60 grams of deionized waters, 0.1319 gram of Diversa is added to this mixture ^tM(PLC enzyme) and 0.2139 gram of Novozymes's ultra (PLA1 enzyme), shears 1 minute by entire mixture.At the temperature of about 45 DEG C, oil mixt is stirred 1 hour with usual dispatch.Then this oil mixt centrifugal; Collect the oil and wet glue that are separated.The technique of this PLC and double strength PLA1 combination enzyme mixture is reacted to generate for 1 hour and is had the degummed oil that 7.0ppm remains phosphorus under pH4.5.

This residual phosphorus value can accept in some applications, but so good not as the value of embodiment 7-9, even if display is under the reaction conditions of PLA1 the best astoundingly, the dosage improving PLA1 does not cause the raising of degumming technology effect.

Embodiment 11

Together with PLC with PLA2, pH4.5,1 little the reaction time-1998.4 grams of crude soybean oil containing 341.2ppm phosphorus are heated to 75-80 DEG C under normal stirring with being separated mixing tank at 45 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 40-45 DEG C, adds the Purifine of 0.1112 gram of Diversa ^tM(PLC lipase B D16449, containing 205U/mg) and 0.2094 gram of ABEnzymes sale mPL (lot number Ch:4738), then adds 60 grams of deionized waters, and by entire mixture shear-mixed 60 seconds.At the temperature of about 40-45 DEG C, oil mixt is stirred 60 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA2 combines the degummed oil that enzyme mixture generates the residual phosphorus with 3.3ppm under neutral ph.

The present embodiment is similar to embodiment 7 above, just substituted for PLA1 with PLA2.Low residue phosphorus level display PLA2 in end product can result from the effect equally good with PLA1 in the method for the invention.

Embodiment 12

Together with PLC with PLA2, pH4.5,4 little the reaction times-1998.4 grams of crude soybean oil containing 341.2ppm phosphorus are heated to 75-80 DEG C under normal stirring with being separated mixing tank at 45 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid of pH4.5.Temperature is maintained 40-45 DEG C.Add the Purifine of 0.1038 gram of Diversa ^tM(PLC lipase B D16449, containing 205U/mg) and 0.2047 gram of ABEnzymes sale mPL (lot number Ch:4738), then adds 60 grams of deionized waters, and by entire mixture shear-mixed 60 seconds.At the temperature of about 40-45 DEG C, oil mixt is stirred 4 hours with usual dispatch.Then the oil of this ferment treatment centrifugal; Collect the oil and wet glue that are separated.PLC and PLA2 combines the degummed oil that enzyme mixture generates the residual phosphorus with 5.8ppm under neutral ph.

Reaction times was increased to 4 hours by 1 hour and does not cause better coming unstuck by the present embodiment display, in fact caused the higher level of residual phosphorus.

Previous embodiment 1-12 the results are summarized in following table 2.

Table 2

Embodiment 13-30

Experimental design (DOE) is carried out, to determine the effect of specific process control variable to this enzymatic degumming technique shown according to the form below 3.

Table 3

-often kind enzyme adds separately in turn, although two kinds of enzymes all contact with oil mixt within least part of reaction times.

-two kinds of enzymes add at same time simultaneously.

PH-enzyme is exposed to the pH of oil.

Temperature-enzyme is exposed to the temperature of oil.

The time of high-speed stirring mixture after churning time-often kind enzyme adds.

Reaction times-at least one enzyme and total time of contacting of oil.

These operational variables are assessed in 18 independent tests, show in embodiment 13-30 herein.The value of each variable tested in embodiment 13-30 is recited in table 4 hereafter.

