EP2118248B1 - 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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EP2118248B1
EP2118248B1 EP08728362.8A EP08728362A EP2118248B1 EP 2118248 B1 EP2118248 B1 EP 2118248B1 EP 08728362 A EP08728362 A EP 08728362A EP 2118248 B1 EP2118248 B1 EP 2118248B1
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oil
enzyme
ppm
enzymes
grams
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EP2118248A1 (fr
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Christopher L.G. Dayton
Erin Marie Rosswurm
Flavio Da Silva Galhardo
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Bunge Oils Inc
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Bunge Oils Inc
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Priority claimed from US11/668,921 external-priority patent/US8956853B2/en
Priority claimed from US11/853,339 external-priority patent/US8460905B2/en
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Priority to PL08728362T priority Critical patent/PL2118248T3/pl
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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

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  • 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.
  • Crude vegetable oils obtained from either pressing or solvent extraction methods are a complex mixture of triacylglycerols, phospholipids, sterols, tocopherols, free fatty acids, trace metals, and other minor compounds. It is desirable to remove the phospholipids, free fatty acids and trace metals in order to produce a quality salad oil with a bland taste, light color, and a long shelf life.
  • phospholipids contain a phosphate group on one of the two ends of the glycerol backbone, whereas a triacylglycerol 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 R1 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 phospholipid's phosphate-containing functional group denoted in FIG. 1 as "X" determines the degree of its hydrophilic nature.
  • the functional group X in FIG. 1 may be any of several of a variety of known 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 phosphatidic 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.
  • Table 1 Typical levels and phospholipid distributions for common oilseeds. Soy Oil Canola Oil Sunflower Oil P (ppm) 400 - 1200 200 - 900 300 - 700 PC (-choline) 12% - 46% 25% - 40% 29% - 52% PE (-ethanolamine) 8% - 34% 15 - 25% 17% - 26% PA (-acid) 2% - 21% 10% - 20% 15% - 30% PI (-inositol) 2% - 15% 2% - 25% 11% - 22%
  • 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 emulsifier).
  • 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 PLA1 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 PI, 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). This 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.
  • This technique is usually applied to crude oils when the goal is the total removal of phospholipids.
  • the oil obtained is usually called “super-degummed” or “totally degummed” in the industry.
  • Crude oil is treated with 250 to 2000 ppm of phosphoric acid or citric acid at 60 - 90 °C with vigorous mixing.
  • the acid is allowed to react with the salts 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 °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 degumming process contain at least one molecule of triacylolycerol (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.
  • This technique is usually applied to crude or water degummed oils when the goal is to remove all of the phospholipids and free fatty acids.
  • 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 the acid-treated oil at 65 - 75 °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 °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 deodorization to produce salad oil.
  • An alternative to water washing is to treat the caustic treated oil with an absorbent silica gel, and filter out the residual soaps and phospholipids not removed in the initial centrifugation.
  • 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 refining 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 deodorization, 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 PLA1, PLA2, PLC, and PLD. The positions on the phospholipid molecule at which the different types of phospholipases react are illustrated in FIG. 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°C while PLC enzymes will denature at about 65°C.
  • One commercial PLA1 enzyme product with phospholipase activity is Novozymes' phospholipase A1 Lecitase® Ultra. This product is known to yield polar lysophospholipids 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 PLA1 selectively hydrolyzes the fatty acid opposite the phosphate functional group on the glycerol backbone, as illustrated in FIG. 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.
  • the PLA1 degumming 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 pH, 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.
  • PLC enzymes react with a phospholipid by selectively hydrolyzing the phosphate functional group, as shown in FIG. 5 .
  • the resulting reaction yields a diacylglycerol ("DAG") and a phosphatidic group.
  • DAG diacylglycerol
  • the diacylglycerol molecule no longer has the phosphate functional group and does not need to be removed.
  • the PLC degumming process reduces the refining loss by retaining the original phospholipid molecule, while removing only the phosphate functional group.
