SILICA ADSORBENT FOR REMOVAL OF CHLOROPHYLL DERIVATIVES FROM TRIACYLGLYCEROL-BASED OILS

Abstract

The present invention relates to an adsorbent for treating an oil comprising a chlorophyll derivative. In particular, the present disclosure relates to an improved silica gel adsorbent for removing impurities, including chlorophyll derivatives and/or trace metals, from an oil, in particular triacylglycerol-based oils. The adsorbent comprises a silica gel treated with an alkali earth metal oxide, such as magnesium oxide, and has a pH of about 7 or greater and a water content of about 3 wt % or greater.

Claims

1. An adsorbent for removing chlorophyll and chlorophyll derivatives from an oil, which adsorbent comprises an amorphous porous silica gel treated with an alkaline earth metal oxide, said adsorbent having a pH of from about 7 or greater, an alkaline earth metal oxide content of at least 0.1 weight percent (wt %), on a dry basis, and a water content of from about 3 wt % or greater.

2. The adsorbent of claim 1, wherein the adsorbent comprises from 1 to about 40 wt % alkaline earth metal oxide, on a dry basis.

3. The adsorbent of claim wherein the alkaline earth metal oxide is selected from the group consisting of magnesium oxide, calcium oxide, barium oxide, beryllium oxide, or combinations thereof.

4. The adsorbent of claim wherein the alkaline earth metal oxide is magnesium oxide.

5. The adsorbent of claim wherein the amount of magnesium oxide ranges from about 5 wt % to about 25 wt % MgO, wherein the magnesium oxide preferably comprises from about 10 to about 20 wt % MgO.

6. (canceled)

7. The adsorbent of claim 1, wherein the adsorbent has a pH of from 7.0 to 10.0, preferably from 7.5 to 9.7, more preferred from 8.0 to 9.5.

8-9. (canceled)

10. The adsorbent of claim 1, wherein the adsorbent has a water content of 10 wt % or greater, preferably from about 25 to about 75 wt %, more preferred from about 40 to about 70 wt %, still more preferred from about 55 to about 65 wt %.

11-13. (canceled)

14. The adsorbent of claim 1, wherein the adsorbent has a median particle size of from about 0.1 to about 2,000 microns, preferably from about 2 to about 500 microns, more preferred from about 5 to about 50 microns.

15-16. (canceled)

17. The adsorbent of claim 1, wherein the adsorbent has a surface area of at least 50 m.sup.2/g, preferably from about 50 to about 800 m.sup.2/g.

18. (canceled)

19. The adsorbent of claim 1, wherein the adsorbent has a pore volume of at least about 0.1 cc/g, preferably of at least about 0.4 cc/g, more preferred from about 0.7 to about 2.0 cc/g.

20-21. (canceled)

22. The adsorbent of claim wherein the adsorbent has a pH of from about 7 to about 10, a magnesium oxide amount of from about 5 to 25 wt % MgO, on a dry basis, and a water content of from about 25 wt % to about 65 wt %, preferably has a pH of from about 8 to about 9.5, a magnesium oxide amount of from about 10 to 20 wt % MgO, on a dry basis, and a water content of from about 55 wt % to about 65 wt %.

23. (canceled)

24. The adsorbent of claim 1, wherein the adsorbent has the capability of removing chlorophyll and chlorophyll derivatives from an oil, wherein the oil preferably comprises a triacylglycerol-based oil.

25. (canceled)

26. The adsorbent of claim 24, wherein the triacylglycerol-based oil is selected from the group consisting of canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hempseed oil, linseed oil, mango kernel oil, meadowfoam oil, neat's foot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, sesame oil, soybean oil, sunflower seed oil, tall oil, tsubaki oil, vegetable oil, and an oil from alga.

27. The adsorbent of claim 24, wherein the oil is a decolorase-treated oil.

28. The adsorbent of claim 27, wherein the silica gel is a hydrogel or a hydrated xerogel.

29. The adsorbent of claim 28, wherein the adsorbent reduces the total concentration of chlorophyll and chlorophyll derivatives in the decolorase-treated oil by at least 5% by weight, compared to the total concentration of chlorophyll and chlorophyll derivatives in the decolorase-treated oil prior to contact with the adsorbent, preferably by at least 50% by weight, compared to the total concentration of chlorophyll and chlorophyll derivatives in the decolorase-treated oil prior to contact with the adsorbent.

30. (canceled)

Description

FIGURES

[0161] FIG. 1: Overview of the conversion of chlorophyll into pheophytin and pyropheophytin and into the respective reaction products chlorophyllide, pheophorbide and pyropheophorbide. The A compounds are shown, which have a methyl group at the C7 position. B compounds have an aldehyde in the C7 group instead of a methyl group. Structures are taken from PubChem, NCBI.

[0162] FIGS. 2-2A: HPLC results of incubation pheophytin a and b and pyropheophytin a and b with different putative chlorophyllases at pH 7 and 50° C., for 24 hours. The amounts of the substrates pheophytin a and b and pyropheophytin a and b and the reaction products pheophorbide a and b and pyropheophorbide a and b are given as peak surface areas. The first two columns show the sum of reaction products and substrates. “nd” means: not detectable.

[0163] FIG. 3: HPLC results of incubation pheophytin a and b and pyropheophytin a and b with different putative chlorophyllases at pH 5 and 50° C., for 24 hours. The amounts of the substrates pheophytin a and b and pyropheophytin a and b and the reaction products pheophorbide a and b and pyropheophorbide a and b are given as peak surface areas. The first two columns show the sum of reaction products and substrates. “nd” means: not detectable.

[0164] FIG. 4: 4A: Chlorophyll derivatives 4B: Phosphor compounds in canola oil after 24 h incubation with CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii.

[0165] FIG. 5: 5A: Chlorophyll derivatives, and 5B: Phosphor compounds in canola oil and soy bean after several incubations with CHL26 enzyme from Hordeum vulgare and/or the reference enzyme ELDC94 from Chlamydomonas reinhardtii, under different reaction conditions and 5C: chlorophyll derivatives in the obtained gums.

[0166] FIGS. 6-6A: Chlorophyll derivatives in canola oil and soybean oil after caustic refining and after incubation with CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii.

[0167] FIG. 7: Schematic presentation of a typical chemical refinery process for triacylglycerol based oils. A process of solvent extraction and/or pressing on an oilseed (rapeseed or soybean), oil fruit plant (palm), or single cell source (algal) to obtain a crude oil. The crude oil this then treated with citric or phosphoric acid to react with the non-hydratable phospholipids and then the addition of sodium hydroxide to neutralize the free fatty acids and form sodium soaps. The sodium soaps form an emulsion with the water present allowing the removal of non-hydratable phospholipids when the oil is centrifuged to produce refined oil. The refined oil may then be washed with hot water and centrifuged to remove the remaining soaps and phospholipids. Alternatively, the refined oil may be treated with acidic silica to adsorb soaps, trace metals and phospholipids. The industrial acidic silicas do not have any capacity to remove chlorophyll or chlorophyll derivatives. The oil is then treated with bleaching earth to remove the soaps, phospholipids, and chlorophyll and chlorophyll derivatives present in the oil. The final step in the deodorization step of steam distillation at elevated temperatures and vacuums of less than 5 mBar. The distillation primarily removes peroxides, aldehydes, ketones and other flavor compounds. It also destroys beta-carotene and removes the remaining free fatty acids (0.1 percent) to reach a level of 0.02 to 0.05% final Free Fatty Acid (FFA).

[0168] FIG. 8: Schematic presentation of a typical enzymatic degumming/physical refining process. The crude oil is treated with phosphoric or citric acid to enable the non-hydratable phospholipids to lose the calcium or magnesium bond to them at a pH of roughly 2. The sodium hydroxide is then added to bring the pH above 4 for citric acid or above 6 for phosphoric acid in order that the phospholipase may work and obtain a very low residual phosphorus<5 ppm) after the enzymatic reaction with the PLAs. Alternatively, the PLAs may be reacted with the PLC and/or PI-PLC to maximize the oil yield and still obtain a very low residual phosphorus allowing for physical refining. The oil is then either washed or treated with an acidic silica followed or in combination with bleaching earth. After the bleaching process with chlorophyll levels of less than 50 ppb, the oil is physically refined in the deodorizer. The high temperature steam distillation removes all of the compounds describe above in FIG. 7, but its primary purpose is the removal of FFA. The FFAs are distilled and collected in the scrubber. Very limited neutral oil is lost in the deodorization process compared to the losses associated from the emulsions formed in water degumming or chemical refining.

[0169] FIG. 9: Schematic presentation of the use of a decolorase enzyme in the water degumming process or the enzyme assisted water degumming process. A decolorase enzyme may be added with the water at 60° C., or with the PLC, or with the combination of PLC and PI-PLC. After two hours of incubation, the oil is heated to 70 to 85° C. and centrifuged to remove the reacted gums and reacted chlorophyll derivatives.

[0170] FIG. 10: Schematic presentation of an enzymatic degumming process modified to include treatment with a decolorase enzyme and a silica adsorbent (such as a MgO-treated adsorbent) of the present disclosure. The crude oil is first treated with citric acid to a pH of roughly 2 to dissociate the bond calcium and magnesium ions, the pH is raised above 4 to enable the PLCs and Decolorase enzymes in a pH that enable them to work efficiently. 1 to 5 percent water is added for the hydrolysis reactions. After the completion of the PLCs and Decolorase incubations, a PLA1 or PLA2 may be added to react with the non-hydratable phospholipids present in the oil. After an additional incubation of 2 to 6 hours, the oil is heated to 70 to 85° C. and centrifuged to remove the reacted gums and chlorophyll derivatives producing an oil with less than 5 ppm residual phosphorus in the oil. The enzymatically degummed oil may then be contacted with a silica adsorbent of the present disclosure, optionally under vacuum, to further remove chlorophyll derivatives and trace metals, as described herein.

[0171] FIG. 11: Schematic presentation of a chemical refining process with a decolorase enzyme, followed by treatment with a silica adsorbent of the present disclosure. The decolorase enzyme may not be added in the acid or caustic addition steps due to the very low pH (roughly 2) and the very high pH (roughly 14) in the early steps of the process. The decolorase enzyme must be added after the initial centrifuge step in the refined oil. It is advantageous to add the decolorase enzyme with the washing step at a temperature suitable for the enzyme (50 to 65° C.). Allow an incubation time of at least two hours followed by heating to 70 to 85° C. prior to centrifugation. The oil would then be further processed by contacting with a silica adsorbent of the present disclosure, optionally under vacuum, to further remove chlorophyll derivatives and/or trace metals, as described herein.

[0172] FIG. 12: Schematic presentation of an enzymatic degumming/physical refining process modified to include treatment with a silica adsorbent of the present disclosure, but without decolorase treatment. The enzymatically degummed oil or the enzymatically degummed and washed oil is contacted with a silica adsorbent of the present disclosure, and optionally with bleaching earth, optionally under vacuum, to remove chlorophyll derivatives and trace metals, as described herein.

