CN103952448B - A kind of method utilizing enzyme-chemically method directional preparation OPO - Google Patents
A kind of method utilizing enzyme-chemically method directional preparation OPO Download PDFInfo
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Abstract
The invention discloses one and utilize enzyme-chemically method directional preparation 1, the method of 3-bis-oleic acid-2-palmitic acid three ester, first be starting raw material with glycerine, under agitation condition, dissolve at the temperature of reacting with oleic acid, add free or immobilized lipase as catalyst, carry out selective esterification reaction, obtain 1,3-position triglyceride; Again by the triglyceride that obtains and palmitinic acid or palmitinic acid derivatives reaction, obtain the thick product of OPO; Finally by molecular distillation, obtain described OPO.The present invention is used for the preparation of 1,3-DAG by the specific lipase of screening, and the content obtaining 1,3-DAG is more than 90%, and yield reaches more than 90%; The content of 1, the 3-bis-oleic acid-triglyceride of final acquisition is up to 95%, and yield reaches more than 95%.
Description
Technical field
The present invention relates to functional lipids research field, be specifically related to a kind of method utilizing enzyme-chemically method directional preparation OPO.
Background technology
Triglyceride level (TAG) is the important composition of fat, and it contains glycerine and three kinds of lipid acid.Different mainly according to lipid acid and acyl group binding site of TAG, can be divided into α (Sn-1,3) position and two kinds, β (Sn-2) position main Types.Owing to there is asymmetric C atom in TAG, the binding site of lipid acid and glycerine can be divided into three kinds: Sn-1 (α) position, Sn-2 (β) and Sn-3 (α) position.Structured lipid (Structuredlipids) is according to a kind of special fat of lipid in vivo designed by digestion and metabolism process, by changing composition and the position of various lipid acid in TAG of lipid acid in natural lipid, and the lipid acid with special nutrition or physiological function is attached to specific position, thus play physics and the functional property of various lipid acid to greatest extent.Due to the difference of different different acid glycerol three fat digestion and metabolism process in vivo, therefore can by changing composition and the position of various lipid acid in TAG of lipid acid in natural lipid, the lipid acid with special nutrition or physiological function is attached to specific position, thus the function such as nutrition and absorption of various lipid acid can be played to greatest extent.
The speed of neonatal lingual lipase hydrolysis Sn-3 (α) position lipid acid wants fast 2 times than the speed of Sn-1 (α) position.The short chain of these digestion and medium chain fatty acid admission port transmucosal, be converted into acetyl-CoA.Acetyl-CoA is as intracellular energy derive, and lipid acid is without the need to being converted into the endoplasmic reticulum of acylcarnitine by cell.In human milk, in TAG, palmitinic acid is mainly in Sn-2 (β) position, and palmitinic acid is conducive to the absorption of small intestine to lipid acid in Sn-2 (β) position.Therefore, newborn infant can absorb energy easily by this mechanism.But with human milk unlike Sn-2 (β) position mainly palmitinic acid in TAG in natural lipid acid and grease, at present in infant formula, in TAG, the general esterification of Sn-1 and Sn-3 is plam oil, is combined in Sn-2 position on a small quantity.In cow's milk, the palmitic acid content of Sn-2 position is than plant innage 40%.Therefore in order to improve the structure of TAG, making it be applied to infant food better, needing to transform the TAG in natural fat and grease, the human milk fat (HMF) of these transformations is called human milk fat replacer (HMFS).
HMFS is structure triacylglycerol, and also known as structured lipid, its structure is similar to HMF, and wherein the Sn-2 position of structured lipid is saturated occupied by lipid acid.Wherein, unsaturated fatty acids accounts at 1, the 3-total palmitinic acid in position of structured lipid and 70% of unsaturated fatty acids.Structured lipid is except as except human milk fat replacer, or obesity controlling disease and reduce effective lipoid of serum cholesterol, has a wide range of applications at field of medicaments.At present, main type is Sn-USU (S refers to saturated fatty acid, and U refers to unsaturated fatty acids), and what be widely used is OPO (1,3-dioleoyl-2-palmitoylglycerol, OPO).
The structured lipid comprising OPO does not exist at nature, needs artificial reconstructed synthesis.If employing chemical synthesis, on glycerol molecule, location certain lipid acid of distribution is also uncontrollable at present, does not therefore have report prepared by chemical method in document.The preparation of current structured lipid realizes mainly through enzyme process or chemo-enzymatic process both at home and abroad.
Lipase, owing to having regioselectivity, on purpose can act on catalyst article, carries out molecular designing from structure, and preparation has high value added product.The oil that Production by Enzymes OPO is mainly rich in Sn-2 position palmitinic acid with a kind of and acry radical donor prepare composite structure fat by transesterify under the catalysis of Sn-1, Sn-3 position specific lipase.The preparation technology of existing OPO mainly from palmitinic acid triglyceride (PPP) for raw material is prepared, simultaneously according to the number of times using lipase in preparation process, a step enzyme method, two step enzyme methods and other method can be divided into, be shown below.
