RU2477746C2 - Enzymatic degumming using mixture of pla and plc phospholipases - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to enzymatic techniques of removing phospholipids from plant oil. The method of degumming an oil composition involves contacting the oil composition containing phospholipids simultaneously with one or more phospholipase A (PLA) enzymes in amount of about 2 ppm of the active enzyme or less and one or more phospholipase C (PLC) enzymes in amount of about 30 ppm of the active enzyme or less. Reaction of the enzymes with phospholipids takes place at pH of about 3-7, at temperature of about 40-80°C and the reaction takes less than one hour. The reaction products are separated from the oil composition. A method of degumming oil is also disclosed.

EFFECT: obtained degummed oil composition has phospholipid content, measured in parts per million of phosphorus, of about 20 ppm or less.

23 cl, 7 dwg, 7 tbl, 38 ex

Description

FIELD OF THE INVENTION

The present application relates to an enzymatic method for removing various phospholipids and lecithins (collectively known as “resins”) from vegetable oils to obtain a tarred oil or fat product that can be used for food and / or non-food applications. More specifically, the present application relates to a method for enzymatically treating and removing various phospholipids and lecithins, where the method can be practiced for either crude oils or water-demineralized oils. In one embodiment, the enzymatic reaction or treatment period may be less than about an hour.

State of the art

Crude vegetable oils obtained by either pressing or solvent extraction methods are a complex mixture of triacylglycerols, phospholipids, sterols, tocopherols, free fatty acids, traces of metals and other minor compounds. It is advisable to remove phospholipids, free fatty acids and traces of metals in order to obtain high-quality salad oil with a mild flavor, light color and long shelf life.

The removal of phospholipids leads to almost all losses associated with the refining of vegetable oils. As illustrated in FIG. 1, phospholipids contain a phosphate group at one of the two ends of the glycerol backbone, while triacylglycerol contains at least one fatty acid.

The phosphate group of the phospholipid is “hydrophilic” or “water-loving”, which means that the functional group X is attracted to water. The fatty acid chains of phospholipids R1 and R2 are “lipophilic” or “lipid-loving”, which means that they are attracted to lipids. Since the phospholipid molecule has both a hydrophilic functional group and lipophilic chains of fatty acids, it is an excellent natural emulsifier.

The phosphate-containing functional group of phospholipids, designated as “X” in FIG. 1, determines the degree of their hydrophilic nature. Functional group X in FIG. 1 may refer to any of several various known types, some of which are illustrated in FIG.

Phospholipids containing functional groups - choline and ethanolamine, have a greater affinity for water, while acids, acid salts (calcium, magnesium and iron) and inositol have less affinity for water. Phosphatidic acid and salts of phosphatidic acid are commonly known as "non-hydratable phospholipids" or NHP. Phospholipids are usually defined in oil as the “phosphorus content” in parts per million. Table 1 contains typical amounts of phospholipids present in the seeds of the main agricultural oil plants, and the distribution of various functional groups as a percentage of phospholipids present in the oil.

The composition of phospholipids

Table 1
Typical levels and distributions of phospholipids for common oilseeds Soybean oil Canola oil Sunflower oil P (ppm) 400-1200 200-900 300-700 PC (-choline) 12-46% 25-40% 29-52% PE (-ethanolamine) 8-34% 15-25% 17-26% PA (-acid) 2-21% 10-20% 15-30% PI (inositol) 2-15% 2-25% 11-22%

Phospholipids can be partially or completely removed from vegetable oils by several different known methods. The most commonly used methods in industry are water malting, acidic degumming, alkaline refining and enzymatic degumming.

Water Resin

This method is usually applied to crude oils containing a large amount of hydratable phospholipids. Due to their mild characteristics, the resulting phospholipids can be used as lecithin (a natural emulsifier). Oil obtained in this way, as a rule, is designated in this area as "tarred", despite the fact that it is only partially tarred. Since water-demineralized oil still contains large amounts of phospholipids, especially non-hydratable phospholipids, the use of other process technologies, such as alkaline refining or degumming with the enzyme PLA1, may be necessary to obtain a finished high-quality oil with high stability and non-intense color.

In the method of aqueous demineralization, water (1-5% w / w) is added to the crude oil at 60-75 ° C with vigorous stirring. Then the oil is gently mixed for 15 to 60 minutes to facilitate hydration of the phospholipids present in the oil. Hydration of phospholipids or “resins” causes swelling and agglomeration of resins in the form of a flocculant. A flocculant is an emulsion or a mixture of hydrated resins and oil. The emulsion has a specific density higher than that of the oil and can be separated by sedimentation, filtration or industrial centrifugation practice. By centrifugation, two streams of oil-free oil and wet resins are obtained. By the method of aqueous degumming, only hydratable phospholipids are mainly removed. The remaining phospholipids (50-250 ppm), defined as salts of phosphatidic acid and / or PI, can be removed in subsequent processing operations.

Separated wet resins are present in an emulsified oil mixture containing at least one triacylglycerol (or oil) molecule for every two phospholipid (or resin) molecules. Such an emulsified oil cannot be physically separated or isolated from the emulsion, and it is considered as loss associated with the method. Resins can be dried and sold as edible lecithin, but they are usually used as a by-product for other applications, such as animal feed or in an industrial process, with reduced economic value.

The loss of oil during emulsification is significant, with a negative contribution to the overall economic balance of the cost of the process for refined oil.

Sour Resin

This method is usually used for crude oils, when the goal is the complete removal of phospholipids. The resulting oil is commonly referred to in the industry as "super-tarred" or "fully tarred."

The crude oil is treated with 250-2000 ppm of phosphoric acid or citric acid at 60-90 ° C with vigorous stirring. The acid is allowed to interact with NHP salts for a period of 10-90 minutes. Acid improves the hydrophilic properties of NHP, thus contributing to their removal. Then water (1-5% w / w) is added to the acid-treated crude oil at 60-75 ° C. with vigorous stirring. Then the oil is carefully mixed for 15 to 60 minutes to facilitate the hydration of phospholipids. Hydration of phospholipids or “resins” causes swelling and agglomeration of resins in the form of a flocculant. A flocculant is an emulsion or a mixture of hydrated resins and oil. The emulsion has a specific density higher than that of the oil and can be separated by sedimentation, filtration or industrial centrifugation practice. By centrifugation, an acidic tarless oil and a wet resin are obtained. Most phospholipids are removed by the method of acidic resins, but there is still enough (25-100 ppm) in the resined oil for the need for additional processing. For use in food industries, tarless oil is typically discolored and deodorized according to a method known in the industry as “physical refining”. Acid treated resins are no longer suitable for edible lecithin.

As in the water-based resin removal method, the separated and anhydrous resins in the acid-resin removal method contain at least one molecule of triacylglycerol (or oil) for every two molecules of phospholipid (or resin). Such emulsified oil cannot be physically separated or isolated, and it is considered as a loss associated with the method, with a negative contribution to the overall economic balance of the cost of the method for refined oil.

Alkaline refining

This method is usually used for crude or oil-less oils, when the goal is to remove all phospholipids and free fatty acids.

Crude or oil-free water is treated with 200-1000 ppm of phosphoric acid or citric acid at 60-90 ° C with vigorous stirring. The acid is allowed to interact with NHP salts for 10 to 90 minutes. Acid improves the hydrophilic properties of NHP, thus contributing to their removal. A dilute sodium hydroxide solution (10-18% w / w) is added to the acid-treated oil at 65-75 ° C. The amount of sodium hydroxide (alkali) is based on the amount of free fatty acids present in the oil, as well as an excess of 0.05-0.20% based on dry matter. An alkaline solution neutralizes free fatty acids (forming sodium soaps), neutralizes excess acid and, with formed sodium soaps, promotes hydration and emulsification of all remaining phospholipids.

The sodium hydroxide / oil solution is stirred for approximately 10 minutes, then separated by precipitation, filtration or industrial centrifugation. By centrifugation, alkali-treated oil and raw materials are obtained to obtain soap. The alkali-treated oil is then “washed” with 10-20% softened water at 90-95 ° C and centrifuged again. Centrifuge oil is known as “once refined,” and water is commonly known as “flush water.” For use in food processing industries, “once refined” oil is usually discolored and deodorized to produce salad oil. An alternative to washing with water is to treat the alkali-treated oil with absorbent silica gel and filter out residual soaps and phospholipids that have not been removed by initial centrifugation.

