TECHNICAL FIELD

The instant disclosure is in the field of chemical sciences, and more particularly relates to vegetable oil refining. The present disclosure provides an enzyme assisted chemical method for refining of vegetable oils.

BACKGROUND OF THE DISCLOSURE

Edible/Vegetable oil refining is a step by step process. Vegetable oil refining is usually performed to remove gums (phospholipids), free fatty acids (FFAs), odors/off-flavors, pigments, metals, waxes and other impurities in the crude oil.

Gums (phospholipids) comprise hydratable gums and non-hydratable gums. Some vegetable oils such as soybean oil, rapeseed oil, sunflower oil and com oil are degummed (i.e. removal of phospholipids) with water to remove hydratable gums before they are subjected to either chemical refining process or for transportation to long distances. This is done primarily to prevent settling of gums in the tankers during transportation and to reduce oil losses during chemical refining. However, water degummed oils still carry considerable quantities of non-hydratable gums. Similarly, vegetable oils extracted through expeller process also contain considerable amount of gums (phospholipids).

Vegetable oils are subjected to refining process before they are marketed for human consumption. Vegetable oils are refined either by chemical process or physical process. Chemical process employs caustic (NaOH) to neutralize free fatty acids (FFAs) and removing/separating the soap from the oil. Physical process employs subjecting the vegetable oil to distillation under very high vacuum coupled with very high temperature to remove free fatty acids. In chemical processes, the aforementioned water degummed or expeller oils are subjected to neutralization of FFAs with caustic and this neutralization process also removes the residual non-hydratable phospholipids along with soap.

However, the existing chemical refining processes have several drawbacks including but not limiting to the following: a) LOSS OF NEUTRAL OIL: Both phospholipids (gums) and soap carry neutral oil with them, when they are removed from the oil using a separation technique such as centrifugation. Therefore, chemical refining process always results in loss of neutral oil. This mixture of gums, soap (formed from FFAs), neutral oil and insolubles is referred to as soap stock.

b) HIGH PHOSPHOROUS LEVELS IN NEUTRALIZED OIL: The neutralized oil coming out of soap removal centrifuge usually has about 8-18 ppm phosphorous and this can often lead to either close to or more than 5 ppm phosphorous in the oil being fed to deodorizer. This can result in more than acceptable level of phosphorous in deodorized oil that may have unwanted effects on shelf stability of RBD (refined, bleached and deodorized) oil.

c) HIGH SOAP LEVELS IN NEUTRALIZED OIL: The neutralized oil after centrifugation to remove soap, still has very high content of soap up to about 400-800 ppm or greater, and it requires further treatments to bring it down to desired level and these treatments increase process costs as well as additional capital cost in a new plant.

d) OXIDATION OF FATTY ACIDS AND NEUTRAL OIL IN SOAP STOCK: The neutral oil lost in soap and the free fatty acids forming the soap are recovered from soap stock by diluting soap stock with water, adding sulfuric acid to it to get a very low pH ~2.0 and boiling for some time to break the emulsion to separate soap stock into aqueous layer and oil layer. The recovered oil layer is known as acid oil. This boiling under strong acidic conditions results in oxidation as well as polymerization of fatty acids as well as tri glycerides and gives very dark color to recovered acid oil. A good amount of phospholipids also come into acid oil phase. Thus, the quality of obtained acid oil is very poor and therefore, it fetches very low price as compared to fatty acid distillate.

e) ENVIRONMENTAL ISSUES: The existing processes also produce highly acidic liquid effluent, acid vapors in air and good amount of steam consumption. In general, the vegetable oil refining industry would be happy if they can improve the overall chemical refining process and still recover the neutral oil and free fatty acids from soap stock subsequently by a simple process. Therefore, there is an immense need to develop a simpler, efficient and cost-effective process of refining of vegetable oils in order to address the aforementioned drawbacks. The present disclosure addresses the said need.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to an enzyme assisted method for chemical refining of vegetable oils. Accordingly, the present disclosure provides a method for refining a vegetable oil comprising: a) subjecting the oil to enzyme treatment to obtain an enzyme treated oil, wherein the enzyme is a phospholipid hydrolysing enzyme;

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil; and c) bleaching the neutral oil to obtain a bleached oil, and deodorizing the bleached oil to obtain the refined vegetable oil,

wherein the reaction of enzyme treated oil and alkali is characterized by:

mixing the enzyme treated oil and alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18% (w/w).

The present disclosure also relates to a method for refining an enzyme treated vegetable oil comprising:

a) reacting the enzyme treated vegetable oil and an alkali to obtain soap and neutral oil, wherein the enzyme is a phospholipid hydrolysing enzyme,

wherein the reaction of enzyme treated vegetable oil and the alkali is characterized by: mixing the enzyme treated oil and alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18% (w/w); and

b) bleaching the neutral oil to obtain a bleached oil, and deodorizing the bleached oil to obtain a refined vegetable oil. The present disclosure further relates to a method for neutralization of an enzyme treated vegetable oil in a process of refining oils, the method comprising reacting the enzyme treated vegetable oil and an alkali to obtain soap and neutral oil, wherein the enzyme is a phospholipid hydrolysing enzyme,

wherein the reaction of enzyme treated vegetable oil and the alkali is characterized by: mixing the enzyme treated oil and alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18% (w/w).

BRIEF DESCRIPTION OF FIGURES

Figure 1 depicts neutralization reaction results of: (A) Simple Mixing at 50°C: Soap settles at bottom and oil was clear even before centrifugation; (B) Simple Mixing at 50°C: Neutral oil after centrifugation at different RPMs i.e. 2000 to 4000; (C) Simple Mixing at 80°C: Soap dispersed in the entire body of oil and the oil was turbid; and (D) Simple Mixing at 80°C: Neutral oil after centrifugation at different RPMs i.e. 2000 to 4000.

Figure 2 depicts neutralization reaction results of: (A) Vigorous Mixing at 50°C: The neutral oil is very turbid even before centrifugation; (B) Vigorous Mixing at 50°C: Neutral oil after centrifugation at different RPMs i.e. 2000 to 4000; (C) Simple Mixing at 50°C: Soap settles at bottom and oil was clear even before centrifugation; and (D) Simple Mixing at 50°C: Neutral oil after centrifugation at different RPMs i.e. 2000 to 4000.

Figure 3 depicts neutralization reaction results. Particularly, the results are with respect to the neutral oil at the end of neutralization reaction carried out with 16% concentration of caustic soda (stoichiometric quantity caustic). The Soap settles at bottom and the neutral oil was clear.

DESCRIPTION OF THE DISCLOSURE

As used herein, the terms/phrases ‘gums’, ‘phospholipids’ and ‘phosphatides’ are employed interchangeably in the present disclosure. As used herein, the term/phrase ‘crude vegetable oil’ refers to vegetable oil comprising phospholipids, free fatty acids, natural pigments, metals, odours and insoluble material. In some embodiments of the present disclosure, the crude vegetable oil is derived from vegetables or plants or their products, such as but not limited to soybean, com, olive, sunflower, rapeseed, rice bran, canola and mustard.

As used herein, the term/phrase ‘water degummed vegetable oil’ refers to a vegetable oil comprising non-hydratable or oil-soluble gums which is obtained after water degumming process. Said water degumming process comprises hydrating the crude vegetable oil with water to remove hydratable gums (hydratable phospholipids) from the oil, and thereafter removing the hydrated gums. Only hydratable gums (hydratable phospholipids) from the crude vegetable oil are removed by water degumming process. The water degummed oil therefore primarily comprises non- hydratable or oil-soluble gums along with some amount of hydratable gums. As used herein, the terms/phrases‘enzyme treatment’,‘enzyme treated’,‘enzyme hydrolysis’, ‘enzyme hydrolysed’,‘enzyme degumming’,‘enzyme degummed’, and‘enzymatic degumming’ are employed interchangeably in the present disclosure.

As used herein, the terms/phrases ‘glycerolphosphates’, ‘phosphoglycerides’, ‘glycerophosphatides’ and‘glycerol phosphatides’ are employed interchangeably in the present disclosure. In some embodiments of the present disclosure, glycerolphosphates, phosphoglycerides, glycerophosphatides or glycerol phosphatides refer to the glycerol-based phosphate molecules produced by reaction of either crude or water degummed oil and phospholipid hydrolysing enzyme(s). In some embodiments of the present disclosure, phospholipid hydrolysing enzyme(s) is in the form of an enzyme solution as described herein.

As used herein, the terms/phrases ‘sodium hydroxide’ and ‘caustic soda’ are employed interchangeably in the present disclosure. As used herein, the terms/phrases‘potassium hydroxide’ and‘caustic potash’ are employed interchangeably in the present disclosure. As used herein, the term/phrase‘stoichiometric quantity’ or‘stoichiometric amount’ means the measure of amount of reagents required in a reaction for stoichiometry. In other words, stoichiometric quantity of a reagent refers to the optimum amount needed for a reaction to proceed to completion wherein: i) all of the reagent is consumed, ii) there is no deficiency of the reagent, and iii) there is no excess of the reagent. In some embodiments of the present disclosure, stoichiometric quantity of the alkali refers to the amount of alkali theoretically needed to completely neutralize the acidity (free fatty acids content) in enzyme treated oil.

As used herein, the term/phrase‘less than stoichiometric quantity’ or‘less than stoichiometric amount’ means the quantity of alkali that is less than the quantity theoretically needed to completely neutralize the acidity (free fatty acids content) in enzyme treated oil”.

One objective of the present disclosure is to obtain high quality refined oils by achieving lower phosphorous and soap levels in the neutral oil during chemical process of oil refining.

Another objective of the present disclosure is to obtain high quality refined oils by achieving reduced free fatty acid (FFA) content and soap content in neutral oil during chemical process of oil refining.

Yet another objective of the present disclosure is to maximize the yield of neutral oils during chemical process of oil refining.

Still another objective of the present disclosure is to achieve lower neutralization losses including reduced loss of neutral oil into soap during chemical process of oil refining.