1) step enzyme method
The preparation of OPO generally uses plam oil (tristearin or Trihexanoylglycerol) to be substrate, 1,3 specific fats enzyme catalysiss issue raw transesterification reaction, in preparation process, because this method Problems existing is containing PPP, PPO and OPP, wherein PPO in product in product, the mol ratio of OPO and PPP is respectively 48:36:16, and OPO ratio is not high.And product needs to be separated by distillation method, cost is higher, and the yield of OPO only has 42.8% (theoretical value should be 66.6%).This method has Uniliver and declares patent, and invested in plantization is produced.
Because this method exists above-mentioned shortcoming, existing a lot of people improves this method at present, as described below:
(1) people (JournaloftheAmericanOilChemistsSociety such as Bulgarian GunchevaM, 2008,129-132) Trihexanoylglycerol (99%) and oleic acid (98%) are synthesized OPO triglyceride level under lipase MC7 (Bacillusstearothermophilus) acts on.Shortcoming: have a large amount of by products in this reaction product, brings certain difficulty to later separation purifying.
(2) people (JournaloftheAmericanOilChemistsSociety such as Taiwan ChenML, 2004,525 – 532) report and obtain by three steps the method for OPO triglyceride level of being rich in: first by obtaining palmitinic acid through fractionation after Hydrolysis of Palm Oil, then palmitinic acid is converted into ethyl palmitate; Secondly ethyl palmitate and glycerine are synthesized PPP under the effect of Novozym435; Finally Trihexanoylglycerol and oleic acid are synthesized OPO triglyceride level under normal hexane system and LipozymeIM60 effect, wherein the insertion rate of oleic acid is 66.1%, Sn-2 palmitinic acid is that 90.7%, OPO purity can to about 74%.
(3) people (JournalofAgriculturalandFoodChemistry such as AntiaS, 2006,5175 – 5181) compare LIPI (Candidarugosalipase) and the LipozymeRMIM lipase effect at normal hexane solvent system catalysis Trihexanoylglycerol and oleic acid and Witconol 2301 synthesis OPO, have studied the impact of the factors such as reaction substrate, temperature of reaction, reaction times on synthesis OPO.Found that the Sn-1 of LIPI, 3 specificitys do not have that LipozymeRMIM's is strong, and the Witconol 2301 insertion rate of doing acry radical donor than oleic acid is high in addition.
(4) Trihexanoylglycerol (92%) and ethyl oleate are synthesized the structural fatty being rich in OPO by the people (NewBiotechnology, 2010,38-45) such as Korea S JEUNGHL under the effect of LipozymeTLIM lipase.Result can obtain the OPO that content is 31.43% at optimum conditions, and wherein Sn-2 palmitinic acid is 80.6%, Sn-1, and 3 oleic acid are 64.9%.
(5) people (BiochemicalEngineeringJournal such as Spain LuisE, 2011,62-69) obtain OPO product by two step enzyme method transesterification reactions: first palm oil stearin and palmitinic acid synthesize Trihexanoylglycerol under non-specific lipase effect; Then by Trihexanoylglycerol from oleic acid at different Sn-1,3 specific lipases are (as the lipaseDFfrom of the Rhizopusoryzae that originates, the Palatase20000L of Mucormiehe, the LipozymeRMIM (Novozymes Company) of Rhizomucormiehei, the lipaseQLC of LipozymeTLIM with Alcaligenessp of Thermomyceslanuginosus) on the impact becoming OPO.
(6) people (JournaloftheAmericanOilChemistsSociety such as ToshihiroN, 2001,167-172) Trihexanoylglycerol (containing 89%C16:0 and 8%C18:0) and oleic acid (88%) are synthesized OPO triglyceride level under the effect of heat stable lipases R275A (Fusariumheterosporum).And investigated the thermostability of immobilized R275A at solvent-free system, find that R275A has very high thermostability solvent-free system 60 DEG C, its transformation period can reach 370d.Be 1 ︰ 2 (mass ratio) in the ratio of Trihexanoylglycerol and oleic acid, temperature of reaction is 50 DEG C, and the reaction times is 24h, the OPO containing 36% in the finished product under the reaction conditionss such as 8% enzyme amount.
(7) publication number is disclose palm oil stearin and oleic acid in the american documentation literature of WO2005036987A1 and WO2007029018A1 at Sn-1, carry out building-up reactions under the effect of 3 lipase, then reaction product is carried out point structural fatty raising the triglyceride level being rich in OPO.
(8) publication number is that to disclose iodine number in the american documentation literature of WO2008104381A1 be 18 ~ 40 two or more plam oil first self random transesterify from palm stearin acid essence, then carries out transesterify with oleic acid or its nonglyceride.Random transesterify can significantly improve palmitinic acid in the distribution of Sn-2 position, thus improves the yield of the finished product.