As with water and acid demineralization methods, the separated and anhydrous resins in the alkaline refining method contain one molecule of triacylglycerol (or oil) for every two molecules of phospholipid (or resin). Such emulsified oil cannot be physically separated or isolated, and it is considered as loss associated with the method. In addition, sodium hydroxide will react with a neutral oil, forming soaps, thereby further reducing the overall oil yield with a negative contribution to the overall economic balance of the cost of the refined oil process.

Enzymatic treatment

Another refining method used in the vegetable oil industry is “enzymatic refining” or “enzymatic degumming”. Enzymatic degumming is used when the goal is to completely remove phospholipids. Typically, enzymatic treatments for the degumming of the prior art have been carried out in practice for oils that have previously been degummed using one of the other methods, usually aqueous degumming. For use in food processing industries, the enzymatically resinless oil is then discolored and deodorized in a manner known in the industry as “physical refining”. Enzymatic degumming provides a better oil yield than aqueous, acidic or alkaline degumming, with improved economic results.

The enzymatic reaction alters the nature of the phospholipid, cleaving some of the parts of the phospholipid. This reduces the emulsifying properties of phospholipids, so that less oil is lost when the resins are separated from the oil, thus preserving the oil. Enzymes with activity against phospholipids are commonly called "phospholipases." Types of phospholipases are based on the position in the molecule of the phospholipid with which the enzyme reacts, and are known as PLA1, PLA2, PLC and PLD. The positions in the phospholipid molecule with which various types of phospholipases react, are illustrated in FIG.

3, it can be seen that different types of phospholipases will form different compounds when interacting with phospholipids. In addition, each type of phospholipase has its own reaction rate and its own reaction conditions with respect to pH,% water and temperature. For PLA when used alone, typically a reaction time of at least about 4 hours is needed, while for PLC when used separately, it usually takes a reaction time of about one hour. It is known that enzymatic treatment should be carried out at a pH of less than or equal to 8 in order to minimize unwanted saponification of the oil, however, 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 stability. PLA enzymes will denature at approximately 50 ° C, while PLC enzymes will denature at approximately 65 ° C.

Sequences of amino acids with phospholipase activity are widely published in the literature and described in patents, and it has been published that some of them have activity against phospholipids present in vegetable oils. All of this is known in the art.

One of the commercial PLA1 enzyme products with phospholipase activity is phospholipase from Novozymes A1 Lecitase® Ultra. This product is known to form polar lysophospholipids and polar fatty acids when mixed with demineralized oil with 1-1.5% citric acid-NaOH buffer in water, at 4.5 <pH <7.0 and 40 ° C <T <55 ° C as described in Novozymes Application Sheet Oils & Fats # 2002-185255-01 and 2002-05894-03. PLA1 selectively hydrolyzes a fatty acid opposite a phosphate functional group on a glycerol backbone, as illustrated in FIG. 4.

The final reaction leads to the formation of lysophospholipid and fatty acid. The lysophospholipid molecule loses one hydrophilic functional group, and the remaining alcohol group in the reaction site is hydrophilic. Now, with two hydrophilic sites, the lysophospholipid molecule is water soluble and loses its emulsifying properties. The PLA1 degumming process thus reduces refining losses due to the fact that it no longer removes any neutral oil with resins, and the only phospholipid molecule is the only loss.

While enzymatic degumming provides significant advantages for oil refineries, it also has certain disadvantages. One of the disadvantages 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 take many hours, depending on such variable reactions as pH, temperature, relative concentrations and mixing conditions. Such long reaction times can have a significant negative effect on the overall economic benefit of enzymatic degumming processes. Enzymatic degumming, due to the slow reaction of PLA, is typically carried out in oil formulations, which were first subjected to aqueous degumming. Thus, the oil must be demineralized twice to obtain a product that has a phosphorus level low enough for its intended purpose.

It is known in the art that PLC enzymes react with a phospholipid by selectively hydrolyzing a phosphate functional group, as shown in FIG. The final reaction leads to the formation of diacylglycerol ("DAG") and phosphatide groups. The diacylglycerol molecule no longer has a phosphate functional group and does not need to be removed. The PLC demineralization method reduces refining losses by preserving the original phospholipid molecule while removing only the phosphate functional group. However, PLC does not react with all phospholipids present in the oil. Typically, PLC does not react with either phosphatidic acid (PA) or phosphatidic inositol (PI), illustrated in FIG. 2. Both PA and PI are still non-hydratable phosphatides, which remain in the oil after aqueous degumming. Thus, the PLC treated oil must be further treated with alkali to remove residual resins.

It is known that specific PLCs will only react with specific phosphatide groups. For example, a PI-specific PLC identified as PI-PLC is known.

Thus, in one aspect of the invention, there is provided a method for enzymatically degreasing oils in which the rate of an enzymatic reaction is greater than in prior art enzymatic degumming methods.

In another aspect of the invention, there is provided a method of increasing the reaction rate of the phospholipase A enzyme used in the enzymatic degumming method.

In another aspect of the present invention, there is provided a method for the degumming of an oil composition in which both hydratable and non-hydratable phospholipids can be processed in one process.

In another aspect of the present invention, enzymatic degumming and processing methods are provided in which the duration of the enzymatic reaction is less than about one hour.

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

US 5,264,367 to Aalrust et al. describes the use of phospholipases A1, A2 or B for the treatment of oil, which is first refined to 50-250 ppm of phosphorus. The technology described in this patent is commercially known as Enzymax®. Aalrust argues that since these enzymes attack lecithin, "it makes no sense to use the method of the invention for oils that are high in lecithin, such as raw soybean oil." The reaction is carried out at a temperature of 20-80 ° C, with citric acid or its salt in the pH range of 3-7. It is argued that the enzyme must be carefully distributed in oil, where the enzyme-water solution is present in the form of drops less than 10 microns in diameter. The form of measurement and calculation of the weighted average is not described. An emulsifier is used to dissolve phospholipases obtained from pancreatin or pancreatic products containing fat. Aalrust claims that since the oil that is released contains less than 5 ppm of phosphorus, it can be adapted for physical refining to edible oil. The details of the technology described by Aalrust were later disclosed in several publications (Dahlke, K. and Eichelsbacher, M., Enzymax® and Alcon® - Lurgi's route to Physical Refining in Proceeding of the World Conference on Oilseed and Edible Oils Processing, Istanbul, Turkey 1996, ed. Kaseoglu, Rhee and Wilson; Dalke, K. et al., First Experiments with Enzymatic Oil Refining, Inform, vol. 6, No. 12, Dec. 1995). The data disclosed in these publications for industrial testing support the use of the technology described in the references for oils with a P content in the range of 40 to 180 ppm and not higher. They also described that “the method does not require any special equipment. All pumps, mixers, mixers and heat exchangers, as well as a centrifuge, have a 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.

US 5,532,163 from Yagi et al. An enzymatic method is described using 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 100-10000 ppm of phosphorus . Then the oil is washed with 30-200% mass parts of water or an acidic aqueous solution per 100 mass parts of oil or fat. The total load of water required to use the method lies in the range from 60% to 400% wt./wt. processed oil. Obtaining such a large amount of wastewater at an industrial enterprise classifies this method as uneconomical.

US 6001640 from Loeffler et al. A method is described where one or more vegetable oils containing phosphorus-containing components are exposed to a phospholipase mixture obtained from Aspergillus, wherein the mixture contains an enzyme having an A1 activity, an A2 activity, or both, and an enzyme having a lysophospholipase activity. The patent states that since phospholipase will attack lecithin, it is impractical to use this method for oils with a high lecithin content, such as crude soybean oil.

Loeffler et al. describe that the enzymatic reaction should be carried out at a pH of less than 4 and with an emulsion droplet size of less than 20 microns. The form of measurement and calculation of the weighted average droplet size of the emulsion is not described. The patent states that the final product will have a residual P of 15 ppm or less. It is known in the art that if an oil is exposed to a pH as low as 4 or lower, the resins present in the oil will be forced to become hydrated and separate from the reaction medium. Hydrated resins will act as emulsifiers, so that when they separate, they will carry oil with them, thus leading to oil losses. No data on oil loss in resins.