Table 4

Embodiment 13

Use separation mixing tank that 1999.1 grams of crude soybean oil containing 769.5ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Separation mixing tank is used normally to stir oil 1 hour.Under usual dispatch stirs, cool this oil, until oil temperature is 40 DEG C, then interpolation 2.4 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 5.0.Temperature is maintained 40 DEG C, add the Purifine of 1.5008 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 30 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.This oil mixt is stirred 60 minutes under usual dispatch.Temperature is maintained 40 DEG C, add 0.2132 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 120 seconds.At the temperature of 40 DEG C, oil mixt is stirred 60 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.After first PLC, the residual phosphorus of PLA1 in turn in degummed oil is 6.5ppm.

Embodiment 14

Use separation mixing tank that 2010.5 grams of crude soybean oil containing 785.1ppm phosphorus are cooled to 60 DEG C under normal stirring.Temperature is maintained 60 DEG C, add the Purifine of 1.5316 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.2073 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 30 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.At the temperature of 60 DEG C, oil mixt is stirred 60 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 109.6ppm under neutral ph.

Embodiment 15

Use separation mixing tank that 1994.5 grams of crude soybean oil containing 785.1ppm phosphorus are cooled to 40 DEG C under normal stirring.Temperature is maintained 40 DEG C, add the Purifine of 0.754 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 60 seconds.This oil mixt is stirred 15 minutes under usual dispatch.Temperature is remained on 40 DEG C.Add 0.2242 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 60 seconds.At the temperature of 40 DEG C, oil mixt is stirred 15 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.After first PLC, the residual phosphorus of PLA1 in turn in degummed oil is 27.4ppm.

Embodiment 16

Use separation mixing tank that 2002.0 grams of crude soybean oil containing 785.1ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under usual dispatch stirs, cool this oil, until oil temperature is 50 DEG C, then interpolation 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 50 DEG C, add the Purifine of 0.7498 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 30 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.This oil mixt is stirred 30 minutes under usual dispatch.Temperature is maintained 50 DEG C, add 0.4064 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 120 seconds.At the temperature of 50 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.After first PLC, the residual phosphorus of PLA1 in turn in degummed oil is 7.6ppm.

Embodiment 17

Use separation mixing tank that 2010.7 grams of crude soybean oil containing 785.1ppm phosphorus are heated to 50 DEG C under normal stirring.Temperature is maintained 50 DEG C, add the Purifine of 1.4981 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 60 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.This oil mixt is stirred 15 minutes under usual dispatch.Temperature is maintained 50 DEG C, add 0.4143 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 45 seconds.At the temperature of 50 DEG C, oil mixt is stirred 15 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.After first PLC, the residual phosphorus of PLA1 in turn in degummed oil is 79.3ppm.

Embodiment 18

Use separation mixing tank that 2005.3 grams of crude soybean oil containing 742.9ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 60 DEG C, then add 2.4 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 5.0.Temperature is maintained 60 DEG C, add the Purifine of 0.7491 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 60 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.This oil mixt is stirred 60 minutes under usual dispatch.Temperature is maintained 60 DEG C, add 0.1220 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 45 seconds.At the temperature of 60 DEG C, oil mixt is stirred 60 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.After first PLC, the residual phosphorus of PLA1 in turn in degummed oil is 2.2ppm.

Embodiment 19

Use separation mixing tank that 2000.4 grams of crude soybean oil containing 742.9ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 60 DEG C, then add 2.4 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 5.0.Temperature is maintained 60 DEG C, add the Purifine of 2.2270 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.3937 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 30 grams of deionized waters, and by entire mixture shear-mixed 60 seconds.At the temperature of 60 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 7.8ppm under pH5.0.

Embodiment 20

Use separation mixing tank that 2006.3 grams of crude soybean oil containing 719.3ppm phosphorus are heated to 60 DEG C under normal stirring.Temperature is maintained 60 DEG C, add the Purifine of 0.7561 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.4098 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.At the temperature of 60 DEG C, oil mixt is stirred 120 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 64.1ppm under neutral ph.

