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WO2008094847A1 - Dégommage enzymatique utilisant un mélange de phospholipases pla et plc - Google Patents

Dégommage enzymatique utilisant un mélange de phospholipases pla et plc Download PDF

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Publication number
WO2008094847A1
WO2008094847A1 PCT/US2008/052162 US2008052162W WO2008094847A1 WO 2008094847 A1 WO2008094847 A1 WO 2008094847A1 US 2008052162 W US2008052162 W US 2008052162W WO 2008094847 A1 WO2008094847 A1 WO 2008094847A1
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WIPO (PCT)
Prior art keywords
oil
enzyme
enzymes
ppm
grams
Prior art date
Application number
PCT/US2008/052162
Other languages
English (en)
Inventor
Christopher L.G. Dayton
Erin Marie Rosswurm
Flavio Da Silva Galhardo
Original Assignee
Bunge Oils, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/668,921 external-priority patent/US8956853B2/en
Priority claimed from US11/853,339 external-priority patent/US8460905B2/en
Priority to PL08728362T priority Critical patent/PL2118248T3/pl
Priority to UAA200908943A priority patent/UA97972C2/ru
Priority to BRPI0808024A priority patent/BRPI0808024B1/pt
Priority to ES08728362.8T priority patent/ES2523300T3/es
Application filed by Bunge Oils, Inc. filed Critical Bunge Oils, Inc.
Priority to MX2009007919A priority patent/MX2009007919A/es
Priority to RU2009132518/10A priority patent/RU2477746C2/ru
Priority to CA2676412A priority patent/CA2676412C/fr
Priority to DK08728362.8T priority patent/DK2118248T3/da
Priority to EP08728362.8A priority patent/EP2118248B1/fr
Publication of WO2008094847A1 publication Critical patent/WO2008094847A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead

Definitions

  • This application relates to an enzymatic method for removing various phospholipids and lecithins (known collectively as "'gums " ') from vegetable oils to produce a degummed oil or fat product that can be used for food production and/or non-food applications. More particularly, this application relates to a method for the enzymatic treatment and removal of various phospholipids and lecithins, which method can be practiced on either crude oils or water-degummed oils. In one embodiment, the enzyme reaction or treatment period can be less than about an hour.
  • phospholipids contain a phosphate group on one of the two ends of the glycerol backbone, whereas a triacy .glycerol contains at least one fatty acid.
  • the phosphate group of the phospholipid is "hydrophilic” or ''water-loving,” meaning that the functional group X is attracted to water.
  • the phospholipid's fatty acid chains Rl and R2 are "lipophilic " ' or "lipid-loving.” meaning that they are attracted to lipids. Since the phospholipid molecule possesses both a hydrophilic functional group and lipophilic fatty acid chains, it is an excellent natural emulsifier.
  • the functional group X in FIG. 1 may be any of several of a variety ofknown types, a few of which are illustrated in FIG. 2.
  • Phospholipids containing the functional groups -choline and -ethanolamine have the greatest affinity for water, while the acids, acid salts (calcium, magnesium, and iron), and - inositol have much lower affinities for water.
  • Phosphatidic acid and the salts of phosphatide acid are commonly known as ''Non Hydratable Phospholipids" or NHPs.
  • Phospholipids are commonly measured in oil as "phosphorous content" in parts per million. Table 1 contains the typical amounts of phospholipids present in the major oilseed crops, and the distribution of the various functional groups as a percentage of the phospholipids present in the oil.
  • Phospholipids can be partially or totally removed from vegetable oils through several different known means.
  • the most commonly used processes in the industry are water degumming, acid degumming, caustic refining and enzymatic degumming.
  • This technique is usually applied to crude oils containing a high amount of hydratable phospholipids. Due to its mild characteristics, the phospholipids obtained can be used as lecithin (a natural emulsif ⁇ er).
  • the oil obtained from this technique is generally referred to in the industry as being "degummed,” despite being only partially degummed. Since water degummed oil still contains high amounts of phospholipids, especially non- hydratable phospholipids, the use of other process techniques, such as caustic refining or PL ⁇ 1 enzyme degumming, can be required to produce a finished, high quality oil having high stability and low color.
  • water degumming process water (1 to 5% w/w) is added to crude oil at 60 - 75°C with vigorous mixing. The oil is then gently mixed from 15 to 60 minutes to aid the hydration of the phospholipids present in the oil.
  • the hydration of the phospholipids or "'gums" causes the gums to swell and agglomerate as a flocculent.
  • the flocculent is an emulsion or mixture of hydrated gums and oil.
  • the emulsion has a specific gravity higher than that of the oil and may be separated by settling, filtration, or the industrial practice of centrifugation.
  • the centrifuge yields two streams, water degummed oil and wet gums.
  • the water degumming process removes predominately only the hydratable phospholipids. The remaining phospholipids (50 to 250 ppm), measured as the salts of phosphatidic acid and/or PL can be removed in subsequent processing operations.
  • the separated wet gums are an emulsified oil mixture containing at least one molecule of triacylglycerol (or oil) for every two molecules of phospholipid (or gum).
  • CHl-1627663vl emulsified oil cannot be physically separated or recovered from the emulsion and is considered a process loss.
  • the gums may be dried and sold as a food grade lecithin, but they are usually used as a by product in other applications such as animal feed or in an industrial process, with reduced economic value.
  • Crude oil is treated with 250 to 2000 ppm of phosphoric acid or citric acid at 60 - 90 0 C with vigorous mixing.
  • the acid is allowed to react with the sails of the NHPs for a period of 10 to 90 minutes.
  • the acid improves the hydrophilic nature of the NHPs, thus aiding in their removal.
  • Water (1 to 5% w/w) is then added to the acid-treated crude oil at 60 - 75 0 C with vigorous mixing.
  • the oil is then gently mixed from 15 to 60 minutes to aid the hydration of the phospholipids.
  • the hydration of the phospholipids or "gums” causes the gums to swell and agglomerate as a flocculent
  • the flocculent is an emulsion or mixture of hydrated gums and oil.