US 6,103,505 from Clausen et al. The discovery and activity of specific phospholipases (A1, A2 or B) for use in the enzymatic removal of phospholipids, and a method for producing enzymes are described. In the enzymatic degumming method, the method described in US 5,264,367 can be used without any additional process steps.

US 6,127,137 from Hasida et al. the discovery and activity of specific phospholipases that are capable of removing both fatty acyl groups present in a phospholipid molecule when mixed with demineralized oil (50-250 ppm phosphorus) with 0.5-5% water, pH 1.5-3, at a temperature of 30-45 ° C and a time of 1-12 hours.

US 6143545 from Clausen et al. The discovery and activity of specific phospholipases (A1, A2 or B) for use in the enzymatic removal of phospholipids, and a method for producing enzymes are described. In the enzymatic degumming method, the method described in US 5,264,367 can be used without any additional process steps.

US 6,548,633 from Edwards et al. cDNA sequences encoding secreted proteins are described. Column 44 of the patent indicates that the protein of this invention can be used in enzymatic degumming of vegetable oils as described in US 6001640, cited above. In addition, the patent states, in the same paragraph, that the protein of this invention can be combined in a “cocktail” with other enzymes to improve the use of animal feed.

In US Patent Application Serial No. 10/556816 from Dayton et al. A method for improved enzymatic degumming is described, where the pH of the buffered reaction mixture for the enzyme is lowered below 4.5 after the completion of the enzymatic reaction, thus eliminating the clogging of equipment, in particular heat exchangers and separation centrifuges, which will result from the precipitation of calcium and magnesium salts at the optimum pH required for enzyme activity.

US 2004/0005399 A1 from Chakrabarti et al. An enzymatic method is described using a single addition of an enzyme and a buffer system and a short retention / reaction time, followed by decolorization with 2-4% bleaching clay and 0-1% activated carbon, and then dewaxing to obtain an oil with a phosphorus content of 5 hours / million Both by bleaching and dewaxing methods, residual phosphorus will be removed from the oil. In addition, Chakrabarti et al. argue that the oil loss in the resins is in the range of 30-40% of the separated resins, suggesting that the enzymatic reaction did not complete, leading to large oil losses due to oil emulsification in the removed phospholipids.

US 2005/0059130 A1 from Bojsen at al. The discovery and activity of specific phospholipases for use in the enzymatic removal of phospholipids, and a method for producing enzymes are described. The publication relates to the processing of vegetable oil to reduce the content of phospholipids, as described in US 5264367.

US 2005/0108789A1 from Gramatikova et al., Currently US Pat. No. 7,226,771, describes phospholipases (e.g., phospholipase A, B, C, D, patatin enzymes) that efficiently cleave glycerophosphate ester bonds in oils such as vegetable oils, to obtain a water extractable phosphorylated base and diglyceride. Paragraph 108 of the application further indicates 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, PLA1 and PLA2 in industrial oil-degumming, such as the ENZYMAX® method where phospholipids are hydrolyzed by PLA1 and PLA2. Paragraph 474 of this application states that PLC can be used separately or together with PLA to remove non-hydratable phospholipids from an oil that has previously been subjected to water-based demineralization but does not present reaction conditions for the use of two enzymes together. Since the optimal reaction conditions for the PLA enzyme and the PLC enzyme are different, this statement in the application without working examples does not explain to the person skilled in the art how to use the PLA and PLC enzymes at the same time. In addition, the application states that phospholipases C, D1 and D2 can be used in enzymatic degumming of pre-tarred and non-tarred (crude) oils and as an aid for alkaline refining.

Summary of the invention

The invention relates to a method for the degumming of an oil composition, comprising

(a) providing an oil composition containing an amount of phospholipids,

(b) contacting said oil composition with one or more phospholipase A enzymes and one or more phospholipase C enzymes, under conditions suitable for the enzymes, to interact with phospholipids to form reaction products with phospholipids, and

(c) separating the reaction products with phospholipids from the oil composition, where the remaining oil composition after separation is an oil-free oil composition,

moreover, during stage (b), the reaction with the specified one or more phospholipase A enzymes proceeds at a higher rate than it would in the absence of the specified one or more phospholipase C enzymes. In a preferred embodiment, the reaction in stage (b) continues for less than about one hour.

The amount of water required for the method of the present invention can be less than about 5%, and it can mainly be reduced to less than about 3% and, preferably, to about 1.5-2.0%.

The pH of the system can be adjusted either before or after adding one or all of the enzymes to the oil composition. The oil yield is maximized based on the composition of phospholipids contained in the crude feed.

In particular, the present invention relates to a method in which both the phospholipase C enzyme (PLC) and the phospholipase A enzyme (PLA) are used together in an enzymatic reaction to remove phospholipids present in the oil. More specifically, the present invention relates to the addition of phospholipase C (PLC) and / or phosphatidyl-inositol-specific phospholipase C (PI-PLC) in combination with phospholipase A1 (PLA1) and / or phospholipase A2 (PLA2) to maximize oil yield and reduce the amount of production waste received. It was unexpectedly discovered that the kinetics of enzymatic reactions is much faster than expected when two enzymes are used together than when each is used separately. In addition, it was found that the reactions are faster than expected, even when the reaction conditions are not optimized for at least one of the enzymes. In addition, it was found that the reaction can take place in less than about one hour, and can take place as quickly as in about thirty minutes.

Advantageously, the oil to be treated may be a crude oil or a water-demineralized oil. Enzymes can be added to the oil either individually or together, but the two enzymes will be in contact with the oil at the same time. In accordance with the present invention, the parameters of the enzymatic reaction, such as the concentration of water, temperature, pH, mixing time and concentration of the enzyme, can be adjusted by optimizing the reaction for a particular combination of enzymes in a particular oil system.

Description of figures

Figure 1 illustrates the chemical structures of common phospholipids and common triacylglycerols.

Figure 2 illustrates the functional groups and structures of well-known phospholipids.

Figure 3 illustrates the positions in the phospholipid molecule with which various types of phospholipases react.

Figure 4 illustrates the reaction of a phospholipid with the enzyme PLA 1 and the final products.

Figure 5 illustrates the reaction of a phospholipid with an enzyme PLC and end products.

Fig.6 is a graph summarizing the results of examples 13-30, in which the average residual phosphorus in the tarless sample is plotted for each level of each estimated experimental factor.

7 is a graph summarizing the results of examples 31-38, in which the average residual phosphorus in the tarless sample is plotted for each level of each estimated experimental factor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an improvement in the method of enzymatic degumming of an oil composition. The inventors have found that, unexpectedly, using a combination of enzymes, the kinetics of the phospholipid cleavage reaction can be improved. In particular, an enzymatic degumming method carried out with a combination of phospholipase C enzyme and phospholipase A enzyme gives an oil-free oil product with a lower phosphorus content in a shorter reaction time than can be achieved with phospholipase A. In addition, it was unexpectedly discovered that the reaction can pass in less than about one hour and can pass as fast as in about thirty minutes. This is especially surprising since PLA when used alone typically requires a reaction time of at least about 4 hours, while for PLC when used alone, it usually takes a reaction time of about one hour. Moreover, PLA has an optimum pH of 4.5 for the reaction, while PLC has an optimal pH of 7.0 for the reaction. Each enzyme also has different thermal stability. The PLA enzyme will denature at approximately 50 ° C, while the PLC enzyme will denature at approximately 65 ° C. In addition, it is known in the art that thermostability of enzymes can be improved through site-specific mutations. Such cloned enzymes can be thermostable at temperatures up to 80 ° C, and the use of such cloned enzymes is provided in the present invention.

Reduced reaction time is confirmed by PLA. When used in combination with PLC, the reaction time is significantly reduced, and in some embodiments, the invention may be less than about 1 hour, even under acidic reaction conditions that are not optimal for PLC. In addition, the inventors have found that under appropriate conditions it is possible to reduce the reaction time to as low as approximately 30 minutes.