  • PLC does not react with all of the phospholipids present in the oil.
  • PLC does not react with either phosphatidic acid (PA) or phosphatidic inositol (PI), illustrated in FIG. 2 .
  • PA and PI are non-hydratable phosphatides that remain in oil after water degumming.
  • the PLC-treated oil must be further treated with caustic to remove the residual gums.
  • PI-PLC PI-specific PLC
  • U.S. 5,264,367 to Aalrust et al. describes the use of phospholipases A1, A2, or B to treat oil that has first been refined to 50 to 250 ppm phosphorous.
  • the technology described in this patent is known commercially as Enzymax®.
  • Aalrust states that since these enzymes attack lecithin, "it would make no sense to use the method of the invention on oils having a high content of lecithin, such as raw soybean oil.” The reaction is carried out at a temperature of 20 - 80 °C, with citric acid or a salt thereof at a pH range of 3-7. It is stated that the enzyme should be thoroughly distributed in the oil, with the enzyme-water solution present as droplets smaller than 10 ⁇ m in diameter.
  • 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® - Lurgi's route to Physical Refining in Proceeding of the World Conference on Oilseed and Edible Oils Processing, Istanbul, Turkey, 1996, ed. Kaseoglu, Rhee and Wils on; Dalke, K. et al..
  • 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 A1, 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 A1 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 calculations of the emulsion drop size weight average were not disclosed. The patent states that the resulting product will have residual P of 15 ppm or less. It is known in the art that submitting the oil to pH as low as 4, or lower, will cause gums present in the oil to become hydrated and to separate from the reaction medium. The hydrated gums will act as emulsifiers. such that when they are separated they will carry oil with them, thus causing oil loss. No data on oil loss in the gums is presented.
  • U.S. 6,103,505 to Clausen et al. discloses the discovery and activity of certain phospholipase (A1, 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. 6,127,137 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 (A1, 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 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 A1 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 A1 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 PLC's of the invention can be used in addition to or in place of PLA1s and PLA2s in commercial oil degumming, such as in the ENZYMAX® process, where phospholipids are hydrolyzed by PLA1 and PLA2.
  • the application states that PLC may be used alone or with PLA to remove non-hydratable phospholipids from oil that previously has 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 phospholipase C, D1 and D2 may be employed in the enzymatic degumming of previously degummed and non-degumnned (crude) oils and as an aid to caustic refining.
  • WO 99/53001 A1 to Clausen et al. discloses a process for enzymatic reducing the content of phosphorus containing components in an edible oil.
  • the method comprises the use of phospholipase and a low amount of water.
  • the enzymatic degumming of oil is performed at a pH from 1.5 to 8 with an aqueous solution of a phospholipase A1, phospholipase A2, or phospholipase B.
  • WO 99/53001 A1 does neither teach that the degumming reaction can be conducted with a combination of phospholipase A and phospholipase C enzymes nor provide reaction conditions for use of the two enzymes together.
  • a vegetable oil to be degummed is adjusted to a pH of 3 to 6 and mixed with an aqueous enzyme solution which contains one of the enzymes phospholipase A1, A2 or B.
  • the enzymes are allowed to act in the oil with stirring at temperatures from 20 to 90°C in a degumming reactor.
  • a separation auxiliary or a solubiliser is added to the liquid drawn off from the degumming reactor at temperatures from 20 to 90°C before or after separating off the degummed oil.
  • a largely sludge-free, solution containing used enzyme is obtained which is at least partly recycled ahead of the degumming reactor.
  • the proportion of used, recycled enzymes in the total amount of enzymes dispersed in the oil is at least 10 %.
  • DE 4339556 C1 does neither teach that the degumming reaction can be conducted with a combination of phospholipase A and phospholipase C enzymes nor provide reaction conditions for use of the two enzymes together.
  • JP 06306386 A to Tsuruoka et al. discloses the enzymatic degumming of oil with phospholipase C enzyme. JP 06306386 A does not provide reaction conditions for use of the combination of the two enzymes phospholipase A and phospholipase C together including the quantity of phospholipase A and phospholipase C enzymes.