  • the emulsion has a specific gravity higher than that of the oil and may be separated by settling, filtration, or the industrial practice of centrifugation.
  • the centrifuge yields acid degummed oil and a wet gum.
  • the acid degumming process removes most of the phospholipids, but enough still remain (25 - 100 ppm) in the degummed oil to require additional processing.
  • the acid degummed oil is usually submitted to bleaching and deodorization, a process known in the industry as "physical refining".
  • the gums treated with acid are no longer usable for a food grade lecithin.
  • the separated and dry gums in the acid degumrning process contain at least one molecule of triacyl glycerol (or oil) for every two molecules of phospholipid (or gum). This emulsified oil cannot be physically separated or recovered and is considered a process loss, with negative economic impact on the overall economic balance of the refined oil process cost.
  • Crude or water degummed oil is treated with 200 to 1000 ppm of phosphoric acid or citric acid at 60 - 90 ° with vigorous mixing.
  • the acid is allowed to react with the salts of the NHPs from 10 to 90 minutes.
  • the acid improves the hydrophilic nature of the NHPs, thus aiding in their removal.
  • a diluted sodium hydroxide solution (10 - 18% w/w) is added to lhe acid-treated oil at 65 - 75 0 C.
  • the amount of sodium hydroxide (caustic) is based on the amount of free fatty acids present in the oil as well as an excess of between 0.05 to 0.20% on a dry basis.
  • the caustic solution neutralizes the free fatty acids (producing sodium soaps), neutralizes the excess acid, and with the sodium soaps created, assists in hydrating and emulsifying all the remaining phospholipids.
  • the sodium hydroxide solution / oil is mixed for approximately 10 minutes then separated by settling, filtration, or industrially by centrifugation.
  • the centrifuge yields a caustic treated oil and soapstock.
  • the caustic treated oil is then "washed' " with 10 to 20 % softened water at 90 - 95 0 C and centrifuged again.
  • the oil from the centrifuge is known as 'Once Refined” and the water is commonly known as ' " Wash Water " '.
  • the "once refined' " oil is usually submitted for bleaching and deodorizat ⁇ on to produce salad oil.
  • An alternative to water washing is to treat the caustic treated oil with an absorbent silica
  • the separated and dry gums in the caustic refining process contain one molecule of triacylglycerol (or oil) for every two molecules of phospholipid (or gum).
  • This emulsified oil cannot be physically separated or recovered and is considered a process loss.
  • the sodium hydroxide will react with the neutral oil to form soaps, thereby further reducing the overall oil yield with negative economic impact in the overall economic balance on the refined oil process cost.
  • Enzymatic degumming is used when the goal is the total removal of phospholipids.
  • enzymatic degumming treatments of the prior art have been practiced on oils that have been degummed previously by one of the other methods, typically water degumming.
  • the enzyme degummed oil is sequentially submitted to bleaching and deodorizalion, a process known in the industry as ' " physical refining.”
  • Enzymatic degumming provides a better oil yield than water, acid, or caustic degumming, with improved economic results.
  • the enzymatic reaction changes the nature of the phospholipid, cleaving some of the phospholipid parts. This reduces the phospholipids' emulsification properties, so that less oil is lost when the gums are separated from the oil, thus saving oil.
  • Enzymes exhibiting activity with phospholipids are commonly called "'phospholipases".
  • the types of phospholipase are based on the position on the phospholipid molecule at which the enzyme reacts, and are known as PLAl, PLA2, PLC, and PLD. The positions on the phospholipid molecule at which the different types of phospholipases react are illustrated in FlG. 3.
  • each type of phospholipase has its own rate of reaction and its own optimal reaction conditions in terms of pH, water % and temperature.
  • PLA when used alone generally requires a reaction time of at least about 4 hours, while PLC when used alone generally requires a reaction time of about one hour.
  • enzymatic treatment should occur at a pH less than or equal to 8, in order to minimize undesirable oil saponification, but PLA has an optimum reaction pH of 4.5, while PLC has an optimum reaction pH of 7.0,
  • Each enzyme also has different thermal tolerances. PLA enzymes will denature at about 50 0 C while PLC enzymes will denature at about 65°C.
  • One commercial PLAl enzyme product with phospholipase activity is Novozymes' phospholipase Al Lecitase® Ultra. This product is known to yield polar lyso- phospholipids and polar fatty acids when mixed with degummed oil with a 1-1.5% water citric acid-NaOH buffer at 4.5 ⁇ pH ⁇ 7.0 and 40°C ⁇ T ⁇ 55°C, as described on Novozymes' Application Sheet Oils & Fats# 2002-185255-01 and 2002-05894-03.
  • the PLAl selectively hydrolyzes the fatty acid opposite the phosphate functional group on the glycerol backbone, as illustrated in FlG. 4.
  • the resulting reaction yields a lyso -phospholipid and a fatty acid.
  • the lyso- phospholipid molecule has lost one hydrophilic functional group, and the remaining alcohol group at the reaction site is hydrophilic. Now with two hydrophilic sites, the lyso-phospholipid molecule is water soluble, and has lost its emulsification properties.
  • CHI- 1627663 v 1 process thus reduces refining losses by no longer removing any neutral oil with the gums, and the only loss is the original phospholipid molecule.
  • enzymatic degumming offers significant advantages to oil processors, it also poses certain disadvantages.
  • One disadvantage is that the reaction of the enzyme with the phospholipids can be slow and time consuming.
  • the reaction of phospholipase A enzymes with phospholipids can take many hours, depending on reaction variables such as pi L temperature, relative concentrations, and mixing conditions. Such prolonged reaction times can have a significant negative impact on the overall economic value of enzymatic degumming processes.
  • enzymatic degumming is typically carried out on oil compositions that have been first been subjected to water degumming. Thus, the oil must be degummed twice to obtain a product that has a phosphorous level low enough for its intended purposes.
  • PLCs will react with only certain phosphatidic groups.
  • PLPLC Pi-specific PLC
  • CHI-I627663vf It is thus one aspect of the invention to provide a method for enzymatic degumming of oils wherein the enzymatic reaction rate is faster than in prior art enzymatic degumming processes.
