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    Method of Enzymatic Process

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    shinta sari
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    © All Rights Reserved
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    United States Patent Brunner et al. (4) METHOD FOR ENZYMATIC 81 OILS AND FATS ING OF (73) Inventors: Karlheinz Brunner, Grosskrotzenbui (DE) Rainer Frische, Frankfurt (DE Dirk Kilian, Maintal (DE) (73) Assignee: T¥P Oleochemie GmbH, Alzensu (DE) (4). Notice: Subject to any disclaimer, the term of this pateat is extended or adjusted uader 35 USCC. 154(b) by 262 days. (21) Appl. No. 1O/170,608 (2) Filed) Jun, 11, 2002 co) Prior Publication Data Us 20020197687 AI Dee. 26, 2002 (30) Foreign Application Priority Data Jun, 21, 2001 (EP) ounsost (1) Im cu! erp 7/64 (2) US.Cl. 134, 435/198 (55) Field of Search 485/134, 198, 66) References Cited U.S. PATENT DOCUMENTS. 44025,62 A * S/1977 Tenthot eta 470 5210733 A 6/1993 Myojo ct 85/52 POREIGN PATENT DOCUMENTS B Os 3 A 9/1985, cues OTHER PUBLICATIONS, Rehm et al, Biotechnology, vol. 2, pp. 470-472, 1988, vers Continuous use of lipases in fat hydmlysis Buchler et al. Proc.—Workd Conf. Biotechnol. Fats Oils Ind, (1988), Meeting Date 1987, pp. 230-237.* 1U$00693313982 US 6,933,139 B2 Aug. 23, 2005 (20) Patent No. (5) Date of Patent: M. Buller etal, “Enzymatische Fetispaltung”, Fat Science Technology, Bal 89, Ne. 4, (1987). M. Bubler et al, “Oleochemicals by Biochemicals Reac- tions?", Far Science Technology, Bal. 94, Nt. 3 (1992), ‘M. Bable tal, “Continuous Use OF Lipsses in Fat Hydeoly- Sis", Fat Science Technology, Bd 89, Nr. 14 (1987). 1 Masanobu t al, “Hydrolysis OF Soybean Oil By Lipase With A Bioreactor Having ‘Two Different Membranes", Journal Of Fermentation And Bioengineering, Bd. 73,.r. 1, pp 53-57 (1993) (Abstract), Gan © et al, “Simullancous Reaction And Separation in Enzymatic Hydrolysis Of High Oleate Sunflower Oit— Evaluation OF Ultrafiltration Performace And Process Syn- ergy”, Chemical Engineering Journal, BA. 71, Nt. 2, pp 87-96 (Dee. 2, 1998) (Absrae). Patent Abstucts OF Japan, vol. O13, No, LIE (C-S77), Mas 16, 1989 & IP 63 287492 A(KAO Corp), Now. 24, 1988. * cited by examiner Primary Examiner—vene Marx (74) Attorney, Agent, or Firm—Quarles & Brady LLP 6 Method for ihe enzymatic spliting of os and fats for obtaining faty acids and glyeerol by using lipases being adkled (0 a mixture containing an oil or fat and water, ‘wherein the spitting reaction Is performed only up to a spliting degree. at which slowing-down of the spiting ‘action is sill below a preset value using ciscontimiously ‘operated loop reactors, wherein he fatty aids tobe obtained fae separated from the reaction misture that seal partially Soli by fst separating an aqueous glyeero-containing phase fom a partially spit organic phase containing spit fay acids, ina self-cleaning ceurfugal separator andy alterwards the fatty aids are separated from the partially splitorgane phase and the residve of the ganic phase freed from the te fatty acids fed back ito the spliting proces. ABSTRACT US 6,933,139 B2 Sheet 1 of 6 Aug. 23, 2005 U.S. Patent vee WH 00 a au 1Old U.S. Patent Aug. 23, 2005 Sheet 2 of 6 US 6,933,139 B2 2 T T T S ' 2 L \ LS \ 2 o — + tS a 5 > i i 2 Lo = & a) = £— = = 2 L Leo a e 5 s S = r& 5 $ oi 9 7 T T T ° 8 o o 2 ° 3 8 s 3 g = S anjea pioe U.S. Patent Aug. 23, 2005 Sheet 3 of 6 US 6,933,139 B2 = T —T ae! a = Ls o | cr +3 5 e 5 = S < 4 < £ oS = r2 3 £ 2 = 2 ° 2r r+ & z = 6 eS 5 sf r& $ a 9 ' 7 7 3 S 3 3S s 8 8 8 2 8 anjea poe US 6,933,139 B2 Sheet 4 of 6 Aug. 23, 2005 U.S. Patent [uy] own Oct O01 08 09 1 1 i. 1. awAzua paiaAooad yim a6e}s Bul ids yey yo} BY) JO UL 00% anjea pie US 6,933,139 B2 Sheet 5 of 6 Aug. 23, 2005 U.S. Patent [ut] awy Oct OOL 08 09 Ov 0g oO rl ! fi i : 0 } FOS r j-OOL L - OSL : 1 : : ‘ ooz aWAZUS paaAodaJ UM aBeys Bt dS }eJ puz By) JO UONeJadO 'g “OI4 anjea poe U.S. Patent Aug. 23, 2005 Sheet 6 of 6 US 6,933,139 B2 2 & & & s can re 2 2 = 3 ZF 4 r& 2 | — 3 E . ce = gr ro i o o € 3 5 3 £7 r< 3S 2 = g g r rN 2 o 9S i ° ot oto omc Oe Oe el anjea poe 200: 180: 160 4 US 6,933,139 B2 1 METHOD FOR ENZYMATIC SPLITTING OF OILS AND FATS: (CROSS-REFERENCE TO RELATED "APPLICATIONS. ‘This application claims the provty benelit of European patent application EP 01 115 (81.0 fied on Jua, 21, 2001, STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [Not Applicable. FIELD OF INVENTION The present invention relates to the enzymatic spiting 0 ‘cleavage of native oils and fats, ie. of the triglycerides of oils an fal, inthe presence of water into glycerol and fay acids BACKGROUND Enzymati splitting of fat or ol hasbeen known fora long time and offers, contrary to 2 pressure spliting mainly practised in te industy, considerable principle advantages. ‘The enzymatic sping canbe performed at aormal pressure and, depending on the enzyme and oll a fat, even at room temperature, has also been known for along time that this method of fat spliting isthe most gentle one. The technical progress in industrial biotechnology also provided enzymes that are available now and suitable for Fat spliting. Such available ‘enzymes are eg. used in aumerous detergents. However, mainly because of the high enzyme consumption, ofthe very ig times and the sulting low ellcieney of the ie fat spliting, the enzymatic spiting did not became an industrial alternative method to the large-scale pressure splitting which is Well established in industry "The main problem that occurred in most of the numerous attempts to lower the enzyme consumption is described in the following publications: “Continuous Use of Lipsses in at Hydeolysis", M, Biller and Che, Wandzey, Fat Seience ‘Technology 89/Dee, 87, pages 598 to 605; “Enzymatische Feuspaltung”, M. Bibles aad Che, Wandrey, Fat Science Technology 89/Nr.4/1987, pages 156 to 164; and “Ole ‘ochemicals by Biochemical Resctions2", M, Buhler and (Chr. Wand, Fat Science Technology 94/No. 3/1992, pages 82 10 94 ‘The enzymatic fat spiting sing enzymes, so-called 5 Tipases, as biocatalysts acting on a wateroll mixture is described in these publications. By means ofthis spliting technique, the oil or fat, respectively, is split into glycerol and frve fatty acids. The glycerol migrates into the water phase whereas the organic phase euriches more and more ‘with free fatty acids until, finaly, only the fre fatty acs remain inthe onganie phase The activity of the used-up enzyme decreases to 8 great ‘extent with time, and the decrease is mostly independent from the amount ofthe catalytically converted product, This reduction ean be compenssted by further additions of| ‘enzyme, however, a time-dependenlly varying enzyme con- sumption cannot be avoided. Inthe course of the spliting reaction, the eaction speed or spliting rate decreases more and more, and the enzyme consumption increases. This is ‘due tothe Fact thatthe enzymatically catalysed hydrolysis is an equilibrium reaction, With an increasing concentration of| o 2 slyeerol in the water phase and of fatty acid in the organ phase, the reaction speed ix slowed down and finaly asymp- fotically approaches the equilibrium concentration. A. desired splitting degree near 1KK®, therefore, ean only be achieved after avery Tong reaction time, This Tong reaction time unavoidably results ina high loss of enzyme activity. The reaction time ean be shortened by lowering the glycerol concentration in the water phase, this, however, implies 2 low eoncentration of the glycerol obtainable in the spiting reaction and, due 10 the higher percentage of the wat phase, the water/gylerine phase bas more enzyme dissolved ‘herein that will be discharged with the water phase and ceannot be used again, ‘The enzymatic splitting reaction takes place a the phase boundary between organic and aqueous phase, and only tenzyme being present at the phase boundary and triglyeer- ides being present at the phase boundary contribute 10 or participate in the splitting reaction. With increasing spiting ‘degree, the occupation density o¢ concentration of fatty acids