ES2523300T3 - Enzymatic degumming using a mixture of PLA and PLC phospholipases - Google Patents

Abstract

A method for degumming an oil composition, a method comprising: (a) Providing an oil composition containing an amount of phospholipids, (b) Contacting said oil composition simultaneously with one or more phospholipase A enzymes in an amount of about 2 ppm of active enzyme or less and one or more phospholipase C enzymes in an amount of approximately 30 ppm of active enzyme or less, under conditions sufficient for enzymes to react with phospholipids to create phospholipid reaction products, and (c) Separate products of phospholipid reaction of the oily composition, the remaining oily composition being after separation a degreased oily composition, whereby during step (b) the reaction of said one or more phospholipase A enzymes proceeds at a faster rate than that it would be in the absence of said one or more phospholipase C enzymes, where the duration of the enzyme reaction s with the phospholipids is less than one hour, and where said reaction of the enzymes with the phospholipids is carried out at a pH of about 3-7 at a temperature of about 40-80 ° C, and the oily composition degraded from the stage ( c) has a phospholipid content, measured as parts per million phosphorus, of approximately 20 ppm or less.

Description

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DESCRIPTION

Enzymatic degumming using a mixture of PLA and PLC phospholipases

Field of the Invention

This application refers to an enzymatic method for separating various phospholipids and lecithins (collectively known as "gums") from vegetable oils to produce a product, oil or degreased fat 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 separation of various phospholipids and lecithins, a method that can be practiced with crude oils or oils degummed with water. In one embodiment, the enzymatic reaction or the enzymatic treatment period may be less than about an hour.

Background of the invention

The raw vegetable oils obtained by 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 separate phospholipids, free fatty acids and trace metals in order to produce a quality salad oil with a smooth taste, light color and long shelf life.

The phospholipid separation generates almost all losses associated with the refining of vegetable oils. As illustrated in Fig. 1, phospholipids contain a phosphate group on one of the two ends of the glycerol chain, while a triacylglycerol contains at least one fatty acid.

The phospholipid phosphate group is "hydrophilic" or "water-loving", which means that functional group X is attracted to water. The R1 and R2 fatty acid chains of phospholipids are "lipophilic" or "lipid lovers", which means 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 functional group containing the phosphate group denoted in Fig. 1 as "X" determines the degree of its hydrophilic nature. The functional group X in Fig. 1 can be any of several of a variety of known types, a few of which are illustrated in Fig. 2.

Phospholipids containing the choline and ethanolamine functional groups have the highest affinity for water, while acids, acid salts (calcium, magnesium and iron) and inositol have a much lower water affinity. Phosphatidic acid and phosphatidic acid salts are commonly known as "Nonhydratable Phospholipids" or NHPs. Phospholipids are commonly measured in oil as "phosphorus content" in parts per million. Table 1 contains the typical amounts of phospholipids present in the main oilseed crops, and the distribution of the various functional groups as a percentage of the phospholipids present in the oil.

Phospholipid Composition

Table 1. Typical concentrations and distributions of phospholipids in common oilseeds

Soy oil

Canola oil Sunflower oil

P (ppm)

400 - 1200 200-900 300-700

PC (-chine)

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 separated from vegetable oils by different known means. The most commonly used procedures in the industry are water degumming, acid degumming, caustic refining and enzymatic degumming.

Degummed with water

This technique is usually applied to crude oils that contain a high amount of hydrable phospholipids. Due to its mild characteristics, the phospholipids obtained can be used as lecithin (a natural emulsifier). In general, the oil obtained by this technique is referred to in the industry as being

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"Degummed", despite being only partially degummed. Since oil degummed with water still contains high amounts of phospholipids, especially non-hydrable phospholipids, the use of other processing techniques, such as caustic refining or degumming with the PLA1 enzyme, may be required to produce a high quality finished oil. It has high stability and little color.

In the water degumming process, water (1 to 5% by weight) is added to a crude oil at 60-75 ° C with vigorous mixing. The oil is then mixed gently for 15 to 60 minutes to help hydrate the phospholipids present in the oil. Hydration of phospholipids or "gums" causes the gums to swell and agglomerate like a flocculant. The flocculant is an emulsion or mixture of hydrated gums and oil. The emulsion has a specific gravity greater than that of the oil and can be separated by sedimentation, filtration

or the industrial practice of centrifugation. The centrifuge gives two streams, oil degummed with water and wet rubber. The degumming process predominantly separates only hydrable phospholipids. The remaining phospholipids (50 to 250 ppm), measured as phosphatidic acid and / or PI salts, can be separated in subsequent processing operations.

Separate wet gums are a mixture of emulsified oil that contains at least one triacylglycerol (or oil) molecule for every two phospholipid (or gum) molecules. This emulsified oil cannot be separated

or recover physically from the emulsion and is considered a loss of processing. The gums can be dried and sold as food grade lecithin, but they are usually used as a byproduct in other applications such as animal feed or in an industrial process, with a lower economic value.

The loss of oil due to emulsion formation is significant, with a negative impact on the overall economic balance on the processing cost of refined oil.

Acid degumming

This technique is usually applied to crude oils when the objective is the total separation of phospholipids. The oil obtained is usually called "super degummed" or "fully degummed" in the industry.

The crude oil is treated with 250 to 2000 ppm 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 NHPs, thus helping their separation. Then, water (1 to 5% by weight) is added to the crude oil treated with acid at 60-75 ° C with vigorous mixing. The oil is then mixed gently for 15 to 60 minutes to help hydrate the phospholipids. Hydration of phospholipids or "gums" causes the gums to swell and agglomerate like a flocculant. The flocculant is an emulsion or mixture of hydrated gums and oil. The emulsion has a specific gravity greater than that of the oil and can be separated by sedimentation, filtration or the industrial practice of centrifugation. The centrifuge gives an acid degummed oil and a wet rubber. The acid degumming process separates most of the phospholipids, but enough still remain (25-100 ppm) in the degummed oil to require further processing. For food applications, acid degummed oil is usually sent to a bleaching and deodorization procedure, a process known in the industry as "physical refining." Gums treated with acid can no longer use food grade lecithin.

As in the water degumming process, the separated and dried gums in the acid degumming process contain at least one triacylglycerol (or oil) molecule for every two phospholipid (or gum) molecules. This emulsified oil cannot be physically separated or recovered and is considered a processing loss, with a negative economic impact on the overall economic balance of the refined oil's processing cost.

Caustic Refining

This technique is usually applied to crude oils or degummed with water when the objective is to separate all phospholipids and fatty acids.

The crude oil or degummed with water is treated with 200 to 1000 ppm phosphoric or citric acid at 60-90 ° C with vigorous mixing. The acid is allowed to react for 10 to 90 minutes with the salts of the NHPs. The acid improves the hydrophilic nature of NHPs, thus helping their separation. A dilute sodium hydroxide solution (10-18% by weight) is added to the acid treated oil at 60-75 ° C. The amount of sodium hydroxide (caustic soda) 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% with respect to dry basis. The caustic solution neutralizes free fatty acids (which produce sodium soaps), neutralizes excess acid and, with the sodium soaps created, helps hydrate and emulsify all remaining phospholipids.

The sodium hydroxide / oil solution is mixed for approximately 10 minutes, then separated by sedimentation, filtration or industrially by centrifugation. The centrifuge gives an oil treated with caustic soda and fatty material for soap making. The oil treated with caustic soda is then "washed" with 10 to 20% softened water at 90-95 ° C and centrifuged again. Centrifugal oil is known as "Once Refined" and water is commonly known as "Wash Water." For food applications, the oil “a

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once refined ”is usually sent for bleaching and deodorization to produce salad oil. An alternative to water washing is to treat the oil treated with caustic soda with an absorbent silica gel and filter out residual soaps and phospholipids not separated in the initial centrifugation.

As with the water and acid degumming procedures, the separated and dried gums of the caustic soda refining process contain at least one molecule of triacylglycerol (or oil) for every two phospholipid (or gum) molecules. This emulsified oil cannot be physically separated or recovered and is considered a loss of processing. Additionally, sodium hydroxide will react with the neutral oil to form soaps, thereby reducing the overall oil yield with a negative economic impact on the overall economic balance on the processing cost of the refined oil.

