Enzymatic degumming of unrefined triglyceride oil

Abstract

The invention relates to a process for enzymatic degumming of unrefined triglyceride oil, said process comprising the following successive steps: (a) providing an unrefined triglyceride oil having a phosphorus content of at least 100 mg per kg of unrefined triglyceride oil; (b) combining the unrefined triglyceride oil with water, an acid and a phospholipase to produce an oil-in-water emulsion having a pH in the range of 2.5 to 4.5; said phospholipase being selected phospholipase A1, phospholipase A2 and combinations thereof; (c) keeping the emulsion at a temperature of 20-90° C. for at least 10 minutes; (d) introducing a base into the emulsion; and (e) separating degummed triglyceride oil from the emulsion. This enzymatic degumming process is extremely effective in removing phospholipids, including non-hydratable phospholipids (NHP), from unrefined vegetable oils and produces degummed vegetable oil in high yield.

Claims

1. A process for enzymatic degumming of unrefined triglyceride oil, said process comprising the following successive steps: a) providing an unrefined triglyceride oil having a phosphorus content of at least 100 mg per kg of unrefined triglyceride oil; b) combining the unrefined triglyceride oil with water, an acid and a phospholipase to produce an oil-and-water emulsion having a pH in the range of 2.5 to 4.5; said phospholipase being selected phospholipase A1, phospholipase A2 and combinations thereof; c) keeping the emulsion at a temperature of 20-90° C. for at least 10 minutes; d) introducing a base into the emulsion; and e) separating degummed triglyceride oil from the emulsion.

2. Process according to claim 1, wherein the acid is selected from citric acid, phosphoric acid, lactic acid and combinations thereof.

3. Process according to claim 1, wherein the base is selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, sodium silicate and combinations thereof.

4. Process according to claim 1, wherein the base is introduced to increase the pH of the emulsion with at least 0.4 pH points.

5. Process according to claim 1, wherein the base is introduced to increase the pH to more than 4.3.

6. Process according to claim 1, wherein the base is introduced to increase the pH to not more than 5.5.

7. Process according to claim 1, wherein the phospholipase is phospholipase A1.

8. Process according to claim 1, wherein the phospholipase has maximum activity at a pH in the range of 2.5-4.0.

9. Process according to claim 1, wherein water is combined with the unrefined triglyceride oil in a total amount of 5-100 ml water per kg or unrefined triglyceride oil.

10. Process according to claim 1, wherein step b) of the process comprises the successive steps of: b1) mixing the unrefined triglyceride oil with an aqueous acid solution; b2) keeping the mixture at a temperature of 20-90° C. for at least 5 minutes; and b3) combining the mixture with an aqueous solution of the enzyme to prepare the oil-and-water emulsion.

11. Process according to claim 1, wherein step c) of the process comprises keeping the emulsion at temperature of 35-85° C. for at least 10 minutes.

12. Process according to claim 1, wherein the degummed triglyceride oil is separated from the emulsion by means of centrifugation.

13. Process according to claim 1, wherein the emulsion has a temperature of 50-100° C. when the degummed triglyceride oil is separated therefrom.

14. Process according to claim 1, wherein the degummed triglyceride oil has a phosphorus content of less than 150 mg per kg of unrefined degummed triglyceride oil.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Accordingly, a first aspect of the invention relates to a process for enzymatic degumming of unrefined triglyceride oil, said process comprising the following successive steps: a) providing an unrefined triglyceride oil having a phosphorus content of at least 100 mg per kg of unrefined triglyceride oil; b) combining the unrefined triglyceride oil with water, an acid and a phospholipase to produce an oil-in-water emulsion having a pH in the range of 2.5 to 4.5; said phospholipase being selected phospholipase A1, phospholipase A2 and combinations thereof; c) keeping the emulsion at a temperature of 20-90° C. for at least 10 minutes; d) introducing a base into the emulsion; and e) separating degummed triglyceride oil from the emulsion.

(2) The term “oil” as used herein refers to a lipid material that can be liquid, solid or semi-solid at ambient temperature (20° C.). The terms “oil” and “fat” are used interchangeably.