  • Aalrust states that because the oil which is recovered contains less than 5 ppm of phosphorous, it is adaptable to be physically refined to edible oil. Later, details of the technology described by Aalrust were disclosed in several publications (Dahlke, K. and Eichelsbacher, M., Enzymax® and Alcon® -
  • U.S. 5,532,163 to Yagi et al. discloses an enzymatic method using at least 30 weight parts water, and preferably 50-200 weight parts water, per 100 weight parts oil or fat, for the reaction of phospholipases Al, A2 or B with oil containing 100 to 10,000 ppm phosphorous. The oil is then washed with a 30% to 200% weight parts water or acidic aqueous solution per 100 weight parts oil or fat. The total water load required to utilize the process ranges from 60% to 400% w/w of oil processed. The production of such a large effluent in an industrial plant renders this method uneconomical.
  • U.S. 6,001.640 to Loeffler et al. discloses a process wherein one or more vegetable oils containing phosphorous-containing components are subjected to a mixture of phospholipases obtained from Aspergillus, the mixture comprising an enzyme having Al activity, A2 activity, or both, and an enzyme having lysophospholipase activity.
  • the patent states that since phospholipase would attack lecithin, it is not practical to use that method with oils with a high lecithin content, such as crude soybean oil.
  • Loeffler et al. disclose that the enzymatic reaction should be run at a pH of less than 4, and with the emulsion drop size being below 20 ⁇ m. The form of measurement and
  • U.S. 6,127,137 to Hasida et al. discloses the discovery and activity of certain phospholipases capable of removing both of the fatty acyl groups present on a phospholipid molecule when mixed with degummed oil (50 to 250 ppm phosphorous) with a 0.5 - 5% water, pH from 1.5 - 3, temperature from 30 - 45°C, and a time of 1 to 12 hours.
  • U.S. 6,143.545 to Clausen et al. discloses the discovery and activity of certain phospholipases (Al, A2, or B) for use in the enzymatic removal of phospholipids, and a method for producing the enzymes.
  • the enzymatic degumming process utilizes the method described in US 5,264,367 without any additional process steps.
  • U.S. 6,548,633 to Edwards et al. discloses sequences of cDNA " s encoding secreted proteins.
  • the patent states that the protein of that invention can be used in the enzyme degumming of vegetable oils as disclosed in U.S. 6,001,640. cited above.
  • the patent further states in the same paragraph that the protein of that invention can be combined in a ''cocktail' " with other enzymes to improve feed utilization in animals.
  • U.S. Patent Application Serial No. 10/556,816 of Dayton et al. discloses an improved enzymatic degumming process wherein the pH of the buffered enzymatic reaction is
  • CHi-1627663V 1 lowered to below 4.5 after the enzymatic reaction is completed, thereby eliminating the fouling of the equipment, particularly the heat exchangers and the separating centrifuge, that would result from precipitation of calcium and magnesium salts at the optimum pH required for the enzyme activity.
  • U.S. 2004/0005399 Al of Chakrabarti et al. discloses an enzymatic method utilizing a single addition of enzyme and buffering system and a short retention / reaction time, followed by bleaching with 2-4% bleaching earth and 0-1% activated carbon, and then dewaxing to achieve an oil with a phosphorus content of 5 ppm. Both the bleaching process and dewaxing process will remove residual phosphorus from the oil. Additionally, Chakrabarti et al. states that the oil lost to the gums is in the range of 30-40% of the gums separated, suggesting that the enzymatic reaction did not go to completion, resulting in high oil losses due to emulsification of oil in the removed phospholipids.
  • U.S. 2005/0059130 Al of Bojsen at al. discloses the discovery and activity of certain phospholipases for use in the enzymatic removal of phospholipids, and a method for producing the enzymes.
  • the publication refers to the treatment of vegetable oil to reduce the content of phospholipids as disclosed in U.S. 5,264,367.
  • the application further states that such phospholipases can be used for enzymatic degumming of vegetable oils, and that the PLCs of the invention can be used in addition to or in place of PLAIs and PLA2s in commercial oil degumming, such as in the ENZYMAX® process, where phospholipids are hydrolyzed by PLAl and PLA2.
  • the application states that PLC ma ⁇ be used alone or with PLA to remove non-hydratable phospholipids from oil that previous! ⁇ has
  • CHI- 1627663% 1 been water degummed. but does not provide reaction conditions for use of the two enzymes together. As the optimum reaction conditions of PLA enzyme and PLC enzyme are different. this statement in the application with no working examples does not teach one skilled in the art how to use PLA and PLC enzymes simultaneously.
  • the application further states that phospholipases C. Dl and D2 may be employed in the enzymatic de gumming of previously degummed and non-degummed (crude) oils and as an aid to caustic refining.
  • the invention relates to a method for degumming an oil composition, the method comprising
  • step (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.
  • the reaction of step (b) continues for a duration of less than about one hour.
  • the amount of water necessary for the process of the present invention can be less than about 5%, and advantageously can be reduced to less than about 3%, and preferably to about 1.5-2.0%.
  • CHI-1627663vl The pH of the system can be adjusted either before or after the addition of one or all of the enzymes to the oil composition.
  • the yield of oil is maximized based on the phospholipid composition contained in the crude.
  • this invention relates to a method in which both a Phospholipase C (PLC) enzyme and a Phospholipase A (PLA) enzyme are used together in an enzyme reaction to remove phospholipids present in oil. More specifically, this invention relates to adding in combination a Phospholipase C (PLC) and / or Phosphatidyl-Inositol specific Phospholipase C (PI-PLC) with Phospholipase Al (PLAl) and / or Phospholipase A2 (PLA2) to maximize oil yield and reduce the amount of waste products produced.
  • PLC Phospholipase C
  • PPA Phospholipase A
  • the kinetics of the enzyme reactions proceed much more rapidly than expected when the two enzymes are used together than when either one is used separately. Further, it has been found that the reactions proceed more rapidly than expected even if the reaction conditions are not optimized for at least one of the enzymes. Further, it has been found that the reaction can proceed in less than about one hour, and can proceed as quickly as about thirty minutes.