Embodiment 21

Use separation mixing tank that 1998.5 grams of crude soybean oil containing 719.3ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 40 DEG C, add the Purifine of 1.4798 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.4018 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 60 grams of deionized waters, and by entire mixture shear-mixed 60 seconds.At the temperature of 40 DEG C, oil mixt is stirred 120 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 5.5ppm under pH4.5.

Embodiment 22

Use separation mixing tank that 2001.3 grams of crude soybean oil containing 719.3ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 2.4 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 5.0.Temperature is maintained 40 DEG C, add the Purifine of 2.2580 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.This oil mixt is stirred 30 minutes under usual dispatch.Temperature is maintained 40 DEG C, add 0.4126 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 45 seconds.At the temperature of 40 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.There is in the degummed oil of PLC and PLA1 processed in sequence the residual phosphorus of 2.1ppm.

Embodiment 23

Use separation mixing tank that 2002.0 grams of crude soybean oil containing 747.3ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 60 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 60 DEG C, add the Purifine of 2.2194 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 60 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.This oil mixt is stirred 15 minutes under usual dispatch.Temperature is maintained 60 DEG C, add 0.2198 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 120 seconds.At the temperature of 60 DEG C, oil mixt is stirred 15 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.There is in the degummed oil of PLC and PLA1 processed in sequence the residual phosphorus of 4.6ppm.

Embodiment 24

Use separation mixing tank that 2000.8 grams of crude soybean oil containing 747.3ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 50 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 50 DEG C, add the Purifine of 2.2500 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.2216 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.At the temperature of 50 DEG C, oil mixt is stirred 120 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal; Collect the oil and wet glue that are separated.PLC and PLA1 combines enzyme mixture and generates the degummed oil with the residual phosphorus of 1.8ppm.

Embodiment 25

1998.9 grams of crude soybean oil containing 747.3ppm phosphorus are separated under mixing tank normally stirs in use and are heated to 75-80 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 50 DEG C, then add 2.4 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 5.0.Temperature is maintained 50 DEG C, add the Purifine of 0.7445 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.2042 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 60 grams of deionized waters, and by entire mixture shear-mixed 60 seconds.At the temperature of 50 DEG C, oil mixt is stirred 60 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal; Collect the oil and wet glue that are separated.PLC and PLA1 combines enzyme mixture and generates the degummed oil with the residual phosphorus of 7.2ppm.

Embodiment 26

1997.3 grams of crude soybean oil containing 810.8ppm phosphorus are separated under mixing tank normally stirs in use and are heated to 75-80 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 60 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 60 DEG C, add the Purifine of 1.5189 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 60 seconds.This oil mixt is stirred 30 minutes under usual dispatch.Temperature is maintained 60 DEG C, add 0.1119 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 60 seconds.At the temperature of 60 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.There is in the degummed oil of PLC and PLA1 processed in sequence the residual phosphorus of 2.2ppm.

Embodiment 27

Use separation mixing tank that 2010.0 grams of crude soybean oil containing 810.8ppm phosphorus are cooled to 50 DEG C under normal stirring.Temperature is maintained 50 DEG C, add the Purifine of 2.2608 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1), then adds 30 grams of deionized waters, and by entire mixture shear-mixed 60 seconds.This oil mixt is stirred 60 minutes under usual dispatch.Temperature is maintained 50 DEG C, add 0.1172 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), and by entire mixture shear-mixed 60 seconds.At the temperature of 50 DEG C, oil mixt is stirred 60 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.There is in the degummed oil that PLC and PLA1 processes in turn under neutral ph the residual phosphorus of 72.6ppm.

Embodiment 28

Use separation mixing tank that 2005.1 grams of crude soybean oil containing 810.8ppm phosphorus are heated to 40 DEG C under normal stirring.Temperature is maintained 40 DEG C, add the Purifine of 2.2622 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1031 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 60 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.At the temperature of 40 DEG C, oil mixt is stirred 60 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 61.5ppm under neutral ph.