Enzymatic treatment

Yet another refining technique used in the vegetable oil industry is "enzymatic refining" or "enzymatic degumming." Enzymatic degumming is used when the goal is total phospholipid separation. In general, enzymatic degumming treatments of the prior art have been practiced on oils that have been previously degummed by one of the other methods, typically degumming with water. For food applications, the enzyme degummed oil is sent sequentially to bleaching and deodorization, a process known in the industry as "physical refining." Enzymatic degumming provides better oil yield than degumming with water, with acid or with caustic soda, with better economic results.

The enzymatic reaction changes the nature of the phospholipid, cleaving some of the parts of the phospholipid. This reduces the emulsification properties of the phospholipid so that less oil is lost when the gums are separated from the oil, thus saving oil. Enzymes that exhibit activity with phospholipids are commonly referred to as "phospholipases." The types of phospholipases are based on the position of the phospholipid molecule in which the enzyme reacts, and are known as PLA1, PLA2, PLC and PLD. The positions on the phospholipid molecule in which the different types of phospholipases react are illustrated in Fig. 3.

It can be seen in Fig. 3 that different types of phospholipases will give different compounds after reacting with the phospholipids. In addition, each type of phospholipase has its own reaction rate and optimal reaction conditions in terms of pH, water% and temperature. When used alone, the PLA generally requires a reaction time of at least about 4 hours, while when used alone the PLC generally requires a reaction time of approximately one hour. It is known that enzymatic treatment must occur at a pH less than or equal to 8, in order to minimize undesirable saponification of the oil, but the PLA has an optimum reaction pH of 4.5, while the PLC has an optimal pH Reaction 7.0. Each enzyme also has different thermal tolerances. PLA enzymes are denatured at approximately 50 ° C while PLC enzymes are denatured at approximately 65 ° C.

The amino acid sequences with phospholipase activity are widely reflected in the literature and described in patents, and it has been reported that some of them have activity on the phospholipids present in vegetable oils. All this is known in the art.

A commercial product with PLA1 enzyme with phospholipase activity is Novozymes phospholipase A1 Lecitase® Ultra. It is known that this product gives polar smooth-phospholipids and polar fatty acids when mixed with degummed oil with a pH buffer solution in water of 1% citric acid-1.5% NaOH at 4.5 <pH <7, 0 and 40 ° C <T <55 ° C, as described in the Application Sheet of Novozymes Oils & Fats Nos. 2002-18525501 and 2002-05894-03. PLA1 selectively hydrolyzes the fatty acid opposite the phosphate functional group on the glycerol chain, as illustrated in Fig. 4.

The resulting reaction gives a smooth-phospholipid and a fatty acid. The lysophospholipid molecule has lost a hydrophilic functional group, and the remaining alcohol group at the reaction site is hydrophilic. Now, with two hydrophilic sites, the smooth-phospholipid molecule is soluble in water, and has lost its emulsifying properties. The degumming process with PLA1 thus reduces the refining losses by no longer separating any neutral oil with the gums, and the only loss is the original phospholipid molecule.

Although enzymatic degumming offers significant advantages to oil processors, it also has certain disadvantages. A disadvantage is that the reaction of the enzyme with phospholipids can be slow and time consuming. In particular, the reaction of phospholipase A enzymes with phospholipids can last 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 procedures. Due to the slowness of the PLA reaction, enzymatic degumming is typically carried out in oil compositions that have been subjected first to degumming with water. Thus, the oil has to be degummed twice to obtain a product that has a phosphorus concentration low enough for its intended purposes.

It is known in the art that PLC enzymes react with a phospholipid by selectively hydrolyzing the phosphate functional group, as shown in Fig. 5. The resulting reaction gives a diacylglycerol ("DAG") and a group

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phosphatidic The diacylglycerol molecule no longer has the phosphate functional group and does not need to be separated. The PLC degumming process reduces the losses of the refining by retaining the original phospholipid molecule, while separating only the phosphate functional group. However, PLC enzymes do not react with all phospholipids present in the oil. In general, PLC enzymes do not react with phosphatidic acid (PA) or phosphatidic inositol (PI), illustrated in Fig. 2. However, both PA and PI are non-hydratable phosphatides that remain in the oil after degumming with Water. Thus, the oil treated with PLC has to be further treated with caustic soda to separate the residual gums.

It is known that certain PLC enzymes will react only with certain phosphatidic groups. For example, a PI-specific PLC enzyme, identified as PI-PLC, is known.

It is therefore an aspect of the invention to provide a method for enzymatic degumming of oils in which the speed of the enzymatic reaction is faster than in the enzymatic degumming procedures of the prior art.

It is another aspect of the invention to provide a method for increasing the reaction rate of a phospholipase A enzyme used in an enzymatic degumming process.

It is yet another aspect of the present invention to provide a method for degumming an oily composition in which both hydrable and non-hydrable phospholipids can be treated in a single process.

It is yet another aspect of the present invention to provide such methods of treatment and enzymatic degumming where the enzymatic reaction lasts less than about an hour.

The following references relate to the technique of enzymatic degumming of oils.

US 5,264,367, by Aalrust et al., Describes the use of phospholipases A1, A2 or B to treat an oil that has been first refined to a phosphorus concentration of 50 to 250 ppm. The technology described in this patent is commercially known as Enzymax®. Aalrust specifies that since these enzymes attack lecithin "it would make no sense to use the method of the invention in oils that have a high lecithin content, such as crude 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 specified that the enzyme should be distributed throughout the oil, with the enzyme-water solution present as droplets of a diameter less than 10 µm. The measurement method and the average weight calculations were not described. To dissolve the phospholipases obtained from the pancreas or products of the pancreas, an emulsifier is used, which contains fat. Aalrust specifies that because the oil contains less than 5 ppm phosphorus, it is adaptable to be physically refined to an edible oil. Subsequently, details of the technology described by Aalrust were described 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 Wilson; Dalke, K. et al., First Experiences with Enzymatic Oil Refining, Inform, vol. 6, N. 12, Dec 1995). The data described in these publications for industrial tests reinforce the use of the technology referred to in oils with a P content ranging from 40 to 180 ppm, and not greater. It is also described that “the procedure does not require any special equipment. All pumps, agitators, mixers and heat exchangers as well as the centrifuge are of standard design and can be purchased from various suppliers. ” 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 Wilson, page 56.

The U.S. document 5,532,163, of Yagi et al., Describes an enzymatic method that uses at least 30 parts by weight of water, and preferably 50-200 parts by weight of water, per 100 parts by weight of oil or fat, for the reaction of phospholipases A1, A2 or B with an oil containing 100 to 10,000 ppm of phosphorus. The oil is then washed with 30% to 200% parts by weight of water or an acidic aqueous solution per 100 parts by weight of oil or grease. The total water load required to use the procedure varies from 60% to 400% w / w of processed oil. The production of such a large effluent in an industrial plant makes this method not economical.

The U.S. document 6,001,640, from Loeffler et al., Describes a procedure in which one or more plant oils containing phosphorus-containing components are subjected to a mixture of phospholipases obtained from Aspergillus, a mixture comprising an enzyme having activity A1, activity A2 , or both, and an enzyme that has lysophospholipase activity. The patent specifies 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. describe that the enzymatic reaction must be run at a pH of less than 4, and with a droplet size in the emulsion that is less than 20 µm. The measurement form and the average weight calculations of the emulsion drop size were not described. The patent specifies that the resulting product will have a residual P concentration of 15 ppm or less. It is known in the art that subjecting the oil to a pH as low as 4, or less, will cause the gums present in the oil to hydrate and separate from the reaction medium. The hydrated gums will act as emulsifying agents such that when separated they will carry the oil with them, thus causing the loss of oil. There is no data on the loss of oil in the gums.

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The U.S. document 6,103,505, by Clausen et al., Describes the discovery and activity of certain phospholipases (A1, A2 or B) for use in the enzymatic separation of phospholipids, and a method for producing enzymes. The enzymatic degumming process uses the method described in US 5,264,367 without any additional process step.

The U.S. document 6,127,137, of Hasida et al., Describes the discovery and activity of certain phospholipases capable of separating both fatty acyl groups present in a phospholipid molecule when mixed with degummed oil (50 to 250 ppm phosphorus) with a content of 0.5-5% water, pH 1.5-3, temperature 30-45 ° C and a time of 1 to 12 hours.

The U.S. document 6,143,545, by Clausen et al., Describes the discovery and activity of certain phospholipases (A1, A2 or B) for use in the enzymatic separation of phospholipids, and a method for producing enzymes. The enzymatic degumming process uses the method described in US 5,264,367 without any additional process step.