  • the invention relates to a method for degumming an oil composition, the method comprising
  • 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%.
  • 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 A1 (PLA) and / or Phospholipase A2 (PA2) to maximize oil yield and reduce the amount of waste products produced.
  • PLC Phospholipase C
  • PPA Phospholipase A2
  • 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.
  • 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.
  • 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.
  • the reaction can proceed in less than about one hour, and can proceed as quickly as about thirty minutes.
  • 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.
  • PLA has an optimum reaction pH of 4.5
  • PLC has an optimum reaction pH of 7.0.
  • Each enzyme also has different thermal tolerances.
  • the PLA enzyme will denature at about 50°C while the PLC enzyme will denature at about 65°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.
  • 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 efficiency of the degumming process.
  • the 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, meadowfoam oil, neat's foot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, soybean oil, sunflower seed oil, tall oil, tsubaki oil, and vegetable oil.
  • canola oil castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hempseed oil, linseed oil, mango kernel oil, meadowfoam oil, neat's foot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, rapeseed oil, rice bran oil, safflower oil, sasanqua
  • the phospholipase A enzyme used in the method of the present invention can be either a phospholipase A1 enzyme or a phospholipase A2 enzyme.
  • the phospholipase C 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 A 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 with both enzymes present in the reaction 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°C, preferably in the range of about 40-60°C, and more preferably in the range of about 45-55°C. It has been found that, surprisingly, under the methods of the present invention PLA 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, 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 PLA1 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 Enzymes located in Germany, and having a concentration of 2000 Units/mg.
  • the PLC enzyme was Purifine TM 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 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 °C under normal agitation.
  • 63 grams of de-ionized water and 0.1123 grams of Diversa's PurifineTM (PLC lipase BD16449 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 °C.
  • the oil was then centrifuged, and the oil and wet gums were collected.
  • 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.
  • Example 7 This example is similar to Example 7 above, but for the substitution of PLA2 for PLA1.
  • the low residual phosphorous level in the finished product demonstrates that PLA2 can function about equally well as PLA1 in the method of the present invention.
  • the oil mixture was agitated at normal speed for 15 minutes at a temperature of 40 °C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC then PLA1 sequential degummed oil was 27.4 ppm.
  • the oil mixture was agitated at normal speed for 15 minutes at a temperature of 50 °C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC then PLA1 sequential degummed oil was 79.3 ppm.
  • PLC 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 °C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC and PLA1 sequential treated degummed oil had a residual phosphorous of 2.1 ppm.
  • the oil mixture was agitated at normal speed for 60 minutes at a temperature of 50 °C.
  • the enzyme treated oil was then centrifuged; and the separated oil and wet gums were collected.
  • the residual phosphorous in the PLC and PLA1 sequential treated degummed oil at a neutral pH had a residual phosphorous of 72.6 ppm.
  • FIG. 6 is a plot of the average final phosphorous amount at each level of each factor.
  • 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.
  • 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°C, but is more preferably less than about 50°C, and surprisingly most preferable at about 40°C.
  • the agitation 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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Claims (13)

  1. Procédé de dégommage d'une composition d'huile, le procédé comprenant les étapes consistant à
    (a) fournir une composition d'huile contenant une quantité de phospholipides,
    (b) mettre en contact ladite composition d'huile simultanément avec une ou plusieurs enzymes phospholipase A en une quantité d'environ 2 ppm d'enzyme active ou moins et une ou plusieurs enzymes phospholipase C en une quantité d'environ 30 ppm d'enzyme active ou moins, dans des conditions suffisantes pour que les enzymes réagissent avec les phospholipides pour créer des produits réactionnels phospholipidiques, et
    (c) séparer les produits réactionnels phospholipidiques de la composition d'huile, la composition d'huile restante après la séparation étant une composition d'huile dégommée,
    grâce auquel, pendant l'étape (b), la réaction desdites une ou plusieurs enzymes phospholipase A a lieu à une vitesse plus rapide qu'en cas d'absence desdites une ou plusieurs enzymes phospholipase C, la durée de la réaction des enzymes avec les phospholipides étant inférieure à une heure, et dans lequel ladite réaction des enzymes avec les phospholipides étant effectuée à un pH d'environ 3 à 7 et à une température d'environ 40 à 80°C, et la composition d'huile dégommée de l'étape (c) présentant une teneur en phospholipides, mesurée en parties par million de phosphore, d'environ 20 ppm ou moins.