Embodiment 29

Use separation mixing tank that 2006.3 grams of crude soybean oil containing 795.3ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 50 DEG C, then add 2.4 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 5.0.Temperature is maintained 50 DEG C, add the Purifine of 1.5373 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1168 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.At the temperature of 50 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 1.9ppm under pH5.0.

Embodiment 30

Be separated mixing tank and 2006.1 grams of crude soybean oil containing 795.3ppm phosphorus are heated to 75-80 DEG C under the normal stirring of use.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 40 DEG C, add the Purifine of 0.7736 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1072 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 30 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.At the temperature of 40 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 13.7ppm under pH4.5.

The phosphorus content of embodiment 13-30 the results are shown in table 5 hereafter.

Table 5

Be shown in Fig. 6 to the summary of above-mentioned experiment, this figure is the figure of the final average phosphorus content under each level of each factor.

Under the neutral pH to PLC enzyme the best, the combination of two kinds of enzymes fails to generate the oil having and allow oil to carry out the accepted phosphorus value of physical refining.But under the acid pH to PLA enzyme the best, the enzyme added in turn or is simultaneously combined in oil to generate and allows it by the acceptable residual phosphorus level of physical refining.When adopting acid pH, only an experiment is failed to generate to have and is less than the oil that 10ppm remains phosphorus.Embodiment 30 has the residual phosphorus level being greater than 10 (13.7ppm), and is generate under with the minimum level of two kinds of enzymes, minimum temperature, minimum water content, the shortest mixing and churning time and the most acid pH.

Between enzyme combination, find synergistic effect, allow reaction to complete being less than in 30 minutes, and PLC enzyme require 1 hour, PLA enzyme require 4 hours.Complete additional measurement to verify the impact of short reaction time; The results are shown in following table 6.In these additional measurements, consider and find that the comparable neutral pH of lower pH generates obviously more favourable result hereinbefore, pH is maintained at 4.5.The amount of PLA1 is maintained at constant 0.5ppm equally, because from showing that PLA amount does not cause more effective coming unstuck higher than during this level above.In addition, consider that enzyme is in these embodiments and adds simultaneously, but not adds in turn by showing that adding enzyme generates better degumming effect than adding enzyme in turn above simultaneously.In addition, in industrial technology, it is favourable for limiting total treatment time, total equipment and specific investment.

Table 6

Embodiment 31

Use separation mixing tank that 2003.6 grams of crude soybean oil containing 784.8ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 40 DEG C, add the Purifine of 1.4603 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1021 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 40 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.At the temperature of 40 DEG C, oil mixt is stirred 120 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal; Collect the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 10.7ppm under pH4.5.

Embodiment 32

Use separation mixing tank that 2004.8 grams of crude soybean oil containing 784.8ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 60 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 60 DEG C, add the Purifine of 0.7509 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1105 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.At the temperature of 60 DEG C, oil mixt is stirred 120 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 6.7ppm under pH4.5.

Embodiment 33

2000.4 grams of crude soybean oil containing 697.7ppm phosphorus are separated under mixing tank normally stirs in use and are heated to 75-80 DEG C.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 40 DEG C, add the Purifine of 0.7530 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1022 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.At the temperature of 40 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 2.2ppm under pH4.5.

Embodiment 34

Use separation mixing tank that 1999.4 grams of crude soybean oil containing 714.2ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 60 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 60 DEG C, add the Purifine of 1.508 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1139 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 40 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.At the temperature of 60 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 16.5ppm under pH4.5.

Embodiment 35

Use separation mixing tank that 1999 grams of crude soybean oil containing 714.2ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 40 DEG C, add the Purifine of 1.5010 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1060 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.At the temperature of 40 DEG C, oil mixt is stirred 120 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 1.9ppm under pH4.5.

Embodiment 36

Use separation mixing tank that 1999 grams of crude soybean oil containing 695.1ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 60 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 60 DEG C, add the Purifine of 1.5296 grams of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1241 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 90 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.At the temperature of 60 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 5.2ppm under pH4.5.