Still another objective of the present disclosure is to achieve complete hydrolysis of phospholipids in the crude oil or water degummed crude oil by splitting both fatty acid chains of the phospholipid molecule and therefore converting the phospholipids into glycerol phosphates and free fatty acids (FFAs).

It is another objective of the present disclosure to eliminate water wash of neutral oil during chemical process of oil refining. An objective of the present disclosure is also to reduce the dosage or requirement of bleaching earth for bleaching the neutral oil during chemical process of oil refining.

Y et another objective of the present disclosure is to provide easier processing of soap stock without employing harsh conditions or high concentration of acids (eg. sulphuric acid).

Accordingly, the present disclosure provides an enzyme assisted method for refining of vegetable oils. In particular, in order to address the limitations as stated in the background and to meet the aforesaid objectives, the present disclosure provides an efficient enzyme assisted chemical refining process of edible or vegetable oils.

The present disclosure provides a method for refining vegetable oil comprising:

a) subjecting oil to enzyme treatment;

b) subjecting the enzyme treated oil to neutralization in presence of an alkali to obtain soap and neutral oil; and

c) bleaching the neutral oil to obtain a bleached oil and deodorizing the bleached oil to obtain the refined vegetable oil.

In some embodiments of the present disclosure, a method for refining vegetable oils is provided comprising:

a) subjecting oil to enzyme treatment to obtain an enzyme treated oil, wherein the enzyme is a phospholipid hydrolysing enzyme;

b) subj ecting the enzyme treated oil to neutralization comprising reacting the enzyme treated oil and an alkali to obtain soap and neutral oil; and

c) bleaching the neutral oil to obtain a bleached oil and deodorizing the bleached oil to obtain the refined vegetable oil.

In some embodiments of the present disclosure, an enzyme assisted method for chemical refining of vegetable oils is provided comprising:

a) subjecting oil to enzyme hydrolysis to hydrolyse gums to free fatty acids and glycerophosphatides; b) subjecting the enzyme hydrolyzed oil to neutralization, said neutralization comprising reacting the enzyme hydrolyzed oil and an alkali to obtain soap and neutral oil; and c) bleaching the neutral oil and deodorizing the bleached oil to obtain chemically refined vegetable oil.

In some embodiments of the present disclosure, the method for refining vegetable oils comprises milder neutralization conditions (i.e. reaction of the enzyme hydrolyzed oil and an alkali) to obtain neutral oil and soap. Therefore, in embodiments of the present disclosure, the reaction of enzyme hydrolyzed oil (or enzyme treated oil) and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18% (w/w).

Accordingly, the present disclosure provides a method for refining a vegetable oil comprising: a) subjecting oil to enzyme treatment to obtain an enzyme treated oil, wherein the enzyme is a phospholipid hydrolysing enzyme;

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil; and c) bleaching the neutral oil to obtain a bleached oil and deodorizing the bleached oil to obtain the refined vegetable oil,

wherein the reaction of enzyme treated oil and alkali is characterized by:

mixing the enzyme treated oil and alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18% (w/w).

In embodiments of the present method, mixing the enzyme treated oil and alkali is at a rate of about 15 RPM, 16 RPM, 17 RPM, 18 RPM, 19 RPM, 20 RPM, 21 RPM, 22 RPM, 23 RPM, 24 RPM, 25 RPM, 26 RPM, 27 RPM, 28 RPM, 29 RPM, 30 RPM, 31 RPM, 32 RPM, 33 RPM, 34 RPM, 35 RPM, 36 RPM, 37 RPM, 38 RPM, 39 RPM or 40 RPM. In some embodiments of the present method, mixing the enzyme treated oil and alkali is at a rate of about 20 RPM to 30 RPM.

In some embodiments of the present method, mixing the enzyme treated oil and alkali is at a rate of about 25 RPM.

In some embodiments of the present method, the mixing of enzyme treated oil and alkali is at a temperature of about 25°C to 70°C.

In some embodiments of the present method, the mixing of enzyme treated oil and alkali is at a temperature of about 25°C to 55°C.

In some embodiments of the present method, the mixing of enzyme treated oil and alkali is at a temperature of about 40°C to 60°C.

In some embodiments of the present method, the mixing of enzyme treated oil and alkali is at a temperature of about 50°C to 55°C.

In some embodiments of the present method, the mixing of enzyme treated oil and alkali is at a temperature of about 50°C, 51°C, 52°C, 53°C, 54°C or 55°C.

In some embodiments of the present method, a stochiometric quantity of alkali is employed in the reaction of enzyme treated oil and alkali. In an embodiment of the present method, the stochiometric quantity of alkali refers to the quantity of alkali theoretically required to completely neutralize the free fatty acids (FFAs) present in the enzyme treated oil.

In some embodiments of the present method, less than stochiometric quantity of alkali is employed in the reaction of enzyme treated oil (or enzyme hydrolyzed oil) and the alkali. In an embodiment of the present method, less than stochiometric quantity of alkali refers to the quantity of alkali that is less than the quantity theoretically needed to completely neutralize the free fatty acids (FFAs) present in the enzyme treated oil. In some embodiments of the present method, the reaction of enzyme treated oil and alkali comprises alkali at a concentration of about 12.5% to 18% (w/w), including values and ranges thereof.

As used in the present disclosure, the alkali concentration of about 12.5% to 18% including all values and ranges thereof employed for the reaction of enzyme treated oil and alkali, is in w/w (weight/weight) basis with respect to the solvent employed. In exemplary embodiments, the solvent is water and the alkali solution at a concentration of about 12.5% to 18% including all values and ranges thereof, is prepared with water.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali comprises alkali at a concentration of about 14% to 18%. In an embodiment of the present method, the mentioned concentration (in % w/w) of alkali is calculated based on the stochiometric quantity or less than stochiometric quantity of the alkali in a solvent. In some embodiments, the solvent is water. In some embodiments of the present method, the mentioned concentration (in % w/w) of alkali is calculated based on the stochiometric quantity or less than stochiometric quantity of the alkali in water. Accordingly, in some embodiments, 14% of alkali refers to the concentration of alkali (NaOH) on weight by weight basis in the solution of caustic soda being 14%. Accordingly, the various alkali % between 12.5% to 18% can be similarly calculated.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali comprises alkali at a concentration of about 15% to 16%.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali comprises alkali at a concentration of about 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5% or 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali comprises alkali selected from a group comprising sodium hydroxide or caustic soda, potassium hydroxide or caustic potash, sodium carbonate, ammonium hydroxide, calcium oxide, calcium hydroxide, any other alkali that is capable of reacting with free fatty acids and form an oil insoluble salt of fatty acid and combinations thereof.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali employs sodium hydroxide, or potassium hydroxide or a combination thereof.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali employs sodium hydroxide.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali employs potassium hydroxide.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 20 RPM to 30 RPM and at a temperature of about 25°C to 70°C, wherein:

- the alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 14% to 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 20 RPM to 30 RPM and at a temperature of about 25°C to 70°C, wherein:

- the alkali is at less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 14% to 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 15 RPM to 40 RPM and at a temperature of about 40°C to 60°C, wherein: - the alkali is at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 15 RPM to 40 RPM and at a temperature of about 50°C to 55°C, wherein:

- the alkali is at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 20 RPM to 30 RPM and at a temperature of about 10°C to 70°C, wherein:

- the alkali is at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 25 RPM and at a temperature of about 10°C to 70°C, wherein:

- the alkali is at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, wherein: - the alkali is at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 14% to 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a mixing rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, wherein:

- the alkali is at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 15% to 16%.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali is carried out for a time period of about 2 minutes to 40 minutes.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali is carried out for a time period of about 5 minutes to 35 minutes.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali is carried out for a time period of aboutlO minutes to 25 minutes.

In some embodiments of the present method, the reaction of enzyme treated oil and alkali is carried out for a time period of about 15 minutes to 20 minutes.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is carried out by:

mixing the enzyme treated oil and alkali at a mixing rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C and a time period of about 5 minutes to 35 minutes, wherein:

- the alkali is at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%. In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is carried out by:

mixing the enzyme treated oil and alkali at a mixing rate of about 20 RPM to 30 RPM and at a temperature of about 40°C to 60°C and a time period of about 10 minutes to 25 minutes, wherein:

- the alkali is at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 14% to 18%.

In some embodiments of the present method, the reaction of enzyme treated oil and the alkali is carried out by:

mixing the enzyme treated oil and alkali at a mixing rate of about 20 RPM to 30 RPM and at a temperature of about 50°C to 55°C and a time period of about 15 minutes to 20 minutes, wherein:

- the alkali is at stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 14% to 18%.

It will be understood by a person of ordinary skill in the art that any of the features/embodiments of the reaction of enzyme treated oil and the alkali described herein including the mixing rate of about 15 RPM to 40 RPM including values and ranges thereof, the temperature of about 10°C to 70°C including values and ranges thereof, the alkali at stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and the alkali at a concentration of about 12.5% to 18% including values and ranges thereof, can be employed in any combination with one or more steps/features of enzyme treatment (reaction of crude vegetable oil or water degummed vegetable oil with phospholipid hydrolysing enzyme(s), bleaching of neutral oil and deodorization of bleached oil as disclosed in the present disclosure.

In various embodiments, method of refining of vegetable oils described in the present disclosure comprises steps/features of enzyme treatment (reaction of oil and phospholipid hydrolysing enzyme(s), bleaching of neutral oil and deodorization of bleached oil which can be combined with one or more features/embodiments of neutralization (i.e. the reaction of enzyme treated oil and the alkali) including the mixing rate of about 15 RPM to 40 RPM including values and ranges thereof, the temperature of about 10°C to 70°C including values and ranges thereof, the alkali at stochiometric quantity or less than stochiometric quantity based on free fatty acids in the enzyme treated oil, and the alkali at a concentration of about 12.5% to 18% including values and ranges thereof, as described herein.

In some embodiments of the present disclosure, the vegetable oil is an edible oil. In some embodiments of the present disclosure, the vegetable oil is a non-edible oil.