2) two step enzyme methods
Although a step enzyme transformation approach can prepare OPO, the triglyceride (DAG) that this method is difficult to all intermediate reactions to produce is converted into OPO, and a step enzyme method can produce a large amount of by products, and it is comparatively unfavorable that this produces OPO to industrialization.At present, two step enzyme methods production OPO are adopted to cause the interest of people.By Trihexanoylglycerol in solvent system; at Sn-1; under the effect of 3 specific lipases; after alcoholysis (alcoholysis) and purification, obtain the Sn-2-hexadecanoic acid direactive glyceride (2-MAG) that purity is greater than 95%, then Sn-2-hexadecanoic acid direactive glyceride and acry radical donor oleic acid are synthesized (trans-esterification) OPO under solvent or solvent-free system.The advantage of this method adopts alcoholysis to replace hydrolysis effectively to prevent acyl group from shifting, thus obtain high purity 2-MAG, but this method also exists certain problem, mainly needs more purification, thus have impact on the industrial applications of this technique.
(1) people (BiotechnologyandBioengineering such as SchmidU; 1999; 678-684) by two-step approach obtain be rich in OPO triglyceride level: first by Trihexanoylglycerol in solvent system; at Sn-1; under the effect of 3 specific lipases; through alcoholysis and purify after obtain purity be greater than 95% Sn-2-hexadecanoic acid direactive glyceride; then Sn-2-hexadecanoic acid direactive glyceride and acry radical donor oleic acid are synthesized OPO triglyceride level under solvent or solvent-free system; product purity can reach 82%, and its Sn-2 palmitinic acid is 96%.
(2) publication number be the European patent of EP0882797 report first by palm oil stearin at Sn-1, the lower Sn-2 hexadecanoic acid direactive glyceride obtaining purity and be greater than 95% of 3 specific lipase RhizopusDelamar effects.Then Sn-2 hexadecanoic acid direactive glyceride and oleic acid are obtained purity under the reaction conditions of immobilized RhizopusDelamar lipase and normal hexane system or solvent-free system and are greater than 90%, yield be greater than 70% OPO.
(3) (the PfefferJan such as JanP, FreundAndreas, Bel-RhlidRachid, etal.Highlyefficientenzymaticsynthesisof2-monoacylglycer idesandstructuredlipidsandtheirproductiononatechnicalsca le [J] .Lipids, 2007,42:947-953.) by small scale pilot equipment plam oil Trihexanoylglycerol and ethanol believed in CaIB (Novozym435) and acetone solvent system to carry through alcoholysis effect and freezing point in Novi and obtain Sn-2 hexadecanoic acid direactive glyceride.Then Sn-2 hexadecanoic acid direactive glyceride and oleic acid are obtained purity 95% under CaIB and normal hexane reaction conditions, yield is the OPO of 90%.
3) other method
(1) people (JournalofAgriculturalandFoodChemistry such as NeseS; 2006; 3717 – 3722 and JournaloftheAmericanOilChemists'Society; 2005; 549-557) Trihexanoylglycerol and hazelnut oil lipid acid (being rich in oleic acid) and EPA, DHA acry radical donor are rich in OPO by synthesizing under the katalysis of Lipozyme RMIM, the structural fatty of EPE and DPD triglyceride level under normal hexane solvent system.This method can solve the fishy smell problem of fish oil.
(2) people (JournaloftheAmericanOilChemistsSociety, 2000,89-93) such as YujiS is by Trihexanoylglycerol and arachidonic acid at Sn-1, synthesizes APA under the effect of 3 specific lipase Rhizopusdelemar.Wherein the molar ratio of palm tree Witepsol W-S 55 and ARA is 1:5, and temperature of reaction is 40 DEG C, time 24h, and result has the ARA of 60% to be combined in the Sn-1 of structural fatty, 3, and the palmitinic acid of 30% is combined in the Sn-2 position of structural fatty.
(3) people (FoodScienceandTechnology such as HuriI, 2011,999-1004) to be synthesized under the effect of LipozymeRMIM with hazelnut oil lipid acid (being rich in oleic acid) and C8:0, C10:0 lipid acid by Trihexanoylglycerol (85% purity) and be rich in OPO, CPC and CyPCy triglyceride structure.
(4) publication number is that the European patent of EP0965578 reports by Trihexanoylglycerol and C8:0, synthesizes CPC triglyceride structure under LipozymeIM60 effect.The method can improve the stability of product.
Can find from above-mentioned research report, with OPO be at present the structure triacylglycerol of main representative preparation method mainly with same acid glycerol three fat and Trihexanoylglycerol for raw material, adopt 1,3 selectivity transesterification enzymes by PPP through 1,3 transesterification synthesis OPO etc. 1,3-position identical fatty acid glycerine three fat, the subject matter that these class methods exist is that product purity is low, 1,3 transesterification rates are low, and in product, foreign matter content is high, the problem such as the high and production stage of product separation cost is numerous and diverse, not only have impact on purity and the yield of product, but also improve the production cost of product.
Summary of the invention
The invention provides one and utilize enzyme-chemically method directional preparation 1, the method of 3-bis-oleic acid-2-palmitic acid three ester, from cheap glycerine, screen efficient lipase, 1 of high yield is obtained through selective esterification reaction, 3-triglyceride intermediate, then prepare OPO through chemical esterification method, product purity reaches as high as 96%.