Embodiment 37

Use separation mixing tank that 2005.2 grams of crude soybean oil containing 695.1ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 40 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 40 DEG C, add the Purifine of 0.7422 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1195 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 40 grams of deionized waters, and by entire mixture shear-mixed 45 seconds.At the temperature of 40 DEG C, oil mixt is stirred 30 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 6.7ppm under pH4.5.

Embodiment 38

Use separation mixing tank that 1998 grams of crude soybean oil containing 695.1ppm phosphorus are heated to 75-80 DEG C under normal stirring.Add the 50%w/w citric acid solution of 2.0 grams, shear 1 minute.Use the normal stirring being separated mixing tank and oil being carried out 1 hour.Under the stirring of usual dispatch, cool this oil, until oil temperature is 60 DEG C, then add 1.8 milliliter of 4 molar sodium hydroxide solution, by mixture shear-mixed 10 seconds.Citric acid and caustic alkali form the weak damping fluid that pH is 4.5.Temperature is maintained 60 DEG C, add the Purifine of 0.7429 gram of Diversa ^tM(PLC lipase, lot number 90BU002A1) and 0.1041 gram of Novozymes's ultra (PLA1 lipase, lot number LYN05007), then adds 40 grams of deionized waters, and by entire mixture shear-mixed 120 seconds.At the temperature of 60 DEG C, oil mixt is stirred 120 minutes with usual dispatch.Then the oil of this ferment treatment centrifugal, collects the oil and wet glue that are separated.PLC and PLA1 combines the degummed oil that enzyme mixture generates the residual phosphorus with 4.4ppm under pH4.5.

Table 7

Fig. 7 sums up embodiment 31-38, maps to the average final phosphorus content of often planting under each level of factor when maintaining the interpolation constant of pH, PLA dosage and combination.

Five factors assessed in embodiment 31-38 are set forth in hereinafter by its effect size order to degumming technology.The described difference (absolute value) acting as average phosphorus content between high and low factor is arranged.

The water-content risen reduces residual phosphorus.

The PLC dosage reduced reduces residual phosphorus.

The temperature of reaction declined reduces residual phosphorus.

The stirring increased reduces residual phosphorus.

The reaction times increased reduces residual phosphorus.

Disclose and used phospholipase A and Phospholipase C to carry out the novel process of degumming of oil simultaneously.Be surprisingly found out that, this combination is more more effective than being used alone any one enzyme, even if wherein react under the reaction conditions of one or another kind of enzyme require beyond this kind of enzyme top condition.In addition, be surprisingly found out that, the level of coming unstuck that under correct condition, by being low to moderate the reaction times of 30 minutes, can obtain being less than about 10ppm phosphorus in end product, being low to moderate about 5ppm phosphorus, being even low to moderate 3ppm phosphorus.In addition, do not wish to be limited to theory, seem the reaction of one of PLC enzyme or its reaction product catalysis PLA enzyme, thus allow the reaction times to be obviously less than the reaction times of any one enzyme.From the known optimum response parameter for PLA and PLC enzyme, these results are beat all.

Those skilled in the art can be recognized by foregoing disclosure, the target as the case may be, can change and implement operating parameters of the present invention, but still within the scope of the present invention.Such as, for determining the concentration of PLA and PLC enzyme in specific reaction batch, can be that minimum possible cost or the highest possible performance are selected according to the target of this batch.If the target of this batch is minimum possible cost, then the concentration of PLA can lower than about 2.0ppm, preferably lower than about 1.0ppm, most preferably lower than about 0.5ppm.So low PLA enzyme concn still can provide in many cases effectively comes unstuck.Contrary, if need maximized performance, then the concentration of PLA is preferably at least about 0.5ppm, more preferably at least about 1.0ppm, and most preferably 2.0ppm.Technician in oil treatment field will understand the enzyme concn how changed in this reaction mixture, to obtain the balance between required cost benefit and product performance.