(3) The term “triglyceride oil” as used herein refers to an oil containing at least 75 wt. % triglycerides.

(4) The term “unrefined triglyceride oil” as used herein refers to a triglyceride oil that has a phosphorus content of at least 100 mg per kg. Crude triglycerides oils that have been extracted from a natural source are an example of unrefined triglyceride oils. Another example of unrefined triglyceride oils are partially degummed triglyceride oils. Partially degummed triglyceride oils may be produced by water-degumming of crude triglyceride oils.

(5) The “phosphorus content” as referred to herein is measured by: ISO 10540-3:2002 (Animal and vegetable fats and oils—Determination of phosphorus content—Part 3: Method using inductively coupled plasma (ICP) optical emission spectroscopy 90.93 ISO/TC 34/SC 11)

(6) The term “phospholipase” as used herein refers to enzyme that hydrolyze phospholipids into fatty acids and other lipophilic substances. There are four major classes, termed A, B, C and D, distinguished by the type of reaction which they catalyze: Phospholipase A Phospholipase A1—cleaves the SN-1 acyl chain Phospholipase A2—cleaves the SN-2 acyl chain Phospholipase B—cleaves both SN-1 and SN-2 acyl chains Phospholipase C—cleaves before the phosphate, releasing diacylglycerol and a phosphate-containing head group. Phospholipase D—cleaves after the phosphate, releasing phosphatidic acid and an alcohol.

(7) The term “acid” as used herein refers to a Brønsted-Lowry acid, i.e. a substance that is a proton (hydrogen ion) donor.

(8) The term “citric acid” as used herein, unless indicated otherwise, refers to citric acid (2-hydroxypropane-1,2,3-tricarboxylic acid) as well as alkali metal salts of citric acid.

(9) The term “phosphoric acid” as used herein, unless indicated otherwise, refers to phosphoric acid (H.sub.3PO.sub.4) as well as alkali metal salts of phosphoric acid.

(10) The term “lactic acid” as used herein, unless indicated otherwise, refers to lactic acid (2-Hydroxypropanoic acid) as well as alkali metal salts of lactic acid.

(11) The term “base” as used herein refers to a Brønsted-Lowry base, i.e. a substance that is a proton (hydrogen ion) acceptor.

(12) The unrefined triglyceride oil that is degummed in the present process preferably has a phosphorus content of at least 150 mg per kg of unrefined triglyceride oil, more preferably of at least 200 mg per kg of unrefined triglyceride oil and most preferably of at least 300 mg per kg of unrefined triglyceride oil. Typically, the phosphorus content of the unrefined triglyceride oil does not exceed 2,000 mg per kg of unrefined triglyceride oil.

(13) The calcium content of the unrefined triglyceride oil preferably is at least 10 mg per kg unrefined triglyceride oil, more preferably at least 30 mg per kg of unrefined triglyceride oil. Typically, the unrefined triglyceride oil contains not more than 200 mg, preferably not more than 150 mg calcium per kg of unrefined triglyceride oil.

(14) The magnesium content of the unrefined triglyceride oil preferably is at least 10 mg per kg of unrefined triglyceride oil, more preferably at least 30 mg per kg of unrefined triglyceride oil. Typically, the unrefined triglyceride oil contains not more than 200 mg, more preferably not more than 150 mg magnesium per kg of unrefined triglyceride oil.

(15) Besides triglycerides and phospholipids, the unrefined triglyceride oil typically contains other lipid component such as diglycerides, monoglycerides, free fatty acids, tocopherols, tocotrienols etc. The unrefined triglyceride oil preferably contains at least 80 wt. %, more preferably at least 85 wt. % triglycerides.

(16) The unrefined triglyceride oil that is degummed in the present process preferably is an unrefined vegetable oil. Examples of unrefined vegetable oils that can suitably be degummed by the present process include unrefined soybean oil, unrefined rapeseed oil, unrefined sunflower oil, unrefined corn oil, unrefined cottonseed oil, unrefined palm oil, unrefined rice bran oil, unrefined arachis oil and combinations thereof. More preferably, the unrefined vegetable oil is selected from unrefined soybean oil, unrefined rapeseed oil, unrefined sunflower oil, unrefined corn oil, unrefined cottonseed oil, unrefined arachis oil and combinations thereof.