  • the oil treated can be either a crude oil or a water-degummed oil.
  • the enzymes can be added to the oil either separately or together, but the two enzymes will be in simultaneous contact with the oil.
  • enzymatic reaction parameters such as water concentration, temperature, pH, agitation time, and enzyme concentration can be controlled to optimize the reaction for a particular enzyme combination in a particular oil system.
  • FIG. 1 is illustrates the chemical structures of generic phospholipids and generic triacylglycerols.
  • FIG. 2 illustrates functional groups and structures for common phospholipids.
  • CHI-1627663V 1 [0053J FlG. 3 illustrates the positions on the phospholipid molecule at which the different types of phospholipases react.
  • FlG. 4 illustrates the reaction of a phospholipid with a PLA 1 enzyme and the resulting products.
  • FIG. 5 illustrates the reaction of a phospholipid with a PLC enzyme and the resulting products.
  • FIG. 6 is a graph summarizing the results of Examples 13-30, in which the average residual phosphorous in the degummed sample is plotted for each level of each experimental factor being evaluated.
  • FlG. 7 is a graph summarizing the results of Examples 31-38, in which the average residual phosphorous in the degummed sample is plotted for each level of each experimental factor being evaluated.
  • the present invention relates to an improvement in a process for enzymatically degumming an oil composition.
  • the inventors have found that, surprisingly, using a combination of enzymes can improve the reaction kinetics of phospholipid cleavage, In particular an enzymatic degumming process conducted with a combination of a phospholipase C enzyme with a phospholipase A enzyme provides a degummed oil product with a lower phosphorus content in a shorter reaction time than would be achieved with phospholipase A alone. Further, it has been found that surprisingly the reaction can proceed in less than about one hour, and can proceed as quickly as about thirty minutes.
  • PLA when used alone generall ⁇ requires a reaction time of at least about 4 hours, while PLC when used alone generally requires a reaction time of about one hour.
  • PLA has an optimum reaction pH of 4.5, while PLC has an optimum reaction pH of 7.0.
  • Each enzyme also has different thermal tolerances. The PLA enzyme will denature at about 50 0 C
  • CHI-1627663vl while the PLC enzyme will denature al about 65 0 C.
  • thermal stability of enzymes can be improved via site specific mutations.
  • Such cloned enzymes can be thermally stable at temperatures as high as 80°C, and the use of such cloned enzymes is contemplated in the present invention,
  • reaction time is evidenced by the PLA, When used in combination with PLC. the reaction time is dramatically reduced, and in some embodiments can be less than about 1 hour, even under acidic reaction conditions which are not optimum for PLC. The inventors further have found that under proper conditions it is possible to reduce the reaction time to as low as about 30 minutes.
  • the water concentration can be adjusted to meet the needs of a particular processing environment.
  • the water concentration can be decreased to about 1-2%, and particularly to about 1.5%, where it is desired to reduce the amount of wastewater produced by the process.
  • the water concentration can be increased to about 4-5%, and particularly to about 4.5%, where it is desired to increase the efficienc; of the degumming process.
  • Oil to be degummed can be either crude oil, or previously degummed by one of the prior art methods. It is a distinct advantage to the oil processor to be able to accomplish the oil degumming in a single step.
  • Oils that can be treated in accordance with the present invention may include but are not limited to the following: canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil. hazelnut oil, hempseed oil, linseed oil, mango kernel oil, meadowfcam oil, neat's foot oil. olive oil, palm oil, palm kernel oil, palm olein, peanut oil. rapeseed oil, rice bran oil, safllower oil, sasanqua oil. soybean oil, sunflower seed oil, tall oil, tsubaki oil. and vegetable oil.
  • the phospholipase A enzyme used in the method of the present im ention can be either a phospholipase Al enzyme or a phospholipase A2 enzyme.
  • CI IH 627663 vl enzyme used in the present invention can be either a phospholipase C or an inositol specific phospholipase C.
  • Many varieties of enzymes in the phospholipase ⁇ and phospholipase C families are available commercially; and it is contemplated that such enzymes and their equivalents will be suitable for use in the present invention.
  • the different phospholipases used together in an enzymatic degumming process of the present invention can be mixed together before being added to the oil to be treated. Alternatively, they can be added to the oil separately, either sequentially or simultaneously. Whether added sequentially or simultaneously, the enzymatic reaction will proceed at some point vvith both enzymes present in the rection mixture.
  • the degumming process of the present invention is carried out at a pH below about 8, preferable between about 3-7, and most preferably between about 4-5.
  • the pH of the enzyme degumming process can be achieved by the addition of known buffers. Citric acid and sodium hydroxide are well known to be suited to this purpose. Other buffering agents can be used as needed to adjust the pH under specific reaction conditions.
  • the temperature of the enzymatic degumming process of the present invention can be in the range of about 40-80 0 C, preferably in the range of about 40-60 0 C, and more preferably in the range of about 45-55°C. Tt has been found that, surprisingly, under the methods of the present invention PL ⁇ degumming can proceed at a temperature above its own optimum of 45°C. and closer to the optimum operating temperature of PLC, without excessive denaturing.
  • the method of the present invention provides a single step degumming process in which the phospholipids content of an oil, even a crude oil, can be reduced to less than 50 ppm P, preferably less than 20 ppm P 5 more preferably less than 10 ppm P, and most preferably less than 5 ppm P.
  • the degummed oil can be subjected to further processing steps known in the art such as bleaching or deodorizing, as may be necessary or desirable depending on the end use for which the degummed oil product is intended.
  • the overhead mixer was a Heidolph mixer model Elector KG with a flat blade paddle; operated at 90 rpm for normal agitation and 350 rpm for vigorous agitation.
  • the centrifuge was a De Laval Gyro - Tester installed with ''The Bowl Unit" for continuous separation. The centrifuge bowl was closed with the plug screws installed. Shear mixing was accomplished with an Ultra-Turrax homogenizer SD-45 with a G450 rotor stator at 10,000 rpm.