[0173] FIG. 13: Schematic presentation of a chemical refinery process for triacylglycerol based oils, modified to include treatment with a silica adsorbent of the present disclosure, but without decolorase treatment. The refined oil or the once refined oil is contacted with a silica-based adsorbent of the present disclosure, and optionally with bleaching earth, optionally under vacuum, to remove chlorophyll derivatives and trace metals, as described herein.

SEQUENCES

[0174] SEQ ID NO: 1=CHL26 polypeptide having decolorase including a pyropheophytinase activity from Hordeum vulgare.

[0175] MASAGDVFDHGRHGTSLARVEQAKNTRCSAASRVDADAQAQQSPPKPLLVAAPCDAGEYPVVVFLHGYLCNNYFYSQ LIQHVASHGFIVVCPQLYTVSGPDTTSEINSAAAVIDWLAAGLSSKLAPGIRPNLAAVSISGHSRGGKVAFALGLGHAKTSL PLAALIAVDPVDGTGMGNQTPPPILAYKPNAIRVPAPVMVIGTGLGELPRNALFPPCAPLGVSHAAFYDECAAPACHLVA RDYGHTDMMDDVITGAKGLATRALCKSGGARAPMRRFVAGAMVAFLNKWVEGKPEWLDAVREQTVAAPVVLSAVEF RDE

[0176] SEQ ID NO: 2: Codon optimized nucleic acid sequence encoding a polypeptide having decolorase including pyropheophytinase activity from Hordeum vulgare CHL26 for expression in Pseudomonas fluorescens.

[0177] SEQ ID NO: 3; CHL25 putative chlorophyllase from Gossypium raimondii

[0178] SEQ ID NO: 4; CHL27 putative chlorophyllase from Phoenix dactylifera

[0179] SEQ ID NO: 5; CHL28 putative chlorophyllase from Wollemia nobilis

[0180] SEQ ID NO: 6; CHL29 putative chlorophyllase from Cucumis sativus

[0181] SEQ ID NO: 7; CHL30 putative chlorophyllase from Tarenaya hassleriana

[0182] SEQ ID NO: 8; CHL31 putative chlorophyllase from Solanum tuberosum

[0183] SEQ ID NO: 9; CHL32 putative chlorophyllase from Populus trichocarpa

[0184] SEQ ID NO: 10; CHL33 putative chlorophyllase from Vigna radiata

[0185] SEQ ID NO: 11; N1 Negative control, Green Fluoresent Protein (GFP)

[0186] SEQ ID NO: 12; P2, Chlamydomonas reinhardtii chlorophyllase having pyropheophytinase activity. SEQ ID NO: 12 is also referred to herein as ELDC94.

[0187] SEQ ID NO: 13; SpeI site and ribosome binding site

[0188] SEQ ID NO: 14; stop codon and XhoI site.

EXAMPLES

Materials and Methods

General

[0189] Standard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3.sup.rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987). Water is Milli-Q water where nothing else is specified.

Analytical Methods:

[0190] pH—stoichiometric addition of acid and base to a water percentage that was added to the oil.

[0191] 2 percent water in a 2000 grams reaction would be 40 grams, adding 2.0 grams of a 50 percent solution of citric acid, plus 1.6 mL of 4 M sodium hydroxide would yield a water solution with a pH of 4.5. The pH of the oil will always remain 7.

[0192] Soap—American Oil Chemists' Society Official Method Cc 13a-43, revised 2017. Free Fatty Acid—American Oil Chemists' Society Official Method Ca 5a-40, revised 2017.

[0193] Color—American Oil Chemists' Society Official Method Ce 13e-92, reapproved 2017. Utilized Tintometer's PFX-950 at 5¼″ cell.

[0194] Phosphorus and trace metals—American Oil Chemists' Society Official Method Ca 17-01-43, revised 2017.

[0195] Phospholipid Compositions For .sup.31P NMR methods (also referred to as 31-P NMR), 10 μL of 10% DOL dispersion was dispersed in 1 mL of an aqueous solvent containing demineralized water with 10% deuterium oxide (D.sub.2O, Cambridge Isotope Laboratories, DLM-4), 25 mg/mL deoxycholic acid (Sigma D2510), 5.84 mg/mL EDTA di Na (Titriplex III, Merck 108418), and 5.45 mg/mL TRIS base (Tris(hydroxymethyl) aminomethane, Merck 108387), of which the pH was adjusted to pH 9 using 4N KOH and to which 2 mg/mL TIP internal standard (tri-isopropylphosphate, Aldrich 554669) (accurately weighed) was added.

[0196] All samples were measured in a Bruker 400 MHz AvanceIII NMR spectrometer with a Prodigy BBO probe. The temperature of the probe head was set at 300K.

[0197] The measurement for quantification was performed with semi-quantitative parameters: 128 scans, 90° pulse, D1=5 sec. Values are reported in μmol/g of dry weight (DOL) of the sample.

[0198] Analysis of green color content by UV/Vis—The AOCS UV/Vis method is used to measure the green color content of oils. The AOCS UV/Vis method is described in Cc 13d-55, reapproved 2017.

[0199] Analysis of Chlorophyll Derivatives by HPLC-FLU

[0200] The analysis of pheophytins A and B, and pyropheophytins A and B, and their phorbides, as well as chlorophyll and chlorophyllide, was performed by HPLC using fluorescence detection, a method developed based on the work of Hwang et al J. Food Hyg. Soc. Japan Vol. 46, No. 2, 45-48, extended by fluorescence detection at λex 410 nm/λem 666 nm for the A compounds, and λex 436 nm/λem 653 nm for the B compounds.

[0201] Analysis for the Water Content of the Adsorbent

[0202] Water content, in wt %, is determined by heating the adsorbent to 1750 F until a constant weight is observed. The water content equals the mass lost divided by the original mass of the material expressed in percentage.

[0203] Sample Preparation

[0204] Oil samples were diluted in acetone, 1 g oil in 9 mL acetone, and centrifuged at 14000 rpm for 5 minutes. The clear supernatants were transferred into injection vials, and 10 μl of a sample was injected into the HPLC. As the chlorophyll levels were so low in all practical oil samples, these were not taken into account in the analysis.

[0205] Data Analysis

[0206] The peak surface areas (in arbitrary units) of the chromatograms indicate the amount of pheophytins, pyropheophytins, pheophorbides and pyropheophorbides present in the oil samples. FIGS. 2 and 3 show the peak surface areas of pheophytins, pyropheophytins, pheophorbides and pyropheophorbides in oil samples after incubation with putative chlorophyllases at pH 5 and pH 7. The sum of the peak surface area of phytines, the sum of peak surface area of phorbides and the peak surface area of the individual compounds are shown. The formation of pheophorbide and pyropheophorbide is a measure for the presence of pheophytinase activity and pyropheophytinase activity, respectively.

Enzymes

[0207] Purifine® Phospholipase C (PLC), and Purifine® PI-PLC and a fungal PLA1 were obtained from DSM.

[0208] Purifine® Phospholipase C comprises amino acids 38-282 of SEQ ID NO: 2, having the amino acid substitutions 63D, 131S and 134D disclosed in WO2005/086900

[0209] Purifine® PI-PLC comprises the mature polypeptide according to SEQ ID NO: 8 disclosed in WO2011/046812.

[0210] Fungal PLA1 comprises the mature amino acid sequence of SEQ ID NO: 1 disclosed in European application no. EP18171015.3

Equipment

[0211] The overhead mixer was an IKA RW 20 Digital with a flat blade paddle.

[0212] The centrifuge was a De Laval Gyro—Tester installed with “The Bowl Unit” for continuous separation. The centrifuge bowl was closed with the plug screws installed. Shear mixing was accomplished with an Ultra-Turrax homogenizer SD-45 with a G450 rotor stator at 10,000 rpm.

Silica Adsorbents

[0213] Test adsorbents SP-2113, SP-2114, SP-2115, SP-2116, SP-2117, and SP-2119 were obtained from W.R. Grace & Co.-Conn. (Columbia, Md.). TRISYL® silica and TRISYL® 300 (W.R. Grace & Co.-Conn., Columbia, Md.) are commercially available. The properties of various silicas and adsorbents used in the Examples are set forth below.

TABLE-US-00001 Na.sub.2O MgO (wt % (wt % Median Base on a on a Water Particle Silica dry dry Content Size Type pH basis) basis) (wt %) (μm) SP-2113 Hydrogel 10.2 5.81 <0.1 51.9 20 SP-2114 Hydrogel 8.6 <0.1 5.0 60.2 20.1 SP-2115 Hydrogel 8.7 <0.1 11.7 58.4 20.1 SP-2116 Acidic 6.1 2.91 <0.1 53.7 21 hydrogel SP-2117 Acidic 6.1 4.88 <0.1 54.6 21 hydrogel SP-2119 Xerogel 9.1 0.10 5.2 11.7 19.1 TriSyl ® Acidic 4.5 <0.1 <0.1 60.0 20.0 silica hydrogel TriSyl ® Acidic 2.5 <0.1 <0.1 60.0 20.0 300 hydrogel Absorbent A Hydrogel 8.9 <0.1 11.7 5.2 — Absorbent B Hydrogel 9.8 <0.1 34.5 48.9 14.3 Absorbent C Xerogel 8.7 0.10 9.4 51.8 19.4 Absorbent D Xerogel 8.3 <0.1 14.7 56.7 151

Example 1

Expression of a Putative Chlorophyllases in Pseudomonas

[0214] Putative chlorophyllases (CHL) as provided in the tables of FIGS. 2 and 3 were expressed in the Pseudomonas system obtained from Dow Global Technologies Inc. (US20050130160, US20050186666 and US20060110747). The 12 synthetic genes based on the protein sequence of the putative chlorophyllases protein sequences as shown in FIG. 2-2A and 3 were designed by optimizing the gene codon usage for Pseudomonas according to the algorithm of DNA2.0 (GeneGPS® technology). For cloning purposes, the DNA sequence contain a SpeI site and ribosome binding site (ACTAGTAGGAGGTAACTAATG) (SEQ ID NO: 13) at the 5′-end and a stop codon and XhoI site (TGATGACTCGAG) (SEQ ID NO: 14) at the 3′-end.

[0215] SEQ ID NO: 2 shows the codon optimized nucleic acid sequence encoding the putative chlorophyllase SEQ ID NO:1 of Hordeum vulgare.

[0216] The DNA sequences were inserted in the pDOW1169 vector (Dow Global Technologies Inc., US20080058262) using SpeI and XhoI restriction enzyme cloning. The pDOW1169 vectors containing the genes encoding the CHL and PPH enzymes under control of a modified tac promotor were then introduced into Pseudomonas fluorescens uracil auxotrophic strain DPfl0. The transformed cells were selected after incubating on M9 minimal medium at 30° C. for 48 hours (Dow Global Technologies Inc., US20050186666) without uracil (Schneider et al. 2005).