It has been found that the concentration of water can be adjusted to suit the needs of a particular work environment. Thus, the concentration of water can be reduced to about 1-2% and, in particular, to about 1.5%, if it is desirable to reduce the amount of wastewater resulting from the method. Alternatively, the concentration of water can be increased to about 4-5% and, in particular, to about 4.5%, if it is desirable to increase the effectiveness of the method of desalination.

An advantage of the present invention is that the oil to be degummed can be either crude oil or previously degummed using one of the methods of the prior art. A separate advantage for the oil refining enterprise is the ability to perform oil de-oiling in one stage. Oils that can be processed in accordance with the present invention may include, without limitation, the following: canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hemp oil, linseed oil, seed oil mangoes, meadow pennic oil, hoofed oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, soybean oil, sunflower Noah oil, tall oil, Japanese camellia and vegetable oil.

The phospholipase A enzyme used in the method of the present invention can be either a phospholipase A1 enzyme or a phospholipase A2 enzyme. The phospholipase C enzyme used in the present invention can be either phospholipase C or an inositol-specific phospholipase C. Many enzyme variants from the phospholipase A and phospholipase C families are commercially available; and provide that such enzymes and their equivalents will be suitable for use in the present invention.

In the method of the invention, various phospholipases used together in the enzymatic degumming method of the present invention can be mixed together before being added to the oil to be treated. Alternatively, they can be added to the oil individually, either sequentially or simultaneously. With sequential or joint addition, the enzymatic reaction will take place at some point when both enzymes are present in the reaction mixture.

The degumming method of the present invention is carried out at a pH below about 8, preferably about 3-7, and most preferably about 4-5. The pH values in the enzymatic degumming process can be achieved by adding known buffers. It is well known that citric acid and sodium hydroxide are suitable for this purpose. Other buffering agents can be used as needed to adjust the pH under specific reaction conditions.

The temperature in the enzymatic degumming method 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. It has been unexpectedly discovered that in the methods of the present invention, PLA degumming can take place at a temperature above its own optimum of 45 ° C and closer to the optimum of the PLC operating temperature, without excessive denaturation.

The method of the present invention provides a one-step method of demineralization, in which the content of phospholipids in the oil, even in the crude oil, can be reduced to less than 50 ppm P, preferably less than 20 ppm P, more preferably less than 10 ppm ppm, and most preferably less than 5 ppm p.

After the enzymatic degumming is completed and the degummed oil is separated from the resins, the degummed oil can be subjected to additional processing steps known in the art, such as discoloration or deodorization, which may be necessary or desirable depending on the end use for which the degummed oil product is intended.

Various preferred embodiments of the invention are indicated in the examples below, together with control examples using the conditions of the prior art. In each of the examples below, the overhead stirrer was a Heidolph mixer of the Elector KG model with flat blades, operating at 90 rpm for normal mixing and 350 rpm for intensive mixing. The centrifuge was a De Laval Gyro - Tester, with a built-in “rotor unit” for continuous separation. The centrifuge rotor was closed by built-in locking screws. Shear mixing was carried out using 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 from Denmark and had a concentration of 11.2 units / mg. The PLA2 enzyme was Rohalase® MPL (batch number Ch: 4738) sold by AB Enzymes in Germany and had a concentration of 2000 u / mg. The PLC enzyme was Purifine ™ sold by Diversa Corporation of San Diego, California. For examples 1-12, PLC was a batch of BD16449, having a concentration of 205 units / mg. For examples 13-38, the PLC was a batch of 90BU002A1 having a concentration of 27.5 units / mg. The amount of phospholipids remaining in the treated oil was determined as ppm P according to the method of American Oil Chemists' Society Official Method Ca 20-99, "Analysis of Phosphorus in Oil by Inductively Coupled Plasma Optical Emission Spectroscopy".

Example 1

Control: water -based desalination - 1965.4 grams of crude soybean oil containing 746 ppm phosphorus was heated to 70-75 ° C with normal stirring using an overhead stirrer. To the warm oil was added 39.4 grams of deionized water with vigorous stirring for 1 minute. The rotation of the stirrer was slowed down to normal speed (90 rpm) to allow the resins to flocculate for 30 minutes. The oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil-free oil was 80.7 ppm.

Example 2

Control: Desalting with a single enzyme with phospholipase A1 (PLA1) - 1997.9 g of crude soybean oil containing 746 ppm phosphorus was heated to 75-80 ° C with stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and the mixture was stirred with a shift for 1 minute. The oil was subjected to normal stirring for one hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40-45 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40-45 ° C, 60.0 grams of deionized water was first added and the mixture was mixed with a shift of 1 minute, then 0.1044 grams of Novozymes' Lecitase® Ultra PLA1 was added and the whole mixture was mixed with a shift for 1 minute. The oil mixture was stirred at normal speed using an overhead stirrer for 4 hours at a temperature in the range 41-48 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the demineralized PLA1 oil was 31.7 ppm.

Example 3

Control: Desalting with a single enzyme with phospholipase C (PLC) - 2011.1 grams of crude soybean oil containing 746 ppm phosphorus was heated to 55-60 ° C with normal stirring using an overhead stirrer. 60.3 grams of deionized water was added and the mixture was sheared for 1 minute. 0.1051 grams of Diversa's Purifine ™ (PLC lipase BD 16449 containing 205 units / mg) was added and the mixture was stirred for 1 minute. The oil mixture was subjected to normal stirring for 1 hour at 50-63 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the demineralized PLC oil was 70.9 ppm.

Example 4

Control: Demineralization of PLC, then PLA - In this control example, an oil sample was reacted with each enzyme separately under the reaction conditions optimal for the enzyme according to the prior art, 2110.5 grams of crude soybean oil containing 560.1 ppm phosphorus, heated to 60 ° C with normal stirring. 63 grams of deionized water and 0.1123 grams of Diversa's Purifine ™ (PLC BD16449 lipase containing 205 units / mg) were added and the mixture was stirred for 1 minute. The oil mixture was stirred at normal speed for 1 hour at 55-56 ° C. The oil was then centrifuged and the oil and wet resins collected. To obtain a buffer with a pH of 4.5, first 2.0 grams of 50% w / w. a citric acid solution was added to the PLC-resin-free oil, the mixture was sheared for 1 minute, and then stirred for one hour at normal speed using an overhead stirrer; then 1.8 milliliters of a 4 molar sodium hydroxide solution was added, and the oil mixture was sheared for 10 seconds. 59 grams of deionized water was added and the mixture was stirred for 1 minute. 0.1061 grams of Novozymes' Lecitase® Ultra PLA1 was added with the resulting buffer, and the whole mixture was sheared for 1 minute. The oil was stirred at normal speed for 4 hours at a temperature of approximately 45 ° C. Then the oil was centrifuged; separated oil and wet resins were collected. The residual phosphorus in the oil sequentially tarred by PLC, then PLA1, was 3.2 ppm.

Example 5

PLC and PLA1 together, a neutral pH with a reaction time of 1 hour at 45 ° C - 2004.9 grams of crude soybean oil containing 560.1 ppm phosphorus, was heated to 45 ° C with normal stirring. At a neutral pH of the oil, 60 grams of deionized water, 0.1037 grams of Diversa's Purifine ™ (PLC enzyme) and 0.1076 grams of Novozymes' Lecitase® Ultra (PLA1 enzyme) were added to the oil, and the whole mixture was sheared for 1 minute. The mixture of oil and enzyme was stirred at normal speed for 1 hour at a temperature of approximately 45 ° C. The oil was then centrifuged and the separated oil and wet resins were collected. An oil-free oil containing 13.2 ppm of residual phosphorus was obtained from an oil treated with a combined mixture of PLC and PLA1 enzymes at a neutral pH and 45 ° C with a reaction time of one hour.

The level of residual phosphorus is significantly lower than the level achieved either with one PLA under optimal conditions for it (example 2), or with one PLC under optimal conditions for it (example 3).