  2. Procédé selon la revendication 1, dans lequel la durée de la réaction des enzymes avec les phospholipides est d'environ trente minutes.
  3. Procédé selon la revendication 1, dans lequel lesdites une ou plusieurs enzymes phospholipase A sont sélectionnées parmi le groupe constitué d'une enzyme phospholipase A1 et une enzyme phospholipase A2.
  4. Procédé selon la revendication 1, dans lequel lesdites une ou plusieurs enzymes phospholipase C sont sélectionnées parmi le groupe constitué d'une enzyme phospholipase C et d'une enzyme phospholipase C spécifique au phosphatidylinositol.
  5. Procédé selon la revendication 1, dans lequel ladite réaction des enzymes avec les phospholipides a lieu à un pH d'environ 4 à 5.
  6. Procédé selon la revendication 1, dans lequel ladite réaction des enzymes avec les phospholipides a lieu à une température d'environ 40 à 60°C, ou à une température d'environ 45 à 55°C.
  7. Procédé selon la revendication 1, dans lequel ladite composition d'huile comprend une huile brute, ou une huile précédemment dégommée.
  8. Procédé selon la revendication 1, dans lequel ladite enzyme PLC est présente en une quantité d'environ 20 ppm d'enzyme active ou moins, ou d'environ 10 ppm d'enzyme active ou moins.
  9. Procédé selon la revendication 1, dans lequel ladite enzyme PLA est présente en une quantité d'environ 1 ppm d'enzyme active ou moins, ou d'environ 0,5 ppm d'enzyme active ou moins.
  10. Procédé selon la revendication 1, dans lequel, pendant l'étape (b), la préparation constituée de la composition d'huile et des enzymes est initialement mélangée avec cisaillement, de manière préférée dans lequel ledit mélange avec cisaillement continue pendant une durée d'au moins environ 45 secondes.
  11. Procédé selon la revendication 1, dans lequel, pendant l'étape (b), une quantité d'eau est ajoutée, de manière préférée dans lequel ladite quantité d'eau est au moins d'environ 1,5 % en poids de la préparation totale, est au moins d'environ 3,0 % en poids de la préparation totale, ou est au moins d'environ 4,5 % en poids de la préparation totale.
  12. Procédé selon la revendication 1, dans lequel la composition d'huile dégommée de l'étape (c) présente une teneur en phospholipides, mesurée en parties par million de phosphore, d'environ 10 ppm ou moins, ou d'environ 5 ppm ou moins.
  13. Procédé de dégommage d'huile comprenant l'étape consistant à faire réagir une enzyme phospholipase A avec des phospholipides dans une composition d'huile, le procédé étant caractérisé en ce que la composition d'huile est mise en contact simultanément avec au moins une enzyme phospholipase A en une quantité d'environ 2 ppm d'enzyme active ou moins et au moins une enzyme phospholipase C en une quantité d'environ 30 ppm d'enzyme active ou moins, afin de réagir avec les phospholipides présents dans la composition d'huile, dans des conditions réactionnelles telles que la réaction de l'enzyme phospholipase A a lieu à une vitesse plus rapide qu'en cas d'absence de l'enzyme phospholipase C, la durée de la réaction des enzymes avec les phospholipides étant inférieure à une heure, et ladite réaction des enzymes avec les phospholipides étant effectuée à un pH d'environ 3 à 7 et à une température d'environ 40 à 80°C, et la composition d'huile dégommée présentant une teneur en phospholipides, mesurée en parties par million de phosphore, d'environ 20 ppm ou moins.