[0217] Correct transformants were pre-cultured in 24 well pre-sterile deep well plates (Axygen, Calif., USA) containing 3 ml M9 medium. Plates were covered by a Breathseal (Greiner bio-one, Frickenhausen, Germany) and incubated at 30° C., 550 rpm and 80% humidity for 16 hours in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland). From these cultures 30 μl was used to inoculate a second 24 well pre-sterile deep well plates (Axygen, Calif., USA) containing 3 ml M9 medium at 30° C., 550 rpm for 24 hours. After 8 hours, the cultures were induced with IPTG (0.3 mM final concentration). Cultures were harvested by centrifugation for 10 minutes at 2750 rpm and the supernatants removed. The cell pellets were stored overnight at −20° C. The cell pellets from the 3 ml cultures were suspended in 1 ml lysis buffer and incubated for one hour at 37° C. Lysis buffer (1 mM EDTA, 50 mM Tris, pH 8, 0.25 mg/ml lysozyme, 10 mg/ml DnaseI, 25 μM MgSO.sub.4 and 0.03% triton). The lysates were centrifuged at 2750 rpm for 10 minutes and the supernatants were removed and stored.

Example 2

Determination of Pyropheophytinase Activity in Cell-Free Extracts in Crude Canola Oil

[0218] Incubation

[0219] Crude canola oil from North American origin, high in pheophytins and pyropheophytins was used to determine activity of the enzyme in the supernatant as produced in Example 1 on pyropheophytin A and B in the following way. Buffer (5% (v/v)) was added to oil under high-shear mixing using a Silverson mixer. For pH 5, a 20 mM citric acid buffer was used. For pH 7 a 20 mM phosphate buffer was used. A 24 wells microtiter plate was filled with 1.425 mL buffer-in-oil dispersion per well, and to each well 75 μL, 5% (v/v) cell-free extract (supernatant) produced in Example 1 was added. A list of tested samples is given in the tables of FIG. 2-2A and FIG. 3, and include a positive reference containing Chlamydomonas reinhardtii pyropheophytinase and negative control Green Fluorescent Protein (GFP). The microtiter plate was covered with plastic foil [Fasson S695]. Each well was stirred with an individual magnetic stirring bar. Incubations were performed at 50° C. using a KBMD microtiter-plate stirrer. Samples were taken after 24 hours and analysed for the presence of pheophytins A and B, and pyropheophytins A and B, and their phorbides using HPLC-FLU as described above.

[0220] The results in FIGS. 2 and 3 show that only CHL26, a putative chlorophyllase from Hordeum vulgare, was able to hydrolyse all pheophytins and pyropheophytins into their respective (pyro)pheophorbides at pH 7 and pH 5.

Example 3

Incubation of Crude Canola Oil with CHL26 Versus Time

[0221] Incubation of crude canola oil with 5% cell free extract of Hordeum vulgare putative chlorophyllase CHL26 produced as described in Example 1, was repeated in the same way as described in Example 2 at pH7. Samples were taken after 30 min, 2 hr, 5 hr, and 24 hr. Pyropheophytin a and b, and pheophytin a and b, pyropheophorbide a and b and pheophorbide a and b were measured by HPLC as described above.

[0222] The formation of the reaction products pyropheophorbide a and b and pheophorbide a and b in Table 1 is expressed as percentage of the amount reaction product (respective phorbide molecule) after 24 hr.

[0223] Table 2 shows the relative amounts of pheophytins and pyropheophytins as a function of time after 0.5, 2 and 5 hr, expressed in percentages relatively to the value at t=0 (average of 4 measurements).

TABLE-US-00002 TABLE 1 Relative HPLC results for all reaction products after incubation for 0.5, 2, 5 and 24 hours at pH 7 and 50° C., in percentages relative to value after 24 hrs. Pheophor- Pyropheophorbide Pheophorbide Pyropheophorbide time bide B B A A [hr] (%) (%) (%) (%) 0.5 53.7 48.8 69.4 56.7 2 85.3 95.5 96.4 90.0 5 92.9 92.9 98.7 94.3 24 100.0 100.0 100.0 100.0

TABLE-US-00003 TABLE 2 Relative HPLC results for all phytin compounds after incubation for 0.5, 2, and 5 hours at pH 7 and 50° C., in percentages relative to value at t = 0. Pheophytin Pyropheophytin Pheophytin Pyropheophytin Sum Time B B A A phytins [hr] (%) (%) (%) (%) (%) 0 100.0 100.0 100.0 100.0 100.0 0.5 34.7 43.8 34.4 46.4 41.4 2 0.0 12.7 0.0 12.9 8.2 5 0.0 6.2 0.0 6.0 3.9

[0224] The results in Table 1 and 2 show that enzyme CHL26 from Hordeum vulgare is able to hydrolyse both pheophytin and pyropheophytin, and both the a and b compounds. After 2 hrs all pheophytins were converted (below detection limit), whereas after 5 hours almost all the pyropheophytins were converted.

Example 4

Production of CHL26 and ELDC94 by 10 L Bioreactor Fermentation

[0225] Strains and Inoculum

[0226] Of a P. fluorescens strain containing CHL26 (SEQ ID NO: 1) and Chlamydomonas reinhardtii (ELDC94; SEQ ID NO: 12) chlorophyllase as described in Example 1 a pre-culture was prepared in one-phase shake flasks with complex medium comprising yeast extract, slats and glycerol as a C-source, which was used as inoculum for the 10 L fermentations with inoculation ratio of 10% described below.

[0227] 10 L Fermentations

[0228] Fermentation process was based on industrial Pseudomonas fluorescens fermentations (fed-batch process, sugar limited, IPTG induced). The fermentation process consisted of biomass production under exponential feed of glucose as C-source followed by production phase under IPTG induction system. After 23 hr fermentation (end of biomass production phase), IPTG was added to a final concentration of 0.125 mM in order to induce enzyme production. The feed rate of C-source (glucose) was reduced to ˜70% of maximum and fermentation prolonged till 48-55 hours after inoculation.

[0229] At the end of fermentation, the broth was killed off and the enzyme release via benzoate treatment followed by pH increase of the fermentation broth.

[0230] Recovery

[0231] The intra-cellular enzyme was released by homogenization. Two passes at 750 bars, with a cooling period of 12-hours in-between was applied. Subsequently the homogenized broth was diluted with 30% water, 15% DBF (Dicalite BF), Calcium Chloride (20 g/kg original broth), and Flocculent C577 (0.1% on original broth) were added. The pH was adjusted to 8, and the material was clarified and ultra-filtrated. The UF was stabilized with 50% glycerol, and to ensure full killing of remaining bacteria MEP (methyl/ethyl paraben in a solution with propene-diol) was added, diluting the product with about 15% v/v.

[0232] Activity

[0233] Activity on p-NP Substrates

[0234] The enzyme activity was determined using the chromogenic substrate 4-nitrophenyl butyrate (Sigma N9874). Substrate stock solution: 50 mM pNP-butyrate in acetonitrile. Substrate solution: Prior to use the substrate stock solution was mixed in ratio 1:4 with 0.1 M phosphate buffer pH 7.0 also containing 0.2% BSA and 2.0% Triton X-100.

[0235] In micro titer plates, 120 μL phosphate buffer (same as above) was mixed with 15 μL substrate solution and equilibrated at 37° C. After starting the reaction by adding 15 μL sample, the OD at 405 nm was measured for 5 minutes. Also, a blank measurement was done by adding 15 μL buffer instead of sample. The slope of the linear part of the curve is used as measure for the activity. Samples were diluted such to assure that the absorbance increase after 5 minutes is less than 1.0.

[0236] Activity is calculated as follows:

U/mL=(ΔAbs/min sample−ΔAbs/min blank)/(ε.sub.pNP×5)×1000×150/15×Df/W

[0237] ε.sub.pNP=Molar Extinction Coefficient of para-nitro-phenol [L.Math.mol-1.Math.cm-1]

[0238] 5=Incubation time [min]

[0239] 1000=factor from mmol to μmol

[0240] 150=assay volume [μL]

[0241] 15=sample volume [μL]

[0242] Df=Dilution factor

[0243] W=weight of sample (g)

[0244] The activity is expressed as the amount of enzyme that liberates 1 micromol p-nitrophenol per minute under the conditions of the test. Calibration is done using a 4-nitrophenol standard solution (Sigma N7660) diluted in the above-mentioned phosphate buffer.

[0245] The activity of the final formulations of CHL26 was 1.4 U/g (0.5 w/w %), and of ELDC94 87 U/g (0.04 w/w %).

Example 5

Incubation of Crude Canola Oil with an Enzyme Having Pyropheophytinase Activity Derived from Hordeum vulgare (CHL26) Compared to Incubation of Crude Canola Oil with a Reference Enzyme (ELDC94) from Chlamydomonas reinhardtii at Various Conditions

[0246] Crude canola oil was incubated with 0.5 w/w % cell free extract of Hordeum vulgare putative chlorophyllase CHL26 and compared to 0.04 w/w % of cell-free extract of Chlamydomonas reinhardtii chlorophyllase (coded ELDC94=Ref) both enzymes produced as described in Example 4. The incubation was performed on 10 g scale (10 g oil in 15 ml glass reaction vessels incubated on a hot plate aluminium reaction block with temperature control. Contents are kept vigorously stirred by magnetic bars), and now at three different temperatures (40, 50 and 60° C.), and under four regimes with varying acidity of the aqueous phase:

[0247] Acidic: 400 ppm citric acid pre-treatment;

[0248] Mildly acidic: pre-treatment with 500 ppm citric acid and 138 ppm caustic (NaOH);

[0249] Neutral: only water;

[0250] Mildly alkaline: pre-treatment with 150 ppm NaOH.

[0251] The total water level during incubation is 3% w/w, which includes enzyme formulation and pre-treatment solutions. Prior to the experiment, the acidity of the aqueous environment was assessed by diluting the pre-treated oil 1:1 with water and then the pH was measured by a pH meter. This resulted in the following pH values, indicative for the acidity of the aqueous environment in the dispersion during reaction: Acidic: pH 3.4; mildly acidic: pH 4.5; Neutral: pH 5.9 and alkalic pH 7.9.

[0252] For pre-treatment with citric acid, the citric acid (as 50% w/w solution) was added to the oil at 70° C., kept stirred at 70° C. for 30 minutes, subsequently the temperature was reduced to incubation temperature and for the mildly acid condition the NaOH (as 2.0% w/w solution) was added. In case of only NaOH addition, the oil was stirred at incubation temperature for 30 minutes.