Example 6

PLC and PLA1 together, a neutral pH with a reaction time of 4 hours at 45 ° C - 2003.7 grams of crude soybean oil containing 560.1 ppm phosphorus, was heated to 45 ° C with normal stirring. 60 grams of deionized water, 0.1040 grams of Diversa's Purfine ™ (PLC enzyme) and 0.1085 grams of Novozymes' Lecitase® Ultra (PLA1 enzyme) were added and the whole mixture was stirred for 1 minute. The oil mixture was stirred at normal speed for 4 hours at a temperature of approximately 45 ° C. The oil was then centrifuged and the separated oil and wet resins were collected. By a method using a combined mixture of PLC and PLA1 enzymes, a tarred oil containing 10.5 ppm of residual phosphorus was obtained with a reaction time of four hours at neutral pH.

The level of residual phosphorus is only slightly improved compared to the level achieved in Example 5, indicating that an increase in the reaction time from one hour to four hours does not lead to a significant difference in the effectiveness of the method of demineralization.

Example 7

PLC and PLA1 together, pH 4.5 with a reaction time of 1 hour at 45 ° C - 2021.4 g of crude soybean oil containing 547.9 ppm of phosphorus were heated to 75-80 ° C with normal stirring using top drive mixers. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift 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 a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. 61.0 grams of deionized water, 0.1184 grams of Diversa's Purfine ™ (PLC enzyme) and 0.1038 grams of Novozymes' Lecitase® Ultra (PLA1 enzyme) were added, and the whole mixture was sheared for 1 minute. The oil mixture was stirred at normal speed for 1 hour at a temperature of approximately 45 ° C. The oil was then centrifuged and the separated oil and wet resins were collected. Using a combined mixture of PLC and PLA1 enzymes, with a reaction time of one hour, at a pH of 4.5 and a temperature of 45 ° C, a tarless oil containing 2.4 ppm of residual phosphorus was obtained.

This level of residual phosphorus is approximately the same, and even slightly better than the level achieved in example 4, where the reaction with each enzyme was carried out separately and under its own optimal conditions. Surprisingly, the degumming efficiency is just as good when the two enzymes act together at a reaction time that is not optimal for the PLA, and at pH and temperature that are not optimal for the PLC, as and when the two enzymes act separately, each in his own optimal conditions.

Example 8

PLC and PLA1 together, pH 4.5 with a reaction time of 4 hours at 45 ° C — 2069.3 g of crude soybean oil containing 547.9 ppm of phosphorus were heated to 75-80 ° C with normal stirring. Added 2.0 grams of 50% wt./wt. citric acid solution and the mixture was stirred with a shift 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 a 4 molar sodium hydroxide solution was added, and the mixture was sheared for 10 seconds. 63 grams of deionized water, 0.1112 grams of Diversa's Purfine ™ (PLC enzyme) and 0.1258 grams of Novozymes' Lecitase® Ultra (PLA1 enzyme) were added, and the whole 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 separated oil and wet resins were collected. By a method using a combined mixture of PLC and PLA1 enzymes, a resin-free oil containing 2.5 ppm of residual phosphorus was obtained with a reaction time of four hours at pH 4.5.

This level of residual phosphorus is approximately the same as that achieved in example 7, indicating that an increase in the reaction time from one hour to four hours did not lead to a significant difference in the effectiveness of the method of demineralization.

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 of phosphorus were heated to 75-80 ° C with normal stirring. Added 2.0 grams of 50% wt./wt. citric acid solution and the mixture was stirred with a shift 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 a 4 molar sodium hydroxide solution was added, and the mixture was sheared for 10 seconds. 63.0 grams of deionized water, 0.1085 grams of Diversa's Purfine ™ (PLC enzyme) and 0.1045 grams of Novozymes' Lecitase® Ultra (PLA1 enzyme) were added, and the whole 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; separated oil and wet resins were collected. Using a combined mixture of PLC and PLA1 enzymes, with a reaction time of one hour, at a pH of 4.5 and a reaction temperature of 55 ° C, a tarless oil containing 2.3 ppm of residual phosphorus was obtained.

This level of residual phosphorus 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 does not lead to a significant difference in the effectiveness of the method of demineralization that normally it can be expected that PLA1 will denature at temperatures above 50 ° C.

Example 10

PLC together with a 2-fold concentration of PLA1, pH 4.5 with a reaction time of 1 hour at 45 ° C - 1992.2 g of crude soybean oil containing 547.9 ppm of phosphorus was heated to 75-80 ° C with stirring at normal speed. Added 2.0 grams of 50% wt./wt. citric acid solution and the mixture was stirred with a shift for 1 minute, then stirred for one hour. The mixture was allowed to cool to 40-45 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added, and the mixture was sheared for 10 seconds. 60 grams of deionized water, 0.1319 grams of Diversa's Purfine ™ (PLC enzyme) and 0.2139 grams of Novozymes' Lecitase® Ultra (PLA1 enzyme) were added to the mixture, and the whole mixture was sheared for 1 minute. The oil mixture was stirred at normal speed for 1 hour at a temperature of about 45 ° C. Then the oil mixture was centrifuged; separated oil and wet resins were collected. Using a combined mixture of PLC enzymes and a double concentration of PLA1, a tarred oil containing 7.0 ppm of residual phosphorus was obtained with a reaction time of one hour at pH 4.5.

This level of residual phosphorus is acceptable for certain applications, but not as good as the level achieved in examples 7-9, indicating that, unexpectedly, increasing the dose of PLA1 does not lead to improved efficiency of the method of demineralization, even under reaction conditions, optimal 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, was heated to 75-80 ° C with normal stirring using top drive mixers. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40-45 ° C, 0.1112 grams of Diversa's Purifine ™ (PLC BD16449 lipase containing 205 units / mg) and 0.2094 grams of Rohalase® MPL (lot number Ch: 4738) sold by AB Enzymes were added, then 60 grams of deionized water and the whole mixture was sheared for 60 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 40-45 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA2 enzymes at neutral pH resulted in a tarless oil containing 3.3 ppm residual phosphorus.

This example is similar to example 7 above, except for replacing PLA1 with PLA2. The low level of residual phosphorus in the final product indicates that PLA2 can function approximately as 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, was heated to 75-80 ° C with normal stirring using top drive mixers. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40-45 ° C, 0.1038 grams of Diversa's Purifine ™ (PLC BD16449 lipase containing 205 units / mg) and 0.2047 grams of Rohalase® MPL (lot number Ch: 4738) sold by AB Enzymes were added, then 60 grams of deionized water and the whole mixture was sheared for 60 seconds. The oil mixture was stirred at normal speed for 4 hours at a temperature of 40-45 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA2 enzymes at neutral pH resulted in an oil-free oil containing 5.8 ppm of residual phosphorus.

This example illustrates that an increase in the reaction time from one to four hours does not lead to a better tarring, but in fact leads to a higher level of residual phosphorus.

The results of the above examples 1-12 are summarized in table 2 below.

table 2 Etc. Enzyme addition PLC (ppm of active enzyme) PLA1 (ppm of active enzyme) PLA2 (ppm of active enzyme) Reaction time (min) Pace. (° C) pH Water (%) Phosphorus (ppm) one No thirty 70-75 7 2.0 80.7 2 Separately 0.6 240 41-48 4,5 3.0 31.7 3 Separately 10.8 60 50-63 7 3.0 70.9 four Sequentially 11.5 0.6 60, 240 55.45 4,5 3,1 3.2 5 At the same time 10.6 0.6 60 45 7 3.0 13,2 6 At the same time 10.7 0.6 240 45 7 3.0 10.5 7 At the same time 12.1 0.6 60 45 4,5 3.0 2,4 8 At the same time 11,4 0.7 240 45 4,5 3,1 2,5 9 At the same time 11.1 0.6 60 55 4,5 3,1 2,3 10 At the same time 13.5 1,2 60 45 4,5 3.0 7.0 eleven At the same time 11,4 209 60 45 4,5 3.0 3.3 12 At the same time 10.6 205 240 45 4,5 3.0 5.8

Examples 13-30

The design of the experiments (DOE) was developed to determine the effects of control variables of a particular method on the method of enzymatic degumming, as shown in table 3 below.