EP08728362.8A 2007-01-30 2008-01-28 Dégommage enzymatique utilisant un mélange de phospholipases pla et plc Active EP2118248B1 (fr)

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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
US11/853,339 US8460905B2 (en) 2007-09-11 2007-09-11 Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases with reduced reaction time
PCT/US2008/052162 WO2008094847A1 (fr) 2007-01-30 2008-01-28 Dégommage enzymatique utilisant un mélange de phospholipases pla et plc

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ES (1) ES2523300T3 (fr)
MX (1) MX2009007919A (fr)
PL (1) PL2118248T3 (fr)
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US6936289B2 (en) 1995-06-07 2005-08-30 Danisco A/S Method of improving the properties of a flour dough, a flour dough improving composition and improved food products
NZ528260A (en) 2001-05-18 2005-09-30 Danisco Method of improving dough and bread quality with the addition of an enzyme that hydrolyses a glycolipid and a phospholipid and incapable of hydrolysing a triglyceride or monoglyceride
MXPA05007653A (es) 2003-01-17 2005-09-30 Danisco Metodo.
EP1791933B1 (fr) 2004-07-16 2011-06-29 Danisco A/S Procede de demucilagination enzymatique
JP5509094B2 (ja) * 2007-12-21 2014-06-04 デュポン ニュートリション バイオサイエンシーズ エーピーエス 脂質アシルトランスフェラーゼを用いる食用油精製のためのプロセス
GB0904787D0 (en) 2009-03-20 2009-05-06 Desmet Ballestra Engineering Sa Improved enzymatic oil recuperation process
DE102009051013A1 (de) 2009-10-28 2011-06-09 Ab Enzymes Gmbh Klonierung, Expression und Verwendung saurer Phospholipasen
BR122020023911B1 (pt) * 2010-11-12 2022-02-15 Novozymes A/S Construto de ácido nucleico, célula microbiana hospedeira recombinante, e, método de produção do polipeptídeo
ES2495991T3 (es) * 2011-11-09 2014-09-18 Alfa Laval Corporate Ab Desengomado enzimático
PL2814924T3 (pl) * 2012-02-17 2018-11-30 Clariant Produkte (Deutschland) Gmbh Sposób enzymatycznego odśluzowywania oleju
US9657319B2 (en) * 2012-06-14 2017-05-23 Bunge Global Innovation Llc Process for production of low saturate oils
UA115886C2 (uk) * 2012-10-31 2018-01-10 Альфа Лавал Корпорейт Аб Ферментативне рафінування гідратацією
WO2014090161A1 (fr) * 2012-12-11 2014-06-19 Novozymes A/S Polypeptides ayant une activité phospholipase c et polynucléotides codant pour ceux-ci
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
EP3143135B1 (fr) 2014-05-15 2019-04-10 Novozymes A/S Compositions comprenant des polypeptides ayant une activité de phospholipase c et leur utilisation
AR104205A1 (es) 2015-04-09 2017-07-05 Dsm Ip Assets Bv Fosfolipasa c
WO2018186734A1 (fr) * 2017-04-06 2018-10-11 Purac Biochem B.V. Démucilagination enzymatique d'huile de triglycéride non raffinée
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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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BRPI0808024B1 (pt) 2017-05-16
EP2118248A1 (fr) 2009-11-18
BRPI0808024A2 (pt) 2014-06-17
DK2118248T3 (da) 2014-11-03
CN105038978A (zh) 2015-11-11
RU2009132518A (ru) 2011-03-10
ES2523300T3 (es) 2014-11-24
PL2118248T3 (pl) 2015-03-31
MX2009007919A (es) 2009-08-27
CN105038978B (zh) 2017-09-29
CA2676412A1 (fr) 2008-08-07
CA2676412C (fr) 2015-10-06
RU2477746C2 (ru) 2013-03-20
WO2008094847A1 (fr) 2008-08-07

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