(17) The unrefined triglyceride oil that is used in the present process is preferably produced by solvent extraction, more preferably by extracting crushed seed or crushed fruit with n-hexane.

(18) In step b) of the present process the unrefined triglyceride oil may be combined with water, acid and enzyme in successive stages. For instance, water may be introduced during a water degumming step which is followed by other process steps in which acid and enzyme (and little or no water) are added.

(19) In step b) water is preferably combined with the unrefined triglyceride oil in a total amount of 5-100 ml water per kg or unrefined triglyceride oil, more preferably of 10-70 ml water per kg or unrefined triglyceride oil and most preferably of 15-60 ml water per kg or unrefined triglyceride oil. The total amount of water that is introduced in step b) includes water that is introduced together with lactic acid and/or the enzyme.

(20) The acid that is applied in step b) is preferably selected from citric acid, phosphoric acid, lactic acid and combinations thereof.

(21) In accordance with a preferred embodiment of the present process, acid is combined with the unrefined triglyceride oil in a total amount of 100-3,000 mg per kg of unrefined triglyceride oil, more preferably in a total amount of 200-2,000 mg per kg of unrefined triglyceride oil and most preferably in a total amount of 350-1,500 mg per kg of unrefined triglyceride oil.

(22) According to a preferred embodiment, the step b) acid is introduced in the form of the protonated acid, i.e. not as a salt. Accordingly, in a preferred embodiment, step b) comprises combining protonated acid with the unrefined triglyceride oil in a total amount of at least 100 mg per kg of unrefined triglyceride oil, more preferably in a total amount of at least 200 mg per kg of unrefined triglyceride oil and most preferably in a total amount of at least 350 mg per kg of unrefined triglyceride oil.

(23) The enzyme employed in step b) of the present process preferably is selected from the group of phospholipase A1, phospholipase A2 and combinations thereof. Most preferably, the phospholipase is phospholipase A1. Quara® LowP is an example of a commercially available phospholipase A1 that can advantageously be employed in the present process.

(24) According to a particularly preferred embodiment, the enzyme employed in the present process has maximum activity at a pH in the range of 2.5-4.0. Examples of phospholipase that have maximum activity within this acid pH range are Quara® LowP and Rohalase PL-Xtra®.

(25) In the present process, the enzyme is typically combined with the unrefined triglyceride oil in the form of an enzyme product that contains pure enzyme and carrier material. Typically, this enzyme product is combined with the unrefined triglyceride oil in a dose of 10-300 mg enzyme per kg of oil. More preferably, the enzyme product is applied in a dose of 20-200 mg enzyme per kg of oil and most preferably in a dose of 30-150 mg enzyme per kg of oil.

(26) The water-and-oil emulsion that is produced in step b) of the present process preferably is water-in-oil emulsion. Even more preferably, the emulsion is a water-in-oil emulsion comprising a dispersed aqueous phase having volume weighted mean diameter of less than 100 microns, more preferably of 5-30 microns. The volume weighted mean diameter of the dispersed phase can suitably be determined by means of laser diffraction.

(27) The production of the water-and-oil emulsion in step b) preferably comprises emulsification in a mixer, preferably a medium or high shear mixer.

(28) The water-and-oil emulsion that is produced in step b) typically contains 0.5-10 wt. %, more preferably 1-7 wt. % and most preferably 1.5-6 wt. % water.

(29) The aqueous phase of the water-and-oil emulsion preferably has a pH in the range of 2 to 6, more preferably of 2.5 to 5.0 and most preferably of 3.0 to 4.5.

(30) In a particularly preferred embodiment of the present invention the unrefined triglyceride oil is combined with the acid some time before addition of the enzyme. By allowing the acid to interact with the non-hydratable phospholipids in the unrefined triglyceride oil these phospholipids are rendered more susceptible for enzymatic hydrolysis. Accordingly, in a particularly preferred embodiment, step b) of the present process comprises the successive steps of:

(31) b1) mixing the unrefined triglyceride oil with an aqueous acid solution;

(32) b2) keeping the mixture at a temperature of 20-90° C. for at least 5 minutes; and

(33) b3) combining the mixture with an aqueous solution of the enzyme to prepare the oil-and-water emulsion.