The invention discloses a kind of method utilizing enzyme-chemically method directional preparation OPO, comprise the steps:
(1) be that the oleic acid of 2.0 ~ 2.2:1 and glycerine add reactor by mol ratio, 35 DEG C ~ 65 DEG C are heated under agitation condition, add lipase, esterification is carried out under vacuum tightness is 300 ~ 500Pa, until system mid-oleic <0.5%, obtain 1,3-bis-Oleic acid diglyceride after filtration;
(2) step (1) is obtained 1,3-bis-Oleic acid diglyceride, triethylamine and solvent, stirring is cooled to 0 ~ 6 DEG C, add palmitinic acid or palmitinic acid derivative again, after insulation reaction 1 ~ 5h, after being warming up to room temperature, continue reaction, until 1, the transformation efficiency >97% of 3-bis-Oleic acid diglyceride, more after filtration, washing, drying obtain the crude product of OPO;
The mol ratio of described triethylamine and 1,3-bis-Oleic acid diglyceride is 1 ~ 2:1;
The mol ratio of described palmitinic acid or palmitinic acid derivative and 1,3-bis-Oleic acid diglyceride is 1 ~ 1.5:1;
(3) crude product that step (2) obtains is carried out molecular distillation, obtain described OPO.
The present invention devises a variation route from the glycerine of low cost, and the technique related to mainly comprises following gordian technique:
1., be first the Sn-1 at glycerine, 3 are carried out selective esterification reaction, carry out under lipase-catalyzed effect, are dewatered or other method makes the balanced deflection target compound of esterification by vacuum distilling, prepare 1,3-DAG intermediate.
2., then glycerine remaining 2 chemically selectivity introduce palmitinic acid, concrete steps are shown below:
In formula: R
1=-CH
3(CH
2)
7cH=CH (CH
2)
7, R
2=-C
16h
31.
Described lipase is free-fat enzyme or immobilized lipase, and the add-on of enzyme is 50 ~ 5000 international unit/every mole of glycerin.
Described lipase is LipasePL, CALA (bacterial classification is Candiaantarctica), TTL (bacterial classification is Thermusthermophilus), RAL (bacterial classification is Rhizopusarrhizus), CLL (bacterial classification is Candialipolytica), LipolaseUltra, CRL (bacterial classification is Candiadarugosa), LipasePS (bacterial classification is Burkholderiacepacia), AmanoAK (bacterial classification is Pseudomonasfluorescens), PPL (bacterial classification is Porcinepancreas), LipSM54 (bacterial classification is StenotrophomonasmaltophiliaCGMCC4254) or LipPA99 (bacterial classification is PseudomonaspseudoalcaligenesCGMCCNo.4405).
Described immobilized lipase can by embedding, absorption or crosslinked acquisition.
As preferably, described lipase is LipSM54, LipolaseUltra or LipPA99.
LipSM54 is free-fat enzyme, and preparation method can refer to the Chinese patent literature that publication number is CN102031237B, is specially:
Be that the Stenotrophomonas of CGMCCNo.4254 joins in substratum and carries out fermentation culture by preserving number, fermented liquid lyophilize is obtained described LipSM54 lipase.
The composition of described substratum is: peptone 1 ~ 25g/L, lactose 1 ~ 15g/L, yeast extract paste 1 ~ 8g/L, ammonium sulfate 0 ~ 10g/L, dipotassium hydrogen phosphate 1 ~ 6g/L, sodium-chlor 0 ~ 2g/L and anhydrous magnesium sulfate 0 ~ 1g/L, and the pH value of described substratum is 5 ~ 10.
Further preferably:
Described lipase LipolaseUltra is the LipolaseUltra of sintered glass absorption, and preparation method is as follows:
By mass percentage for 65:27:8 takes SiO
2, B
2o
3and Na
2o, in retort furnace, 1000 DEG C are heated 2 hours, 1500 DEG C are incubated 12 hours, the glass of melting is pulverized, then makes separant with magnesium oxide, be heated to 850 DEG C, be incubated after 1 hour and take out, cooling, sieve removing magnesium oxide, obtains glass microballon, then to select diameter be the glass microballon of 1.5 ~ 2mm is raw material.
Take 1g porous glass beads and be placed in 250mL Erlenmeyer flask, add the LipolaseUltra lipase buffered soln of 50mL, described damping fluid is the 100mLKH of pH=7.0
2pO
4-K
2hPO
4buffered soln, vacuum suction 15min, vibrate after absorption 1 ~ 4h in the water-bath constant temperature oscillator of 4 ~ 8 DEG C, pour out residual enzyme liquid, after the abundant cleaning glass microballon of the Sodium phosphate dibasic-citrate buffer solution of pH=6.5 ~ 7.0, vacuum-drying is the LipolaseUltra of sintered glass absorption, is immobilized enzyme.
Wherein, the LipolaseUltra as raw material can by commercially available acquisition.
Described lipase LipPA99 is the LipPA99 of calcium alginate embedded LipPA99 or ion-exchange absorption.