In some embodiments of the present disclosure, the vegetable oil is selected from a group comprising soybean oil, sunflower oil, rapeseed oil, com oil, rice bran oil, canola oil, olive oil, mustard oil and combinations thereof.

In some embodiments of the present disclosure, the vegetable oil subjected to the present method is crude vegetable oil or water degummed vegetable oil.

In some embodiments of the present disclosure, the crude vegetable oil comprises hydratable and non-hydratable phospholipids. In some embodiments of the present disclosure, the water degummed vegetable oil comprises predominantly non-hydratable phospholipids and a smaller proportion of hydratable phospholipids. In some embodiments of the present disclosure, the water degummed vegetable oil comprises non-hydratable phospholipids and a substantially less/negligible amount of hydratable phospholipids.

In some embodiments of the present method, the oil subjected to enzyme treatment is a crude soybean oil, crude sunflower oil, crude rapeseed oil, crude com oil, crude rice bran oil, crude canola oil, crude olive oil, crude mustard oil and combinations thereof.

In some embodiments of the present method, the oil subjected to enzyme treatment is a water degummed soybean oil, water degummed sunflower oil, water degummed rapeseed oil, water degummed com oil, water degummed rice bran oil, water degummed canola oil, water degummed olive oil, water degummed mustard oil and combinations thereof. In some embodiments of the present method, the enzyme treatment is carried out at a temperature of about 50°C to 55°C for a time-period of about 30 minutes to 6 hours to obtain the enzyme treated oil.

In some embodiments of the present method, the enzyme treatment is carried out at a temperature of about 50°C to 55°C for a time-period of about 60 minutes to 5 hours to obtain the enzyme treated oil.

In some embodiments of the present method, the enzyme treatment comprises reacting the crude vegetable oil or the water degummed vegetable oil with an enzyme solution.

In some embodiments of the present method, the enzyme treatment comprises reacting the crude vegetable oil or water degummed vegetable oil with an enzyme solution comprising a phospholipid hydrolysing enzyme(s), a pH regulator, and water.

In some embodiments of the present method, the phospholipid hydrolysing enzyme is selected from a group comprising phospholipase A1 (PL-A1), phospholipase A2 (PL-A2), phospholipase C (PL-C), phospholipase B (PL-B), lyso-phospholipase (LPL), lipid acyl transferase (LAT) and combinations thereof.

In some embodiments of the present method, the phospholipid hydrolysing enzyme is a combination selected from:

(i) phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(ii) phospholipase A1 (PL-A1) and lyso-phospholipase (LPL),

(iii) phospholipase A1 (PL-A1) and phospholipase B (PL-B),

(iv) phospholipase A2 (PL-A2) and phospholipase B (PL-B),

(v) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(vi) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and phospholipase B (PL-B), and

(vii) phospholipase C (PL-C) and one or more enzyme selected from phospholipase A1 (PL- Al), phospholipase A2 (PL-A2), lyso-phospholipase (LPL) and phospholipase B (PL-B).

In some embodiments of the present method, the phospholipid hydrolysing enzyme is the combination of phospholipase A2 (PL-A2) and lyso-phospholipase (LPL). In some embodiments of the present method, the phospholipid hydrolysing enzyme is the combination of phospholipase A1 (PL-A1) and lyso-phospholipase (LPL).

In some embodiments of the present method, the phospholipid hydrolysing enzyme is the combination of phospholipase A1 (PL-A1) and phospholipase B (PL-B).

In some embodiments of the present method, the phospholipid hydrolysing enzyme is the combination of phospholipase A2 (PL-A2) and phospholipase B (PL-B).

In some embodiments of the present method, the phospholipid hydrolysing enzyme is the combination of phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and lyso-phospholipase (LPL).

In some embodiments of the present method, the phospholipid hydrolysing enzyme is the combination of phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and phospholipase B (PL- B).

In some embodiments of the present method, the phospholipid hydrolysing enzyme is the combination of phospholipase C (PL-C) and one or more enzyme selected from phospholipase A1 (PL-A1), phospholipase A2 (PL-A2), lyso-phospholipase (LPL) and phospholipase B (PL-B)

In some embodiments of the present method, the pH regulator in the enzyme solution is an organic acid or a buffer. In some embodiments of the present method, the organic acid is selected from a group comprising citric acid, acetic acid, phthalic acid, and combinations thereof. In some embodiments of the present method, the organic acid is citric acid or citric acid monohydrate. In some embodiments of the present method, the buffer is any buffer solution which maintains a pH of about 3 to 8. In some embodiments of the present method, the buffer is an acidic buffer. In some embodiments of the present method, the buffer contains an alkali. In some embodiments of the present method, the acidic buffer contains an alkali. In some embodiments of the present method, the buffer or acidic buffer contains an alkali selected from a group comprising sodium hydroxide or caustic soda, potassium hydroxide or caustic potash, dibasic sodium phosphate, monobasic sodium phosphate, sodium bicarbonate and combinations thereof. In some embodiments of the present method, the alkali in the buffer of the enzyme solution is sodium hydroxide.

In some embodiments of the present method, pH of the enzyme solution is about 3 to 8.

In some embodiments of the present method, pH of the enzyme solution is about 3.5 to 7.5.

In some embodiments of the present method, pH of the enzyme solution is about 4 to 7.

In some embodiments of the present method, pH of the enzyme solution is about 3.8 to 6.

In some embodiments of the present method, the enzyme treatment comprises employing enzyme solution at a dosage of about 0.5% to 3.0% by weight of the oil.

In some embodiments of the present method, the enzyme treatment comprises employing enzyme solution at a dosage of about 1.0 % to 1.5 % by weight of the oil.

In some embodiments of the present disclosure, wherein the enzymes in the enzyme solution are phospholipase A2 and lysophospholipase, wherein the phospholipase A2 with an activity of 10,000 PLU/gm is at a dosage of about 30 gm to 50 gm per ton of the oil, and the lysophospholipase with an activity of 10,000 PLU/gm is at a dosage of about 50 gm to 100 gm per ton of the oil, depending upon the phospholipid content of the crude or water degummed vegetable oils.

In some embodiments of the present method, the enzyme treatment hydrolyses phospholipids in the oil to yield glycerophosphatides and free fatty acids in the enzyme treated oil.

In some embodiments of the present method, the enzyme treatment hydrolyses the phospholipids in the oil completely to yield glycerophosphatides and free fatty acids in the enzyme treated oil.

In some embodiments of the present method, the enzyme treatment hydrolyses the phospholipids in the oil completely by splitting both fatty acid chains from phospholipid molecule and therefore phospholipids get converted into glycerophosphatides and free fatty acids. In some embodiments, the glycerophosphatides obtained after enzyme treatment are completely water soluble and totally hydrophilic. Therefore, said glycerophosphatides do not carry neutral oil with it, when the glycerophosphatides are taken out of oil during a separation technique to remove neutral oil and soap. In some embodiments of the present method, the free fatty acids released from phospholipids after enzyme treatment carry less oil as compared to phospholipids. Therefore, the present method employing enzyme treatment to completely hydrolyse the phospholipids to release fatty acids is extremely advantageous for the subsequent neutralization step (i.e. reaction of enzyme treated oil and an alkali).

In some embodiments of the present method, the soap and the neutral oil obtained by reacting the enzyme treated oil and an alkali are separated before bleaching.

In some embodiments of the present method, the soap and the neutral oil obtained by reacting the enzyme treated oil and an alkali are separated by a separation technique selected from a group comprising centrifugation, filtration, decantation, settling, and combinations thereof.

In some embodiments of the present method, the bleaching of neutral oil comprises treating the neutral oil with a bleaching agent to obtain the bleached oil.

In some embodiments of the present method, the bleaching of neutral oil comprises treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95 °C for a time- period of about 30 minutes to 45 minutes, to obtain the bleached oil.

In some embodiments of the present method, the deodorizing of bleached oil comprises subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230 °C to 250 °C for a time-period of about 60 minutes to 120 minutes, to obtain the refined oil product.

In some embodiments of the present method, the method of refining a vegetable oil comprises: a) subjecting crude vegetable oil or water degummed vegetable oil to enzyme treatment at a temperature of about 50°C to 55°C for a time-period of about 30 minutes to 6 hours to obtain enzyme treated oil, wherein the enzyme treatment comprises reacting the oil with an enzyme solution comprising phospholipid hydrolysing enzyme(s), pH regulator, and water; b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C for a time-period ranging from about 5 minutes to 30 minutes,

and wherein said alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 12.5% to 18%; and

c) bleaching the neutral oil to obtain bleached oil and deodorizing the bleached oil to obtain the refined vegetable oil.

In some embodiments of the present method, the method of refining oil comprises:

a) subjecting crude vegetable oil or water degummed vegetable oil to enzyme treatment at a temperature of about 50°C to 55°C for a time-period of about 30 minutes to 6 hours to obtain enzyme treated oil, wherein the enzyme treatment comprises reacting the oil with an enzyme solution comprising phospholipid hydrolysing enzyme(s), pH regulator, and water;

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 20 RPM to 30 RPM and at a temperature of about 25 °C to 70°C for a time-period ranging from about 10 minutes to 20 minutes,

and wherein said alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 14% to 18%; and

c) bleaching the neutral oil to obtain bleached oil and deodorizing the bleached oil to obtain the refined vegetable oil.

In some embodiments of the present method, the method of refining oil comprises:

a) subjecting crude vegetable oil or water degummed vegetable oil to enzyme treatment at a temperature of about 50°C to 55°C for a time-period of about 30 minutes to 6 hours to obtain enzyme treated oil,

wherein the vegetable oil is selected from a group comprising soybean oil, sunflower oil, rapeseed oil, com oil, rice bran oil, canola oil, olive oil, mustard oil and combinations thereof, wherein the enzyme treatment comprises reacting the oil with an enzyme solution having a pH of about 3 to 8 and comprising phospholipid hydrolysing enzyme(s), pH regulator, and water,

and wherein the phospholipid hydrolysing enzyme is selected from a group comprising phospholipase A1 (PL-A1), phospholipase A2 (PL-A2), phospholipase C (PL-C), phospholipase B (PL-B), lyso-phospholipase (LPL), lipid acyl transferase (LAT) and combinations thereof;

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C for a time-period ranging from about 10 minutes to 20 minutes,

wherein said alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 12.5% to 18%,

and wherein said alkali is selected from a group comprising sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide, sodium bicarbonate, ammonium bicarbonate, potassium bicarbonate, calcium hydroxide and combinations thereof; and

c) bleaching the neutral oil by treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes to obtain bleached oil, and

deodorizing the bleached oil by subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230 °C to 250 °C for a time-period of about 60 minutes to 120 minutes to obtain the refined vegetable oil.