(34) The acid solution that is mixed with the triglyceride oil in step b1) preferably contains 5-92 wt. %, more preferably 10-90 wt. % and most preferably 35-88 wt. % acid. The acid solution might suitably be buffered. An examples of such a buffered acid solution is a solution comprising a combination of citric acid and sodium hydroxide.

(35) Step b2) preferably comprises keeping the mixture at a temperature of 35-95° C. for at least 5 minutes, more preferably at a temperature of 45-90° C. for at least 5 minutes, most preferably at a temperature of 45-85° C. for at least 10 minutes. Preferably, the mixture is stirred while being kept at elevated temperature in step b2).

(36) Step c) of the degumming process of the present invention preferably comprises keeping the emulsion at temperature of 35-85° C. for at least 10 minutes, preferably for at least 15 minutes. More preferably, step c) comprises keeping the emulsion at temperature of 40-80° C. for at least 10 minutes, more preferably for at least 15 minutes and most preferably for 30-360 minutes. Preferably, the emulsion is stirred while being kept at elevated temperature in step c).

(37) Step d) of the present process comprises the introduction of a base into the emulsion. The base that is introduced into the emulsion is preferably selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium silicate and combinations thereof. More preferably, the base is selected from sodium hydroxide, sodium carbonate and combinations thereof.

(38) The base is preferably introduced into the emulsion in step d) to increase the pH of the emulsion with at least 0.2 pH points, more preferably with at least 0.4 pH points and most preferably with 0.6 to 1.5 pH points.

(39) Typically, the base is introduced in step d) to increase the pH to more than 4.3, more preferably to increase the pH to 4.4-5.5, most preferably to increase the pH to 4.5-5.0.

(40) According to another preferred embodiment, the base is introduced into the emulsion in the form of an aqueous solution. This aqueous solution preferably contains 0.05 to 12 mol/L of base, more preferably 0.1 to 5 mol/L of base and most preferably 0.5 to 2 mol/L of base.

(41) In the present process the degummed oil is separated from the emulsion in step e). This separation typically comprises separation of the oil phase and the aqueous phase of the emulsion. The aqueous phase contains phospholipids and hydratable phospholipid breakdown products that were produced in step c) by removing the aqueous phase a degummed oil is obtained having a reduced phospholipid content.

(42) The degummed triglyceride oil can be separated from the emulsion using separation techniques known in the art, such as centrifugation, decantation etc. Preferably, the degummed triglyceride oil is separated from the emulsion by means of centrifugation.

(43) Typically, the phosphorus content of the degummed triglyceride oil (in mg per kg) that is obtained in the present process is less than 30%, more preferably less than 20% of the phosphorus content of the unrefined triglyceride oil (in mg per kg).

(44) The calcium content of the degummed triglyceride oil (in mg per kg) is typically less than 40%, more preferably less than 30% of the calcium content of the unrefined triglyceride oil (in mg per kg).

(45) The magnesium content of the degummed triglyceride oil (in mg per kg) is typically less than 40%, more preferably less than 30% of the magnesium content of the unrefined triglyceride oil (in mg per kg).

(46) The degummed triglyceride oil that is obtained by the present process preferably has a phosphorus content of less than 150 mg per kg of degummed triglyceride oil. More preferably, the degummed triglyceride oil has a phosphorus content of less than 100 mg per kg of degummed triglyceride oil, even more preferably of less than 50 mg per kg of degummed triglyceride oil. Most preferably, the degummed triglyceride oil has a phosphorus content of less than 20 mg per kg of degummed triglyceride oil.

(47) The degummed triglyceride oil that is obtained by the present process preferably has a calcium content of less than 50 mg per kg of degummed triglyceride oil. More preferably, the degummed triglyceride oil has a calcium content of less than 20 mg per kg of degummed triglyceride oil, even more preferably of less than 10 mg per kg of degummed triglyceride oil. Most preferably, the degummed triglyceride oil has a calcium content of less than 5 mg per kg of degummed triglyceride oil.