[0253] During incubation, samples were taken after 0.5, 2, 4 and 24 hours, and analysed by HPLC-Flu as described in Example 2, now against a set of standards with known concentration. Concentrations of all substrates (chlorophyll, pheophytins, pyropheophytin—a and b) and all reaction products (chlorophyllide, pheophorbide, pyropheophorbide—a and b) were summed into total substrates and total reaction products, respectively, in mg/kg oil. All results are given in percentage of substrates and reaction products in the table below.

[0254] The results in Tables 3, 4 and 5 show that the Hordeum vulgare enzyme CHL26 according to SEQ ID NO: 1 has a wider application range in the presence of acid and caustic and is active at a higher temperature than the reference chlorophyllase from Chlamydomonas reinhardtii.

TABLE-US-00004 TABLE 3 Chlorophyll derivatives (wt %) in crude canola oil after incubation with the CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at 40° C. CHL26 Reference Sum Sum 40° C. Time Sum reaction Sum reaction Condition [hr] substrates products substrates products — 0 94.9 5.1 94.9 5.1 Acidic 0.5 66.5 33.5 80.6 19.4 2 56.4 43.6 78.1 21.9 4 51.9 48.1 75.2 24.8 24 45.4 54.6 75.2 24.8 Mildly acidic 0.5 29.1 70.9 35.2 64.8 2 10.0 90.0 28.7 71.3 4 3.4 96.6 19.0 81.0 24 0.0 100.0 15.4 84.6 Neutral 0.5 30.9 69.1 3.2 96.8 2 10.0 90.0 1.7 98.3 4 4.4 95.6 1.8 98.2 24 3.0 97.0 1.7 98.3 Mildly alkaline 0.5 71.8 28.2 60.7 39.3 2 72.5 27.5 55.2 44.8 4 57.4 42.6 38.2 61.8 24 14.8 85.2 32.8 67.2

TABLE-US-00005 TABLE 4 Chlorophyll derivatives (wt %) in crude canola oil after incubation with the CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at 50° C. CHL26 Reference Sum Sum 50° C. Time Sum reaction Sum reaction Condition [hr] substrates products substrates products — 0 94.9 5.1 94.9 5.1 Acidic 0.5 87.7 12.3 89.9 10.1 2 87.7 12.3 92.6 7.4 4 88.5 11.5 93.4 6.6 24 88.0 12.0 92.6 7.4 Mildly acidic 0.5 27.5 72.5 21.3 78.7 2 9.7 90.3 11.7 88.3 4 2.7 97.3 8.7 91.3 24 2.2 97.8 2.1 97.9 Neutral 0.5 28.5 71.5 5.8 94.2 2 13.7 86.3 3.9 96.1 4 5.5 94.5 2.0 98.0 24 0.6 99.4 0.6 99.4 Mildly alkaline 0.5 66.8 33.2 54.6 45.4 2 66.4 33.6 62.1 37.9 4 51.1 48.9 51.8 48.2 24 10.9 89.1 41.0 59.0

TABLE-US-00006 TABLE 5 Chlorophyll derivatives (wt %) in crude canola oil after incubation with the CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at 60° C. CHL26 Reference Sum Sum 60° C. Time Sum reaction Sum reaction Condition [hr] substrates products substrates products — 0 94.9 5.1 94.9 5.1 Acidic 0.5 90.5 9.5 94.7 5.3 2 90.5 9.5 95.0 5.0 4 90.7 9.3 92.7 7.3 24 90.5 9.5 94.9 5.1 Mildly acidic 0.5 13.5 86.5 60.4 39.6 2 2.5 97.5 65.7 34.3 4 4.1 95.9 66.4 33.6 24 2.0 98.0 68.5 31.5 Neutral 0.5 29.0 71.0 11.9 88.1 2 10.1 89.9 7.4 92.6 4 5.2 94.8 4.6 95.4 24 0.0 100.0 1.5 98.5 Mildly alkaline 0.5 57.7 42.3 52.5 47.5 2 65.1 34.9 80.4 19.6 4 67.4 32.6 80.4 19.6 24 33.0 67.0 93.0 7.0

Example 6

Incubation of Crude Canola Oil with an Enzyme from Chlamydomonas reinhardtii (ELDC94), Followed by Treatment with Various Silicas

[0255] 1,500 grams of crude canola oil was placed into a 2 liter jacketed glass beaker with an overhead mixer with a square paddle and mixed at 90 revolutions per minute (rpm). The jacket temperature was set at 65° C. 20 mL of ELDC94 Chlamydomonas (alga) (prepared as described in Example 4) and 100 grams of deionized water were added to the oil once the oil temperature had reached the set point. The material was shear mixed for 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered with plastic wrap. The material was mixed covered for 24 hours at 250 rpm.

[0256] 1.5 grams of 50% (wt. %) citric acid was added to the mixing oil. The set point of the jacket was reduced to 55° C. Once the material reached 55° C., the oil was moved to the shear mixer. 1.2 mL of 4 N NaOH was added to the oil and shear mixed 30 seconds. 0.3 grams of Purifine® Phospholipase C (PLC) and 30 grams of deionized water were added. The oil was shear mixed for 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 2 hours at 55° C. at 250 rpm.

[0257] The beaker was moved back to the high shear mixer and 0.1 grams of Phospholipase A1 (PLA1) enzyme (Lecitase Ultra) was added to the oil and shear mixed 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 2 hours at 55° C. at 250 rpm. Increased the set point of the water bath to 75° C. Once the oil reached 75° C., the oil was centrifuged utilizing Gyro-Centrifuge with the bowl with holes closed. Samples of the oil were collected. The gums were discarded.

[0258] The above reaction was repeated 11 times and the oil was combined and labelled as “control”.

[0259] Six 500 gram samples of the above enzyme treated canola oil “control” were added to six 1000 mL round bottom flask. The oils were heated to 80° C. and 0.25, 1.0, 2.0, 4.0, 6.0, and 8.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. It was unexpected that the oil turned dark green during the adsorptive process with the test silica. In previous experiments using industrial silicas (TRISYL® (Grace Davison, Columbia, Md.), or, SORBSIL® silicas (INEOS Silicas, Joliet, Ill.) the color of the oil did not change. The vacuum was broken and the material filtered with a Buchner Funnel. The filter paper disc was a dark green color, but when using industrial silicas, the filter disc and cakes were always yellow. The filter disc and cake were a dark green color.

[0260] Two 500 gram samples of the above enzyme treated canola oil “control” from above were split and added to two 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 1.0 and 2.0 grams of silica SP-2116 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The color of the oil during the trial did not change from the original color. The vacuum was broken and the material filtered with a Buchner Funnel. The filter disc and cake were a yellow color.

[0261] Two 500 gram samples of the above enzyme treated canola oil “control” from above were split and added to two 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 1.0 and 2.0 grams of silica SP-2117 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The color of the oil during the trial did not change from the original color. The vacuum was broken and the material filtered with a Buchner Funnel. The filter disc and cake were a yellow color.

[0262] The content of the oils is set forth in Tables 6 and 7.

TABLE-US-00007 TABLE 6 P, Ca, Mg, and Fe content in ELDC94 treated oils following further treatment with various silicas Phosphorus Calcium Magnesium Iron (ppm) (ppm) (ppm) (ppm) Crude Canola 836 161 119 1.6 Enzyme treated 14.2 11.42 1.99 0.42 Control (ELDC94) SP-2115-0.25 g 9.34 6.19 1.19 0.19 SP-2115-1 g 9.34 7.32 1.43 0.25 SP-2115-2 g 4.52 3.86 1.17 0.13 SP-2115-4 g 3.24 3.26 0.90 0.14 SP-2115-6 g 5.24 4.37 1.53 0.16 SP-2115-8 g 1.96 2.12 0.88 0.05 SP-2116-0.25 g 9.99 6.81 1.25 0.21 SP-2116-1 g 9.43 7.01 1.42 0.22 SP-2117-0.25 g 10.06 7.32 1.31 0.23 SP-2117-1 g 9.55 7.81 1.57 0.16

TABLE-US-00008 TABLE 7 Content of chlorophyll derivatives in ELDC94 treated oils following further treatment with various silicas Chlorophyll and Chlorophyll Derivatives “A” “B” CHYL PYN PPYN POB PPOB CHYL PYN Total UV/Vis (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) Crude Canola 0.31 5.48 11.72 0.06 0.94 0.56 0.79 19.86 53.60 Control b.d. 0.49 2.79 1.49 2.95 b.d. 0.34 8.06 38.49 SP-2115 - 0.25 g b.d. 0.47 2.73 1.3  2.95 b.d. 0.46 7.91 36.29 SP-2115 - 1 g b.d. 0.31 1.65 0.98 1.62 b.d. b.d. 4.56 25.65 SP-2115 - 2 g b.d. 0.2  1.43 b.d. 0.89 b.d. b.d. 2.52 20.36 SP-2115 - 4 g b.d. b.d. 0.83 b.d. 0.61 b.d. b.d. 1.44 14.32 SP-2115 - 6 g b.d. 0.39 0.68 0.32 b.d. b.d. b.d. 1.39 11.04 SP-2115 - 8 g b.d. 0.34 0.58 b.d. b.d. b.d. b.d. 0.92 8.93 SP-2116 - 0.25 g b.d. 0.41 2.78 1.59 3.39 b.d. 0.44 8.61 39.03 SP-2116 - 1 g b.d. 0.52 2.46 1.42 2.99 b.d. 0.39 7.78 36.51 SP-2117 - 0.25 g b.d. 0.38 2.53 1.4  3.04 b.d. 0.34 7.69 38.66 SP-2117 - 1 g b.d. 0.53 2.48 1.43 3.01 b.d. 0.39 7.84 36.66 Control deodorized b.d. 0.11 1.02 1.58 n.d. b.d. b.d. 2.71 19.31 SP-2115 - 6 g b.d. b.d. b.d. b.d. b.d. b.d. b.d. b.d. 3.84 deodorized CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide b.d. = below detection n.d. = not determined

[0263] ELDC94 Chlamydomonas (alga) enzyme decreases the amount of chlorophyll and chlorophyll derivates from 19.86 ppm to 8.06 ppm (53.6 to 38.49 ppm via the AOCS UV/VIS method) after 24 hours. However, it is not great enough to significantly enable the process in an industrial process. An enzyme with a greater ability to hydrolyze chlorophyll and chlorophyll derivatives is required as well as a process for greater removal of those generated derivatives.

[0264] The above data demonstrates that silica SP-2115 has the greatest capacity to remove metals, chlorophyll, and chlorophyll derivatives compared to the other two silicas. The UV/Vis method demonstrates that treatment with the low doses (i.e., 0.25 g and 1 g) of SP-2115 reduces the chlorophyll by 2.20 and 12.84 ppm, respectively, as compared to the amount of chlorophyll in the control (i.e., 38.49 ppm). In comparison, the amount of chlorophyll following treatment with the lowest dosages (i.e., 0.25 g) of SP-2116 and SP-2117 increased by 0.54 ppm and 0.17 ppm, respectively, while the amount of chlorophyll following treatment with the highest dosages (i.e., 1 g) of SP-2116 and SP 2117 decreased 1.98 ppm and 1.83 ppm, respectively, as compared to control. The HPLC test method demonstrates the same pattern of limited reduction at the highest dosage for SP-2116 and SP-2117. The data also demonstrates that the HPLC method for chlorophyll and chlorophyll derivatives needs to be improved by finding additional standards and response factors in order to bring it closer in line with the AOCS method for measuring the green color in vegetable oils. Additional work on the method has been completed and is encompassed in the following examples.