In some embodiments of the present method, the method of refining oil comprises:

a) subjecting crude vegetable oil or water degummed vegetable oil to enzyme treatment at a temperature of about 50°C to 55°C for a time-period of about 30 minutes to 6 hours to obtain enzyme treated oil,

wherein the vegetable oil is selected from a group comprising soybean oil, sunflower oil, rapeseed oil, com oil, rice bran oil, canola oil, olive oil, mustard oil and combinations thereof, wherein the enzyme treatment comprises reacting the oil with an enzyme solution having a pH of about 3 to 8 and comprising phospholipid hydrolysing enzyme(s), pH regulator, and water,

and wherein the phospholipid hydrolysing enzyme is a combination selected from

(i) phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(ii) phospholipase A1 (PL-A1) and lyso-phospholipase (LPL),

(iii) phospholipase A1 (PL-A1) and phospholipase B (PL-B),

(iv) phospholipase A2 (PL-A2) and phospholipase B (PL-B),

(v) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(vi) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and phospholipase B (PL- B), and

(vii) phospholipase C (PL-C) and one or more enzyme selected from phospholipase A1 (PL-A1), phospholipase A2 (PL-A2), lyso-phospholipase (LPL) and phospholipase B (PL-B);

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 20 RPM to 30 RPM and at a temperature of about 25 °C to 70°C for a time-period ranging from about 10 minutes to 20 minutes,

wherein said alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 14% to 18%,

and wherein said alkali is selected from a group comprising sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, calcium hydroxide and combinations thereof; and

c) bleaching the neutral oil by treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes to obtain bleached oil, and

deodorizing the bleached oil by subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230 °C to 250 °C for a time-period of about 60 minutes to 120 minutes to obtain the refined vegetable oil. In some embodiments of the present method, the method of refining oil comprises:

a) subjecting crude vegetable oil or water degummed vegetable oil selected from a group comprising soybean oil, sunflower oil, rapeseed oil, com oil, rice bran oil, canola oil, olive oil, mustard oil and combinations thereof to enzyme treatment to obtain enzyme treated oil, wherein the enzyme treatment comprises:

(i) raising temperature of the crude vegetable oil or the water degummed crude vegetable oil to about 50°C to 55°C, and

(ii) reacting the oil with an enzyme solution comprising phospholipid hydrolysing enzyme(s), pH regulator, and water, for a time-period of about 30 minutes to 6 hours, to obtain the enzyme treated oil,

and wherein the phospholipid hydrolysing enzyme is a combination selected from:

(i) phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(ii) phospholipase A1 (PL-A1) and lyso-phospholipase (LPL),

(iii) phospholipase A1 (PL-A1) and phospholipase B (PL-B),

(iv) phospholipase A2 (PL-A2) and phospholipase B (PL-B),

(v) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(vi) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and phospholipase B (PL- B), and

(vii) phospholipase C (PL-C) and one or more enzyme selected from phospholipase A1

(PL-A1), phospholipase A2 (PL-A2), lyso-phospholipase (LPL) and phospholipase B (PL-B);

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 15°C to 70°C for a time-period ranging from about 10 minutes to 20 minutes,

wherein said alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 12.5% to 18%,

and wherein said alkali is selected from a group comprising sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, calcium hydroxide and combinations thereof; c) separated the soap and the neutral oil by a separation technique selected from a group comprising centrifugation, fdtration, distillation, decantation, settling and combinations thereof; and

d) bleaching the neutral oil by treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes to obtain bleached oil, and

deodorizing the bleached oil by subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230 °C to 250 °C for a time-period of about 60 minutes to 120 minutes to obtain the refined vegetable oil.

In some embodiments of the present method, the method of refining oil comprises:

a) subjecting crude vegetable oil or water degummed vegetable oil selected from a group comprising soybean oil, sunflower oil, rapeseed oil, com oil, rice bran oil, canola oil, olive oil, mustard oil and combinations thereof to enzyme treatment to obtain enzyme treated oil, wherein the enzyme treatment comprises:

(i) raising temperature of the crude vegetable oil or the water degummed crude vegetable oil to about 50°C to 55°C, and

(ii) reacting the oil with an enzyme solution comprising phospholipid hydrolysing enzyme(s), pH regulator, and water, for a time-period of about 30 minutes to 6 hours, to obtain the enzyme treated oil,

and wherein the phospholipid hydrolysing enzyme is a combination selected from:

(i) phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(ii) phospholipase A1 (PL-A1) and lyso-phospholipase (LPL),

(iii) phospholipase A1 (PL-A1) and phospholipase B (PL-B),

(iv) phospholipase A2 (PL-A2) and phospholipase B (PL-B),

(v) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(vi) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and phospholipase B (PL- B), and

(vii) phospholipase C (PL-C) and one or more enzyme selected from phospholipase A1 (PL-A1), phospholipase A2 (PL-A2), lyso-phospholipase (LPL) and phospholipase B (PL-B); b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 20 RPM to 30 RPM and at a temperature of about 50°C to 55°C for a time-period ranging from about 10 minutes to 20 minutes,

wherein said alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 15% to 16%,

and wherein said alkali is selected from a group comprising sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, calcium hydroxide and combinations thereof;

c) separated the soap and the neutral oil by a separation technique selected from a group comprising centrifugation, fdtration, distillation, decantation, settling and combinations thereof; and

d) bleaching the neutral oil by treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes to obtain bleached oil, and

deodorizing the bleached oil by subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230 °C to 250 °C for a time-period of about 60 minutes to 120 minutes to obtain the refined vegetable oil.

In some embodiments of the present method, the method of refining oil comprises:

a) subjecting crude vegetable oil or water degummed vegetable oil selected from a group comprising soybean oil, sunflower oil, rapeseed oil, com oil, rice bran oil, canola oil, olive oil, mustard oil and combinations thereof to enzyme treatment to obtain enzyme treated oil, wherein the enzyme treatment comprises:

(i) raising temperature of the crude vegetable oil or the water degummed crude vegetable oil to about 50°C to 55°C, and

(ii) reacting the oil with an enzyme solution having a pH of about 4 to 7 and comprising phospholipid hydrolysing enzyme(s), pH regulator, and water, for a time-period of about 30 minutes to 6 hours, to obtain the enzyme treated oil, and wherein the phospholipid hydrolysing enzyme is a combination selected from: (i) phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(ii) phospholipase A1 (PL-A1) and lyso-phospholipase (LPL),

(iii) phospholipase A1 (PL-A1) and phospholipase B (PL-B),

(iv) phospholipase A2 (PL-A2) and phospholipase B (PL-B),

(v) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and lyso-phospholipase (LPL),

(vi) phospholipase A1 (PL-A1), phospholipase A2 (PL-A2) and phospholipase B (PL-

B),

(vii) phospholipase C (PL-C) and one or more enzyme selected from phospholipase A1 (PL-A1), phospholipase A2 (PL-A2), lyso-phospholipase (LPL) and phospholipase B (PL-B);

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 15°C to 70°C for a time-period ranging from about 10 minutes to 20 minutes,

wherein said alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 12.5% to 18%,

and wherein said alkali is sodium hydroxide or potassium hydroxide;

c) separating the soap and the neutral oil by centrifugation;

d) bleaching the neutral oil by treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes to obtain bleached oil; and

e) deodorizing the bleached oil by subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230 °C to 250 °C for a time-period of about 60 minutes to 120 minutes to obtain the refined vegetable oil.

In some embodiments of the present method, the method of refining oil comprises:

a) subjecting crude vegetable oil or water degummed vegetable oil selected from a group comprising soybean oil, sunflower oil, rapeseed oil, com oil, rice bran oil, canola oil, olive oil, mustard oil and combinations thereof to enzyme treatment to obtain enzyme treated oil, wherein the enzyme treatment comprises: (i) raising temperature of the crude vegetable oil or the water degummed crude vegetable oil to about 50°C to 55°C, and

(ii) reacting the oil with an enzyme solution comprising phospholipid hydrolysing enzymes phospholipase A2 (PL-A2) and lyso-phospholipase (LPL), pH regulator, and water, for a time -period of about 30 minutes to 6 hours, to obtain the enzyme treated oil;

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 15°C to 70°C for a time-period ranging from about 10 minutes to 20 minutes,

wherein said alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 12.5% to 18%,

and wherein said alkali is selected from a group comprising sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, calcium hydroxide and combinations thereof;

c) separated the soap and the neutral oil by a separation technique selected from a group comprising centrifugation, fdtration, distillation, decantation, settling and combinations thereof; and

d) bleaching the neutral oil by treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes to obtain bleached oil, and

deodorizing the bleached oil by subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230 °C to 250 °C for a time-period of about 60 minutes to 120 minutes to obtain the refined vegetable oil.