  • the PLAl enzyme was Lecitase® Ultra (lot number LYN050070) sold by Novozymes A/S of Denmark, and having a concentration of 11.2 Units/mg.
  • the PLA2 enzyme was Rohalase® MPL (Lot number Ch: 4738) sold by AB Knzymes located in Germany, and having a concentration of 2000 Units/mg.
  • the PLC enzyme was Purifine 1 M sold by Diversa Corporation of San Diego, California. For examples 1-12, the PLC was Lot BD16449, having a concentration of 205 Units/mg. For Examples 13-38, the PLC was Lot 90BU002A1 , having a concentration of 27.5 Units/mg.
  • the amount of phospholipids remaining in the treated oil was measured as ppm P in accordance with the method of American Oil Chemists' Society Official Method Ca 20-99, "'Analysis of Phosphorus in Oil by Inductively Coupled Plasma Optical Emission Spectroscopy.”
  • Control Water Degumming - 1965.4 grams of crude soybean oil containing 746 ppm phosphorous was heated to 70 - 75 0 C under normal agitation utilizing an overhead mixer. To the warm oil, 39.4 grams of de-ionized water was added with vigorous agitation for
  • Control Single enzyme degumming with Phospholipase Al (PLAl)-- 1997.9 g of crude soybean oil containing 746 ppm phosphorous was heated to 75 - 80 0 C under agitation utilizing an overhead mixer. 2.0 grams of 50 % Ww solution of citric acid was added and the mixture was sheared for 1 minute. The oil underwent normal agitation for one hour with an overhead mixer. The oil was allowed to cool with agitation at normal speed until the oil temperature was at 40 - 45 0 C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was shear mixed for 10 seconds. The citric acid and caustic formed a weak buffer with a pH of 4.5.
  • Control Single enzyme degumming with Phospholipase C (PLC)- 201 1.1 grams of crude soybean oil containing 746 ppm phosphorous was heated to 55 - 60 0 C under normal agitation utilizing an overhead mixer. 60.3 grams of de-ionized water was added and the mixture was shear mixed for 1 minute. 0.1051 grams of Diversa ' s PurifmeTM (PLC lipase BD 16449 containing 205 U/mg) was added and the mixture was sheared for 1 minute. The oil
  • CHI-1627663vi mixture underwent normal agitation for 1 hour at 50 - 63 0 C.
  • the enzyme treated oil was then centrifuged, and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC degummed oil was 70,9 ppm.
  • Control PLC followed by PLA Degumming -
  • the oil sample is reacted with each enzyme separately under the reaction conditions optimum for that enzyme, in accordance with the prior art, 2110.5 grams of crude soybean oil containing 560.1 ppm phosphorous was heated to 60 0 C under normal agitation.
  • 63 grams of dc-ionizcd water and 0.1123 grams of Diversa's PurifineTM (PLC lipase BDl 6449 containing 205 U/mg) were added and the mixture sheared for 1 minute.
  • the oil mixture was agitated at normal speed for 1 hour at 55 - 56 0 C.
  • the oil was then centrifuged, and the oil and wet gums were collected.
  • PLC and P LA 1 together, neutral pi I with a 4 hour reaction time at 45°C ⁇ 2003.7 grams of crude soybean oil containing 560.1 ppm phosphorus was heated to 45 0 C under normal agitation. 60 grams of de-ionized water, 0,1040 grams of Diversa ' s Purf ⁇ neTM (PLC enzyme) and 0.1085 grams of Novozymes * Lecitase® Ultra (PLAl enzyme) were added and the entire mixture was sheared for 1 minute. The oil mixture was agitated at normal speed for 4 hours at a temperature of approximately 45 0 C. The oil was then centrifuged, and the separated oi! and wet gums were collected. The process using the PLC and PLAl combined enzyme mixture with four hours of reaction time at a neutral pH produced a degummed oil with a residual phosphorous of 10.5 ppm,
  • This residual phosphorous value is about the same as that achieved in Example 7 indicating that an increase of the reaction time from one hour to four hours did not make a significant difference in the efficacy of the degumming process.
  • This residual phosphorous value is about the same as that achieved in Examples 7 and 8. indicating that an increase of the reaction temperature from about 45°C to about 55°C
  • the oil mixture was agitated at normal speed for 60 minutes at a temperature of 40 — 45 0 C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC and PL ⁇ 2 combined enzyme mixture at neutral pH produced a de gummed oil with a residual phosphorous of 3.3 ppm.
  • Example 7 This example is similar to Example 7 above, but for the substitution of PLA2 for PLAl .
  • the low residual phosphorous level in the finished product demonstrates that PLA2 can function about equally well as PLAl in the method of the present invention.
  • CHI-1627663V I mixture was agitated at normal speed for 60 minutes at a temperature of 40 0 C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC then PLAl sequential degummed oil was 6.5 ppm.
  • the oil mixture was agitated at normal speed for 15 minutes at a temperature of 40 0 C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC then PLAl sequential degummed oil was 27.4 ppm.
  • CHI-1627663vl for 45 seconds.
  • the oil mixture was agitated at normal speed for 15 minutes at a temperature of 50 0 C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC then PLAl sequential deguramed oil was 79.3 ppm.
  • CHl- 1627663V underwent normal agitation for 1 hour with an overhead mixer.
  • the oil was allowed to cool with agitation at normal speed until the oil temperature was 60 0 C, then 2.4 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was shear mixed for 10 seconds.
  • the citric acid and caustic formed a weak buffer with a pH of 5.0.
  • CHM 627663V 1 (PLAl lipase lot number LYN05007) was added and the entire mixture was shear mixed for 45 seconds. The oil mixture was agitated at normal speed for 30 minutes at a temperature of 40 0 C. The enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected. The residual phosphorous in the PLC and PLAl sequential treated degummed oil had a residual phosphorous of 2.1 ppm.
  • CHJ-1627663V 1 number LYN05007 was added followed by 60 grams of de-ionized water was and the entire mixture was shear mixed for 60 seconds.