Table 3 Variable Levels Enzyme addition Sequentially At the same time The number of PLC (ppm active) 10 twenty thirty The number of PLA (ppm active) 0.5 1,0 2.0 Mixing time (s) 45 60 120 pH 4,5 5,0 7.0 Water (%) 1,5 3.0 4,5 Temperature (° C) 40 fifty 60 Reaction time (min) thirty 60 120

In sequence, each enzyme was added separately, although both enzymes were in contact with the oil mixture for at least part of the reaction time.

At the same time, both enzymes were added at the same time.

pH - pH at which the oil is exposed to enzymes.

Temperature - the temperature at which the oil is exposed to enzymes.

Mixing time is the time during which the mixture is stirred at high speed after adding each enzyme.

Reaction time is the total time during which at least one enzyme is in contact with the oil.

These working variables were evaluated in eighteen separate trials, presented here as examples 13-30. The values of each variable tested in each example 13-30 are shown in table 4 below.

Table 4 Etc. Enzyme addition PLC (ppm of active enzyme) PLA1 (ppm of active enzyme) Reaction time (min) Pace. (° C) pH Water (%) Mixing time (s) 13 Sequentially twenty one 120 40 5 1,5 120 fourteen At the same time twenty one 60 60 7 1,5 45 fifteen Sequentially 10 one thirty 40 7 4,5 60 16 Sequentially 10 2 60 fifty 4,5 1,5 120 17 Sequentially twenty 2 thirty fifty 7 3.0 45 eighteen Sequentially 10 0.5 120 60 5 3.0 45 19 At the same time thirty 2 thirty 60 5 1,5 60 twenty At the same time 10 2 120 60 7 4,5 120 21 At the same time twenty 2 120 40 4,5 3.0 60 22 Sequentially thirty 2 60 40 5 4,5 45 23 Sequentially thirty one thirty 60 4,5 3.0 120 24 At the same time thirty one 120 fifty 4,5 4,5 45 25 At the same time 10 one 60 fifty 5 3.0 60 26 Sequentially twenty 0.5 60 60 4,5 4,5 60 27 Sequentially thirty 0.5 120 fifty 7 1,5 60 28 At the same time thirty 0.5 60 40 7 3.0 120 29th At the same time twenty 0.5 thirty fifty 5 4,5 120 thirty At the same time 10 0.5 thirty 40 4,5 1,5 45

Example 13

1999.1 grams of crude soybean oil containing 769.5 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 2.4 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 5.0. Maintaining a temperature of 40 ° C, 1.5008 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 30 grams of deionized water, and the whole mixture was sheared for 120 seconds. The oil mixture was stirred at normal speed for 60 minutes. Maintaining a temperature of 40 ° C, 0.2132 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, batch number LYN05007) was added and the whole mixture was sheared for 120 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil sequentially tarred by PLC, then PLA1 was 6.5 ppm.

Example 14

2010.5 grams of crude soybean oil containing 785.1 ppm phosphorus was cooled to 60 ° C. using normal mixing and an overhead stirrer. Maintaining a temperature of 60 ° C, 1.5316 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.2073 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 30 grams of deionized water were added and the whole mixture was mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at neutral pH resulted in an oil-free oil containing 109.6 ppm of residual phosphorus.

Example 15

1994.5 grams of crude soybean oil containing 785.1 ppm phosphorus was cooled to 40 ° C. using normal stirring and an overhead stirrer. Maintaining a temperature of 40 ° C, 0.754 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 90 grams of deionized water, and the whole mixture was sheared for 60 seconds. The oil mixture was stirred at normal speed for 15 minutes. Maintaining a temperature of 40 ° C, 0.2242 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) was added and the whole mixture was sheared for 60 seconds. The oil mixture was stirred at normal speed for 15 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil sequentially tarred by PLC, then PLA1, was 27.4 ppm.

Example 16

2002.0 grams of crude soybean oil containing 785.1 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 50 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 50 ° C, 0.7498 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 30 grams of deionized water, and the whole mixture was sheared for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes. Maintaining a temperature of 50 ° C, 0.4064 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, batch number LYN05007) was added and the whole mixture was sheared for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 50 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil sequentially tarred by PLC, then PLA1, was 7.6 ppm.

Example 17

2010.7 grams of crude soybean oil containing 785.1 ppm phosphorus was heated to 50 ° C. using normal mixing and an overhead stirrer. Maintaining a temperature of 50 ° C., 1.4981 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 60 grams of deionized water, and the whole mixture was sheared for 45 seconds. The oil mixture was stirred at normal speed for 15 minutes. Maintaining a temperature of 50 ° C, 0.4143 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) was added and the whole mixture was mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 15 minutes at a temperature of 50 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil sequentially tarred by PLC, then PLA1, was 79.3 ppm.

Example 18

2005.3 grams of crude soybean oil containing 742.9 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 60 ° C, then 2.4 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 5.0. Maintaining a temperature of 60 ° C, 0.7491 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 60 grams of deionized water, and the whole mixture was sheared for 45 seconds. The oil mixture was stirred at normal speed for 60 minutes. Maintaining a temperature of 60 ° C, 0.1220 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) was added and the whole mixture was mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil sequentially tarred by PLC, then PLA1, was 2.2 ppm.

Example 19

2000.4 grams of crude soybean oil containing 742.9 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 60 ° C, then 2.4 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 5.0. Maintaining a temperature of 60 ° C, 2.2270 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.3937 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 30 grams of deionized water were added and the whole mixture was mixed with a shift for 60 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 5.0 resulted in an oil-free oil containing 7.8 ppm of residual phosphorus.

Example 20

2006.3 grams of crude soybean oil containing 719.3 ppm phosphorus was heated to 60 ° C. using normal stirring and an overhead stirrer. Maintaining a temperature of 60 ° C, 0.7561 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.4098 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 90 grams of deionized water and the whole mixture was mixed with a shift for 120 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at neutral pH resulted in an oil-free oil containing 64.1 ppm of residual phosphorus.

Example 21

1998.5 grams of crude soybean oil containing 719.3 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40 ° C, 1.47798 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added and 0.4018 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) was added, then 60 grams of deionized water and the whole mixture mixed with a shift for 60 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 5.5 ppm of residual phosphorus.

Example 22

2001.3 grams of crude soybean oil containing 719.3 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 2.4 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 5.0. Maintaining a temperature of 40 ° C, 2.2580 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 90 grams of deionized water, and the whole mixture was sheared for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes. Maintaining a temperature of 40 ° C, 0.4126 grams of Novozymes' Lecitase © Ultra (PLA1 lipase, lot number LYN05007) was added and the whole mixture was mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil-free oil sequentially treated with PLC and PLA1 was 2.1 ppm of residual phosphorus.

Example 23

2002.0 grams of crude soybean oil containing 747.3 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 60 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 60 ° C, 2.2194 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 60 grams of deionized water, and the whole mixture was sheared for 120 seconds. The oil mixture was stirred at normal speed for 15 minutes. Maintaining a temperature of 60 ° C, 0.2198 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, batch number LYN05007) was added and the whole mixture was sheared for 120 seconds. The oil mixture was stirred at normal speed for 15 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil-free oil sequentially treated with PLC and PLA1 was 4.6 ppm of residual phosphorus.

Example 24

2000.8 grams of crude soybean oil containing 747.3 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 50 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 50 ° C, 2.2500 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added and 0.2216 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) was added, then 90 grams of deionized water and the whole mixture mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 50 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes resulted in an oil-free oil containing 1.8 ppm of residual phosphorus.

Example 25

1998.9 grams of crude soybean oil containing 747.3 ppm phosphorus was heated to 75-80 ° C with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 50 ° C, then 2.4 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 5.0. Maintaining a temperature of 50 ° C, 0.7445 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added and 0.2042 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) was added, then 60 grams of deionized water and the whole mixture mixed with a shift for 60 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 50 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes resulted in an oil-free oil containing 7.2 ppm of residual phosphorus.

Example 26

1997.3 grams of crude soybean oil containing 810.8 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 60 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 60 ° C, 1.5189 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 90 grams of deionized water, and the whole mixture was sheared for 60 seconds. The oil mixture was stirred at normal speed for 30 minutes. Maintaining a temperature of 60 ° C, 0.1119 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) was added and the whole mixture was sheared for 60 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil-free oil sequentially treated with PLC and PLA1 was 2.2 ppm of residual phosphorus.