In some embodiments of the present method, the method of refining oil comprises:

a) subjecting crude vegetable oil or water degummed vegetable oil selected from a group comprising soybean oil, sunflower oil, rapeseed oil, com oil, rice bran oil, canola oil, olive oil, mustard oil and combinations thereof to enzyme treatment to obtain enzyme treated oil, wherein the enzyme treatment comprises: (i) raising temperature of the crude vegetable oil or the water degummed crude vegetable oil to about 50°C to 55°C, and

(ii) reacting the oil with an enzyme solution having a pH of about 3 to 8 and comprising phospholipid hydrolysing enzymes phospholipase A2 (PL-A2) and lyso-phospholipase (LPL), pH regulator, and water, for a time-period of about 30 minutes to 6 hours, to obtain the enzyme treated oil;

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 25 RPM to 30 RPM and at a temperature of about 50°C to 55°C for a time-period ranging from about 10 minutes to 20 minutes,

wherein said alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 15% to 16%,

and wherein said alkali is selected from a group comprising sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, calcium hydroxide and combinations thereof;

c) separating the soap and the neutral oil by a separation technique selected from a group comprising centrifugation, fdtration, distillation, decantation, settling and combinations thereof;

d) bleaching the neutral oil by treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes to obtain bleached oil; and

e) deodorizing the bleached oil by subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230°C to 250°C for a time-period of about 60 minutes to 120 minutes to obtain the refined vegetable oil.

In some embodiments of the present disclosure, a mixing rate of about 15 RPM to 40 RPM or about 25 RPM to 30 RPM or about 25 RPM during the reaction of enzyme treated oil and alkali is achieved by employing a suitable agitator or stirrer, in a neutralizer or neutralization tank where said reaction occurs. In some embodiments, the agitator or stirrer comprises axial flow or hydrofoil kind of impeller that can provide efficient mixing with very less shear. In some embodiments, a known agitator or stirrer is employed to achieve a mixing rate of about 15 RPM to 40 RPM or about 25 RPM to 30 RPM or about 25 RPM during the reaction of enzyme treated oil and alkali. In some embodiments, an axial flow type agitator is employed to achieve a mixing rate of about 15 RPM to 40 RPM or about 25 RPM to 30 RPM or about 25 RPM during the reaction of enzyme treated oil and alkali. In some embodiments, a mixing rate of about 15 RPM to 40 RPM or about 25 RPM to 30 RPM or about 25 RPM during the reaction of enzyme treated oil and alkali provides homogenous mixing without generating shear such that all the fee fatty acids are completely neutralized by alkali without generating any shear force while mixing.

In some embodiments of the present disclosure, a method of refining an enzyme treated oil is provided, the method comprising:

a) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil,

wherein the enzyme is a phospholipid hydrolysing enzyme,

wherein the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%; and

b) bleaching the neutral oil to obtain a bleached oil, and deodorizing the bleached oil to obtain a refined vegetable oil.

In some embodiments of the present method of refining an enzyme treated oil, the reaction of enzyme treated oil and alkali is characterized by:

mixing the enzyme treated oil and alkali at a rate of about 20 RPM to 30 RPM and at a temperature of about 25°C to 70°C for a time-period ranging from about 10 minutes to 20 minutes, wherein:

- the alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 14% to 18%;

and wherein the alkali is selected from a group comprising sodium hydroxide or caustic soda, potassium hydroxide or caustic potash, sodium carbonate, ammonium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, calcium hydroxide and combinations thereof.

In some embodiments of the present disclosure, a method for neutralization of an enzyme treated oil in a process of refining vegetable oils is provided, the method comprising: reacting the enzyme treated oil and an alkali to obtain soap and neutral oil,

wherein the enzyme is a phospholipid hydrolysing enzyme,

wherein the reaction of enzyme treated oil and the alkali is characterized by:

mixing the enzyme treated oil and alkali at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%.

In some embodiments of the method of refining an enzyme treated oil or the method for neutralization of an enzyme treated oil in a process of refining vegetable oils, the enzyme treated oil is obtained by subjecting a crude vegetable oil or a water degummed vegetable oil to an enzyme treatment, and wherein said enzyme treatment hydrolyses phospholipids in the oil into glycerophosphatides and free fatty acids.

In some embodiments, the additional/specific features of the method of refining an enzyme treated oil or the method for neutralization of an enzyme treated oil in a process of refining oils is provided under various embodiments of the method of refining vegetable oils described herein and such features alone or in any combination form part of said methods.

In some embodiments of the present method, the method of refining vegetable oil comprises:

Step a): Subjecting the vegetable oil to enzyme treatment to hydrolyse phospholipids (gums) (Enzyme Hydrolysis Step):

In embodiments of the present disclosure, the step comprising subjecting the vegetable oil to enzyme hydrolysis comprises: (i) raising temperature of water degummed vegetable oil or crude vegetable oil to about 25°C to 80°C. In an embodiment, the temperature of the oil is raised to about 30°C to 75 °C. In another embodiment, the temperature of the oil is raised to about 45 °C to 60°C. In yet another embodiment, the temperature of the oil is raised to about 50°C to 55°C; and

(ii) adding enzyme solution into the oil and allowing the enzyme to react with the oil for a time period ranging from about 30 minutes to 6 hours.

In some embodiments, the temperature of the water degummed vegetable oil or crude vegetable oil is raised to about 25°C to 80°C by taking the oil into a heat exchanger, such as a plate heat exchanger. In some embodiments, the oil from heat exchanger is then taken to a homogenizer selected from a group comprising shear mixer, high shear mixer, conventional homogenizer, and combinations thereof. In an exemplary embodiment, high shear mixer of the make similar to Ross or Bran & Lubbe or Silverson or Alfa Laval MX mixer, Dynamic shear mixer of Desmet Ballastra or any other similar high shear mixer is employed.

In some embodiments, the enzyme solution is added into the oil in the homogenizer, and the enzyme is allowed to react with the oil for a time period ranging from about 30 minutes to 6 hours.

In other embodiments, the enzyme solution is added into the oil in the homogenizer, and the enzyme mixed oil is taken to an enzyme reaction tank wherein, gentle mixing at a rate of about 30 RPM to 40 RPM for a time period ranging from about 30 minutes to 6 hours is carried out allowing the enzyme to react with the oil.

In some embodiments of the present disclosure, the enzyme solution comprises phospholipid hydrolyzing enzymes, a pH regulator and water. In some embodiments, the pH regulator of the enzyme solution is any suitable buffer solution of desired pH for optimum function of enzyme or combination of enzymes which are used according to the present disclosure. In some embodiments, the buffer solution contains sodium hydroxide or potassium hydroxide or any other suitable alkali for maintaining optimum function of enzyme or combination of enzyme blends which are used according to the present disclosure. In some embodiments, the enzyme can be any enzyme that is capable of hydrolyzing phospholipids in vegetable oils. In some embodiments, the enzyme in the enzyme solution is selected from a group comprising phospholipase A1 (PL-A1), phospholipase A2 (PL-A2), phospholipase C (PL- C), phospholipase B (PL-B), lyso-phospholipase (LPL), lipid acyl transferase (LAT) and combinations thereof. In other embodiments, the enzyme is combination selected from: (1) phospholipase A2 and lyso-phospholipase; (2) PL-A1 and LPL; (3) PL-A1 and PLB; or (4) PL- A2 and PLB, (5) PL-A1, PL-A2 and LPL, (6) PL-A1, PL-A2 and PLB, and (7) PLC and one or more enzyme selected from PL-A1 or PL-A2 or LPL or PLB. In exemplary embodiments, the enzyme employed in the enzyme solution is a combination of phospholipase A2 and lyso- phospholipase.

In some embodiments of the present disclosure, the pH regulator employed in the enzyme solution is an organic acid selected from a group comprising citric acid, acetic acid, phthalic acid, and combinations thereof. In some embodiments, the pH regulator in the enzyme solution is citric acid. In other embodiments, the pH regulator is a suitable buffer which provides optimum pH between 3 to 8 for the function of enzyme/enzyme combinations employed in present invention.

In some embodiments of the present disclosure, the enzyme solution is prepared by taking appropriate quantity of water and dissolving an appropriate quantity of organic acid, followed by adding an appropriate quantity of alkali to obtain a pH of about 3.8 to 4.2 in the solution. This is followed by adding the enzyme(s) to prepare the final enzyme solution.

In an embodiment of the present disclosure, the dosage of enzyme(s) employed for enzyme treatment (or enzyme hydrolysis) is dependent upon the types and activities of enzymes being employed. It is also possible to vary the enzyme dosages by taking highly active and very concentrated, or very dilute enzymes. In some embodiments, about 15 gm to 50 gm of phospholipase A2 with an activity of 10,000 PLU /gm and about 30 gm to 100 gm of lyso- phospholipase with an activity of 10,000 LPL/gm is employed per ton of oil.

In some embodiments of the present disclosure, the quantity of water employed to prepare the enzyme solution is dependent upon the level of phospholipids in the crude oil or water degummed crude oil, the type of enzyme being used and the type of oil being used. In some embodiments, the water employed per ton of oil is in the range of about 5 liters to 50 liters. In some embodiments, the water employed per ton of oil is in the range of about 7.5 liters to 30 liters. In other embodiments, the water employed per ton of oil is in the range of about 10 liters to 20 liters. Further, higher levels of phospholipids or natural emulsifiers in crude oil requires higher water levels of 1.5% or more by weight of the oil. For example, if crude soybean oil containing about 2.5 to 3.5% phospholipids or crude rice bran oil containing about l% to 1.5% of phospholipids is used, about 2.5 to 3.0% water by weight of crude oil is employed. In another example, if a crude vegetable oil such as canola, rapeseed, mustard, com or sunflower oil containing 1 to 1.5% of phospholipids is employed, a concentration of 1.5% or more of water by weight of crude vegetable oil is used.

In some embodiments of the present disclosure, the quantity of pH regulator, employed to prepare the enzyme solution is in the range of about 200 gm to 1000 gm per ton of oil. In some embodiments, the quantity of pH regulator employed to prepare the enzyme solution is in the range of about 500 gm to 900 gm per ton of oil. In other embodiments, the quantity of pH regulator employed to prepare the enzyme solution is in the range of about 600 gm to 800 gm per ton of oil. In some embodiments, the quantity of pH regulator is dependent upon the quality of crude oil, the hardness level of water used for enzyme solution and the optimum pH of enzyme(s) which are used for hydrolysis of phospholipids. In some embodiments, the pH regulator is organic acid. In some embodiments, the pH regulator is citric acid. In some embodiments, the pH regulator is citric acid monohydrate. In some embodiments, the pH regulator is a buffer solution. In some embodiments, the pH regulator is an acidic buffer solution containing an alkali.