  • the oil mixture was agitated at normal speed for 60 minutes at a temperature of 50 0 C.
  • the enzyme treated oil was then cenlriiuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC and PLAl combined enzyme mixture produced a degummed oil with a residual phosphorous of 7.2 ppm.
  • the oil mixture was agitated at normal speed for 60 minutes at a temperature of 50 0 C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC and PLA 1 sequential treated degummed oil at a neutral pH had a residual phosphorous of 72.6 ppm.
  • CHI-1627663V 1 LYN05007 was added followed b ⁇ 30 grams of de-ionizcd water and the entire mixture was shear mixed for 45 seconds.
  • the oil mixture was agitated at normal speed for 30 minutes at a temperature of 40 0 C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC and PLAl combined enzyme mixture at a pH of 4.5 produced a degummed oil with a residual phosphorous of 13.7 ppm.
  • FiG. 6 is a plot of the average final phosphorous amount at each level of each factor.
  • Ci ⁇ -1627663vl combination of enzymes added sequentiall ⁇ or together produced acceptable residual phosphorous levels in oils that would allow them to be physically refined.
  • an acidic pll is employed, only one of the experimental runs failed to produce an oil with a residual phosphorous of less than 10 ppm.
  • Example 30 had a residual phosphorous level greater than 10 (13.7 ppm), and was produced with the lowest levels of both enzymes, the lowest temperature, the lowesl percentage of water, the shortest mixing and agitation times, and the most acidic pH.
  • CHI-1627663v S maintained at 60 0 C, 0.7509 grams of Diversa's PurifmeTM (PLC lipase lot number 90BU002A1) and 0.1105 grams Novozymes : Lecitase® Ultra (PLAl lipase lot number LYN05007) was added followed by 90 grams of de-ionized water and the entire mixture was shear mixed for 45 seconds. The oil mixture was agitated at normal speed for 120 minutes at a temperature of 60 0 C. The enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected. The residual phosphorous in the PLC and PLAl combined enzyme mixture at a pH of 4.5 produced a degummed oil with a residual phosphorous of 6.7 ppm.
  • the oil mixture was agitated at normal speed for 30 minutes at a temperature of 40 0 C, The enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected, lhe residual phosphorous in the PLC and PLAl combined enzyme mixture at a pH of 4.5 produced a degummed oil with a residual phosphorous of 2.2 ppm.
  • CHI- i 627663V I grams of 50 % w/w solution of citric acid was added and sheared for 1 minute. The oil underwent normal agitation for 1 hour with an overhead mixer. The oil was allowed to cool with agitation at normal speed until the oil temperature was 60 0 C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was shear mixed for 10 seconds. The citric acid and caustic formed a weak buffer with a pH of 4.5.
  • CHI-1627663vl maintained at 40 0 C, 0.7422 grams of Diversa's PurifmeTM (PLC lipase lot number 90BU002A! ) and 0.1 195 grams Novo/ymes' Lecitase® Ultra (PLAl lipase lot number LYN05007) was added followed by ⁇ O grains of de-ionized water and the entire mixture was shear mixed for 45 seconds. The oil mixture was agitated at normal speed for 30 minutes at a temperature of 40 0 C. The enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected. The residual phosphorous in the PLC and PLAl combined enzyme mixture at a pH of 4.5 produced a degummed oil with a residual phosphorous of 6.7 ppm.
  • FIG. 7 is a chart summarizing Examples 31-38. plotting the average final phosphorous amount at each level of each factor holding pH, PLA dosage, and combined addition constant,
  • CHI-] 627663 vl ppm phosphorous in the final product could be achieved under the proper conditions with reaction times as low as about thirty minutes. Further, without wishing to be bound by theory. it appears that either the PLC enzyme or one of its hydrolysis reaction products is catalyzing the reaction of the PLA enzyme, allowing for the reaction time to be significantly less than the reaction time for either of the single enzymes. These results are unexpected based on the known optimum reaction parameters for PLA and PLC enzymes.
  • the concentrations of the PLA and PLC enzymes to be used in a particular run the choice will depend on whether the goal is to run at the lowest possible cost or the greatest possible performance. If the goal is to run at the lowest possible cost, then the concentration of PLA can be less than about 2,0 ppm, preferably less than about 1.0 ppm, and most preferably less than about 0.5 ppm. Such a low concentration of the PLA enzyme can still provide effective degumming in many situations.
  • the concentration of PLA is preferably at least about 0.5 ppm, more preferably at least about 1.0 ppm, and most preferably 2.0 ppm.
  • the pH can be about 7.0, while pH of about 5.0 is preferable and pH of about 4.5 is presently preferred.
  • the concentration of water in the system can be generally about 3.0%, but can be as low as about 1.5% if reduced wastewater is desired, or as high as about 4.5% if greater degumming efficiency is desired.
  • the reaction temperature can be as high as about 60 0 C, but is more preferably less than about 50 0 C, and surprisingly most preferable at about 40 0 C.
  • CHi-l ⁇ 27663vl time during initial mixing is can be about 45 seconds, is more preferably about 60 seconds, and is most preferably about 120 seconds
  • the duration of the enzyme reaction can be greatly reduced, in some embodiments to be advantageously less than about 60 minutes, and preferably about 30 minutes.

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Abstract

L'invention concerne un procédé de dégommage d'une composition huileuse qui comprend les étapes consistant à (a) obtenir une composition huileuse contenant une quantité de phospholipides, (b) mettre en contact ladite composition huileuse simultanément avec une ou plusieurs enzymes de phospholipase A et une ou plusieurs enzymes de phospholipase C, dans des conditions suffisantes pour que les enzymes réagissent avec les phospholipides pour créer des produits de réaction de phospholipide, et (c) séparer les produits de réaction de phospholipides de la composition huileuse, la composition huileuse restante après la séparation étant une composition huileuse dégommée de sorte que, pendant l'étape (b), la réaction de ladite ou desdites enzymes de phospholipase A a lieu à une vitesse plus rapide qu'en l'absence de ladite ou desdites enzymes de phospholipase C. Dans un mode de réalisation préféré, la réaction de l'étape (b) se poursuit pendant une durée inférieure à environ une heure.