The U.S. document 6,548,633, from Edwards et al., Describes sequences of secreted proteins encoding cDNA. In column 44, the patent specifies that the protein of that invention can be used in the enzymatic phospholipid of vegetable oils described in US 6,001,640, cited above. The patent also specifies 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.

The US Patent Application Serial No. 10 / 556,816, by Dayton et al., describes an improved enzymatic degumming process in which the pH of the buffered enzymatic reaction is decreased below 4.5 after the enzymatic reaction has ended, eliminating from it. mode the fouling of the equipment, particularly of the heat exchangers and the separation centrifuge, which would be produced by the precipitation of the calcium and magnesium salts at the optimum pH required for the enzymatic activity.

The US Patent Application Serial No. 2004/0005399 A1, by Chakrabarti et al., describes an enzymatic method that uses a simple enzyme addition and pH buffer system and a short retention / reaction time, followed by bleaching with a bleaching earth at 2- 4% and 0-1% activated carbon, and then dewaxed to get an oil with a phosphorus content of 5 ppm. Both the bleaching process and the dewaxing process will separate residual phosphorus from the oil. Additionally, Chakrabarti et al. they specify that the loss of oil in the gums is in the range of 30-40% of the separated gums, suggesting that the enzymatic reaction does not end, resulting in high oil losses due to emulsification of the oil in the separated phospholipids.

The U.S. document 2005/0059130 A1, by Bojsen et al., Describes the discovery and activity of certain phospholipases for use in the enzymatic separation of phospholipids, and a method for producing enzymes. The publication refers to the treatment of vegetable oils to reduce the phospholipid content described in US 5,264,367.

The U.S. document 2005/0108789 A1, from Gramatikova et al., Now US Pat. No. 7,226,771, is intended to describe phospholipases (e.g., phospholipase enzymes A, B, C, D with patatin domains) that efficiently cleave glycerol phosphate bonds in oils, such as vegetable oils, to generate a water-removable phosphorylated base and a diglyceride In paragraph 108, the application further specifies 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 instead of PLA1s and PLA2s in commercial degumming of oils, such as in the ENZYMAX® process, in which the phospholipids are hydrolyzed by PLA1 and PLA2. In paragraph 474, the request specifies that the PLC enzyme can be used alone or with the PLA enzyme to separate non-hydrable phospholipids from oil that has been previously degummed with water, but does not provide reaction conditions for the joint use of the two enzymes. . As the optimal reaction conditions of the PLA enzyme and the PLC enzyme are different, this statement in the application without any working example does not show a person skilled in the art how to simultaneously use the PLA and PLC enzymes. The application also specifies that phospholipases C, D1 and D2 can be used in the enzymatic degumming of previously degummed or ungummed (crude) oils and as an aid in refining with caustic soda.

WO 99/53001 A1, by Clausen et al., Describes a method for enzymatically reducing the content of phosphorus-containing components in an edible oil. The method comprises the use of a phospholipase and a low amount of water. The enzymatic degumming of the oil is carried out at a pH of 1.5 to 8 with an aqueous solution of a phospholipase A1, a phospholipase A2, or a phospholipase B. Document WO 99/53001 A1 does not even show that the degumming reaction can carried out with a combination of phospholipase A and phospholipase C enzymes nor gives reaction conditions for the joint use of the two enzymes.

DE 4339556 C1, of Buchold et al., Describes that a vegetable oil to be degummed is adjusted to a pH of 3 to 6 and mixed with an aqueous solution of an enzyme containing one of the phospholipase enzymes A1, A2 or B Enzymes are allowed to act in the oil with stirring at temperatures of 20 to 90 ° C in a degumming reactor. A separation aid or solubilizing agent is added to the liquid extracted from the reactor of

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degummed at temperatures of 20 to 90 ° C before or after separating the degummed oil. A largely sludge-free solution is obtained containing the enzyme used which is recycled, at least partially, before the degumming reactor. The proportion of recycled enzymes used in the total amount of enzymes dispersed in the oil is at least 10%. DE 4339556 C1 does not show that the degumming reaction can be carried out with a combination of phospholipase A and phospholipase C enzymes nor does it give reaction conditions for the joint use of the two enzymes.

JP 06306386 A, by Tsuruoka et al., Describes the enzymatic degumming of oil with the phospholipase C enzyme. JP 06306386 A does not give reaction conditions for the use of the combination of the two phospholipase A and phospholipase C enzymes together. including the amount of phospholipase A and phospholipase C enzymes.

Summary of the invention

The invention relates to a method for degumming an oily composition, a method comprising:

(to)

Provide an oily composition containing an amount of phospholipids,

(b)

Contacting said oily composition simultaneously with one or more phospholipase A enzymes in an amount of about 2 ppm of active enzyme or less and one or more phospholipase C enzymes in an amount of about 30 ppm of active enzyme or less, under conditions sufficient to Enzymes react with phospholipids to create phospholipid reaction products, and

(C)

Separate the phospholipid reaction products from the oily composition, the remaining oily composition being after the separation a degreased oily composition,

Whereby during step (b) the reaction of said one or more phospholipase A enzymes proceeds at a faster rate than would be given in the absence of said one or more phospholipase C enzymes, where the duration of the reaction of the enzymes with the phospholipids it is less than one hour, and where said reaction of the enzymes with the phospholipids is carried out at a pH of about 3-7 at a temperature of about 4080 ° C, and the degreased oil composition of step (c) has a phospholipid content, measured as parts per million phosphorus, of approximately 20 ppm or less. In a preferred embodiment, the reaction duration of step (b) continues for approximately 30 minutes.

The amount of water necessary for the process of the present invention can be less than about 5% and can advantageously be reduced to less than about 3%, and preferably about 1.5-2.0%.

The pH of the system can be adjusted before or after the addition of one or all enzymes to the oil composition. The oil yield is maximized on the basis of the phospholipid composition contained in the crude product.

Specifically, 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 enzymatic reaction to separate phospholipids present in the oil. More specifically, this invention relates to adding in combination a phospholipase C (PLC) and / or a specific phosphatidyl inositol phospholipase C (PI-PLC) with phospholipase A1 (PLA1) and / or phospholipase A2 (PLA2) to maximize yield of oil and reduce the amount of waste products produced. Surprisingly, it has been found that the kinetics of enzyme reactions run much faster than expected when the two enzymes are used together than when each is used separately. In addition, it has been found that the reactions run faster than expected even if the reaction conditions are not optimized for at least one of the enzymes. In addition, it has been found that the reaction can take place in less than about an hour, and it can take place as quickly as in about thirty minutes.

Advantageously, the treated oil may be a crude oil or a water degummed oil. Enzymes can be added to the oil separately or together, but the two enzymes will be in simultaneous contact with the oil. According to the invention, the parameters of the enzymatic reaction, such as water concentration, pH, stirring time and enzyme concentration, can be controlled to optimize the reaction for a particular combination of enzymes in a particular oil system.

Description of the figures

Fig. 1 illustrates the chemical structures of generic phospholipids and generic triacylglycerols.

Fig. 2 illustrates functional groups and structures of common phospholipids.

Fig. 3 illustrates the positions on the phospholipid molecule in which the different types of phospholipases react.

Fig. 4 illustrates the reaction of a phospholipid with a PLA1 enzyme and the resulting products.

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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 phosphorus in the degummed sample is represented for each level of each experimental factor being evaluated.

Fig. 7 is a graph summarizing the results of examples 31-38, in which the average residual phosphorus in the degummed sample is represented for each level of each experimental factor that is evaluated.

Detailed description of the invention

The present invention relates to an improvement in a method for enzymatically degumming an oily composition. The inventors have found that, surprisingly, using a combination of enzymes the reaction kinetics of phospholipid cleavage can be improved. In particular, an enzymatic degumming process carried out with a combination of a phospholipase C enzyme with a phospholipase A enzyme provides an oily degummed product with a lower phosphorus content in a shorter reaction time than would be achieved with phospholipase A alone. In addition, surprisingly it has been found that the reaction can take place in less than about an hour, and may proceed as quickly as in about thirty minutes. This is particularly unexpected because when PLA is used alone in general it requires a reaction time of at least about 4 hours, while when PLC is used alone in general it requires a reaction time of approximately one hour. On the other hand, PLA has an optimal reaction pH of 4.5, while PLC has an optimal reaction pH of 7.0. Each enzyme also has different thermal tolerances. The PLA enzyme is denatured at about 50 ° C while the PLC enzyme is denatured at about 65 ° C. Furthermore, it is known in the art that the 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.