In some embodiments of the present disclosure, the quantity of alkali employed to prepare the enzyme solution is in the range of about 50 gm to 500 gm per ton of oil. In some embodiments, the quantity of alkali employed to prepare the enzyme solution is in the range of about 100 gm to 300 gm per ton of oil. In other embodiments, the quantity of alkali employed to prepare the enzyme solution is in the range of about 150 gm to 250 gm per ton of oil. In some embodiments, the quantity of alkali is dependent upon the purity of oil, the hardness level of water used for enzyme solution and the optimum pH of enzyme(s) which are used for hydrolysis of phospholipids. In some embodiments, the alkali is caustic soda. In some embodiments of the present disclosure, the pH of enzyme solution is in the range of about 3.0 to 8.0. In some embodiments, the pH of enzyme solution is in the range of about 3.5 to 7.5. In some embodiments, the pH of enzyme solution is in the range of about 4.0 to 7.0. In some embodiments, the pH of enzyme solution is in the range of about 3.8 to 6.0. In some embodiments, the pH used for the enzyme solution is dependent upon the optimum pH of the enzyme (s) which are used in the enzyme treatment (or enzyme hydrolysis).

In some embodiments of the present disclosure, the enzyme solution is added into the oil and the enzyme(s) is allowed to react with the oil for a time period of about 30 minutes. In some embodiments of the present disclosure, the enzyme solution is added into the oil and the enzyme(s) is allowed to react with the oil for a time period of about 4 hours.

In exemplary embodiments of the present disclosure, the enzymes in the present enzyme solution hydrolyze the phospholipids (gums) in the oil as depicted in Scheme 1 below:

Scheme 1 : Principle of enzyme treatment of phospholipids according to the present method

In some embodiments of the present disclosure, the product (i.e. enzyme treated oil) obtained post enzyme treatment is directly subjected to neutralization.

Step b): Subjecting the enzyme treated oil (or enzyme hydrolyzed oil) to neutralization in presence of an alkali to obtain soap and neutral oil rNeutralization Step!

In some embodiments of the present disclosure, the enzyme treated oil/enzyme hydrolyzed oil obtained is subjected to neutralization in presence of an alkali selected from a group comprising caustic soda (sodium hydroxide), caustic potash (potassium hydroxide), sodium carbonate, ammonium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, calcium hydroxide and combinations thereof to yield soap stock and neutral oil.

In embodiments of the present disclosure, the neutralization step comprising reaction of enzyme treated oil and alkali is performed under mild reaction parameters or conditions.

In some embodiments of the present disclosure, the neutralization step comprising reaction of enzyme treated oil and alkali is carried out at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%.

In some embodiments of the present disclosure, the neutralization comprising reaction of enzyme treated oil and alkali is carried out at a rate of about 20 RPM to 30 RPM and at a temperature of about 25°C to 70°C, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids in the enzyme treated oil, and

- the alkali is at a concentration of about 14% to 16%.

In some embodiments of the present disclosure, the neutralization comprising reaction of enzyme treated oil and alkali is carried out at a rate of about 25 RPM and at a temperature of about 40°C to 60°C, and wherein: - the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids in the enzyme treated oil, and

- the alkali is at a concentration of about 15% to 16%.

In some embodiments of the present disclosure, the neutralization comprising reaction of enzyme treated oil and alkali is carried out at a rate of about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C for a time-period ranging from about 5 minutes to 40 minutes, and wherein:

- the alkali is at a stochiometric quantity or less than stochiometric quantity based on free fatty acids content in the enzyme treated oil, and

- the alkali is at a concentration of about 12.5% to 18%.

In embodiments of the present disclosure, mild mixing or gentle mixing comprises mixing at a rate of about 15 RPM to 40 RPM.

In embodiments of the present disclosure, mild mixing or gentle mixing comprises mixing at a rate of about 15 RPM, 16 RPM, 17 RPM, 18 RPM, 19 RPM, 20 RPM, 21 RPM, 22 RPM, 23 RPM, 24 RPM, 25 RPM, 26 RPM, 27 RPM, 28 RPM, 29 RPM, 30 RPM, 31 RPM, 32 RPM, 33 RPM, 34 RPM, 35 RPM, 36 RPM, 37 RPM, 38 RPM, 39 RPM or 40 RPM.

In some embodiments of the present disclosure, a mixing rate of about 15 RPM to 40 RPM including values and ranges thereof during the reaction of enzyme treated oil and alkali is achieved by employing a suitable agitator or stirrer such as an axial flow or hydrofoil kind of impeller that can provide efficient mixing with very less shear in a neutralizer or neutralization tank where said reaction occurs. In some embodiments, a known agitator or stirrer is employed to achieve a mixing rate of about 15 RPM to 40 RPM including values and ranges thereof during the neutralization reaction of enzyme treated oil and alkali. In some embodiments, an axial flow or a suitable impeller that can provide efficient mixing without causing turbulence/shear is employed to achieve a mixing rate of about 15 RPM to 40 RPM including values and ranges thereof during the neutralization reaction of enzyme treated oil and alkali. In some embodiments, a mixing rate of about 15 RPM to 40 RPM including values and ranges thereof during the neutralization reaction of enzyme treated oil and alkali provides efficient contact between free fatty acids and alkali. In some embodiments of the present disclosure, employing the various neutralization conditions described herein is advantageous over the traditional neutralization processes which employ harsh conditions with intact phospholipids in the oil during neutralization. In particular, employing harsh conditions during neutralization process, especially in presence of intact phospholipids, trigger increased emulsification of neutral oil with soap stream due to the emulsification power of: (a) soap and (b) unhydrolysed phospholipids. Thus, employing harsh conditions during neutralization such as the use of: (a) high temperature of 80°C or more during short mix neutralization process, (b) use of very vigorous mixing during neutralization at a high temperature of 80°C or more to try and achieve efficient mixing of sodium hydroxide (alkali) with free fatty acids, and/or (c) use of dynamic shear mixer (vigorous mixing) during long mix neutralization process, could result in increased emulsification of neutral oil with soap stream and thereby neutral oil losses. In other words, the stronger the emulsion formed during neutralization, would be the soap content and phosphorous content in neutral oil formed. Additionally, the harsher the conditions of neutralization, stronger is the emulsification and hence higher the oil carried by soap stock (a mixture of soap, oil, water and phospholipids) during separation of neutral oil and soap. Added to this, intact phospholipids carry higher level of neutral oil with them during neutralization and soap removal and this quantity of oil carried by phospholipids is quite significant loss to refiners.

In some embodiments of the present disclosure, a neutralization tank that comprises an impeller which can provide efficient mixing without generating any turbulence/shear, is employed for carrying out neutralization reaction. In some embodiments of the present disclosure, a neutralization tank that comprises an axial flow impeller or hydrofoil kind of impeller is employed for carrying out neutralization reaction.

In some embodiments of the present disclosure, a stoichiometric quantity of caustic soda or caustic potash is added to the enzyme hydrolyzed oil to neutralize the acidity (free fatty acids) of said oil.

In some embodiments of the present disclosure, slightly less than the stoichiometric quantity of caustic soda or caustic potash is added to the enzyme hydrolyzed oil to neutralize the acidity (free fatty acids) of said oil. Preparing the caustic soda solution or caustic potash solution by calculating the concentrations of alkali (caustic soda solution or caustic potash) of about 12.5% to 18% is carried out according to the stochiometric quantity or less than stochiometric quantity of alkali as described in the preceding embodiments.

For the ease of understanding, an exemplary embodiment for calculating the concentrations of alkali based on stochiometric quantity of free fatty acids content is provided. For instance, if free fatty acids content of oil is 0.7% and considering the free fatty acids content to be linoleic acid, this means that one ton of oil has 7 kg of linoleic acid in it. The molecular weight of linoleic acid is 280 and the molecular weight of caustic soda (NaOH) is 40. The quantity of caustic soda (alkali) to react with linoleic acid means one gram mole of caustic soda will react with one gram mole of linoleic acid. In other words, 40 gm of caustic soda will react with 280 gm of linoleic acid. Therefore, to neutralize 0.7% of free fatty acid content (i.e. 7 kg of linoleic acid) in 1 ton oil, the stochiometric quantity of caustic soda required is 1 kg per ton of oil. Accordingly, if 14% caustic soda solution has to be made, then 1 kg caustic soda has to be dissolved in 6.143 kg of water and this will give 7.143 kg of 14% caustic soda solution. Therefore, 7.143 kg caustic soda solution (14% caustic soda solution) has to be dosed in 1 ton of oil containing 0.7% of linoleic acid. This will be the stoichiometric quantity of caustic soda (alkali) to neutralize the free fatty acids content in 1 ton of oil.

In some embodiments of the present disclosure, about 12.5% to 18% caustic soda solution or caustic potash at stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, about 12.5% to 18% caustic soda solution or caustic potash at less than stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, about 14% to 18% caustic soda solution or caustic potash at stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, about 14% to 18% caustic soda solution or caustic potash at less than stochiometric amounts is employed for neutralization. In some embodiments of the present disclosure, about 14% to 16% caustic soda solution or caustic potash at stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, about 14% to 16% caustic soda solution or caustic potash at less than stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, about 15% to 16% caustic soda solution or caustic potash at stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, about 15% to 16% caustic soda solution or caustic potash at less than stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, alkali at a concentration of about 12% to 18% at stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, alkali at a concentration of about 12% to 18% at less than stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, alkali at a concentration of about 12% to 16% at stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, alkali at a concentration of about 12% to 16% at less than stochiometric amounts is employed for neutralization.

In some embodiments of the present disclosure, the enzyme hydrolyzed oil is subjected to neutralization to obtain soap and neutral oil by using stoichiometric quantity of caustic soda or caustic potash wherein the neutralization is carried out in a neutralization tank at a mixing rate of about 15 RPM to 40 RPM for efficient contact between free fatty acids and caustic soda or caustic potash.