The preparation method of calcium alginate embedded LipPA99:
Take 5 ~ 10gLipPA99 enzyme powder, be dissolved in the 100mLKH of pH=7.0
2pO
4-K
2hPO
4in buffered soln, filter and obtain enzyme liquid.In enzyme liquid, add 0.2 ~ 1mol/L sodium alginate soln, mix the rear CaCl utilizing pulsed electrical field micro-ball preparing instrument to instill 20mg/mL
2in the aqueous solution, solidification 15 ~ 30min obtains calcium alginate embedded LipPA99, is immobilized enzyme.
The preparation method of the LipPA99 of ion-exchange absorption:
Take the ROHM AND HAAS AMBERLITEFPA42Cl ion exchange resin of 10g after cleaning, immersion in 250mL Erlenmeyer flask, add the KH of the pH=7.0 containing 1g enzyme powder (lipase activity is greater than 50 units/g)
2pO
4-K
2hPO
4damping fluid 100mL, control temperature 0 ~ 6 DEG C is constant, rotating speed 110r/min, and in constant temperature oscillator, absorption is fixing.After fixedly completing, pipette a certain amount of supernatant liquor, suction filtration, with the deionized water wash of 4 DEG C, obtaining the lipase LipPA99 of described ion-exchange absorption, is immobilized enzyme.
Wherein, the preparation method as the lipase LipPA99 of raw material can refer to the Chinese patent literature that publication number is CN102154166A, is specially:
Be that the Pseudomonas alcaligenes of CGMCCNo.4405 joins in substratum and carries out fermentation culture by preserving number, described Pseudomonas alcaligenes is 1 ~ 10% (volume) relative to the inoculum size of substratum, fermentation time is 12 ~ 48h, temperature is 15 ~ 50 DEG C, then fermented liquid lyophilize is obtained described LipPA99 lipase.
The composition of described substratum is: casein 1 ~ 10g/L, tween-80 1 ~ 15g/L, yeast extract paste 1 ~ 8g/L, ammonium sulfate 0 ~ 10g/L, dipotassium hydrogen phosphate 1 ~ 6g/L, calcium chloride 0 ~ 0.5g/L and anhydrous magnesium sulfate 0 ~ 1g/L, and the pH value of described substratum is 5 ~ 10.
Further preferred, the add-on of described lipase is 500 ~ 1500 international unit/every mole of glycerin.
As preferably, described solvent to be carbon number be 5 ~ 12 aliphatic hydrocarbon, sherwood oil or carbon number be the chloroparaffin of 1 ~ 6.
As preferably, the temperature of described molecular distillation is 180 ~ 220 DEG C, vacuum tightness <40Pa, and input speed is 5 ~ 10g/min.
Compared with prior art, tool of the present invention has the following advantages:
1, the present invention with the glycerine be cheaply easy to get for starting raw material, utilize the selectivity of lipase, at the Sn-1 of glycerine, 3 reaction generations 1,3-triglyceride (1,3-DAG), adopts chemical esterification reaction to produce OPO further, be a kind of New Policy and novel process of synthesizing this series products, have no bibliographical information at present.
2, the present invention carries out the preparation of 1,3-, bis-oleic acid-triglyceride by the highly selective lipase that screening obtains, and wherein the content of 1,3-DAG is more than 90%, and yield also reaches more than 90%; Adopt the methods such as embedding (microcapsule or alginate calcium or carrageenin), covalent cross-linking, porous material (as sintered glass or porous ceramics) absorption to after being fixed of lipase, 1, the content more than 95% of 3-bis-oleic acid-triglyceride, yield reaches more than 95%, and immobilized enzyme can recycle more than 20 times.
3, the enzyme-chemically method set up by the present invention synthesizes the structured lipid product obtained, not only product yield is high, and be further purified product purity without molecular distillation and just can be greater than 85% (w/w), the ratio that wherein Sn-2 position saturated fatty acid accounts for all saturated fatty acids is greater than more than 65%, the content of product purity and Sn-2 position saturated fatty acid exceedes national standard all, is better than currently reported technique.
Embodiment
Embodiment 1:
A, resolvase legal system are for 1,3-bis-Oleic acid diglyceride:
In the there-necked flask of a 500mL, add free oleic acid 282g (1mol), glycerine 46g (0.5mol), LipSM54 lipase 10g.Stir, heat 55 DEG C, system vacuum pump evacuation, maintenance system, at 300 ~ 500Pa, reclaims the water of reaction generation with cold hydrazine before vacuum pump.Reaction approximately continues 12h, detects, and after the content <0.5% of oleic acid, stopped reaction, crosses and filter lipase, and use for next time, product is pale yellow filtrate, and the content of yield 97%, 1,3-bis-Oleic acid diglyceride is about 96%.