(48) The degummed triglyceride oil that is obtained by the present process preferably has a magnesium content of less than 50 mg per kg of degummed triglyceride oil. More preferably, the degummed triglyceride oil has a magnesium content of less than 15 mg per kg of degummed triglyceride oil, even more preferably of less than 7 mg per kg of degummed triglyceride oil. Most preferably, the degummed triglyceride oil has a magnesium content of less than 3 mg per kg of degummed triglyceride oil.

(49) The degummed triglyceride oil that is obtained in the present process may suitably be further processed to produce a refined triglyceride oil. Examples of further processing steps that may be employed include neutralization, bleaching and deodorization. Preferably, the degummed oil is further processed to produce a refined triglyceride oil, said further processing comprising deodorization of the triglyceride oil. Most preferably the further processing comprises bleaching of the degummed triglyceride oil followed by deodorization of the bleached triglyceride oil.

(50) The refined triglyceride oil that is obtained by a process that comprises the aforementioned additional processing preferably has a free fatty acid content of less than 0.25%, more preferably of less than 0.05%.

(51) Another aspect of the invention relates to a triglyceride oil that is obtained by a process as defined herein.

(52) The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

(53) Crude soybean oils were degummed using phosphoric acid in combination with phospholipase (Quara® LowP, ex Novozymes). The following degumming procedure was used: 100 grams of crude soybean oil was homogenized and heated to 70° C.; phosphoric acid was added in the form of a 85% (w/w) aqueous solution in an amount equivalent to 900 mg (dry) phosphoric acid per kg of oil; the combination of crude oil and aqueous acid solution was mixed in high shear mixer for 15 seconds to produce an emulsion; the emulsion was kept at 70° C. for 40 minutes under stirring; next, a solution containing 3 grams of water and 5 mg of Quara® LowP was added to the hot oil; the mixture was submitted to high shear mixing for 60 seconds in order to generate proper emulsion/dispersion of water in oil, and kept for 210 min under constant stirring and constant temperature of 70° C.

(54) Next, prior to centrifugal separation, the emulsion was processed in three different ways: No addition of base (pH of emulsion was 4.2) Addition of 100 mg base (sodium carbonate) per kg of oil (pH of emulsion was 4.8) Addition of 200 mg base (sodium carbonate) per kg of oil (pH of emulsion was 5.1)

(55) Base was added to the emulsion by mixing the emulsion with 0.3 g of an aqueous solution of sodium carbonate (33 or 66 mg/mL) using a magnetic stirrer. The emulsion was kept at 90° C. for 2 minutes. Next, the aqueous phase was separated from the degummed oil phase by centrifugation. The oil yields achieved by the three different enzymolysis procedures were very similar.

(56) Before and after degumming the soybean oil was analysed to determine phosphor content and the concentration of calcium, magnesium. The results are shown in Table 1.

(57) TABLE-US-00001 TABLE 1 P Ca Mg (mg/kg) (mg/kg) (mg/kg) Crude oil 1,080 140 95 No base added after enzymolysis 51 4.4 4.5 100 ppm base added after enzymolysis 19 1.7 1.7 200 ppm base added after enzymolysis 16 2.4 1.6

Example 2

(58) Example 1 was repeated, except that this time, instead of 900 mg phosphoric acid per kg of oil, 800 mg lactic acid was added per kg of crude soybean oil in the form of a 85% (w/w) aqueous solution.

(59) The emulsion that had been prepared without addition of base had a pH of 4.1. The emulsion to which 100 ppm sodium carbonate had been added, had a pH of 5.1. The emulsion to which 200 ppm sodium carbonate had been added, had a pH of 5.4.

(60) Again, The oil yields achieved by the three different enzymolysis procedures were very similar

(61) The results from the analysis are summarized in Table 2.

(62) TABLE-US-00002 TABLE 2 P Ca Mg (mg/kg) (mg/kg) (mg/kg) Crude oil 1,080 140 95 No base added after enzymolysis 66 7.5 8.1 100 ppm base added after enzymolysis 23 4.7 3.0 200 ppm base added after enzymolysis 11 1.6 1.2