Example 7

Incubation of Solvent Extracted Crude Canola Oil with an Enzyme Having Pyropheophytinase Activity Derived from Hordeum vulgare (CHL26) Compared to a Reference Enzyme from Chlamydomonas reinhardtii (ELDC94)

[0265] A 35-pound container of solvent extracted crude canola oil was poured into large stainless-steel container and made uniform with IKA mixer.

[0266] After mixing, approximately 1.5 kg of crude canola was placed into a 2 liter jacketed glass beaker with an overhead mixer with a square paddle and mixed at 90 revolutions per minute (rpm). The jacket temperature was set at 65° C. 0.7 grams of enzyme ELDC94 (reaction 1) or 7.5 grams of CHL26 (reaction 2), produced as described in Example 4, were added to the oil together with 100 grams of deionized water once the oil temperature had reached the set point. The material was shear mixed for 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered with plastic wrap. The materials were incubated with the enzymes for 24 hours at 250 rpm.

[0267] 1.5 grams of 50% (wt. %) citric acid was added to the mixing oil. The set point of the jacket was reduced to 55° C. Once the material reached 55° C., the oil was moved to the shear mixer. 1.2 mL of 4 N NaOH was added to the oil and shear mixed 30 seconds. 0.3 grams of Purifine® Phospholipase C (PLC) and 30 grams of deionized water were added. The oil was shear mixed for 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 2 hours at 55° C. at 250 rpm.

[0268] The beaker was moved back to the high shear mixer and 0.1 grams of a fungal phospholipase A.sub.1 (PLA.sub.1) enzyme was added to the oil and shear mixed 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 2 hours at 55° C. at 250 rpm. Increased the set point of the water bath to 75° C. Once the oil reached 75° C., the oil was centrifuged utilizing a Gyro-Centrifuge with the bowl with holes closed. Samples of the oil and gums were collected and analysed for the presence of P, Ca, Mg and Fe and chlorophyll derivatives (using HPLC) as described above.

[0269] The mixture of oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The oil from the decanted bowl and in the tubes was discarded and liquid heavy phases were combined.

[0270] The results in Table 8 and FIG. 4 a) show that the CHL26 enzyme having pyropheophytinase activity according SEQ ID NO: 1 is able to reduce chlorophyll derivatives in solvent extracted crude canola oil. Chlorophyll substrates are chlorophyll, pheophytin, and pyropheophytin and chlorophyll products are chlorophyllide, pheophorbide and pyropheophorbide.

TABLE-US-00009 TABLE 8 Compounds (in ppm) in crude canola oil after treatment with enzymes CHL26 and the reference enzyme ELDC94 Chlorophyll derivatives (HPLC) (ppm) Enzyme P Ca Mg Fe Total Substrates Products None* 903.0 243.0 127 9.89 15.40 14.72 0.50 ELDC94 88.5 80.9 14.6 1.49 8.39 0.21 8.18 CHL26 82.0 77.3 14.1 1.58 9.15 1.26 7.89 *Starting material (crude canola oil)

[0271] The results in FIG. 4 b) show that there are still unreacted phospholipids present in the collected heavy phase, which is an indication that the phospholipase reactions were too short to come to completion.

Example 8

Incubation of Pressed Crude Canola Oil with the CHL26 Enzyme at Varying Conditions, and Treatment with Silica SP-2115

[0272] A 35-pound container of pressed crude canola oil was poured into large stainless-steel container and made uniform with IKA mixer

Reaction 3—CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 2 hr Incubation with PLA1

[0273] About 1.5 kg of crude canola was placed into a 2 liter jacket glass beaker with an overhead mixer with a square paddle. The oil was mixed at 90 rpm. The jacket temperature was set at 70° C. 1.5 grams of 50% (wt. %) citric acid was added to the mixing oil and shear mixed 1 minute. The set point of the jacket was reduced to 60° C. Once the material reached 60° C., the oil was moved to the shear mixer. 1.2 mL of 4 N NaOH was added to the oil and shear mixed 30 seconds. 0.3 grams of Purifine PLC (LR79.14 February 2018), 0.02 grams of Purifine PI-PLC, 7.5 grams of CHL26 enzyme [Hordeum vulgare var. distichum (barley, plant)], produced as described in Example 4, and 100 grams of deionized water. The material was shear mixed for 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 2 hours at 60° C. at 250 rpm.

[0274] The jacketed glass beaker was again moved to the hear mixer where 0.075 grams of PLA, (notebook, 074362) was added and the oil was shear mixed 1 minute. The jacketed glass beaker was moved back to the overhead and covered with plastic wrap. The oil was mixed and the reactions were allowed to continue for 2 hours at 250 rpm. Increased the set point of the water bath to 75° C. Once the oil reached 75° C., the oil was centrifuged utilizing Gyro-Centrifuge with the bowl with holes closed. Samples of the oil and gums were collected.

[0275] The mixture of oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The oil from the decanted bowl and in the tubes was discarded and liquid heavy phases were combined.

Reaction 4—ELDC94 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 2 hr Incubation with PLA.sub.1

[0276] The same procedure from reaction 1 above was employed for enzyme ELDC94, using 0.61 grams of the formulated enzyme solution (produced as described in Example 4).

[0277] Two 450 gram samples of the Reaction 4 enzyme treated canola oil were split and added to two 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 1.0 and 2.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The oil turned dark green during the adsorptive process with the test silica. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green color.

Reaction 5—CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 4 hr Incubation with PLA.sub.1

[0278] The same procedure was followed as reaction 1, but the PLA1 reaction was allowed to react for 4 hours instead of only 2 hours.

[0279] Two 450 grams samples of the Reaction 5 enzyme treated canola oil were split and added to two 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 2.0 and 3.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green color

Reaction 6—CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 4 hr Incubation with PLA.sub.1

[0280] The same procedure was followed as reaction 3, except twice the amount of CHL26 (15 grams total) was added to the reaction.

[0281] Two 450 grams samples of the Reaction 6 enzyme treated canola oil were split and added to two 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 1.0 and 2.0 g of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green color.

Reaction 7—CHL26 Incubation with PLC and PI-PLC at Neutral pH for 2 hr, Followed by a 4 hr Incubation with PLA.sub.1

[0282] The same procedure was followed as reaction 1, except no pH adjustment was made.

Reaction 8—SBO CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 2 hr Incubation with PLA.sub.1

[0283] The same procedure as reaction 1 was followed, but the oil was a solvent extracted crude soybean oil (SBO).

[0284] Three 450 g samples of the Reaction 8 enzyme treated soybean oil were split and added to three 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 0.25, 0.5 and 1.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green color.

[0285] In Table 9 and FIG. 5b), the phosphorus (P), calcium (Ca), magnesium (Mg), and iron (Fe) contents of the oils and the respective gums before and after enzyme treatments according to reactions 1 to 6 are shown. At neutral pH, a higher amount of P remained in the oil as compared to reaction at pH 4.5. Table 9 also shows P, Ca, Mg, and Fe contents of the oils following treatment with silica SP-2115. This data demonstrates the silica treatment removes trace phosphorus and metals to levels sufficient to meet industrial standards for bleached oils without the use of bleaching earth. They did not lose their capacity to adsorb these impurities when the MgO was added.

[0286] The results in Table 10 and FIG. 5a) show that the CHL26 enzyme converts a higher amount of chlorophyll derivatives in crude canola oil as compared to ELDC94, when the enzymes are incubated under the same conditions (reactions 1 and 2). Table 10 also shows that contacting the enzyme treated oils with silica SP-2115 reduces the amount of both chlorophyll substrates and chlorophyll products in the oils, as compared the amount of chlorophyll substrates and products in enzyme treated oils that are not further contacted with the silica.

[0287] In the present example the CHL26 enzyme converted a higher amount of chlorophyll substrates into the respective chlorophyll products in crude canola oil under neutral conditions as compared to acid conditions (pH 4.5) (compare reaction 7 with reactions 3, 5 and 6).

[0288] The CHL26 enzymes also converts chlorophyll substrates in soybean oil into the respective chlorophyll products (reaction 8).

[0289] The results in Table 10 also show that a higher amount of chlorophyll products were found in the gums (heavy phase) when the oil was reacted with the CHL26 enzyme as compared to the reaction with the ELDC94 enzyme.

TABLE-US-00010 TABLE 9 Compounds in canola oil (Can) or soybean oil (SBO) after treatment with the CHL26 enzyme compared to reference enzyme ELDC94 and/or no enzyme treatment and/or after silica treatment Silica SP- 2115 P Ca Mg Fe Reaction Oil pH (grams) (ppm) None, Crude Can — — 210 90.5 36.7 0.90 Rxn 3 - CHL26 Can 4.5 — 10.7 7.9 1.7 0.20 Rxn 4 - ELDC94 Can 4.5 — 4.4 3.3 0.9 0.07 Rxn 4 1 1.5 2.3 0.3 0.05 Rxn 4 2 1.4 2.3 0.3 0.05 None, Crude Can — — 210 90.5 36.7 0.90 Rxn 5 - CHL26 Can 4.5 — 3.9 2.8 0.6 0.16 Rxn 5 2 1.6 1.8 0.5 0.10 Rxn 5 3 b.d. 0.3 Tr 0.02 Rxn 6 - CHL26 Can 4.5 — 2.0 1.5 0.4 0.10 Rxn 6 1 0.5 0.9 0.2 0.07 Rxn 6 2 0.6 0.8 0.2 0.06 Rxn 7 - CHL26 Can Neu- — 103 80.9 10.5 0.88 tral None, Crude SBO — — 773 66.2 64.3 0.76 Rxn 8 - CHL26 SBO 4.5 — 5.8 0.5 0.7 0.04 Rxn 8   0.25 0.6 0.2 0.1 0.03 Rxn 8   0.5 0.7 0.2 0.2 0.03 Rxn 8 1 b.d. 0.1 0.1 0.02 b.d.—below detection Tr—trace