Also other reaction conditions can be changed.PH is about 7.0, but preferred about 5.0, current preferred about 4.5.In system, the concentration of water is about 3.0% usually, but can be low to moderate about 1.5% when needs reduce waste water, or need higher come unstuck efficiency time up to about 4.5%.Temperature of reaction up to about 60 DEG C, but can be more preferably less than about 50 DEG C, most surprisingly preferably is about 40 DEG C.Churning time when initial mixing is about 45 seconds, more preferably from about 60 seconds, most preferably from about 120 seconds.Finally, the time of enzyme reaction can be greatly reduced, and advantageously can be less than about 60 minutes in certain embodiments, preferably about 30 minutes.

Although list the preferred embodiments of the invention herein, other embodiment comprising the inventive method is apparent to those skilled in the art, and all these embodiments and equivalent way thereof are all contained by by the application, and be included in the claim of the application.

Claims (19)

1. the soybean oil composition method of coming unstuck, comprising:

A () provides the soybean oil composition containing a certain amount of phosphatide,

B described soybean oil composition is being enough to contact under the condition that described enzyme and described phosphatide are reacted with about 2ppm organized enzyme or one or more phospholipase As of less amount and one or more Phospholipase Cs of about 30ppm organized enzyme or less amount by () simultaneously, to generate phosphatide reaction product, and

C () is separated described phosphatide reaction product from oil compositions, after being separated, remaining oil compositions becomes degummed soybean oil compositions,

Wherein in step (b), the speed of reaction of one or more phospholipase As described is faster than the speed of reaction of this phospholipase A when lacking one or more Phospholipase Cs described, the reaction times of wherein said enzyme and described phosphatide is less than one hour, wherein said enzyme and phosphatide react under pH is about 3-7 and carry out at the temperature of about 40-80 DEG C, and the phospholipids content of the oil compositions of degummed soybean described in step (c) is measured by millionth phosphorus and is about 20ppm or less.

2. the method for claim 1, the reaction times of wherein said enzyme and described phosphatide is about 30 minutes.

3. the method for claim 1, one or more phospholipase As wherein said are selected from phospholipase A1 and Phospholipase A2.

4. the method for claim 1, one or more Phospholipase Cs wherein said are selected from Phospholipase C and phosphatidyl-inositol specific phospholipase C.

5. the method for claim 1, the described reaction of wherein said enzyme and described phosphatide is carried out at about 40-60 DEG C temperature.

6. the method for claim 1, wherein said soybean oil composition comprises crude soybean oil.

7. the method for claim 1, wherein said soybean oil composition comprises the soybean oil of once carrying out coming unstuck.

8. the method for claim 1, wherein said PLC enzyme exists with about 20ppm organized enzyme or less amount.

9. method as claimed in claim 8, wherein said PLC enzyme exists with about 10ppm organized enzyme or less amount.

10. the method for claim 1, wherein said PLA enzyme exists with about 1ppm organized enzyme or less amount.

11. methods as claimed in claim 10, wherein said PLA enzyme exists with about 0.5ppm organized enzyme or less amount.

12. the method for claim 1, wherein in step (b), the mixture of described soybean oil composition and described enzyme is sheared mixing at first.

13. methods as claimed in claim 12, wherein said shear-mixed continued at least about 45 seconds.

14. the method for claim 1, wherein with the addition of a certain amount of water in step (b).

15. methods as claimed in claim 14, wherein said a certain amount of water be total mixture weight at least about 1.5%.

16. methods as claimed in claim 15, wherein said a certain amount of water be total mixture weight at least about 3.0%.

17. methods as claimed in claim 16, wherein said a certain amount of water be total mixture weight at least about 4.5%.

18. the method for claim 1, wherein said phospholipids content is about 10ppm or less.

19. methods as claimed in claim 18, wherein said phospholipids content is about 5ppm or less.