The reduction in reaction time is evidenced by the PLA enzyme. When used in combination with the PLC enzyme, the reaction time is drastically reduced, and in some embodiments it may be less than about 1 hour, even under acidic reaction conditions that are not optimal for the PLC enzyme. The inventors have also found that under appropriate conditions it is possible to reduce the reaction time to one as low as about 30 minutes.

It has been found that the water concentration can be adjusted to meet the needs of a particular process environment. Therefore, when it is desired to reduce the amount of wastewater produced by the process, the concentration of water can be decreased to about 1-2%, and particularly to about 1.5%. Alternatively, when it is desired to increase the efficiency of the degumming process, the water concentration can be increased to about 4-5%, and particularly to about 4.5%.

It is an advantage of the present invention that the oil to be degummed may be a crude oil, or previously degummed by one of the prior art methods. It is a distinct advantage for the oil processor to be able to achieve degumming of the oils in a single stage. Oils that can be treated according to 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 , hemp oil, flaxseed oil, mango seed oil, prairie grass oil, ox leg oil, olive oil, palm oil, palm kernel oil, palm oil, rapeseed oil, oil Rice bran, safflower oil, camellia oil, soybean oil, sunflower seed oil, resin oil, tsubaki oil and vegetable oil.

The phospholipase A enzyme used in the method of the present invention may be a phospholipase A1 enzyme or a phospholipase A2 enzyme. The phospholipase C enzyme used in the present invention can be a phospholipase C or a specific inositol phospholipase C. Many varieties of enzymes of the phospholipase A and phospholipase C families are commercially available; and it is contemplated that such enzymes and their equivalents will be suitable for use in the present invention.

In the method of the 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 separated oil, or sequentially or simultaneously. Whether added sequentially or simultaneously, the enzymatic reaction will take place at some time with both enzymes present in the reaction mixture.

The degumming process of the present invention is carried out at a pH of less than about 8, preferably between about 3-7, and most preferably about 4-5. The pH of the enzymatic degumming process can be achieved by the addition of known pH buffer solutions. It is well known that citric acid and sodium hydroxide are suitable for this purpose. When pH adjustment is required under specific reaction conditions, other pH buffer solutions can be used.

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The temperature of the enzymatic degumming process of the present invention may 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. Surprisingly, it has been found that, in the methods of the present invention, the degumming with PLA can proceed at a temperature above its own optimum of 45 ° C, and closer to the optimum PLC operating temperature, without excessive denaturation.

The method of the present invention provides a single stage degumming process in which the phospholipid 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 of P, and most preferably less than 5 ppm of P.

After the enzymatic degumming has finished and the degummed oil has been separated from the gums, the degummed oil may undergo more processing steps known in the art such as bleaching or deodorization, when it may be necessary or desirable depending on the intended end use. for the degummed oil product.

In the following examples several preferred embodiments of the invention are revealed, together with control examples using prior art conditions. In each of the following examples, the suspended mixer was a Heidolph Elector KG model mixer with a flat blade vane; operated at 90 rpm for normal agitation and 350 rpm for vigorous agitation. The centrifuge was one of Laval Gyro - Tester installed with "The Bowl Unit" for continuous separation. The centrifuge bowl was closed with the closing screws installed. Shear mixing was achieved with an Ultra-Turrax SD-45 homogenizer 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 had a concentration of 11.2 units / mg. The PLA2 enzyme was Rohalase® MPL (batch number Ch: 4738) sold by AB Enzymes located in Germany, and had a concentration of 2000 units / mg. The PLC enzyme was PurifineTM sold by Diversa Corporation of San Diego, California. For examples 1-12, the PLC enzyme was Lot BD16449, which had a concentration of 205 units / mg. For examples 13-38, the PLC enzyme was Lot 90BU002A1, which had a concentration of 27.5 units / mg. The amount of phospholipids that remained in the treated oil was measured as ppm of P according to the method of the American Oil Chemists' Society Official Method Ca 20-99, "Analysis of Phosphorous in Oil by Inductively Coupled Plasma Optical Emission Spectroscopy".

Example 1

Witness: Degummed with water - 1965.4 grams of crude soybean oil containing 746 ppm phosphorus were heated to 70-75 ° C, with normal stirring using a suspended mixer. To the hot oil was added 39.4 grams of deionized water with vigorous stirring for 1 minute. The mixer decelerated at normal speed (90 rpm) to allow the gums to flocculate for 30 minutes. Next, the oil was centrifuged, and the oil and wet gums were collected separately. The residual phosphorus concentration in the degummed oil was 80.7 ppm.

Example 2

Witness: Degummed with a single enzyme with phospholipase A1 (PLA1) - 1997.9 g of crude soybean oil containing 746 ppm phosphorus were heated to 75-80 ° C, with stirring using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and the mixture was sheared for 1 minute. The oil was subjected to normal stirring for one hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40-45 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 40-45 ° C, first 60.0 grams of deionized water were added and the mixture was mixed by shear for 1 minute, then 0.1044 grams of Novozymes PLA1 Lecitase® Ultra and the whole mixture were added She was sheared for 1 minute. The oil mixture was stirred at normal speed with a mixer suspended for 4 hours in a temperature range of 41-48 ° C. Next, the enzyme treated oil was centrifuged; and the oil and the wet gums were collected separately. The concentration of residual phosphorus in the oil degummed with PLA1 was 31.7 ppm.

Example 3

Witness: Degummed with a single enzyme with phospholipase C (PLC) - They were heated at 55-60 ° C 2011.1 grams of crude soybean oil containing 746 ppm phosphorus, with normal stirring using a suspended mixer. 60.3 grams of deionized water were added and the mixture was sheared for 1 minute. 0.1051 grams of PurifineTM (PLC lipase BD16449 containing 205 U / mg) of Diversa was added and the mixture was sheared for 1 minute. The oil mixture was subjected to normal stirring for 1 hour at 50-63 ° C. Then, the oil treated with the enzyme was centrifuged, and the oil and the wet gums separated were collected. The concentration of residual phosphorus in the degummed oil with PLC was 70.9 ppm.

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Example 4

Witness: PLC degumming followed by PLA - In this control example, the oil sample is reacted with each enzyme separately under the optimal reaction conditions for that enzyme, according to the prior art. 2110.5 grams of crude soybean oil containing 560.1 ppm phosphorus were heated at 60 ° C with normal stirring. 63 grams of deionized water were added and 0.123 grams of PurifineTM (PLC lipase BD16449 containing 205 U / mg) of Diversa were added and the mixture was sheared for 1 minute. The oil mixture was stirred at normal speed for 1 hour at 55-56 ° C. Next, the oil was centrifuged, and the oil and wet gums were collected. To create a pH buffer of 4.5, 2.0 grams of 50% w / w citric acid solution were first added to the PLC degummed oil, the mixture was sheared for 1 minute and, at then, it was stirred for one hour at normal speed with a suspended mixer; then, 1.8 milliliters of a 4 molar sodium hydroxide solution was added, and the oil mixture was mixed by shear for 10 seconds. 59 grams of deionized water were added and the mixture was mixed by shear for 10 seconds. With the buffer solution of the established pH, 0.1061 grams of Novozymes PLA1 Lecitase® Ultra was added and the entire mixture was sheared for 1 minute. The oil was stirred at normal speed for 4 hours in a temperature range of approximately 45 ° C. Then the oil was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus concentration in the degummed oil sequentially with PLC and then PLA1 was 3.2 ppm.

Example 5

PLC and PLA1 together, neutral pH with a reaction time of 1 hour at 45 ° C - 2004.9 grams of crude soybean oil containing 560.1 ppm phosphorus with normal stirring were heated to 45 ° C. With the oil at a neutral pH, 60 grams of deionized water were added to the oil and 0.1037 grams of Diversa PurfineTM (PLC enzyme) and 0.1076 grams of Novozymes Lecitase® Ultra (PLA1 enzyme) and the mixture were added full shear shed for 1 minute. The mixture of oil and enzymes was stirred at normal speed for 1 hour at a temperature of approximately 45 ° C. Then the oil was centrifuged; and the oil and the wet gums were collected separately. The oil treated with the mixture of PLC and PLA1 enzymes combined at a neutral pH and 45 ° C with a reaction time of one hour produced a degummed oil with a residual phosphorus concentration of 13.2 ppm.