PCT/US2008/052162 2007-01-30 2008-01-28 Dégommage enzymatique utilisant un mélange de phospholipases pla et plc WO2008094847A1 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
EP08728362.8A EP2118248B1 (fr) 2007-01-30 2008-01-28 Dégommage enzymatique utilisant un mélange de phospholipases pla et plc
DK08728362.8T DK2118248T3 (da) 2007-01-30 2008-01-28 Enzymatisk degummiering under anvendelse af en blanding af PLA- og PLC-phospholipaser
UAA200908943A UA97972C2 (ru) 2007-01-30 2008-01-28 Ферментативное обессмоливание масляной композиции с использованием смеси фосфолипаз pla и plc
BRPI0808024A BRPI0808024B1 (pt) 2007-01-30 2008-01-28 método para desengomar uma composição de óleo
ES08728362.8T ES2523300T3 (es) 2007-01-30 2008-01-28 Desgomado enzimático utilizando una mezcla de fosfolipasas PLA y PLC
PL08728362T PL2118248T3 (pl) 2007-01-30 2008-01-28 Enzymatyczne odśluzowywanie wykorzystujące mieszaninę fosfolipaz PLA i PLC
MX2009007919A MX2009007919A (es) 2007-01-30 2008-01-28 Desgomado enzimatico que utiliza una mezcla de fosfolipasas pla y plc.
RU2009132518/10A RU2477746C2 (ru) 2007-01-30 2008-01-28 Ферментативное обессмоливание с использованием смеси фосфолипаз pla и plc
CA2676412A CA2676412C (fr) 2007-01-30 2008-01-28 Degommage enzymatique utilisant un melange de phospholipases pla et plc

Applications Claiming Priority (4)

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US11/668,921 US8956853B2 (en) 2007-01-30 2007-01-30 Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases
US11/668,921 2007-01-30
US11/853,339 2007-09-11
US11/853,339 US8460905B2 (en) 2007-09-11 2007-09-11 Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases with reduced reaction time

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WO2011051322A1 (fr) 2009-10-28 2011-05-05 Ab Enzymes Gmbh Clonage, expression et utilisation de phospholipases acides
USRE43135E1 (en) 2001-05-18 2012-01-24 Danisco A/S Method of improving dough and bread quality
USRE43341E1 (en) 1995-06-07 2012-05-01 Danisco A/S Method of improving the properties of a flour dough, a flour dough improving composition and improved food products
WO2012062817A1 (fr) * 2010-11-12 2012-05-18 Novozymes A/S Polypeptides ayant une activité phospholipase c et polynucléotides codant pour ceux-ci
US8278062B2 (en) 2003-01-14 2012-10-02 Dupont Nutrition Biosciences Aps Method of using lipid acyltransferase
US8435766B2 (en) 2009-03-19 2013-05-07 N.V. Desmet Ballestra Engineering S.A. Enzymatic oil recuperation process
EP2592133A1 (fr) * 2011-11-09 2013-05-15 Alfa Laval Corporate AB Dégommage enzymatique
WO2013121047A1 (fr) 2012-02-17 2013-08-22 Clariant Produkte (Deutschland) Gmbh Composition pour la démucilagination enzymatique d'huiles
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EP2792735A1 (fr) 2013-04-16 2014-10-22 Clariant Produkte (Deutschland) GmbH Procédé d'amélioration de la démucilagination enzymatique aqueuse d'huiles végétales
EP2799531A1 (fr) 2013-05-03 2014-11-05 Clariant Produkte (Deutschland) GmbH Utilisation de phosphatases pour la démucilagination enzymatique de triglycérides
US8889371B2 (en) 2004-07-16 2014-11-18 Dupont Nutrition Biosciences Aps Lipolytic enzyme: uses thereof in the food industry
EP2910129A1 (fr) 2014-02-21 2015-08-26 Clariant Produkte (Deutschland) GmbH Composition pour la démucilagination enzymatique d'huiles
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WO2015140275A1 (fr) * 2014-03-19 2015-09-24 Novozymes A/S Polypeptides présentant une activité phospholipase c et polynucléotides codant pour ceux-ci
WO2015173426A1 (fr) 2014-05-15 2015-11-19 Novozymes A/S Compositions comprenant des polypeptides ayant une activité phospholipase c et leur utilisation
WO2016162456A1 (fr) 2015-04-09 2016-10-13 Dsm Ip Assets B.V. Phospholipase c

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WO2018186734A1 (fr) * 2017-04-06 2018-10-11 Purac Biochem B.V. Démucilagination enzymatique d'huile de triglycéride non raffinée
CN108085131A (zh) * 2017-12-25 2018-05-29 无锡德合食品科技有限公司 低温技术集成制备高品质菜籽油的方法
CN115678674A (zh) * 2022-10-31 2023-02-03 武汉轻工大学 脱胶米糠毛油及利用吸附剂和复合磷脂酶精炼米糠油的脱胶方法

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Cited By (41)

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USRE43341E1 (en) 1995-06-07 2012-05-01 Danisco A/S Method of improving the properties of a flour dough, a flour dough improving composition and improved food products
USRE43135E1 (en) 2001-05-18 2012-01-24 Danisco A/S Method of improving dough and bread quality
US8278062B2 (en) 2003-01-14 2012-10-02 Dupont Nutrition Biosciences Aps Method of using lipid acyltransferase
US8927036B2 (en) 2004-07-16 2015-01-06 Dupont Nutrition Biosciences Aps Enzymatic oil-degumming method