TABLE-US-00011 TABLE 10 Chlorophyll derivatives in canola oil or soybean oil and the separated gums after treatment with the CHL26 enzyme compared to reference enzyme ELDC94 and/or no enzyme treatment and/or after silica treatment Chlorophyll Chlorophyll derivatives derivatives in the oil in the gums (ppm) (ppm) Oil Substrates Products Substrates Products Crude Canola 13.13 0.90 — — Rxn 3-CHL26, pH 4.5 4.19 7.97 0.06 6.39 Rxn 4-ELDC94, pH 4.5 10.28 2.62 0.18 3.06 Rxn 4-SP-2115, 1 g 3.46 b.d. — — Rxn 4-SP-2115, 2 g 6.27 0.22 — — Crude Canola 13.13 0.90 — — Rxn 5, CHL26, pH 4.5 6.85 5.69 0.30 2.68 Rxn 5-SP-2115, 2 g 4.38 0.57 — — Rxn 5-SP-2115, 3 g 0.78 b.d. — — Rxn 6-CHL 26, pH 4.5 6.01 6.41 0.19 1.99 Rxn 6-SP-2115, 1 g 3.88 1.22 — — Rxn 6-SP-2115, 2 g 2.77 0.53 — — Rxn 7-CHL26, neutral pH 1.26 10.02 b.d. 5.02 Crude SBO 0.31 b.d. — — Rxn 8-CHL26, pH 4.5 0.28 b.d. b.d. 0.56 Rxn 8-SP-2115, 0.25 g 0.24 b.d. — — Rxn 8-SP-2115, 0.5 g 0.14 b.d. — — Rxn 8-SP-2115, 1 g 0.87 b.d. — — b.d.—below detection,

Example 9

Use of CHL26 Enzyme and Silica Treatment in Caustic Refining Application of Canola Oil and Soybean Oil

[0290] The following experiments are an evaluation of the CHL26 in a caustic refining application where the oil has been treated with a phosphoric acid and sodium hydroxide, as occurs in industrial processes of canola and soybean oils. A “once refined” product is an oil that was treated with phosphoric acid, then treated with sodium hydroxide to convert the Free Fatty Acids (FFA) into sodium soaps that are water soluble and removed in water or “heavy” phase of the “refining” centrifuge. The oil was then washed with water (2 to 10 percent w/w) to remove the remaining soaps and residual phospholipids present in the oil. Optionally, the enzymes were evaluated after the refining centrifuge in the water washing step, but at a much lower temperature.

[0291] A five-gallon plastic pail of Once Refined Canola (ORCAN) oil was mixed with a high shear mixer to make uniform. 2-3 kg samples were pulled for use in the experiments below.

Reaction 9—ELDC94-Comparative

[0292] 2 kg of once refined canola was placed into a 4 liter glass beaker on a hot plate with overhead mixing at 90 rpm. The oil was heated to 60° C. under agitation. Once the material reached 60° C., the beaker was moved to the shear mixer. 0.8 grams of enzyme ELDC94 (produced as described in Example 4) and 100 grams of deionized water were added to the oil. The material was shear mixed for 1 minute while covered with plastic wrap to minimize water loss. The glass beaker was moved back to the overhead mixer and covered with plastic wrap. The oil was mixed for 4 hours at 60° C. at 250 rpm. The temperature was increased to 75° C. The oil was centrifuged utilizing Gyro-Centrifuge. The separated oil was collected.

[0293] The mixture of oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The oil from the decanted bowl and in the tubes was discarded and liquid heavy phases were combined. The heavy phase was a dark green.

Reaction 10—ELDC94-Comparative

[0294] Reaction 10 was a repeat of reaction 9, except 3 kg of oil was used and 2.0 grams of ELDC94 (produced as described in Example 4).

[0295] After the analyses of the oils from reaction 9 and 10, the oils were combined mixed and analysed again.

Reaction 11—CHL26

[0296] Reaction 11 was a repeat of reaction 9, except that 10.1 grams of CHL26 (produced as described in Example 4) was used instead of ELDC94. The heavy phase was a lighter green than reactions 9 and 10.

Reaction 12—CHL26

[0297] Reaction 12 was a repeat of reaction 10, except that 20 grams of CHL26 was utilized.

[0298] After analyses, the oils of reaction 11 and 12 were combined and mixed and after mixing analysed again.

Reaction 13—ELDC94-Comparative

[0299] 3 kg of once refined soybean oil (ORSBO) was pulled from a caustic refining production line number 1 after the water washing centrifuge. The oil was placed into a 4 liter glass beaker and placed onto a hot plate with overhead mixing with a square mixing paddle (90 rpm). Once the material cooled 60° C., the beaker was moved to a shear mixer. 1.0 grams of ELDC94 enzyme produced as described in Example 4) and 150 grams of deionized water were added to the oil. The material was shear mixed for 1 minute while covered with a plastic wrap to minimize moisture loss. The glass beaker was moved back to the overhead mixer and again covered with a plastic wrap. The oil was mixed for 4 hours at 60° C. at 250 rpm. The temperature was increased to 75° C. and then the oil was centrifuged utilizing Gyro-Centrifuge.

[0300] Collected oil and heavy samples for further analyses.

[0301] The remaining oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The remaining oil in the tubes was discarded and liquid heavy phases were combined. The heavy phase was colorless, no discernible color pigments.

Reaction 14—CHL26

[0302] Reaction 14 was a repeat of reaction 13, except that 15 grams of CHL26 (produced as described in Example 4) was utilized instead of ELDC94.

Reaction 15—EDLC94-Comparative

[0303] 3 kg grams of once refined soybean oil (ORSBO) was pulled from a caustic refining production line number 1 after the water washing centrifuge. The oil was placed into a 4 liter glass beaker and placed onto a hot plate with overhead mixing with a square mixing paddle (90 rpm). Once the material cooled 60° C., the beaker was moved to a shear mixer. 1.2 grams of ELDC94 enzyme produced as described in Example 4 and 150 grams of deionized water were added to the oil. The material was shear mixed for 1 minute while covered with a plastic wrap to minimize moisture loss. The glass beaker was moved back to the overhead mixer and again covered with a plastic wrap. The oil was mixed for 4 hours at 60° C. at 250 rpm. The temperature was increased to 75° C. and then the oil was centrifuged utilizing Gyro-Centrifuge.

[0304] Collected oil and heavy phase (gums) samples for further analyses.

[0305] The remaining oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The remaining oil in the tubes was discarded and liquid heavy phases were combined. The heavy phase was colorless, no discernible color pigments.

Reaction 16—CHL26

[0306] Reaction 16 was a repeat of reaction 15, except that 15 grams of CHL26 was utilized.

[0307] The results of reactions 9 to 16 and the results of the combined and mixed oils from reaction 9 and 10 and from reactions 11 and 12 are shown in Table 11 and FIGS. 6-6A.

[0308] The results in Table 9 and FIGS. 6-6A show that the enzyme CHL26, having pyropheophytinase converts a higher amount of chlorophyll substrates (chlorophyll, pheophytin and pyropheophytin) to its chlorophyll products (chlorophyllide, pheophorbide, pyropheophorbide) than the reference chlorophyllase enzyme ELDC94.

TABLE-US-00012 TABLE 11 Chlorophyll derivatives (substrates and products) in once refined canola oil (ORCAN) and once refined soybean oil (ORSBO) after caustic refining and after treatment with the CHL26 enzyme and the ELDC94 (reference) enzyme Chlorophyll derivatives in oil (ppm) Enzyme reaction Substrates Products None: ORCAN 27.38 b.d. Rxn 9-ELDC94 7.87 4.99 Rxn 10-ELDC94 19.37 0.42 Combined 9 & 10 18.71 0.39 Rxn 11-CHL26 11.60 4.96 Rxn 12-CHL26 n.m. n.m. Combined 11 & 12 12.00 6.19 None: ORSBO 3.85 b.d. Rxn 13-ELDC94 1.09 0.06 None: ORSBO 3.90 b.d. Rxn 14-CHL26 1.12 0.18 None: ORSBO 3.90 b.d. Rxn 15-ELDC94 2.05 b.d. Rxn 16-CHL26 1.77 0.07 b.d. = below detection n.m. = not measured

[0309] The results of Table 12 show the contents of free fatty acids (FFA), soap and phosphor and Ca, Mg, Fe, and/or chlorophyll in once refined canola oil and once refined soybean oil after enzymatic treatments described above.

TABLE-US-00013 TABLE 12 Composition of once refined canola oil (ORCAN), once refined soybean (ORSBO) oil after caustic refining and after treatment with the CHL26 enzyme and the ELDC94 (reference) enzyme Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC * Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow ORCAN 0.05 195 4.5 0.9 0.2 0.03 12047 27.38 t.d. t.d. Rxn 9 - 0.05 b.d. 0.5 0.7 tr 0.02 10461 12.86 t.d. t.d. ELDC94 Rxn 10 - 0.07 b.d. 0.6 1.8 tr 0.03  9377 19.70 t.d. t.d. ELDC94 Combined 0.06 b.d. 0.6 0.7 tr 0.07 — — — — 9 & 10 Rxn 11 - 0.06 tr 1.6 2.4 0.1 0.11 11079 16.56 t.d. t.d. CHL26 Rxn 12 - 0.06 b.d. 1.7 2.9 0.1 0.07 11536 n.m. t.d. t.d. CHL26 Combined 0.06 tr 1.7 2.7 0.1 0.09 — — — — 11 & 12 ORSBO 0.12  20 0.3 0.2 b.d. b.d. — — — — Rxn 13 - 0.10 b.d. 0.2 0.1 b.d. b.d. — — — — ELDC94 ORSBO 0.06  27 1.0 0.4 tr b.d. — — — — Rxn 14 - 0.05 b.d. 0.2 0.1 b.d. b.d. — — — — CHL26 ORSBO 0.03 242 2.8 0.7 0.2 tr — — — — Rxn 15 - 0.02 tr 0.3 0.2 b.d. 0.1  — — — — ELDC94 ORSBO 0.05 396 3.3 0.9 0.2 b.d. — — — — Rxn 16 - 0.03 tr b.d. 0.2 b.d. b.d. — — — — CHL26 tr = trace b.d. = below detection n.m. = not measured t.d. = too dark to measure * = HPLC was a measurement of total chlorophyll derivatives

Reactions 9 and 10—Silica Treatment

[0310] Oils from reactions 9 and 10 were combined and then split into three 500 gram samples of the enzyme treated ORCAN oil and added to three 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 1.0, 2.0 and 3.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The oil turned dark green during the adsorptive process with the test silica. The vacuum was broken and the material filter with a Buchner Funnel. The filter paper disc was a dark green color. The oils were labeled as 9101, 9102, and 9103 respectively. Oil labeled as 9100 was the sample of reaction 9 and reaction 10 combined.

[0311] The canola oil treated, 9102 (436 grams) and 9103 (448 grams) were combined and placed in a 3 L Claisen flask. The oil was sparged with nitrogen for approximately 2 minutes. The vacuum was initiated and the nitrogen sparge was discontinued and water vapor from the steam generator was allowed to begin the deodorization process. The vacuum achieved was between 0.82-0.98 mBar during the deodorization process. The oil was heated under vacuum and water sparge (3 wt. %) to 230° C. The sparge and temperature were maintained for two hours. The heat was discontinued and the oil was allowed to cool under vacuum and water sparge. The vacuum was broken with nitrogen at approximately 100° C. and allowed to further cool to 70° C. before opening to the air. The oil was a dark greenish/grey tint. The oil was labeled as 91023-DEO.