In some embodiments of the present disclosure, the soap and neutral oil obtained after the neutralization process are separated and processed separately. In some embodiments, the soap and neutral oil are separated by a separation technique selected from a group comprising centrifugation, fdtration, distillation, decantation, settling and combinations thereof.

Step c): Bleaching the neutral oil and deodorizing the bleached oil to obtain refined oil.

In some embodiments of the present disclosure, the neutral oil is taken to a bleacher and bleached in presence of a bleaching agent at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes.

In some embodiments of the present disclosure, the bleaching agent is bleaching earth. In some embodiments, about 0.8% of acid activated bleaching earth is employed in the bleaching step/process of the present disclosure as against 1% of the same quality acid activated bleaching earth that is normally employed in traditional chemical refining processes of oils. Such less amount/concentration of acid activated bleaching earth is made possible to be employed in the bleaching step of the present method because of the combination of various neutralization step/process conditions leading to reduced/negligible phospholipid content in neutralized oil.

In embodiments of the present disclosure, the bleached oil is subjected to deodorization.

In some embodiments of the present disclosure, the bleached oil is deodorized in a conventional/traditional ways to obtain bleached, deodorized and refined oil product.

In some embodiments of the present disclosure, deodorization is carried out by distillation using steam as carrier, in order to remove residual free fatty acids, unwanted odor, unwanted taste, natural color of oil, oxidation products, chlorophyll or any combinations thereof from the neutralized and bleached oil to produce a high quality final refined oil product.

In some embodiments of the present disclosure, the method for enzyme assisted chemical refining of vegetable oils is illustrated as follows: crude vegetable oil or water degummed vegetable oil enzyme hydrolysis

(i.e. phospholipids present in the oil are subjected to complete enzyme hydrolysis)

enzyme hydrolysed oil taken to a neutralizer for neutralization

Neutralization

(“stoichiometric quantity of caustic soda” at 14% to 18% concentration is added to the enzyme hydrolysed oil to neutralize the acidity/free fatty acids of the oil, to obtain soap and neutral oil)

separating the soap and the neutral oil by separation technique

subjecting the neutral oil to bleaching

followed by deodorization to obtain the final refined oil product

In some embodiments of the present disclosure, the enzyme hydrolysis process of the present method for enzyme assisted chemical refining of vegetable oils is illustrated as follows: raising the temperature of crude vegetable oil or

water degummed vegetable oil in a heat exchanger

taking the oil to a homogenizer

adding enzyme solution into the oil through the homogenizer allowing the enzyme to react with the oil

for about 30 minutes to 6 hours in an enzyme reaction tank

In some embodiments of the present disclosure, a method for chemical refining of crude vegetable oil or water degummed vegetable oil including soybean oil, sunflower oil, com oil, rapeseed oil, mustard oil, rice bran oil or palm oil is provided, the method comprising:

a) subjecting crude soybean oil or water degummed soybean oil to enzyme treatment at a temperature of about 50°C to 55°C for a time -period of about 4 hours to obtain enzyme treated oil,

wherein the enzyme treatment comprises reacting the oil with an enzyme solution having a pH of about 3 to 8 and comprising phospholipid hydrolysing enzymes phospholipase A2 (PL-A2) and lyso-phospholipase (LPL), citric acid, sodium hydroxide and water;

b) reacting the enzyme treated oil and an alkali to obtain soap and neutral oil, wherein said reaction of enzyme treated oil and alkali comprises mixing the enzyme treated oil and the alkali at a rate of about 20 RPM to 30 RPM and at a temperature of about 50°C to 55°C for a time-period ranging from about 20 minutes,

wherein said alkali is at a stochiometric quantity based on free fatty acids content in the enzyme treated oil, and concentration of the alkali is about 15% to 16%,

and wherein said alkali is sodium hydroxide, potassium hydroxide, or a combination thereof; and

c) bleaching the neutral oil by treating the neutral oil with a bleaching agent in a bleacher at a temperature of about 90°C to 95°C for a time-period of about 30 minutes to 45 minutes to obtain bleached oil; and

d) deodorizing the bleached oil by subjecting the bleached oil to steam distillation in a deodorizer at a temperature of about 230°C to 250°C for a time-period of about 60 minutes to 120 minutes to obtain the refined soybean oil.

In some embodiments of the present disclosure, the crude vegetable oil batches or water degummed vegetable oil batches are subjected to enzyme hydrolysis using a mixture of phospholipase A2 (PL-A2) and lyso-phospholipase (LPL) to achieve complete hydrolysis of phospholipids into glycerophosphatide and free fatty acids, and the free fatty acids present in the oil are neutralized using stoichiometric quantity of caustic soda or caustic potash (or slightly less than stoichiometric quantity of caustic soda or caustic potash) under mild neutralization parameters or conditions described herein. The neutralized oil is thereafter centrifuged to separate soap from the neutral oil, and the neutral oil is subjected to estimation of phosphorous content and free fatty acid (FFA) content. The neutral oil so obtained is further bleached using 0.50% or 0.75% of bleaching earth by weight of neutral oil followed by deodorization to obtain the final refined oil product.

Thus, the present disclosure provides refined vegetable oil products based on the enzyme assisted chemical method of refining vegetable oil described above. The present enzyme assisted chemical method of refining vegetable oils has several advantages over the traditional/known methods as described above and further discussed in the foregoing paragraphs.

The present disclosure also provides a possible method of processing the soap stock obtained during the enzyme assisted chemical method of refining vegetable oil as described above. In some embodiments, the present disclosure provides a possible method for processing the soap stock obtained after neutralization step (i.e. reaction of enzyme treated oil and alkali) of the enzyme assisted chemical method of refining vegetable oil as described above.

In some embodiments of the present disclosure, the method of processing of soap stock comprises:

a) diluting the soap with acidic water at a temperature of about 80°C to 95 °C and a pH of about 4 to obtain a mixture, and subjecting the mixture to mixing to obtain an acidified soap; and b) holding the acidified soap at a temperature of about 80°C to 95°C for about 30 minutes to 60 minutes to obtain an acid water layer comprising glycerophosphatide and an oil layer comprising free fatty acids and oil.

In some embodiments of the method of processing of soap stock as described above, salt or an alkali at suitable concentrations is added to the acidified soap mixture and mixed well before holding the acidified soap at a temperature of about 80°C to 95°C for about 30 minutes to 60 minutes to separate aqueous phase (acid water layer) and oil phase (oil layer). In some embodiments of the method of processing of soap stock as described above, caustic soda (sodium hydroxide) at suitable concentrations is added to the acidified soap mixture and mixed well before holding the acidified soap at a temperature of about 80°C to 95°C for about 30 minutes to 60 minutes. In some embodiments of the method of processing of soap stock as described above, sodium chloride at suitable concentrations is added to the acidified soap mixture and mixed well before holding the acidified soap at a temperature of about 80°C to 95°C for about 30 minutes to 60 minutes.

In some embodiments of the present disclosure, the method of processing of soap comprises: a) diluting the soap with hot acidic water (i.e. water and acid) at a temperature of about 80°C to 95 °C and a pH of about 4 and passing the mixture through a homogenizer, to obtain acidified soap; and

b) holding the acidified soap at a temperature of about 80°C to 95°C in a separation tank, for about 30 minutes to 60 minutes, to separate an acid water layer comprising glycerophosphatides and an oil layer comprising free fatty acids and oil.

In some embodiments of the method of processing of soap described above, the acid employed is a mineral acid and is selected from a group comprising sulfuric acid, hydrochloric acid and a combination thereof.

In some embodiments of the method of processing of soap described above, any other mineral acid or strong organic acids such as citric acid, acetic acid or any similar organic acids can also be employed for diluting the soap with hot acidic water.

In some embodiments of the method of processing of soap described above, the acid is sulfuric acid.

In some embodiments of the method of processing of soap described above, the homogenizer is a high shear mixer.

In some embodiments of the method of processing of soap described above, the separation tank is a batch separation tank. In some embodiments of the method of processing of soap described above, a small portion of pitch is present between acid water layer and oil layer depending upon the level of seed particles and quality of crude vegetable oil employed.

In some embodiments of the method of processing of soap described above, a continuous centrifuge is used to separate/recover acid water layer and oil layer continuously.

Reference throughout this specification to“one embodiment” or“some embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases“in one embodiment” or“in some embodiments” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination of embodiments.

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well- known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides few examples illustrating the above described embodiments. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES

EXAMPLE 1:

General method of refining vegetable oils according to the present disclosure The method of refining crude vegetable oils or water degummed vegetable oils comprised taking 250 gm of water degummed soybean oil or water degummed sunflower oil with a phosphorous content between 80-140 ppm in a beaker. The temperature was raised to 55°C in a water bath and a mixture (enzyme solution) comprising about 3.75 ml distilled water, about 162.5 mg citric acid monohydrate and about 41.25 mg NaOH with pH of solution at 4.0, and about 7.5 mg of a commercially available phospholipase A2 (PL-A2) and about 12.5 mg of a commercially available lysophosphobpase (LPL) was added slowly while mixing the oil with laboratory scale homogenizer. The oil was then kept in a shaker water bath at 55°C for 5 hours while taking care to provide sufficient agitation to the oil and preventing the evaporation of water added to the oil. At the end of 5 hours, the enzyme treated oil was taken for neutralization.

The enzyme treated oil was neutralized with stoichiometric quantity of caustic soda with a concentration ranging from about 12.5% to 18% in water, by using a magnetic stirrer. Other neutralization parameters/conditions comprised a mixing rate of the enzyme treated oil and caustic soda at about 15 RPM to 40 RPM and at a temperature of about 10°C to 70°C. The time- period of neutralization reaction was about 10 minutes to 30 minutes.

The neutralized oil was centrifuged at 2000 RPM to 4000 RPM for 5 minutes to 15 minutes at 80°C to obtain neutral oil and soap. The neutral oil so obtained was analyzed for soap content and phosphorous (P) content which is indicative of the efficiency of the method described herein.