sill chemically bonded as glyeerdes, in comparison to tree Tay acids, decreases. at the phase boundary so that the reaction is slowed down, "The reaction speed can he accelerated by increasing the interface boundary surface. However, this requires 10 jnerease the enzyme amount such thatthe occupation den- sily or concentration of the enzyme atthe phase boundary remains unchanged, The elfect of increasing the reaction speed by addition of enzyme is, however, limited. At a ‘maximum concentration, any further addition of enzyme ‘does not contribute to aceelerate the eexetion. The enzyme consumption is, however, noticeably increased thereby, so ‘thatan optimal adjustment ofthe enzyme quantity and ofthe surface area of the phase boundary cannot be readily obiained Moreover, during 4 separation step for separating the organic, fay acid containing phase and the glycerol con- taining water phase enzyme amounis are discharged and cannot be recovered for futher use tis tue thatthe seaction speed canbe increased by increasing the interface boundary Surfice by intensively mixing the organic and aqueous psc as well as the aclded enzyme amount, but phase separation, recovery and re-usage of the enzyme become more dificult thereby’ In the above mentioned publications, the enzymatic spi ting reaction takes place in a continuous multistage coun- terflow system of water and oil which isto be subjected 10 spliting. When separating the aqueous phase containing alyecrol and the organic phase containing the spit fre fatty acids, an intermediate or interfacial layer is generated. This interfacial layer is emulsion-lke and contains most of the enzyme. In order to recover this enzyme forthe process and to reduce the enzyme consumption, the process according t0 the aforementioned publication “Continuous Use of Lipases in Fat Hydrolysis" is conducted as follows: frst oil is continuously split in mixing reactor. The reaction product Which, besides free fatty acids, contains water, glycerol, nono- and diglyeerde, not yet split oil as well as enzyme, is given to a solid wall howl centrifuge. The centrifuge is adjusted such that the interfacial layer between the aqueous {lyoerol phase and onganie phase is discharged together with the organic phase ‘The organic phase containing the interfacial layer is fed into a second mising reactor that is supplied with a fresh Waterienzyme mixture, The reation peesict of the second reactor is fed into a further solid wall Bow centrifuge that i, however, adjusted in such & mane thatthe interfacial layer US 6,933,139 B2 7 is discharged together with the aqueous phase containing slyccrol and such thatthe discharged fee Latty acids are free ‘of the interfacial emulsion layer. The aqueous phase is recycled into the frst reactor (mixer), s0 that the enzyme amounts contained inthe interfacial emulsion layer are again “supplied or back-aded! ta the process. The splitting degree achieved inthe second reactor is up 10 98, s0 that the Yield ff five falty acids afler disillation of the end reaction product obtained form the second reactor is considerably high. However, even this kind of reaction scheme does nol provide + process being actually competitive vis-a-vis the ‘established large-scale pressure splitting process, [BRIEF SUMMARY OF THE INVENTION ‘The present invention provides a method for the enzy= matic split of ois and fats for obtaining fatty acids and _lycerol by using lipases being added to a mixture contain= Jing an oil or fat and water, wherein the spiting reaction is performed only up to a spiting degree at which slowing- ‘down ofthe sping reaction is sill helow a preset value using discontinuously operated loop reactors, Wherein the fatty acids to be obtained are separated from the reaction mixture that is only partially split, by frst separating an aqueous glyeerol-containing, phase from a partially split ‘organic phase containing split fatty acids, in a self-cleaning, centrifugal separator and, afterwards, the fatty acids are ‘separated from the partly split organic phase and the residue