US8889371B2 (en) 2004-07-16 2014-11-18 Dupont Nutrition Biosciences Aps Lipolytic enzyme: uses thereof in the food industry
WO2009081094A2 (fr) * 2007-12-21 2009-07-02 Danisco A/S Procédé
WO2009081094A3 (fr) * 2007-12-21 2010-03-04 Danisco A/S Procédé
US9228211B2 (en) 2007-12-21 2016-01-05 Dupont Nutrition Biosciences Aps Process of water degumming an edible oil
EA020035B1 (ru) * 2007-12-21 2014-08-29 ДюПон НЬЮТРИШН БАЙОСАЙЕНСИЗ АпС Способы
US8435766B2 (en) 2009-03-19 2013-05-07 N.V. Desmet Ballestra Engineering S.A. Enzymatic oil recuperation process
US9045713B2 (en) 2009-10-28 2015-06-02 Ab Enzymes Gmbh Cloning, expression and use of acid phospholipases
US9322003B2 (en) 2009-10-28 2016-04-26 Ab Enzymes Gmbh Cloning, expression and use of acid phospholipases
DE102009051013A1 (de) 2009-10-28 2011-06-09 Ab Enzymes Gmbh Klonierung, Expression und Verwendung saurer Phospholipasen
WO2011051322A1 (fr) 2009-10-28 2011-05-05 Ab Enzymes Gmbh Clonage, expression et utilisation de phospholipases acides
CN103314091A (zh) * 2010-11-12 2013-09-18 诺维信公司 具有磷脂酶c活性的多肽及其编码多核苷酸
US9538771B2 (en) 2010-11-12 2017-01-10 Novozymes A/S Polypeptides having phospholipase C activity and polynucleotides encoding same
US9279110B2 (en) 2010-11-12 2016-03-08 Novozymes A/S Polynucleotides encoding polypeptides having phospholipase C activity
WO2012062817A1 (fr) * 2010-11-12 2012-05-18 Novozymes A/S Polypeptides ayant une activité phospholipase c et polynucléotides codant pour ceux-ci
EP2592133A1 (fr) * 2011-11-09 2013-05-15 Alfa Laval Corporate AB Dégommage enzymatique
WO2013121047A1 (fr) 2012-02-17 2013-08-22 Clariant Produkte (Deutschland) Gmbh Composition pour la démucilagination enzymatique d'huiles
US9677027B2 (en) * 2012-02-17 2017-06-13 Clariant Produkte (Deutschland) Gmbh Composition for enzymatic oil degumming
US20150017708A1 (en) * 2012-02-17 2015-01-15 Clariant Produkte (Deutschland)Gmbh Composition for enzymatic oil degumming
US10329514B2 (en) 2012-10-31 2019-06-25 Alfa Laval Corporate Ab Enzymatic degumming
WO2014067569A1 (fr) * 2012-10-31 2014-05-08 Alfa Laval Corporate Ab Démucilagination enzymatique
US11180742B2 (en) 2012-12-11 2021-11-23 Novozymes A/S Polypeptides having phospholipase C activity and polynucleotides encoding same
CN104884464A (zh) * 2012-12-11 2015-09-02 诺维信公司 具有磷脂酶c活性的多肽以及对其进行编码的多核苷酸
CN104884464B (zh) * 2012-12-11 2021-02-02 诺维信公司 具有磷脂酶c活性的多肽以及对其进行编码的多核苷酸
US10745676B2 (en) 2012-12-11 2020-08-18 Novozymes A/S Polypeptides having phospholipase C activity and polynucleotides encoding same
WO2014170021A1 (fr) 2013-04-16 2014-10-23 Clariant Produkte (Deutschland) Gmbh Procédé d'amélioration de la démucilagination enzymatique aqueuse d'huiles végétales
EP2792735A1 (fr) 2013-04-16 2014-10-22 Clariant Produkte (Deutschland) GmbH Procédé d'amélioration de la démucilagination enzymatique aqueuse d'huiles végétales
EP2799531A1 (fr) 2013-05-03 2014-11-05 Clariant Produkte (Deutschland) GmbH Utilisation de phosphatases pour la démucilagination enzymatique de triglycérides
EP2910129A1 (fr) 2014-02-21 2015-08-26 Clariant Produkte (Deutschland) GmbH Composition pour la démucilagination enzymatique d'huiles
US10351795B2 (en) 2014-03-19 2019-07-16 Novozymes A/S Polypeptides having phospholipase C activity and polynucleotides encoding same
WO2015140275A1 (fr) * 2014-03-19 2015-09-24 Novozymes A/S Polypeptides présentant une activité phospholipase c et polynucléotides codant pour ceux-ci
US10954469B2 (en) 2014-03-19 2021-03-23 Novozymes A/S Polypeptides having phospholipase C activity and polynucleotides encoding same
EP4163354A2 (fr) 2014-03-19 2023-04-12 Novozymes A/S Polypeptides présentant une activité phospholipase c et polynucléotides codant pour ceux-ci
EP4163354A3 (fr) * 2014-03-19 2023-08-30 Novozymes A/S Polypeptides présentant une activité phospholipase c et polynucléotides codant pour ceux-ci
US11851630B2 (en) 2014-03-19 2023-12-26 Novozymes A/S Polypeptides having phospholipase C activity and polynucleotides encoding same
CN106459934A (zh) * 2014-05-15 2017-02-22 诺维信公司 包括具有磷脂酶c活性的多肽的组合物及其应用
WO2015173426A1 (fr) 2014-05-15 2015-11-19 Novozymes A/S Compositions comprenant des polypeptides ayant une activité phospholipase c et leur utilisation
WO2016162456A1 (fr) 2015-04-09 2016-10-13 Dsm Ip Assets B.V. Phospholipase c

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BRPI0808024B1 (pt) 2017-05-16
EP2118248A1 (fr) 2009-11-18
BRPI0808024A2 (pt) 2014-06-17
RU2009132518A (ru) 2011-03-10
CN105038978A (zh) 2015-11-11
CN105038978B (zh) 2017-09-29
PL2118248T3 (pl) 2015-03-31
EP2118248B1 (fr) 2014-08-27
CA2676412A1 (fr) 2008-08-07
MX2009007919A (es) 2009-08-27
RU2477746C2 (ru) 2013-03-20
CA2676412C (fr) 2015-10-06
DK2118248T3 (da) 2014-11-03

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