The neutral oil obtained herein can be further processed by bleaching and deodorization steps to prepare a final refined oil product fit for end-use.

EXAMPLE 2:

Method of refining vegetable oil according to the present disclosure

250 gm of water degummed soybean oil with a phosphorous content of about 130 to 140 ppm was refined by following enzyme treatment and neutralization procedure as described in Example 1. The neutral oil and soap were separated by centrifugation. The results of soap content and phosphorous content in the obtained neutral oil based on different temperatures of neutralization reaction i.e. mixing enzyme treated oil and alkali (caustic soda) is shown in Table 1 and Figure 1 Table 1 : Effect of mixing temperature during neutralization

The above results indicate the effect of neutralization temperatures and shows that when refining of vegetable oils is carried out by employing the features of neutralization reaction according to the present disclosure, a superior efficacy is achieved as demonstrated by less soap content and phosphorous content in the neutral oil obtained after centrifugation. Particularly, when mixing temperature of 50°C, mixing rate of 40 RPM and stoichiometric quantity of caustic soda of 16% was employed for neutralization, the soap content and the phosphorous content in the neutral oil was significantly lesser compared to a neutralization reaction comprising mixing temperature of 80°C, mixing rate of 40 RPM and stoichiometric quantity of caustic soda of 16%.

The above results additionally demonstrate the significant advantage of the present method in terms of reducing neutralization losses and the associated savings in the subsequent processing steps of neutral oil.

EXAMPLE 3:

Method of refining vegetable oil according to the present disclosure 250 gm of water degummed soybean oil with a phosphorous content of about 130 to 140 ppm was refined by following enzyme treatment and neutralization procedure as described in Example 1. The neutral oil and soap were separated by centrifugation. The effect of degree of mixing (or mixing rate) during neutralization on soap content and phosphorous content in the neutral oil is shown in Table 2 and Figure 2.

Table 2: Effect of degree of mixing during neutralization

The above experiments show the effect of degree of mixing during neutralization. The results show that when refining of vegetable oils is carried out by employing the neutralization features according to the present disclosure, a superior efficacy is achieved as demonstrated by less soap content and phosphorous content in the obtained neutral oil. Particularly, when simple mixing at 40 RPM at 50°C and stoichiometric quantity of caustic soda of 16% was employed for neutralization, the soap content and the phosphorous content in the neutral oil was significantly lesser compared to a neutralization reaction employing vigorous mixing at 10,000 RPM in a homogenizer for 2 minutes at 50°C and stoichiometric quantity of caustic soda of 16%.

The above results additionally demonstrate the significant advantage of the present method in terms of reducing neutralization losses and the associated savings in the subsequent processing steps of neutral oil.

EXAMPLE 4:

Method of refining vegetable oil according to the present disclosure

250 gm of water degummed soybean oil with a phosphorous content of about 130 to 140 ppm was refined by following enzyme treatment and neutralization procedure as described in Example 1. The neutral oil and soap were separated by centrifugation. The effect of different concentrations of alkali employed during neutralization on soap content and phosphorous content of the neutral oil is shown in Table 3 and Figure 3.

Table 3 : Effect of concentrations of alkali during neutralization

The above experiments show the effect of various concentrations of alkali (caustic soda) during neutralization. The results show that when refining of vegetable oils is carried out by employing the neutralization features according to the present disclosure, a superior efficacy is achieved as demonstrated by less soap content and phosphorous content in the obtained neutral oil. Particularly, when stoichiometric quantity of caustic soda of 14%, 16% or 18% along with simple mixing (40 RPM) at 50°C was employed for neutralization, desired/acceptable results for the soap content and the phosphorous content in the neutral oil were achieved. These results further indicate the importance of employing 12.5% to 18% alkali concentrations for neutralization. Also, it is hypothesized that if more lower concentrations of caustic solution (for instance, below 12%) are employed which is an indication of the caustic solution being more diluted (high water content), more emulsification during neutralization may occur and thereby more loss of oil into water in the form of emulsion. The above results additionally demonstrate the significant advantage of the present method in terms of reducing neutralization losses and the associated savings in the subsequent processing steps of neutral oil.

EXAMPLE 5:

Comparison with traditional/prior art methods of chemical refining of vegetable oils

The following Tables 4 and 5 describe experiments conducted using traditional/known methods of chemical refining of vegetable oils.

Table 4: Long Mix and Short Mix processes of neutralization employed in traditional chemical methods of vegetable oil refining

The results of Table 4 indicate that when traditional non-enzymatic chemical refining method comprising acid degumming and neutralization is employed, both the long mix and short mix neutralization conditions lead to higher levels of phosphorous and soap in the neutral oil when compared to the method according to the present disclosure. This further indicates that in traditional chemical refining processes, a stronger emulsion is formed during neutralization leading to oil losses and greater difficulty in separation of phosphorous as well as soap.

Table 5: Comparison of present enzyme assisted vegetable oil refining method with traditional vegetable oil refining method and other variations in the method

The above experiments of Table 5 were conducted under commercial trials with crude soybean oil having a phosphorous content of 128 ppm. Soap separation was carried out using an Alfa Laval SRG 610 separator. The results under SI. Nos. 1, 2 and 4 show that greater intensity of mixing (dynamic shear mixer) at lower or higher temperatures (50°C or 80°C) along with using stoichiometric quantity of caustic yields higher soap content and phosphorous content in neutral oil indicating a stronger emulsification during neutralization and hence greater loss of neutral oil into soap. On the other hand, in the present enzyme assisted chemical refining (SI. No. 3) using stoichiometric quantity of caustic along with mild/simple mixing of caustic at a temperature of 50°C, the neutral oil obtained had a highly reduced soap content and phosphorous content indicating significantly improved neutralization efficiency.

Thus, the present method of enzyme assisted chemical refining of vegetable oils described herein provide efficient enzyme hydrolysis and neutralization steps which lead to an enhancement in the overall efficiency of the refined oil product. In particular, the neutralization process (i.e. reaction of the enzyme treated oil and alkali) including a combination of features such as the mixing rate of about 15 RPM to 40 RPM, temperature of about 10°C to 70°C, stochiometric quantity or less than stochiometric quantity of alkali at 12.5% to 18% act synergistically to achieve high quality neutral oil containing significantly less soap and phosphorous contents. The above examples/results further indicate that any variations in the neutralization process of the method described herein or the traditionally employed non-enzymatic chemical refining of vegetable oils lead to inferior efficacy/quality of neutral oil.

Further, according to the embodiments of the present disclosure, the products of enzyme hydrolysis of gums and the components of enzyme solution including pH regulator and water, are not removed from the enzyme treated oil prior to neutralization and are subjected to the neutralization step as described herein. Additionally, in some embodiments of the present disclosure, the enzyme treatment step of the present method leads to complete hydrolysis of gums and the enzyme treated oil is directly taken for neutralization step as described herein.

The method of enzyme assisted chemical refining of vegetable oils described in the present disclosure provides several advantages including but not limiting to the following:

(a) the present method results in both process and economic benefits and results in complete hydrolysis of non-hydratable gums/phospholipids in crude vegetable oils or water degummed oils.

(b) complete hydrolysis of phospholipids during enzyme treatment helps in reducing effluent load, increases the quantity and quality of enzyme treated oil for neutralization, reduces oil losses in neutralization and at the same time reduces consumption of bleaching earth for bleaching step.

(c) the neutralization process of the present method leads to high quality neutral oils with significantly low soap and phosphorous contents, and thereby reduced oil losses during neutralization.

(d) higher yield of neutral oil is obtained in the present method to the extent of 0.2 to 0.4%.

This increase in yield/efficiency results in substantial cost savings/benefits during large- scale refining of vegetable oils.

(e) processing of soap stock after separation from neural oil: The processing of soap stock according to the present disclosure has several benefits compared to traditional processing of soap stock. Some of the benefits include -

- no need to adjust the pH of soap stock to highly acidic pH of 2.0, resulting in savings in mineral acid (eg. sulphuric acid). - no need to boil the soap stock to split the soap which results in savings in steam consumption.

- quality of acid oil and fatty acids recovered during processing of soap stock is high.

- the acid water layer separated during processing of soap stock has much less mineral acidity in it and it can be easily treated.

- lower acid water effluent generation.

(f) the present method can be employed for any vegetable oil.

(g) the present method is cost effective/economical and highly profitable when employed in commercial scale due to the above-mentioned features.

Thus, the method described herein is successful in meeting the primary objectives of the present disclosure including:

i) to obtain high quality refined oil with lower phosphorous levels in the neutral oil during chemical process of oil refining.

ii) to obtain high quality refined oils with reduced free fatty acid (FFA) content and soap content in neutral oil and reduced phosphorous content in bleached oil during chemical process of oil refining.

iii) to maximize the yield of neutral oils during chemical process of oil refining.

iv) to achieve lower neutralization losses including reduced loss of neutral oil into soap during chemical process of oil refining.

v) to eliminate water wash of neutral oil during chemical process of oil refining. Said water wash of neutral is necessitated by high soap content of neutral oil post separation/centrifugation. Since the neutral oil obtained by the method of the present disclosure contains very low soap content, the need for water wash is eliminated which translates into an additional neutral oil yield of about 0.10% to 0.15% along with significant cost benefits.

vi) to reduce the dosage of bleaching earth for bleaching the neutral oil during chemical process of oil refining. Reduced consumption of bleaching earth is achieved because of the very low phosphorous content of neutral oil obtained by the present method. vii) to provide easier processing of soap stock without employing harsh conditions or high concentration of mineral acids. viii) to achieve complete hydrolysis of gums (phospholipid) in the crude oil or water degummed oil by splitting both fatty acid chains of the phospholipid molecule and therefore converting the phospholipids into glycerophoshates and free fatty acids (FFAs).

The foregoing description of the specific embodiments reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The use of the expression“at least” or“at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. Throughout this specification, the word“comprise”, or variations such as“comprises” or“comprising” or “containing” or“has” or“having”, or“including but not limited to” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications, and/or patent applications (if any) cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.