Example 27

2010.0 grams of crude soybean oil containing 810.8 ppm phosphorus was cooled to 50 ° C. using normal stirring and an overhead stirrer. Maintaining a temperature of 50 ° C, 2.2608 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) was added, then 30 grams of deionized water, and the whole mixture was sheared for 60 seconds. The oil mixture was stirred at normal speed for 60 minutes. Maintaining a temperature of 50 ° C, 0.1172 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, batch number LYN05007) was added and the whole mixture was sheared for 60 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 50 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. The residual phosphorus in the oil-free oil sequentially treated with PLC and PLA1 at neutral pH was 72.6 ppm of residual phosphorus.

Example 28

2005.1 grams of crude soybean oil containing 810.8 ppm phosphorus was heated to 40 ° C. using normal stirring and an overhead stirrer. Maintaining a temperature of 40 ° C, 2.2622 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1031 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 60 grams of deionized water were added and the whole mixture was mixed with a shift for 120 seconds. The oil mixture was stirred at normal speed for 60 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at neutral pH resulted in a tarless oil containing 61.5 ppm of residual phosphorus.

Example 29

2006.3 grams of crude soybean oil containing 795.3 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 50 ° C, then 2.4 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 5.0. Maintaining a temperature of 50 ° C, 1.5373 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1168 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 90 grams of deionized water were added and the whole mixture was mixed with a shift for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 50 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 5.0 resulted in an oil-free oil containing 1.9 ppm residual phosphorus.

Example 30

2006.1 grams of crude soybean oil containing 795.3 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40 ° C, 0.7736 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1072 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 30 grams of deionized water and the whole mixture was mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 13.7 ppm of residual phosphorus.

The results of examples 13-30 in relation to the achieved level of phosphorus are shown in table 5 below.

Table 5 Etc. Enzyme addition PLC (ppm of active enzyme) PLA1 (ppm of active enzyme) Reaction time (min) Pace. (° C) pH Water (%) Mixing time (s) Phosphorus (ppm) 13 Sequentially 20.6 1,2 120 40 5 1,5 120 6.5 fourteen At the same time 21.1 1,2 60 60 7 1,5 45 109.6 fifteen Sequentially 10,4 1.3 thirty 40 7 4,5 60 27.4 16 Sequentially 10.3 2,3 60 fifty 4,5 1,5 120 7.6 17 Sequentially 20.6 2,3 thirty fifty 7 3.0 45 79.3 eighteen Sequentially 10.3 0.7 120 60 5 3.0 45 2.2 19 At the same time 30.6 2.2 thirty 60 5 1,5 60 7.8 twenty At the same time 10,4 2,3 120 60 7 4,5 120 64.1 21 At the same time 20.3 2,3 120 40 4,5 3.0 60 5.5 22 Sequentially 31,0 2,3 60 40 5 4,5 45 2.1 23 Sequentially 30.5 1,2 thirty 60 4,5 3.0 120 4.6 24 At the same time 30.9 1,2 120 fifty 4,5 4,5 45 1.8 25 At the same time 10,2 1.4 60 fifty 5 3.0 60 7.2 26 Sequentially 20.9 0.6 60 60 4,5 4,5 60 2.2 27 Sequentially 31.1 0.7 120 fifty 7 1,5 60 72.6 28 At the same time 31.1 0.6 60 40 7 3.0 120 61.5 29th At the same time 21.1 0.7 thirty fifty 5 4,5 120 1.9 thirty At the same time 10.6 0.6 thirty 40 4,5 1,5 45 13.7

A generalization of the above experimental runs is illustrated in the graph shown in FIG. 6, which is a graph of the average amount of final phosphorus at each level of each factor.

At a neutral pH that is optimal for the PLC enzyme, with a combination of two enzymes, it was not possible to obtain an oil with acceptable phosphorus levels that allow physical refining of the oil. However, at an acidic pH optimal for the PLA enzyme, with a combination of enzymes added sequentially or together, acceptable levels of residual phosphorus in the oils were obtained that would allow their physical refinement. When using acidic pH in only one of the experimental runs, it was not possible to obtain an oil with a residual phosphorus content of less than 10 ppm. In Example 30, a residual phosphorus level of more than 10 (13.7 ppm) was obtained, and it was obtained with the lowest levels of both enzymes, the lowest temperature, the lowest percentage of water, the shortest mixing and stirring time, and the most acidic pH.

A synergistic effect was found between the combination of enzymes, allowing the reaction to proceed to completion in less than 30 minutes, compared with 1 hour for PLC enzymes or 4 hours for PLA enzymes. Additional testing was performed 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, taking into account the aforementioned detection that significantly lower favorable results were obtained at lower pH than at neutral pH. The amount of PLA1 was also kept constant at 0.5 ppm, since it was previously determined that increasing the amount of PLA1 above this level does not lead to a more efficient demineralization. In addition, in each of these examples, enzymes were added simultaneously, and not sequentially, taking into account the above definition that when adding enzymes at the same time get better resins than when sequentially adding. In addition, in an industrial method, it is advantageous to limit the total time of the method, general equipment and specialized devices.

Table 6 Example Enzyme addition PLC (ppm of active enzyme) PLA1 (ppm of active enzyme) Reaction time (min) Pace. (° C) pH Water (%) Mixing time (s) 31 At the same time twenty 0.5 120 40 4,5 2.0 120 32 At the same time 10 0.5 120 60 4,5 4,5 45 33 At the same time 10 0.5 thirty 40 4,5 4,5 120 34 At the same time twenty 0.5 thirty 60 4,5 2.0 45 35 At the same time twenty 0.5 120 40 4,5 4,5 45 36 At the same time twenty 0.5 thirty 60 4,5 4,5 120 37 At the same time 10 0.5 thirty 40 4,5 2.0 45 38 At the same time 10 0.5 120 60 4,5 2.0 120

Example 31

2003.6 grams of crude soybean oil containing 784.8 ppm phosphorus was heated to 75-80 ° C with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40 ° C, 1.4603 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1021 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 40 grams of deionized water and the whole mixture was mixed with a shift for 120 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 10.7 ppm of residual phosphorus.

Example 32

2004.8 grams of crude soybean oil containing 784.8 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 60 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 60 ° C, 0.7509 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1105 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 90 grams of deionized water was added and the whole mixture was mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 6.7 ppm of residual phosphorus.

Example 33

2000.4 grams of crude soybean oil containing 697.7 ppm phosphorus was heated to 75-80 ° C. under normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40 ° C, 0.7530 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1022 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 90 grams of deionized water and the whole mixture was mixed with a shift for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 2.2 ppm of residual phosphorus.

Example 34

1999.4 grams of crude soybean oil containing 714.2 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 60 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 60 ° C, 1.508 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1139 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 40 grams of deionized water were added and the whole mixture was sheared for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 16.5 ppm of residual phosphorus.

Example 35

1999 grams of crude soybean oil containing 714.2 ppm phosphorus was heated to 75-80 ° C under normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40 ° C, 1.5010 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1060 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 90 grams of deionized water were added and the whole mixture was mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 1.9 ppm residual phosphorus.

Example 36

1999 grams of crude soybean oil containing 695.1 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 60 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 60 ° C, 1.5296 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1241 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 90 grams of deionized water were added and the whole mixture was mixed with a shift for 120 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 5.2 ppm of residual phosphorus.

Example 37

2005.2 grams of crude soybean oil containing 695.1 ppm phosphorus was heated to 75-80 ° C. with normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 40 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 40 ° C, 0.7422 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1195 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN05007) were added, then 40 grams of deionized water and the whole mixture was mixed with a shift for 45 seconds. The oil mixture was stirred at normal speed for 30 minutes at a temperature of 40 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 6.7 ppm of residual phosphorus.