This residual phosphorus value is significantly lower than that achieved with PLA alone in its optimal conditions (Example 2), or PLC alone in its optimal conditions (Example 3).

Example 6

PLC and PLA1 together, neutral pH with a reaction time of 4 hours at 45 ° C - 2003.7 grams of crude soybean oil containing 560.1 ppm phosphorus with normal stirring were heated to 45 ° C. 60 grams of deionized water were added and 0.1040 grams of PurfineTM (PLC enzyme) from Diversa and 0.1085 grams of Lecitase® Ultra (PLA1 enzyme) from Novozymes were added and the entire mixture was sheared for 1 minute. The oil mixture was stirred at normal speed for 4 hours at a temperature of approximately 45 ° C. Then the oil was centrifuged; and the oil and the wet gums were collected separately. The process using the mixture of PLC and PLA1 enzymes combined with a reaction time of four hours at a neutral pH produced a degummed oil with a residual phosphorus concentration of 10.5 ppm.

This residual phosphorus value is only a slight improvement over that achieved in Example 5, indicating that an increase in reaction time from one hour to four hours did not produce a significant difference in the effectiveness of the degumming process.

Example 7

PLC and PLA1 together, pH 4.5 with a reaction time of 1 hour at 45 ° C - 2021.4 grams of crude soybean oil containing 547.9 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer . 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil mixture was stirred at normal speed for one hour. The oil was allowed to cool until the temperature reached 40-45 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added and the mixture was mixed by shear for 10 seconds. 61.0 grams of deionized water, 0.1184 grams of PurfineTM (PLC enzyme) from Diversa and 0.1038 grams of Lecitase® Ultra (PLA1 enzyme) from Novozymes were added and the entire mixture was sheared for 1 minute. The oil mixture was stirred at normal speed for 1 hour at a temperature of approximately 45 ° C. Next, the oil was centrifuged, and the oil and wet gums were collected separately. The process using the mixture of PLC and PLA1 enzymes combined with a reaction time of one hour at a pH of 4.5 and a temperature of 45 ° C produced a degummed oil with a residual phosphorus concentration of 2.4 ppm.

This residual phosphorus value is approximately the same, and even slightly better, than that achieved in Example 4 in which each enzyme was reacted separately and in its own optimal conditions. Surprisingly, the effectiveness of degumming is just as good when the two enzymes are made to work together

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at a non-optimal reaction time for PLA and at a pH and temperature not optimal for PLC, that when the two enzymes are operated separately, each in its own optimal conditions.

Example 8

PLC and PLA1 together, pH 4.5 with a reaction time of 4 hours at 45 ° C - 2069.3 grams of crude soybean oil containing 547.9 ppm phosphorus with normal stirring were heated to 75-80 ° C. 2.0 grams of 50% w / w citric acid solution were added and the mixture was sheared for 1 minute, and then stirred at normal speed for one hour. The mixture was allowed to cool to 40-45 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added and the mixture was mixed by shear for 10 seconds. 63 grams of deionized water, 0.1112 grams of PurfineTM (PLC enzyme) from Diversa and 0.1258 grams of Lecitase® Ultra (PLA1 enzyme) from Novozymes were added and the entire mixture was sheared for 1 minute. The oil mixture was stirred at normal speed for 4 hours at a temperature of approximately 45 ° C. Then, the oil mixture was centrifuged, and the oil and the wet gums were collected. The process using the mixture of PLC and PLA1 enzymes combined with a reaction time of four hours at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 2.5 ppm.

This residual phosphorus value is approximately the same as that achieved in Example 7, indicating that an increase in reaction time from one hour to four hours did not produce a significant difference in the effectiveness of the degumming process.

Example 9

PLC and PLA1 together, pH 4.5 with a reaction time of 1 hour at 55 ° C - 1985.2 g of crude soybean oil containing 547.9 ppm phosphorus with normal stirring were heated to 75-80 ° C. 2.0 grams of 50% w / w citric acid solution were added and the mixture was sheared for 1 minute, then stirred at normal speed for one hour. The mixture was allowed to cool to 40-45 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added and the mixture was mixed by shear for 10 seconds. 63.0 grams of deionized water, 0.1085 grams of PurfineTM (PLC enzyme) from Diversa and 0.1045 grams of Lecitase® Ultra (PLA1 enzyme) from Novozymes were added and the entire mixture was sheared for 1 minute. The oil mixture was stirred at normal speed for 1 hour at a temperature of 55 ° C. Then the oil was centrifuged; oil and wet gums were collected separately. The process using the mixture of PLC and PLA1 enzymes combined with a reaction time of one hour at a pH of 4.5 and a reaction temperature of 55 ° C produced a degummed oil with a residual phosphorus concentration of 2.3 ppm.

This residual phosphorus value is approximately the same as that achieved in Examples 7 and 8, indicating that an increase in the reaction temperature from approximately 45 ° C to approximately 55 ° C did not produce a significant difference in the effectiveness of the degumming process, even though it would be normally expect PLA1 to become denatured at a temperature above 50 ° C.

Example 10

PLC and 2 times the concentration of PLA1 together, pH 4.5 with a reaction time of 1 hour at 45 ° C - 1992.2 grams of crude soybean oil containing 547.9 ppm phosphorus with stirring were heated to 75-80 ° C at normal speed 2.0 grams of 50% w / w citric acid solution were added and the mixture was sheared for 1 minute, then stirred for one hour. The mixture was allowed to cool to 40-45 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added and the mixture was mixed by shear for 10 seconds. 60 grams of deionized water, 0.1319 grams of PurfineTM (PLC enzyme) from Diversa and 0.2139 grams of Lecitase® Ultra (PLA1 enzyme) from Novozymes were added to the mixture and the entire mixture was sheared for 1 minute. The oil mixture was stirred at normal speed for 1 hour in a temperature range of 45 ° C. Then the oil was centrifuged, the oil and the wet gums were collected separated. The process using mixed PLC combined enzymes and twice the concentration of PLA1 with a reaction time of one hour at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 7.0 ppm.

This residual phosphorus value is acceptable for certain applications but not as good as that achieved in Examples 7-9, indicating that, surprisingly, the increase in the dosage of PLA1 does not lead to an improvement in the effectiveness of the degumming process, even under optimal reaction conditions for PLA1.

Example 11

PLC and PLA2 together, pH 4.5 with a retention time of 1 hour at 45 ° C - 1998.4 grams of crude soybean oil containing 341.2 ppm phosphorus with normal stirring were heated using a suspended mixer . 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Keeping the

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temperature at 40-45 ° C, 0.1112 grams of PurifineTM (PLC lipase BD16449 containing 205 U / mg) of Diversa and 0.2094 grams of Rohalase® MPL (lot number Ch: 4738) sold by AB Enzymes followed by 60 grams of deionized water and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 40-45 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA2 enzymes combined at neutral pH produced a degummed oil with a residual phosphorus concentration of 3.3 ppm.

This example is similar to Example 7 above, except for the substitution of PLA 1 for PLA 2. The concentration of residual phosphorus in the finished product demonstrates that PLA2 can function approximately equally well as PLA1 in the method of the present invention.

Example 12

PLC and PLA2 together, pH 4.5 with a retention time of 4 hours at 45 ° C - 1998.4 grams of crude soybean oil containing 341.2 ppm phosphorus with normal stirring were heated using a suspended mixer . 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 40-45 ° C, 0.1038 grams of PurifineTM (PLC lipase BD16449 containing 205 U / mg) of Diversa and 0.2047 grams of Rohalase® MPL (lot number Ch: 4738) sold by AB Enzymes were added followed by 60 grams of deionized water and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 4 hours at a temperature of 40-45 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA2 enzymes combined at neutral pH produced a degummed oil with a residual phosphorus concentration of 5.8 ppm.

This example illustrates that increasing the reaction time from one to four hours did not result in a better degumming, and in fact resulted in a higher concentration of residual phosphorus.

The results of examples 1-12 are summarized below in Table 2.

Table 2

Ex.