1,3-bis-oleic acid-2-palmitic acid three ester (OPO) and 1,3-bis-Oleic acid diglyceride (1,3-Di (cis-9-octadecenoyl) glycerol) measuring method be the Gas chromatography contrasted with standard model: (OPO and 1,3-bis-Oleic acid diglyceride standard specimen are bought in Sigma)
Adopt high temperature gas chromatography (HT-GC) direct analysis free fatty acids and various monoglyceride, diester, three esters.Concrete grammar is: take the trimethyl carbinol as solvent, is interior mark with trilaurin, configures glyceryl ester to be measured and interior target solution, and determinand and interior mark concentration are 1molL
-1.Sample introduction analysis in gas-chromatography.Sample size 0.5 μ L; Chromatographic column: DB-17ht; Post case temperature: 300 DEG C, vaporizing chamber (sampler) temperature: 340 DEG C, detector temperature: 340 DEG C; Carrier gas (hydrogen) flow velocity: 240mLmin
-1, air velocity: 170mLmin
-1.Monoglyceride appearance time: 1.7min, inside marks peak time 5.3min, 1,2-diester 8.4min, 1,3-diester 9.1min, OPO tri-ester: 14.1min.
OPO is prepared in the transesterification of B, palmitic acid chloride chemistry:
In the there-necked flask of a 1000mL, add sherwood oil 300ml, filtrate in triethylamine 53g (0.503mol) and steps A, stirring is cooled to 0 DEG C, maintain with this understanding, drip the petroleum ether solution (200ml) of palmitic acid chloride (0.5mol), dropwise, insulation reaction 3h, slowly rise to room temperature, continue reaction 10h, transformation efficiency to diester reaches more than 97% stopped reaction, cross the triethylamine hydrochloride filtering generation, filtrate with a small amount of water washing for several times, pH to system is neutral, anhydrous sodium sulfate drying, vacuum distillation recovered solvent is to dry, obtain yellow liquid product, the content of OPO is about 94%.
Refining of C, product:
In a small-sized molecular distillation equipment, the vacuum tightness <40Pa of system during work, the temperature of heated oil controls at about 200 DEG C, OPO crude product above, molecular distillation is carried out by the speed of 5g/min according to equipment, collecting product is flaxen oily liquids, and the content of OPO is 96%.
Embodiment 2:
A, using embedding immobilization enzyme process prepare 1,3-bis-Oleic acid diglyceride:
In the there-necked flask of a 500mL, add oleic acid 280g (1mol), glycerine 46g (0.5mol), adds calcium alginate embedded LipPA9910g.Stir, heat 55 DEG C, system vacuum pump evacuation, maintenance system, at 300 ~ 500Pa, reclaims the water of reaction generation with cold hydrazine before vacuum pump.Reaction approximately continues 12h, detects, and after the content <0.5% of oleic acid, stopped reaction, crosses and filter immobilized lipase, and use for next time, product is pale yellow filtrate, and the content of yield 98%, 1,3-Oleic acid diglyceride is about 97%.
OPO is prepared in the transesterification of B, palmitic anhydride chemistry:
In the there-necked flask of a 1000mL, add normal hexane 300ml, triethylamine 53g (0.503mol) and filtrate above, stirring is cooled to 0 DEG C, maintain with this understanding, drip the hexane solution of palmitic anhydride (0.5mol is dissolved in 100ml normal hexane), dropwise, insulation reaction 3h, slowly rise to room temperature, continue reaction 10h, transformation efficiency to diester reaches more than 97% stopped reaction, cross the triethylamine hydrochloride filtering generation, filtrate with a small amount of water washing for several times, pH to system is neutral, anhydrous sodium sulfate drying, vacuum distillation recovered solvent is to dry, obtain yellow liquid product, the content of OPO is about 93%.
Refining of C, product:
In a small-sized molecular distillation equipment, the vacuum tightness <40Pa of system during work, control the temperature of thermal oil, the a small amount of palmitinic acid of first time Distillation recovery, then the temperature raising heated oil carries out molecular distillation, collecting product is flaxen oily liquids, and the content of OPO is 95%.
Embodiment 3:
A, using embedding immobilization enzyme process prepare 1,3-bis-Oleic acid diglyceride:
In the there-necked flask of a 500mL, add oleic acid 280g (1mol), glycerine 46g (0.5mol), add the calcium alginate embedded LipPA9910g reusing 20 times.Stir, heat 55 DEG C, system vacuum pump evacuation, maintenance system, at 300 ~ 500Pa, reclaims the water of reaction generation with cold hydrazine before vacuum pump.Reaction approximately continues 12h, detects, and after the content <0.5% of oleic acid, stopped reaction, crosses and filter immobilized lipase, and use for next time, product is pale yellow filtrate, and the content of yield 96%, 1,3-Oleic acid diglyceride is about 95%.
OPO is prepared in the transesterification of B, palmitic anhydride chemistry:
In the there-necked flask of a 1000mL, add normal hexane 300ml, triethylamine 53g (0.503mol) and filtrate above, stirring is cooled to 0 DEG C, maintain with this understanding, drip the hexane solution of palmitic anhydride (0.5mol is dissolved in 100ml normal hexane), dropwise, insulation reaction 3h, slowly rise to room temperature, continue reaction 10h, transformation efficiency to diester reaches more than 97% stopped reaction, cross the triethylamine hydrochloride filtering generation, filtrate with a small amount of water washing for several times, pH to system is neutral, anhydrous sodium sulfate drying, vacuum distillation recovered solvent is to dry, obtain yellow liquid product, the content of OPO is about 91%.