Example 38

1998 grams of crude soybean oil containing 695.1 ppm phosphorus was heated to 75-80 ° C under normal stirring using an overhead stirrer. Added 2.0 grams of 50% wt./wt. citric acid solution and stirred with a shift for 1 minute. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was allowed to cool with stirring at normal speed until the oil temperature reached 60 ° C, then 1.8 milliliters of a 4 molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. Maintaining a temperature of 60 ° C, 0.7429 grams of Diversa's Purifine ™ (PLC lipase, lot number 90BU002A1) and 0.1041 grams of Novozymes' Lecitase® Ultra (PLA1 lipase, lot number LYN 05007) were added, then 40 grams of deionized water and the whole mixture mixed with a shift for 120 seconds. The oil mixture was stirred at normal speed for 120 minutes at a temperature of 60 ° C. The enzyme treated oil was then centrifuged and the separated oil and wet resins were collected. Residual phosphorus in a combined mixture of PLC and PLA1 enzymes at pH 4.5 resulted in an oil-free oil containing 4.4 ppm of residual phosphorus.

Table 7 Example Enzyme addition PLC (ppm of active enzyme) PLA1 (ppm of active enzyme) Reaction time (min) Pace. (° C) pH Water (%) Mixing time (s) Phosphorus (ppm) 31 At the same time 20.9 0.7 120 40 4,5 2.0 120 10.7 32 At the same time 10.3 0.7 120 60 4,5 4,5 45 6.7 33 At the same time 10,4 0.7 thirty 40 4,5 4,5 120 2.2 34 At the same time 20.7 0.6 thirty 60 4,5 2.0 45 16.5 35 At the same time 20.7 0.6 120 40 4,5 4,5 45 1.9 36 At the same time 21.0 0.7 thirty 60 4,5 4,5 120 5.2 37 At the same time 10,2 0.6 thirty 40 4,5 2.0 45 6.7 38 At the same time 10,2 0.6 120 60 4,5 2.0 120 4.4

7 is a graph summarizing examples 31-38, in which the average final amount of phosphorus is plotted for each level of each factor, maintaining the pH, dose of PLA and the combined addition constant.

The five factors evaluated in these examples 31-38 are listed below in order of magnitude of their effect on the method of tarring. The effects are differences in average phosphorus levels (in absolute terms) between high and low factor settings.

Increasing the dose of water reduces residual phosphorus.

Reducing the dose of PLC reduces residual phosphorus.

Reducing the reaction temperature reduces residual phosphorus.

Increased mixing reduces residual phosphorus.

An increase in reaction time reduces residual phosphorus.

This is described in a new method for the degumming of oils using the phospholipase A enzyme and the phospholipase C enzyme at the same time. It was unexpectedly discovered that such a combination works better than each enzyme separately, even when, if necessary, the reaction with one or the other of the enzymes takes place under reaction conditions below the optimum for this enzyme. Surprisingly, it has also been found that degumming to levels of less than about 10 ppm of phosphorus, as low as about 5 ppm of phosphorus, and even as low as about 3 ppm of phosphorus, can be achieved in the final product suitable conditions with reaction times as short as about thirty minutes. In addition, without the desire to be bound by any theory, it seems that either the PLC enzyme or one of the products of its hydrolysis reaction catalyzes the reaction of the PLA enzyme, allowing the reaction time to be significantly shorter than the reaction time for any of the individual enzymes. These results are unexpected, based on the known optimal reaction parameters for the enzymes PLA and PLC.

Specialists in this field will be clear from the foregoing description that various operating parameters can be changed in the practical implementation of the present invention, depending on the objectives of a particular situation, while remaining within the scope of the invention. For example, when determining the concentrations of PLA and PLC enzymes to be used in a particular run, the choice will depend on whether the goal is to run at the lowest possible cost or the highest possible performance. If the goal is to run 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 situations. On the contrary, if maximum productivity is desired, 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. Specialists in the field of oil processing will understand how to change the concentration of enzymes in the reaction mixture to achieve the desired balance between economic efficiency and product productivity.

Changes to other processing conditions are also possible. The pH may be about 7.0, while a pH of about 5.0 is preferred and a pH of about 4.5 is currently preferred. The concentration of water in the system, as a rule, can be approximately 3.0%, but can be as low as approximately 1.5% if a reduction in wastewater volume is desired, or as high as approximately 4.5% if more is desired high efficiency of demineralization. The reaction temperature may be as high as about 60 ° C, but more preferably less than about 50 ° C and, unexpectedly, most preferably about 40 ° C. The mixing time during initial mixing may be approximately 45 seconds, more preferably approximately 60 seconds, and most preferably approximately 120 seconds. Finally, the duration of the enzymatic reaction can be significantly reduced; in some embodiments, it can advantageously be less than about 60 minutes and preferably about 30 minutes.

While preferred embodiments of the present invention are described herein, other embodiments including an inventive method will be readily apparent to those skilled in the art, and all such embodiments and their equivalents are intended to be protected by this application and covered by its claims inventions.

Claims (23)

1. A method for the degumming of an oil composition, comprising
(a) providing an oil composition containing phospholipids,
(b) contacting said oil composition simultaneously with one or more enzymes phospholipase A (PLA) in an amount of about 2 million ^-1 of active enzyme or less, and one or more enzymes phospholipase C (PLC) in an amount of about 30 million active enzyme ^-1 or less and
(c) separating the reaction products with phospholipids from the oil composition, where the remaining oil composition after separation is an oil-free oil composition,
where the duration of the reaction of enzymes with phosphoslipids is less than one hour, and where the specified reaction of enzymes with phospholipids occurs at a pH of about 3-7, at a temperature of about 40-80 ° C, and the oil-free oil composition from step (c) has a phospholipid content, measured in ppm phosphorus, about 20 million ^-1 or less. 2. The method according to claim 1, where the duration of the reaction of enzymes with phospholipids is approximately 30 minutes 3. The method according to claim 1, wherein said one or more phospholipase A enzymes are selected from the group consisting of phospholipase A1 enzyme and phospholipase A2 enzyme. 4. The method according to claim 1, wherein said one or more phospholipase C enzymes are selected from the group consisting of phospholipase C enzyme and phosphatidyl-inositol-specific phospholipase C enzyme. 5. The method according to claim 1, where the specified reaction of enzymes with phospholipids occurs at a pH of about 4-5. 6. The method according to claim 1, where the specified reaction of enzymes with phospholipids occurs at a temperature of approximately 40-80 ° C. 7. The method according to claim 6, where the specified reaction of enzymes with phospholipids occurs at a temperature of approximately 40-60 ° C. 8. The method according to claim 7, where the specified reaction of enzymes with phospholipids occurs at a temperature of approximately 45-55 ° C. 9. The method according to claim 1, where the specified oil composition contains a crude oil. 10. The method according to claim 1, where the specified oil composition contains pre-tarred oil. 11. The method of claim 1, wherein said PLC enzyme is present in an amount of about 20 million ^-1 of active enzyme or less. 12. The method of claim 11, wherein said PLC enzyme is present in an amount of about 10 million ^-1 of active enzyme or less. 13. The method according to claim 1, where the specified PLA enzyme is present in an amount of approximately 1 million ^-1 active enzyme or less. 14. The method of claim 13, wherein said PLA enzyme is present in an amount of about 0.5 million ^-1 of active enzyme or less. 15. The method according to claim 1, where during stage (b) the mixture of the oil composition and enzymes is first mixed with a shift. 16. The method of claim 15, wherein said shear mixing continues for at least about 45 seconds. 17. The method according to claim 1, where during stage (b) add a certain amount of water. 18. The method according to 17, where the specified amount of water is at least about 1.5 wt.% From the total mixture. 19. The method according to p, where the specified amount of water is at least about 3.0 wt.% From the total mixture. 20. The method according to claim 19, where the specified amount of water is at least about 4.5 wt.% From the total mixture. 21. The method according to claim 1, where the specified content of phospholipid is approximately 10 million ^-1 or less. 22. The method according to item 21, where the specified content of phospholipid is approximately 5 million ^-1 or less. 23. A method of degumming an oil comprising the step of reaction of phospholipase A enzyme with phospholipids in an oil composition, wherein the method is characterized in that the oil composition is contacted simultaneously with at least one phospholipase A enzyme in an amount of about 2 million ^-1 of active enzyme or less, and with at least one phospholipase C enzyme in an amount of about 30 million ^-1 or less active enzyme to react with the phospholipids in an oil composition, wherein said reaction occurs at a pH approximate but 3-7, at a temperature of approximately 40-80 ° C.

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