Adding Enzymes PLC (ppm of active enzyme) PLA1 (ppm active enzyme) PLA2 (ppm active enzyme) Reaction time (min) Temp. (ºC) pH Water (%) Phosphorus (ppm)

one

Any 30 70-75 7 2.0 80.7

2

A 0.6 240 41-48 34.5 3.0 31.7

3

A 10.8 60 50-63 7 3.0 70.9

4

Sequential 11.5 0.6 20, 240 55, 45 4,5 3.1 3.2

5

Simultaneous 10.6 0.6 60 Four. Five 7 3.0 13.2

6

Simultaneous 10.7 0.6 240 Four. Five 7 3.0 10.5

7

Simultaneous 12.1 0.6 60 Four. Five 4,5 3.0 2.4

8

Simultaneous 11.4 0.7 240 Four. Five 4,5 3.1 2.5

9

Simultaneous 11.1 0.6 60 Four. Five 4,5 3.1 2.3

10

Simultaneous 13.5 1.2 60 Four. Five 4,5 3.0 7.0

eleven

Simultaneous 11.4 209 60 Four. Five 4,5 3.0 3.3

12

Simultaneous 10.6 205 240 Four. Five 4,5 3.1 5.8

Examples 13-30

An experiment design (DOE) was established to determine the effects of certain process control variables for the enzymatic degumming procedure, as shown in Table 3 below.

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Table 3

Variable

Levels

Enzyme addition

Sequential Simultaneous

Amount of PLC (ppm of active enzyme)

10 twenty 30

Amount of PLA (ppm of active enzyme)

0.5 1.0 2.0

Agitation time (s)

Four. Five 60 120

pH

4,5 5.0 7.0

Water (%)

1.5 3.0 4,5

Temperature (ºC)

40 fifty 60

Reaction time (min)

30 60 120

Sequence - Each enzyme was added separately, although both enzymes were in contact with the oil mixture for at least part of the reaction time. 5 Simultaneous - Both enzymes were added at the same time. pH - The pH at which enzymes are exposed to oil. Temperature - Temperature at which enzymes are exposed to oil. Stirring time - The time the mixture is stirred at high speed after the addition of each enzyme. Reaction time - Total time that at least one enzyme is in contact with the oil.

10 These operating variables were evaluated in eighteen separate tests, presented herein in examples 13-30. The values of each variable tested in each example 13-30 are shown in Table 4 below.

Table 4

Ex.

Enzyme addition PLC (ppm of active enzyme) PLA1 (ppm active enzyme) Reaction time (minutes) Temp. (ºC) pH Water (%) Stirring time (seconds)

13

Sequential twenty one 120 40 5 1.5 120

14

Simultaneous twenty one 60 60 7 1.5 Four. Five

fifteen

Sequential 10 one 30 40 7 4,5 60

16

Sequential 10 2 60 fifty 4,5 1.5 120

17

Sequential twenty 2 30 fifty 7 3.0 Four. Five

18

Sequential 10 0.5 120 60 5 3.0 Four. Five

19

Simultaneous 30 2 30 60 5 1.5 60

twenty

Simultaneous 10 2 120 60 7 4,5 120

twenty-one

Simultaneous twenty 2 120 40 4,5 3.0 60

22

Sequential 30 2 60 40 5 4,5 Four. Five

2. 3

Sequential 30 one 30 60 4,5 3.0 120

24

Simultaneous 30 one 120 fifty 4,5 4,5 Four. Five

25

Simultaneous 10 one 60 fifty 5 3.0 60

26

Sequential twenty 0.5 60 60 4,5 4,5 60

27

Sequential 30 0.5 120 fifty 7 1.5 60

28

Simultaneous 30 0.5 60 40 7 3.0 120

29

Simultaneous twenty 0.5 30 fifty 5 4,5 120

30

Simultaneous 10 0.5  30 40 4,5 1.5 Four. Five

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Example 13

1999.1 grams of crude soybean oil containing 769.5 ppm of phosphorus were heated at 75-80 ° C with normal stirring using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 2.4 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 5.0. Maintaining the temperature at 40 ° C, 1,5008 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa were added followed by 30 grams of deionized water and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 60 minutes. Maintaining the temperature at 40 ° C, 0.2132 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) of Novozymes was added and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus concentration in the degummed oil with sequential addition of PLC and after PLA1 was 6.5 ppm.

Example 14

2010.5 grams of crude soybean oil containing 785.1 ppm of phosphorus were cooled to 60 ° C with normal stirring using a suspended mixer. Maintaining the temperature at 60 ° C, 1.5316 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.2073 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes were added followed by 30 grams of deionized water and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at neutral pH produced a degummed oil with a residual phosphorus concentration of 109.6 ppm.

Example 15

1999.5 grams of crude soybean oil containing 785.1 ppm phosphorus were cooled to 40 ° C with normal stirring using a suspended mixer. Maintaining the temperature at 40 ° C, 0.754 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa was added followed by 90 grams of deionized water and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 15 minutes. Maintaining the temperature at 40 ° C, 0.2242 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) of Novozymes was added and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 15 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus concentration in the degummed oil with sequential addition of PLC and after PLA1 was 27.4 ppm.

Example 16

2002.2 grams of crude soybean oil containing 785.1 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 50 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 50 ° C, 0.7498 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa was added followed by 30 grams of deionized water and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes. Maintaining the temperature at 50 ° C, 0.4064 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) of Novozymes were added and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 50 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus concentration in the degummed oil with sequential addition of PLC and after PLA1 was 7.6 ppm.

Example 17

2010.7 grams of crude soybean oil containing 785.1 ppm of phosphorus were heated at 50 ° C. with normal stirring using a suspended mixer. Maintaining the temperature at 50 ° C, 1,4981 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa was added followed by 60 grams of deionized water and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 15 minutes. Maintaining the temperature at 50 ° C, 0.4143 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) of Novozymes was added and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 15 minutes at a temperature of 50 ° C. Then the oil

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treated with enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus concentration in the degummed oil with sequential addition of PLC and after PLA1 was 79.3 ppm.

Example 18

2005.3 grams of crude soybean oil containing 742.9 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 60 ° C, then 2.4 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 5.0. Maintaining the temperature at 60 ° C, 0.7491 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa was added followed by 60 grams of deionized water and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 60 minutes. Maintaining the temperature at 60 ° C, 0.1220 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) from Novozymes was added and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus concentration in the degummed oil with sequential addition of PLC and then PLA1 was 2.2 ppm.

Example 19

2000.4 grams of crude soybean oil containing 742.9 ppm of phosphorus were heated at 75-80 ° C with normal stirring using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 60 ° C, then 2.4 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 5.0. Maintaining the temperature at 60 ° C, 2.2270 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.3937 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 30 grams of deionized water were added and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at pH 5.0 produced a degummed oil with a residual phosphorus concentration of 7.8 ppm.

Example 20

2006.3 grams of crude soybean oil containing 719.3 ppm of phosphorus were heated at 60 ° C. with normal stirring using a suspended mixer. Maintaining the temperature at 60 ° C, 0.7561 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.4098 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 90 grams of deionized water were added and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at neutral pH produced a degummed oil with a residual phosphorus concentration of 64.1 ppm.

Example 21

1998.5 grams of crude soybean oil containing 719.3 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 40 ° C, 1.4798 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.4018 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes were added followed by 60 grams of deionized water and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at pH 4.5 produced a degummed oil with a residual phosphorus concentration of 5.5 ppm.

Example 22

2001.3 grams of crude soybean oil containing 719.3 ppm of phosphorus were heated at 75-80 ° C with normal stirring using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a mixer

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discontinued. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 2.4 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 5.0. Maintaining the temperature at 40 ° C, 2.2580 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa were added followed by 90 grams of deionized water and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes. Maintaining the temperature at 40 ° C, 0.4126 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) of Novozymes were added and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the degummed oil treated with sequential addition of PLC and PLA1 had a residual phosphorus concentration of 2.1 ppm.

Example 23

2002.0 grams of crude soybean oil containing 747.3 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 60 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 60 ° C, 2.2194 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa were added followed by 60 grams of deionized water and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 15 minutes. Maintaining the temperature at 60 ° C, 0.2198 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) of Novozymes were added and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 15 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the degummed oil treated sequentially with the PLC and PLA1 enzymes had a residual phosphorus concentration of 4.6 ppm.

Example 24

2000.8 grams of crude soybean oil containing 747.3 ppm phosphorus were heated at 75-80 ° C with normal stirring using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 50 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 50 ° C, 2,2500 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.2216 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 90 grams of deionized water were added and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 50 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of combined PLC and PLA1 enzymes produced a degummed oil with a residual phosphorus concentration of 1.8 ppm.

Example 25

1998.9 grams of crude soybean oil containing 747.3 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 50 ° C, then 2.4 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 5.0. Maintaining the temperature at 50 ° C, 0.7445 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.2042 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 60 grams of deionized water were added and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 50 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of combined PLC and PLA1 enzymes produced a degummed oil with a residual phosphorus concentration of 7.2 ppm.