Refining of C, product:
In a small-sized molecular distillation equipment, the vacuum tightness <40Pa of system during work, control the temperature of thermal oil, the a small amount of palmitinic acid of first time Distillation recovery, then the temperature raising heated oil carries out molecular distillation, collecting product is flaxen oily liquids, and the content of OPO is 93%.
Embodiment 4:
A, covalent cross-linking immobilized enzyme method prepare 1,3-bis-Oleic acid diglyceride:
In the there-necked flask of a 500mL, add oleic acid 280g (1mol), glycerine 46g (0.5mol), the lipase LipPA9910g of spent ion exchange resin absorption.Stir, heat 55 DEG C, system vacuum pump evacuation, maintenance system, at 300 ~ 500Pa, reclaims the water of reaction generation with cold hydrazine before vacuum pump.Reaction approximately continues 12h, detects, and after the content <0.5% of oleic acid, stopped reaction, crosses and filter lipase, and use for next time, product is pale yellow filtrate, and the content of yield 97%, 1,3-bis-Oleic acid diglyceride is about 95%.
OPO is prepared in the transesterification of B, palmitinic acid chemistry:
In the there-necked flask of a 1000mL, add methylene dichloride 300ml, triethylamine 53g (0.503mol) and filtrate above, stirring is cooled to 0 DEG C, maintain with this understanding, drip the petroleum ether solution of palmitinic acid (0.5mol is dissolved in 100ml sherwood oil), dropwise, insulation reaction 3h, slowly rise to room temperature, continue reaction 10h, transformation efficiency to diester reaches more than 97% stopped reaction, cross the triethylamine hydrochloride filtering generation, filtrate with a small amount of water washing for several times, pH to system is neutral, anhydrous sodium sulfate drying, vacuum distillation recovered solvent is to dry, obtain yellow liquid product, the content of OPO is about 90%.
Refining of C, product:
In a small-sized molecular distillation equipment, the vacuum tightness <40Pa of system during work, control the temperature of thermal oil, the a small amount of palmitinic acid of first time Distillation recovery, then the temperature raising heated oil carries out molecular distillation, collecting product is flaxen oily liquids, and the content of OPO is 92%.
Embodiment 5:
A, absorption method immobilized lipase prepare 1,3-bis-Oleic acid diglyceride:
In the there-necked flask of a 500mL, add free oleic acid 282g (1mol), glycerine 46g (0.5mol), the LipolaseUltra lipase 10g of sintered glass absorption.Stir, heat 55 DEG C, system vacuum pump evacuation, maintenance system, at 300 ~ 500Pa, reclaims the water of reaction generation with cold hydrazine before vacuum pump.Reaction approximately continues 12h, detects, and after the content <0.5% of oleic acid, stopped reaction, crosses and filter lipase, and use for next time, product is pale yellow filtrate, and the content of yield 96%, 1,3-oleic acid diester is about 95%.
B, palmitic anhydride legal system are for OPO:
In the there-necked flask of a 1000mL, add normal hexane 300ml, triethylamine 53g (0.503mol) and filtrate above, stirring is cooled to 0 DEG C, maintain with this understanding, drip the hexane solution of palmitic anhydride (0.5mol is dissolved in 100ml normal hexane), dropwise, insulation reaction 3h, slowly rise to room temperature, continue reaction 10h, transformation efficiency to diester reaches more than 97% stopped reaction, cross the triethylamine hydrochloride filtering generation, filtrate with a small amount of water washing for several times, pH to system is neutral, anhydrous sodium sulfate drying, vacuum distillation recovered solvent is to dry, obtain yellow liquid product, the content of OPO is about 90%.
Refining of C, product:
In a small-sized molecular distillation equipment, the vacuum tightness <40Pa of system during work, controls the temperature of thermal oil, Distillation recovery palmitinic acid for the first time, then the temperature raising heated oil carries out molecular distillation, and collecting product is flaxen oily liquids, and the content of OPO is 91%.
Embodiment 6:
A, LipozymeRMIM enzyme process prepares 1,3-bis-Oleic acid diglyceride:
In the there-necked flask of a 500mL, add oleic acid 280g (1mol), glycerine 46g (0.5mol), free-fat enzyme LipozymeRMIM10g.Stir, heat 55 DEG C, system vacuum pump evacuation, maintenance system, at 300 ~ 500Pa, reclaims the water of reaction generation with cold hydrazine before vacuum pump.Reaction approximately continues 12h, detects, and after the content <0.5% of oleic acid, stopped reaction, crosses and filter lipase, and use for next time, product is pale yellow filtrate, yield 78%, and wherein the content of 1,3-Oleic acid diglyceride is 75%.