[0312] The results in Table 13 show the contents of free fatty acids (FFA), soap, P, Ca, Mg, Fe, and chlorophyll in the combined reaction 9 and 10 oils following silica treatment as described above.

TABLE-US-00014 TABLE 13 Composition of once refined canola oil (ORCAN), after enzyme treatment or after enzyme and silica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow 9100 0.06 b.d. 0.6 0.7 tr 0.07 n.m. 19.10 t.d. t.d. 9101 n.m. b.d. 0.4 0.3 b.d. tr 8273 19.52 8.0 70 9102 n.m. n.m. 0.3 0.2 b.d. b.d. 6848 17.13 7.6 70 9103 n.m. n.m. b.d. 0.1 b.d. b.d. 5514 14.04 7.1 70 91023-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 1833 3.20 n.m. n.m. tr = trace b.d. = below detection n.m. = not measured t.d. = too dark to measure *= HPLC was a measurement of total chlorophyll derivatives

[0313] The results in Table 14 show the chlorophyll substrates and products in the combined reaction 9 and 10 oils following silica treatment as described above.

TABLE-US-00015 TABLE 14 Chlorophyll substrate and product composition in oils after enzyme and silica treatment a a′ b CHYL PYN PPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm 9100 0.19 5.97 6.85 0.18 0.15 1.85 0.05 0.12 1.64 9101 0.10 4.78 5.85 0.03 0.02 3.31 b.d. 0.11 1.32 9102 0.10 4.50 4.14 b.d. b.d. 3.07 b.d. 0.11 1.34 9103 0.10 3.73 2.73 b.d. b.d. 2.65 b.d. 0.11 1.23 91023-Deo 0.05 0.48 1.43 b.d. b.d. b.d. b.d. 0.17 0.12 b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm 9100 1.58 b.d. b.d. b.d. 0.50 b.d. 19.10 18.71 0.39 9101 3.14 b.d. b.d. b.d. 0.85 b.d. 19.52 19.46 0.06 9102 2.98 b.d. b.d. b.d. 0.90 b.d. 17.13 17.13 b.d. 9103 2.65 b.d. b.d. b.d. 0.86 b.d. 14.04 14.04 b.d. 91023-Deo 0.96 b.d. b.d. b.d. b.d. b.d. 3.20 3.20 b.d. CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = below detection

[0314] The feed material from the combined samples from reactions using ELDC94 of 18.71 ppm as reported from the HPLC method shows a dramatic reduction in substrates following treatment with the SP-2115 to 14.04 ppm and a complete removal of the products from 0.39 ppm in the combined samples to below detection limit following treatment with SP-2115.

Reactions 11 and 12—Silica Treatment

[0315] Oils from reactions 11 and 12 were combined and then split into three 500 gram samples of the enzyme treated ORCAN oil and added to three 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 1.0, 2.0 and 3.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green. The oils were labeled as 11121, 11122, and 11123 respectively. Oil labeled as 11120 was the combined sample of reaction 11 and reaction 12 oil.

[0316] The canola oil treated, 11122 (449 grams) and 11123 (451 grams) were combined and placed in a 3 L Claisen flask and assembled according the deodorization procedure. The oil was sparged with nitrogen for approximately 2 minutes. The vacuum was initiated and the nitrogen sparge was discontinued and water vapor from the steam generator was allowed to begin the deodorization process. The vacuum achieved was between 0.28-0.56 mBar during the deodorization process. The oil was heated under vacuum and water sparge (3 wt. %) to 230° C. The sparge and temperature were maintained for two hours. The heat was discontinued and the oil was allowed to cool under vacuum and water sparge. The vacuum was broken with nitrogen at approximately 100° C. and allowed to further cool to 70° C. before opening to the air. The oil was a light greenish/grey tint. The oil was labeled as 111223-DEO.

[0317] The results in Table 15 show the contents of free fatty acids (FFA), soap, P, Ca, Mg, Fe, and chlorophyll in the combined reaction 11 and 12 oils following silica treatment as described above.

TABLE-US-00016 TABLE 15 Composition of once refined canola oil (ORCAN), after enzyme treatment or after enzyme and silica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow 11120 0.06 tr 1.7 2.7 0.1 0.09 n.m. 18.19 t.d. t.d. 11121 n.m. b.d. 1.2 1.8 tr 0.07 6180 12.05 8.1 70 11122 n.m. n.m. 0.8 1.1 b.d. 0.03 4267 9.38 8.9 70 11123 n.m. n.m. 0.2 0.3 b.d. 0.01 3576 4.12 7.1 70 111223-DEO 0.02 n.m. n.m. n.m. n.m. n.m. 1741 2.41 n.m. n.m. tr = trace b.d. = below detection n.m. = not measured t.d. = too dark to measure *= HPLC was a measurement of total chlorophyll derivatives

[0318] The results in Table 16 show the chlorophyll substrates and products in the combined reaction 11 and 12 oils following silica treatment as described above.

TABLE-US-00017 TABLE 16 Chlorophyll substrate and product composition in oils after enzyme treatment or after enzyme and silica treatment a a′ b CHYL PYN PPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm 11120 0.10 2.22 3.18 3.03 1.29 3.16 0.28 0.12 0.73 11121 0.10 2.40 2.98 0.43 0.21 2.49 0.08 0.12 0.67 11122 0.10 2.26 1.97 0.12 0.05 1.96 0.04 b.d. 0.63 11123 0.09 0.98 0.68 0.05 0.04 0.85 b.d. b.d. 0.31 111223-DEO 0.04 0.40 1.12 b.d. b.d. b.d. b.d. 0.16 b.d. b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm 11120 1.73 0.86 0.64 0.12 0.64 0.08 18.19 12.00 6.19 11121 1.72 0.13 0.10 b.d. 0.60 0.03 12.05 11.07 0.98 11122 1.57 0.04 0.06 b.d. 0.58 b.d. 9.38 9.06 0.31 11123 0.74 0.03 0.05 b.d. 0.29 b.d. 4.12 3.95 0.17 111223-DEO 0.69 b.d. b.d. b.d. b.d. b.d. 2.41 2.41 b.d. CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = below detection

[0319] The feed material from the combined samples from reactions using CHL26 of 18.19 ppm as reported from the HPLC method shows a dramatic reduction in substrates following treatment with the SP-2115 of from 12.00 to 3.95 ppm, and a reduction of the products from 6.19 ppm to 0.17 ppm. It is clear that SP-2115 has a capacity for both the substrates and products of the enzymatic reaction of CHL26 for their removal in an adsorptive process.

Reactions 5 and 13—Silica Treatment. Comparison of SP-2114 and SP-2115

[0320] Three 500 gram samples of the enzyme treated refined soybean oil from reaction 13 were split and added to three 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 0.5, 1.0 and 2.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green color. The oils were labeled as 135, 1310, and 1320 respectively. Oil labeled as 130 was the sample from reaction 13.

[0321] A 500 gram sample of the enzyme treated refined soybean oil from reaction 5 was added to a 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oil was heated to 80° C. and 2.0 grams of silica SP-2114 was mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The color change was greenish/brown in the oil was observed during the adsorption process. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were light green. The sample was labeled as 132-2114.

[0322] The results in Table 17 show the contents of free fatty acids (FFA), soap, P, Ca, Mg, Fe, and chlorophyll in the reaction 5 and reaction 13 oils following silica treatment as described above.

TABLE-US-00018 TABLE 17 Composition of oils after enzyme treatment or after enzyme and silica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow ORSBO 0.12 20 0.3 0.2 b.d. b.d. 331 3.85 11.1 70 130 0.10 b.d. 0.2 0.1 b.d. b.d. 298 1.15 9.9 70 135 b.d. b.d. b.d. 0.1 b.d. b.d. 138 1.06 9.1 70 1310 n.m. b.d. b.d. 0.1 b.d. b.d. 90 1.05 9 70 1320 n.m. b.d. b.d. b.d. b.d. b.d. 41 1.03 8.6 70 132-2114 n.m. n.m. n.m. n.m. n.m. n.m. 104 1.01 8.3 70 b.d. = below detection n.m. = not measured *= HPLC was a measurement of total chlorophyll derivatives

[0323] The results in Table 18 show the chlorophyll substrates and products in the reaction 5 and 13 oils following silica treatment as described above.

TABLE-US-00019 TABLE 18 Chlorophyll substrate and product composition in oils after enzyme treatment or after enzyme and silica treatment a a′ b CHYL PYN PPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm ORSBO 0.21 0.70 0.24 b.d. ND 1.26 b.d. 0.28 0.24 130 0.17 0.31 0.05 0.03 0.03 b.d. b.d. 0.22 0.11 135 0.17 0.31 0.04 b.d. b.d. b.d. b.d. 0.21 0.11 1310 0.17 0.31 0.03 b.d. b.d. b.d. b.d. 0.22 0.11 1320 0.17 0.31 0.02 b.d. b.d. b.d. b.d. 0.22 0.11 132-2114 0.16 0.27 0.03 b.d. b.d. 0.17 b.d. 0.22 ND b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm ORSBO 0.49 b.d. b.d. b.d. 0.43 b.d. 3.85 3.85 b.d. 130 0.23 b.d. b.d. b.d. b.d. b.d. 1.15 1.09 0.06 135 0.22 b.d. b.d. b.d. b.d. b.d. 1.06 1.06 b.d. 1310 0.22 b.d. b.d. b.d. b.d. b.d. 1.05 1.05 b.d. 1320 0.22 b.d. b.d. b.d. b.d. b.d. 1.03 1.03 b.d. 132-2114 0.16 b.d. b.d. b.d. b.d. b.d. 1.01 1.01 b.d. CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = below detection ND = not detected

[0324] In a direct comparison of SP-2114 and SP-2115, SP-2114 was not as good as SP-2115, but was able to reduce the chlorophyll from 298 ppb in the reaction 13 oil to 104 ppb, as compared to 41 ppb achieved using SP-2115, as reported from the UV/Vis method.

Reaction 14—Silica Treatment. Comparison of SP-2113, SP-2115, and SP-2119

[0325] Three 500 gram samples of the enzyme treated refined soybean oil from reaction 14 were split and added to three 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 0.5, 1.0 and 2.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green color. The oils were labeled as 145, 1410, and 1420 respectively. Oil labeled as 140 was the sample of reaction 14.

[0326] A 500 gram sample of the enzyme treated refined soybean oil from reaction 14 was added to a 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oil was heated to 80° C. and 2.0 grams of silica SP-2113 was mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. No change in the color of the oil was observed during the adsorption process. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were yellow. The sample was labeled as 142-2113.