Example 26

1997.3 grams of crude soybean oil containing 810.8 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a mixer

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discontinued. The oil was allowed to cool with stirring at normal speed until the oil temperature was 60 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 60 ° C, 1.5189 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa were added followed by 90 grams of deionized water and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 30 minutes. Maintaining the temperature at 60 ° C, 0.1119 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) of Novozymes were added and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the degummed oil treated sequentially with the PLC and PLA1 enzymes had a residual phosphorus concentration of 2.2 ppm.

Example 27

2010.0 grams of crude soybean oil containing 810.8 ppm phosphorus were cooled to 50 ° C with normal stirring using a suspended mixer. Maintaining the temperature at 50 ° C, 2,2608 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa were added followed by 30 grams of deionized water and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 60 minutes. Maintaining the temperature at 50 ° C, 0.1772 grams of Lecitase® Ultra (Lipase PLA1 lot number LYN05007) of Novozymes was added and the entire mixture was mixed by shear for 60 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 50 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the degummed oil treated sequentially with the PLC and PLA1 enzymes at neutral pH had a residual phosphorus concentration of 72.6 ppm.

Example 28

2005.1 grams of crude soybean oil containing 810.8 ppm phosphorus were heated at 40 ° C. with normal stirring using a suspended mixer. Maintaining the temperature at 40 ° C, 2.2622 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1031 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes were added followed by 60 grams of deionized water and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at neutral pH produced a degummed oil with a residual phosphorus concentration of 61.5 ppm.

Example 29

2006.3 grams of crude soybean oil containing 795.3 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 50 ° C, then 2.4 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 5.0. Maintaining the temperature at 50 ° C, 1.5373 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1168 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 90 grams of deionized water were added and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 50 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 5.0 produced a degummed oil with a residual phosphorus concentration of 1.9 ppm.

Example 30

2006.1 grams of crude soybean oil containing 795.3 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 40 ° C, 0.7736 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1072 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 30 grams of deionized water were added and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and wet gums were collected

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separated. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 13.7 ppm.

The results of examples 13-30 in terms of phosphorus concentration achieved are shown in Table 5 below.

Table 5

Ex.

Adding Enzymes PLC (ppm of active enzyme) PLA1 (ppm active enzyme) Reaction time (min) Temp. (ºC) pH Water (%) Stirring time (seconds) Phosphorus (ppm)

13

Sequential 20.6 1.2 120 40 5 1.5 120 6.5

14

Simultaneous 21.1 1.2 60 60 7 1.5 Four. Five 109.6

fifteen

Sequential 10.4 1.3 30 40 7 4,5 60 27.4

16

Sequential 10.3 2.3 60 fifty 4,5 1.5 120 7.6

17

Sequential 20.6 2.3 30 fifty 7 3.0 Four. Five 79.3

18

Sequential 10.3 0.7 120 60 5 3.0 Four. Five 2.2

19

Simultaneous 30.6 2.2 30 60 5 1.5 60 7.8

twenty

Simultaneous 10.4 2.3 120 60 7 4,5 120 64.1

twenty-one

Simultaneous 20.3 2.3 120 40 4,5 3.0 60 5.5

22

Sequential 31.0 2.3 60 40 5 4,5 Four. Five 2.1

2. 3

Sequential 30.5 1.2 30 60 4,5 3.0 120 4.6

24

Simultaneous 30.9 1.2 120 fifty 4,5 4,5 Four. Five 1.8

25

Simultaneous 10.2 1.4 60 fifty 5 3.0 60 7.2

26

Sequential 20.9 0.6 60 60 4,5 4,5 60 2.2

27

Sequential 31.1 0.7 120 fifty 7 1.5 60 72.6

28

Simultaneous 31.1 0.6 60 40 7 3.0 120 61.5

29

Simultaneous 21.1 0.7 30 fifty 5 4,5 120 1.9

30

Simultaneous 10.6 0.6  30 40 4,5 1.5 Four. Five 13.7

A summary of the previous experiments is illustrated in the graph shown in Fig. 6, which is a representation of the final average amount of phosphorus at each level of each factor.

At a neutral pH, optimal for the PLC enzyme, the combination of the two enzymes failed to produce an oil with

10 acceptable phosphorus concentrations that allowed the oil to be physically refined. However, at an acidic pH, optimal for the PLA enzyme, the combination of enzymes added sequentially or together produced acceptable residual phosphorus concentrations in the oils allowing them to be physically refined. When an acidic pH was used, only one of the experiments failed to produce an oil with a residual phosphorus concentration of less than 10 ppm. Example 30 had a residual phosphorus concentration greater than 10 (13.7

15 ppm) and was produced with the lowest concentrations of both enzymes, the lowest temperature, the lowest percentage of water, the shortest mixing and stirring times and the most acidic pH.

A synergistic effect was discovered among the combination of enzymes that allowed the reaction to end in less than 30 minutes, compared with 1 hour for PLC enzymes or 4 hours for PLC enzymes. Additional tests were completed to verify the effect of a very short reaction time; The results are shown in Table 6 below. In these additional tests, the pH was maintained at 4.5, in view of the previous finding that a lower pH produced significantly more favorable results than a neutral pH. The amount of PLA1 was also kept constant at 0.5 ppm, since it was previously determined that the increase in the amount of PLA1 above this value did not result in more effective degumming. In addition, in each of these examples the enzymes were added simultaneously more than sequentially, in view of the determination

25 above that simultaneous addition of enzymes produced better degumming results than sequential addition. In addition, in an industrial process it is advantageous to limit the total time of the process, the total equipment and the dedicated assets.

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Table 6

Example

Adding Enzymes PLC (ppm of active enzyme) PLA1 (ppm active enzyme) Reaction time (minutes) Temp. (ºC) pH Water (%) Stirring time (seconds)

31

Simultaneous twenty 0.5 120 40 4,5 2.0 120

32

Simultaneous 10 0.5 120 60 4,5 4,5 Four. Five

33

Simultaneous 10 0.5 30 40 4,5 4,5 120

3. 4

Simultaneous twenty 0.5 30 60 4,5 2.0 Four. Five

35

Simultaneous twenty 0.5 120 40 4,5 4,5 Four. Five

36

Simultaneous twenty 0.5 30 60 4,5 4,5 120

37

Simultaneous 10 0.5 30 40 4,5 2.0 Four. Five

38

Simultaneous 10 0.5 120 60 4,5 2.0 120

Example 31

2003.6 grams of crude soybean oil containing 784.8 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 40 ° C, 1.4603 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1021 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes were added followed by 40 grams of deionized water and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 10.7 ppm.

Example 32

2004.8 grams of crude soybean oil containing 784.8 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 60 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 60 ° C, 0.7509 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1105 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 90 grams of deionized water were added and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 6.7 ppm.

Example 33

2000.4 grams of crude soybean oil containing 697.7 ppm phosphorus were heated at 75-80 ° C with normal stirring using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 40 ° C, 0.7530 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1022 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 90 grams of deionized water were added and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 2.2 ppm.

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Example 34

1999.4 grams of crude soybean oil containing 714.2 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 60 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 60 ° C, 1,508 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1139 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes were added followed by 40 grams of deionized water and the Complete mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 16.5 ppm.

Example 35

1999 grams of crude soybean oil containing 714.2 ppm phosphorus were heated to 75-80 ° C with normal stirring using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 40 ° C, 1.5010 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1060 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes were added followed by 90 grams of deionized water and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 1.9 ppm.

Example 36

1999 grams of crude soybean oil containing 695.1 ppm phosphorus were heated at 75-80 ° C with normal stirring using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 60 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 60 ° C, 1,5296 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1241 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes were added followed by 90 grams of deionized water and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 5.2 ppm.

Example 37

2005.2 grams of crude soybean oil containing 695.1 ppm phosphorus with normal stirring were heated to 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 40 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 40 ° C, 0.7422 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1195 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 40 grams of deionized water were added and the entire mixture was mixed by shear for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 40 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 6.7 ppm.