of the organie phase freed from the Ire Istly acs 8 fed buck into the spliting proces. A general objective of the present invention isto provide an enzymatic oil or fat spitting method which allows for & profliable competitive large-scale process. ‘This objective is achieved by means of the features of ‘each one of the indepenlent claims. Advantageous further ‘embodiments are defined ia the sub-claims, The inventive features provide a short reaction time and a reduced enzyme ‘consumption These and sill other objectives and advantages of the present invention will be apparent from the description Which follows, Inthe detailed description below, prefered ‘embodiments ofthe invention will be described in reference to the accompanying drawings. These embodiments do n0t represent the full scope of the invention, Rather the inven- tion may be employed in other embodiments, Reference should therefore be made to the claims herein for interpet ing the bread of the invention. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a schematic drawing of an example for a0 Industrial process showing an embodiments secording to the invention; and FIGS. 2to 6 show increasing acid values ofthe converted oil that oscur during dierent stages ofthe process. DETAILED DESCRIPTION OF THE INVENTION During their numerous attempts for finding solutions of the above object, the inventors of the present application ‘evaluated that i¢ Was necessary to depart [rom tbe process ‘scheme shown in the above mentioned publications in several basic and essential aspects, According 10 a fis solution aspect of the present invention, the inventors departed from obtaining the desired yield of free fatty acids feudy with the enzymatic splitting as sich which conven- tionally was conducted up to a high splitting or conversion % 4 degree. Instead, the enzymatic splitting according © this aspect ofthe present invention isony run up oor conducted ‘ntl reaching a comparatively sonal splitting degree which corresponds to 2 spliting degree at which n0 considerable Sslow-down of the process cecurs. In other words, the sli {ing reaction is topped when the reaction eae falls below a preset value. Such a spliting degree normally les between about 80% up to 90% and consequently below the spiting ‘opree values that were hitherto considered necessary for a fcommercally applicable process. Free fatty acids are then separated from the organic phase freed from enzyme, andthe residue which sil contains fatty acids chemically bonded as ylycerides is fed buck or recycled and mixed with fresh oil or fat to be subjected to spliting. In this manner, recycled glycerides are thea sub- jected lo a further enzymatic splitting process. Ifthe enzy~ ‘matie fat oro splitting is run up 1o only a splitting degree fof about 80%, This is possible in a very short time, If alterwards the fre fatty acids are extracted from anol or fat partially split in such a manner and ifthe chemically bound Tiaty acs (triglycerides) are returned or fed hack into the spliting process, the enzyme consumption canbe drastically redveed, without having f0 renounce to a final complete spliting ofthe oil or fat, The enzymatic splitting process does not get into the above mentioned eritcal process conditions in which the reaction speed, dic 1o lack of alvecrides at the phase boundary between aqueous and fonganie phase, i remarkably slowed down, ‘The spliting degree is determined as the ratio of the measured acid vale divided by the theoretically possible acid value which can be computed for a given oil or fat. Preferably, the acid value is measured by means of itration ocording to standard common methods, Alternatively, the Sensity of the aqueous glycerol phase can be taken a & measure for the splitting deree In order to separate the free fatty acids from the partially split tating product of the enzymatic spliting, preferably a vacuum distillation method is applied, that is, prelerably @ rild short path distillation (sometimes called molecular

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