B, palmitic acid chloride prepare OPO:
In the there-necked flask of a 1000mL, add sherwood oil 300ml, triethylamine 53g (0.503mol) and filtrate above, stirring is cooled to 0 DEG C, maintain with this understanding, drip the petroleum ether solution of palmitic acid chloride (0.5mol is dissolved in 100ml sherwood oil), dropwise, insulation reaction 3h, slowly rise to room temperature, continue reaction 10h, transformation efficiency to diester reaches more than 97% stopped reaction, cross the triethylamine hydrochloride filtering generation, filtrate with a small amount of water washing for several times, pH to system is neutral, anhydrous sodium sulfate drying, vacuum distillation recovered solvent is to dry, obtain yellow liquid product, the content of OPO is about 75%.
Refining of C, product:
In a small-sized molecular distillation equipment, the vacuum tightness <40Pa of system during work, the temperature of heated oil controls at about 200 DEG C, OPO crude product is above carried out molecular distillation according to equipment by the speed of certain charging, collecting product is flaxen oily liquids, and the content of OPO is 79%.
Claims (7)
1. utilize a method for enzyme-chemically method directional preparation OPO, it is characterized in that, comprise the steps:
(1) be that the oleic acid of 2.0 ~ 2.2:1 and glycerine add reactor by mol ratio, 35 DEG C ~ 65 DEG C are heated under agitation condition, add lipase, esterification is carried out under vacuum tightness is 300 ~ 500Pa, until system mid-oleic <0.5%, obtain 1,3-bis-Oleic acid diglyceride after filtration;
(2) step (1) is obtained 1,3-bis-Oleic acid diglyceride, triethylamine and solvent, stirring is cooled to 0 ~ 6 DEG C, add palmitinic acid or palmitinic acid derivative again, after insulation reaction 1 ~ 5h, after being warming up to room temperature, continue reaction, until 1, the transformation efficiency >97% of 3-bis-Oleic acid diglyceride, more after filtration, washing, drying obtain the crude product of OPO;
The mol ratio of described triethylamine and 1,3-bis-Oleic acid diglyceride is 1 ~ 2:1;
The mol ratio of described palmitinic acid or palmitinic acid derivative and 1,3-bis-Oleic acid diglyceride is 1 ~ 1.5:1;
(3) crude product that step (2) obtains is carried out molecular distillation, obtain described OPO;
Described lipase is LipSM54, LipolaseUltra or LipPA99;
LipSM54 is free-fat enzyme, and preparation method is specially: be that the Stenotrophomonas of CGMCCNo.4254 joins in substratum and carries out fermentation culture by preserving number, fermented liquid lyophilize is obtained described LipSM54 lipase;
The preparation method of lipase LipPA99 is specially: be that the Pseudomonas alcaligenes of CGMCCNo.4405 joins in substratum and carries out fermentation culture by preserving number, described Pseudomonas alcaligenes is volume percentage 1 ~ 10% relative to the inoculum size of substratum, fermentation time is 12 ~ 48h, temperature is 15 ~ 50 DEG C, then fermented liquid lyophilize is obtained described LipPA99 lipase;
Described palmitinic acid derivative refers to palmitic anhydride or palmitic acid chloride.
2. method according to claim 1, is characterized in that, the add-on of described lipase is 50 ~ 5000 international unit/every mole of glycerin.
3. method according to claim 1, is characterized in that, described lipase LipolaseUltra is the LipolaseUltra of sintered glass absorption.
4. method according to claim 3, is characterized in that, described lipase LipPA99 is the LipPA99 of calcium alginate embedded LipPA99 or ion-exchange absorption.
5. method according to claim 1, is characterized in that, the add-on of described lipase is 500 ~ 1500 international unit/every mole of glycerin.
6. method according to claim 1, is characterized in that, described solvent to be carbon number be 5 ~ 12 aliphatic hydrocarbon, sherwood oil or carbon number be the chloroparaffin of 1 ~ 6.
7. method according to claim 1, is characterized in that, the temperature of described molecular distillation is 180 ~ 220 DEG C, vacuum tightness <40Pa, and input speed is 5 ~ 10g/min.
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| WO2019030002A1 (en) * | 2017-08-11 | 2019-02-14 | Nestec S.A. | Synthesis of 1,3-olein-2-palmitin (opo) |
| CN108823255B (en) * | 2018-07-04 | 2021-01-05 | 嘉必优生物技术（武汉）股份有限公司 | Preparation method of tri-saturated fatty acid glyceride |
| CN108796000A (en) * | 2018-07-04 | 2018-11-13 | 嘉必优生物技术（武汉）股份有限公司 | Three saturated fatty acid glycerides of one kind and USU type triglycerides |
| CN110724716B (en) * | 2018-07-16 | 2021-07-20 | 浙江贝家生物科技有限公司 | A method for preparing composition containing 1, 3-dioleoyl-2-palmitic acid triglyceride |
| CN114181982A (en) * | 2021-07-13 | 2022-03-15 | 江南大学 | Method for preparing structured fat by enzyme method |
| CN114317136A (en) * | 2021-12-23 | 2022-04-12 | 常州中天气雾制品有限公司 | Thick grease cleanser for kitchen and preparation method thereof |
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