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Example 38

1998 grams of crude soybean oil containing 695.1 ppm phosphorus with normal stirring were heated at 75-80 ° C using a suspended mixer. 2.0 grams of 50% w / w citric acid solution were added and sheared for 1 minute. The oil was subjected to normal stirring for 1 hour with a suspended mixer. The oil was allowed to cool with stirring at normal speed until the oil temperature was 60 ° C, then 1.8 milliliters of 4 molar sodium hydroxide solution was added, and the mixture was mixed by shear for 10 seconds. Citric acid and caustic soda formed a weak pH buffer with a pH of 4.5. Maintaining the temperature at 60 ° C, 0.7429 grams of PurifineTM (PLC lipase lot number 90BU002A1) from Diversa and 0.1041 grams of Lecitase® Ultra (lipase PLA1 lot number LYN05007) from Novozymes followed by 40 grams of deionized water were added and the entire mixture was mixed by shear for 120 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 60 ° C. Next, the oil treated with the enzymes was centrifuged; and the oil and the wet gums were collected separately. The residual phosphorus in the mixture of PLC and PLA1 enzymes combined at a pH of 4.5 produced a degummed oil with a residual phosphorus concentration of 4.4 ppm.

Table 7

Ex.

Adding Enzymes PLC (ppm of active enzyme) PLA1 (ppm active enzyme) Reaction time (minutes) Temp. (ºC) pH Water (%) Stirring time (seconds) Phosphorus (ppm)

31

Simultaneous 20.9 0.7 120 40 4,5 2.0 120 10.7

32

Simultaneous 10.3 0.7 120 60 4,5 4,5 Four. Five 6.7

33

Simultaneous 10.4 0.7 30 40 4,5 4,5 120 2.2

3. 4

Simultaneous 20.7 0.6 30 60 4,5 2.0 Four. Five 16.5

35

Simultaneous 20.7 0.6 120 40 4,5 4,5 Four. Five 1.9

36

Simultaneous 21.0 0.7 30 60 4,5 4,5 120 5.2

37

Simultaneous 10.2 0.6 30 40 4,5 2.0 Four. Five 6.7

38

Simultaneous 10.2 0.6 120 60 4,5 2.0 120 4.4

Fig. 7 is a graph summarizing examples 31-38, which represents the average final amount of phosphorus at each level of each factor keeping the pH, the PLA dosage and the combined addition constant.

The five factors evaluated in these Examples 31-38 are listed below in the order of the importance of their effects on the degumming procedure. The effects are the differences in the average phosphorus concentration (in absolute value) between the high and low factor settings

Increasing the dosage of water decreases residual phosphorus.

The decrease in PLC dosage decreases residual phosphorus.

The decrease in reaction temperature decreases residual phosphorus.

Increasing agitation decreases residual phosphorus.

Increasing reaction time decreases residual phosphorus.

A new process for degumming oils has been described using simultaneously a phospholipase A enzyme and a phospholipase C enzyme. Surprisingly, it has been found that a combination works better than each enzyme separately, even when one enzyme or another is necessarily needed. they react under reaction conditions that are less than optimal for that enzyme. It was also a surprise to find that degumming could be achieved with concentrations of less than approximately 10 ppm phosphorus, as low as approximately 5 ppm phosphorus, and even as low as approximately 3 ppm phosphorus in the final product, under appropriate conditions at times of reaction as low as approximately thirty minutes. In addition, without wishing to link to the theory, it seems that the PLC enzyme or one of its hydrolysis reaction products is catalyzing the reaction of the PLA enzyme, allowing the reaction time to be significantly shorter than the reaction time for any of the enzymes alone. These results are unexpected based on the optimal reaction parameters known for the PLA and PLC enzymes.

Those skilled in the art will recognize from the foregoing description that in the practice of the present invention several operating parameters can be varied, depending on the objectives of a particular situation, while still remaining within the scope of the invention. For example, in determining the concentrations to be used of the PLA and PLC enzymes in a particular experiment, the selection will depend on whether the

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objective is to operate at the lowest possible cost or as effectively as possible. If the objective is to operate at the lowest possible cost, then the concentration of PLA may 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

5 situations On the contrary, if it is desired to maximize efficiency, 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. Those skilled in the oil processing techniques will understand how to vary the concentrations of the enzymes in the reaction mixture to obtain the desired balance of cost efficiency and product yield.

10 Variations of other processing conditions are also possible. The pH may be about 7.0, while a pH of about 5.0 is preferable and a pH of about 4.5 is currently preferred. The concentration of water in the system can in general be about 3.0%, but it can be as low as about 1.5% if it is desired to reduce wastewater, or as high as about 4.5% if higher degumming efficiency. The reaction temperature can be as high as

About 60 ° C, but is more preferably less than about 50 ° C, and surprisingly the most preferable at about 40 ° C. The stirring time during initial mixing can be approximately 45 seconds, is more preferably approximately 60 seconds, and is most preferably approximately 120 seconds. Finally, the duration of the enzymatic reaction can be greatly reduced, in some embodiments it is advantageously less than about 60 minutes, and preferably

20 approximately 30 minutes.

Claims (13)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty E08728362 11-05-2014 1. A method for degumming an oily composition, a method comprising:

(to)

Provide an oily composition containing an amount of phospholipids,

(b)

Contacting said oily composition simultaneously with one or more phospholipase A enzymes in an amount of about 2 ppm of active enzyme or less and one or more phospholipase C enzymes in an amount of about 30 ppm of active enzyme or less, under conditions sufficient to Enzymes react with phospholipids to create phospholipid reaction products, and

(C)

Separate the phospholipid reaction products from the oily composition, the remaining oily composition being after the separation a degreased oily composition,

Whereby during step (b) the reaction of said one or more phospholipase A enzymes proceeds at a faster rate than would be in the absence of said one or more phospholipase C enzymes, where the duration of the reaction of the enzymes with the phospholipids is less than one hour, and where said reaction of the enzymes with the phospholipids is carried out at a pH of about 3-7 at a temperature of about 40-80 ° C, and the oily composition degummed from step (c) It has a phospholipid content, measured as parts per million phosphorus, of approximately 20 ppm or less.

2.

The method according to claim 1, wherein the duration of the reaction of the enzymes with the phospholipids is approximately thirty minutes.

3.

The method according to claim 1, wherein said one or more phospholipase A enzymes are selected from the group consisting of a phospholipase A1 enzyme and a phospholipase A2 enzyme.

Four.

The method according to claim 1, wherein said one or more phospholipase C enzymes are selected from the group consisting of a phospholipase C enzyme and a specific phosphatidyl inositol phospholipase C enzyme.

5.

The method according to claim 1, wherein said reaction of the enzymes with the phospholipids occurs at a pH of about 4-5.

6.

The method according to claim 1, wherein said reaction of the enzymes with the phospholipids occurs at a temperature of about 40-60 ° C, or at a temperature of about 45-55 ° C.

7.

The method according to claim 1, wherein said oily composition comprises a crude oil, or a previously degummed oil.

8.

The method according to claim 1, wherein said PLC enzyme is present in an amount of about 20 ppm of active enzyme or less, or of about 10 ppm of active enzyme or less.

9.

The method according to claim 1, wherein said PLC enzyme is present in an amount of about 1 ppm of active enzyme or less, or of about 0.5 ppm of active enzyme or less.

10.

The method according to claim 1, wherein during step (b) the mixture of the oily composition and the enzymes is initially mixed by shear, preferably where said shear mixture continues for a duration of at least about 45 seconds.

eleven.

The method according to claim 1, wherein during step (b) an amount of water is added, preferably wherein said amount of water is at least about 1.5% by weight of the total mixture, is at least about 3.0% by weight of the total mixture, or is at least about 4.5% by weight of the total mixture.

12.

The method according to claim 1, wherein the degreased oil composition of step (c) has a phospholipid content, measured as parts per million phosphorus, of about 10 ppm or less, or is about 5 ppm or less.

13.

A process for degumming oils comprising the step of reacting a phospholipase A enzyme with phospholipids in an oily composition, a method characterized in that the oily composition is simultaneously contacted with at least one phospholipase A enzyme in an amount of about 2 ppm enzyme activates or less and at least one phospholipase C enzyme in an amount of about 30 ppm of active enzyme or less, so that they react with the phospholipids of the oily composition, under reaction conditions such that the reaction with the phospholipase A enzyme proceeds at a faster rate than would be given in the absence of the phospholipase C enzyme, where the duration of the reaction of the enzymes with the phospholipids is less than one hour, and where said reaction of the enzymes with the phospholipids is carried out at a pH of about 3-7 at a temperature of about 40-80 ° C, and the degreased oil composition has a content of C phospholipids, measured as parts per million phosphorus, of approximately 20 ppm or less.

2. 3