[0327] A 500 gram sample of the enzyme treated refined soybean oil from reaction 14 was added to a 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oil was heated to 80° C. and 2.0 grams of silica SP-2119 was mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. No change in the color of the oil was observed during the adsorption process. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were yellow. The sample was labeled as 142-2119.

[0328] The once refined soybean oil enzyme treated and silica treated samples, 1420 (471 grams) and 1410 (461 grams) were combined and placed in a 3 L Claisen flask and assembled according the deodorization procedure. The oil was sparged with nitrogen for approximately 2 minutes. The vacuum was initiated and the nitrogen sparge was discontinued and water vapor from the steam generator was allowed to begin the deodorization process. The vacuum achieved was between 0.28-0.56 mBar during the deodorization process. The oil was heated under vacuum and water sparge (3 wt. %) to 230° C. The sparge and temperature were maintained for two hours. The heat was discontinued and the oil was allowed to cool under vacuum and water sparge. The vacuum was broken with nitrogen at approximately 100° C. and allowed to further cool to 70° C. before opening to the air. The oil was colorless with no green tint. The oil was labeled as 14101420-DEO.

[0329] The results in Table 19 show the content of free fatty acids (FFA), soap, P, Ca, Mg, Fe, and chlorophyll in the reaction 14 oil following silica treatment as described above.

TABLE-US-00020 TABLE 19 Composition of ORSBOs after enzyme treatment or after enzyme and silica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow ORSBO 0.06 27 1.0 0.4 tr b.d. 319 3.90 9.2 70 140 0.05 b.d. 0.2 0.1 b.d. b.d. 302 1.46 9.6 70 145 n.m. n.m. b.d. 0.1 b.d. b.d. n.m. 1.09 n.m. n.m. 1410 n.m. n.m. b.d. 0.1 b.d. b.d. 94 1.07 9.2 70 1420 n.m. n.m. b.d. 0.1 b.d. b.d. 52 1.04 8.7 70 142-2113 n.m. n.m. n.m. n.m. n.m. n.m. 309 1.13 8.7 70 142-2119 n.m. n.m. n.m. n.m. n.m. n.m. 305 1.16 8.9 70 14101420-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 55 0.46 0.1 2.4 b.d. = below detection n.m. = not measured *= HPLC was a measurement of total chlorophyll derivatives

[0330] The results in Table 20 show the chlorophyll substrates and products in the reaction 14 oil following silica treatment as described above.

TABLE-US-00021 TABLE 20 Chlorophyll substrate and product composition in ORSBOs after enzyme treatment or after enzyme and silica treatment a a′ b CHYL PYN PPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm ORSBO 0.22 0.72 0.25 b.d. b.d. 1.27 b.d. 0.26 0.24 140 0.17 0.31 0.08 0.05 0.05 b.d. 0.03 0.22 0.11 145 0.17 0.31 0.05 b.d. b.d. b.d. b.d. 0.22 0.11 1410 0.17 0.31 0.03 b.d. b.d. b.d. b.d. 0.22 0.11 1420 0.17 0.31 0.02 b.d. b.d. b.d. b.d. 0.22 0.11 142-2113 0.16 0.25 0.08 b.d. b.d. 0.18 b.d. 0.22 b.d. 142-2119 0.16 0.28 0.08 b.d. b.d. 0.18 b.d. 0.22 b.d. 14101420-Deo 0.09 b.d. 0.02 b.d. b.d. b.d. b.d. 0.16 b.d. b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm ORSBO 0.52 b.d. b.d. b.d. 0.43 b.d. 3.90 3.90 b.d. 140 0.23 0.03 0.05 0.11 b.d. b.d. 1.46 1.28 0.18 145 0.23 b.d. b.d. b.d. b.d. b.d. 1.09 1.09 b.d. 1410 0.23 b.d. b.d. b.d. b.d. b.d. 1.07 1.07 b.d. 1420 0.22 b.d. b.d. b.d. b.d. b.d. 1.04 1.04 b.d. 142-2113 0.24 b.d. b.d. b.d. b.d. b.d. 1.13 1.13 b.d. 142-2119 0.24 b.d. b.d. b.d. b.d. b.d. 1.16 1.16 b.d. 14101420-Deo 0.18 b.d. b.d. b.d. b.d. b.d. 0.46 0.46 b.d. CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = below detection

[0331] Unlike samples treated with SP-2115, samples treated with SP-2113 or SP-2119 did not demonstrate any activity for the removal of chlorophyll as measured by the UV/Vis method. This method is accepted by the industry.

Reaction 15—Silica Treatment and/or Deodorization

[0332] Three 500 g samples of the enzyme treated refined soybean oil from reaction 15 were split and added to three 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 0.5, 1.0 and 2.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green color. The oils were labeled as 155, 1510, and 1520 respectively. Oil labeled as 150 was the sample of reaction 15.

[0333] The once refined soybean oil enzyme treated and silica treated samples, 1520 (457 grams) and 1510 (461 grams) were combined and placed in a 3 L Claisen flask and assembled according the deodorization procedure. The oil was sparged with nitrogen for approximately 2 minutes. The vacuum was initiated and the nitrogen sparge was discontinued and water vapor from the steam generator was allowed to begin the deodorization process. The vacuum achieved was between 0.57-0.97 mBar during the deodorization process. The oil was heated under vacuum and water sparge (3 wt. %) to 230° C. The sparge and temperature were maintained for two hours. The heat was discontinued and the oil was allowed to cool under vacuum and water sparge. The vacuum was broken with nitrogen at approximately 100° C. and allowed to further cool to 70° C. before opening to the air. The oil was colorless with no green tint. The oil was labeled as 15101520-DEO.

[0334] 618 grams of the enzyme treated oil from reaction 15, without any adsorbent treatment, was placed in a 3 L Claisen flask and assembled according the deodorization procedure. The oil was sparged with nitrogen for approximately 2 minutes. The vacuum was initiated and the nitrogen sparge was discontinued and water vapor from the steam generator was allowed to begin the deodorization process. The vacuum achieved was between 1.15-1.40 mBar during the deodorization process. The oil was heated under vacuum and water sparge (3 wt. %) to 230° C. The sparge and temperature were maintained for two hours. The heat was discontinued and the oil was allowed to cool under vacuum and water sparge. Broke vacuum with nitrogen at 100° C. and allowed to cool to 70° C. before opening to the air. The oil was colorless without any green tint. The oil was labeled as 150-DEO.

[0335] The results in Table 21 show the contents of free fatty acids (FFA), soap, P, Ca, Mg, Fe, and chlorophyll in the reaction 15 oil following deodorization and/or silica treatment as described above.

TABLE-US-00022 TABLE 21 Composition of oils after enzyme treatment or after enzyme and silica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow Refined 0.03 242 2.8 0.7 0.2 tr 321 3.90 n.m. n.m. 150 0.02 tr 0.3 0.2 b.d. 0.1 282 2.05 9.3 70 155 n.m. b.d. 0.1 0.2 b.d. b.d. 219 1.94 8.7 70 1510 n.m. b.d. 0.1 0.1 b.d. b.d. 133 1.79 8.9 70 1520 n.m. b.d. 0.1 0.1 n.m. b.d. 33 1.04 8.4 70 15101520-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 40 0.82 0.0 2.1 150-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 176 0.88 0.3 3.2 tr = trace b.d. = below detection n.m. = not measured *= HPLC was a measurement of total chlorophyll derivatives

[0336] The results in Table 22 show the chlorophyll substrates and products in the reaction 15 oil following deodorization and/or silica treatment as described above. The results demonstrate the need for an adsorbent to remove the final lower color.

TABLE-US-00023 TABLE 22 Chlorophyll substrate and product composition in oils after enzyme treatment or after enzyme and silica treatment a a′ b CHYL PYN PPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm Refined 0.20 0.71 0.26 b.d. b.d. 1.27 b.d. 0.27 0.23 150 0.17 0.33 0.13 b.d. b.d. 0.62 b.d. 0.22 0.11 155 0.18 0.32 0.10 b.d. b.d. 0.61 b.d. 0.22 0.11 1510 0.17 0.32 0.05 b.d. b.d. 0.61 b.d. 0.22 0.11 1520 0.17 0.31 0.02 b.d. b.d. b.d. b.d. 0.22 0.11 15101520-Deo 0.10 0.31 0.03 b.d. b.d. b.d. b.d. 0.15 b.d. 150-Deo 0.06 0.31 0.13 b.d. b.d. b.d. b.d. 0.13 b.d. b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm Refined 0.52 b.d. b.d. b.d. 0.43 b.d. 3.90 3.90 b.d. 150 0.25 b.d. b.d. b.d. 0.21 b.d. 2.05 2.05 b.d. 155 0.25 b.d. b.d. b.d. b.d. b.d. 1.79 1.79 b.d. 1510 0.25 b.d. b.d. b.d. 0.21 b.d. 1.94 1.94 b.d. 1520 0.22 b.d. b.d. b.d. b.d. b.d. 1.04 1.04 b.d. 15101520-Deo 0.23 b.d. b.d. b.d. b.d. b.d. 0.82 0.82 b.d. 150-Deo 0.24 b.d. b.d. b.d. b.d. b.d. 0.88 0.88 b.d. CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = below detection

Reaction 16—Silica Treatment or Deodorization

[0337] Three 500 gram samples of the enzyme treated refined soybean oil from reaction 16 were split and added to three 1000 mL round bottom flask with the configuration of Adsorbent procedure. The oils were heated to 80° C. and 0.5, 1.0 and 2.0 grams of silica SP-2115 were mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. The filter disc and cake were a dark green color. The oils were labeled as 165, 1610, and 1620. The oil labeled as 160 was the sample from reaction 16.

[0338] 825.3 grams of the enzyme treated oil, without any adsorbent treatment, was placed in a 3 L Claisen flask and assembled according the deodorization procedure. The oil was sparged with nitrogen for approximately 2 minutes. The vacuum was initiated and the nitrogen sparge was discontinued and water vapor from the steam generator was allowed to begin the deodorization process. The vacuum achieved was between 1.15-1.40 mBar during the deodorization process. The oil was heated under vacuum and water sparge (3 wt. %) to 230° C. The sparge and temperature were maintained for two hours. The heat was discontinued and the oil was allowed to cool under vacuum and water sparge. Broke vacuum with nitrogen at 100° C. and allowed to cool to 70° C. before opening to the air. The color was expected to be greening, but was very slightly red with no greenish tint. The sample was labeled as 160-Deo.

[0339] The results in Table 23 show the contents of free fatty acids (FFA), soap, P, Ca, Mg, Fe, and chlorophyll in the reaction 16 oil following deodorization or silica treatment as described above.

TABLE-US-00024 TABLE 23 Composition of oils after enzyme treatment or after enzyme and silica treatment Lovibond FFA Soap P Ca Mg Fe UV/Vis HPLC* Color (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppb) (ppm) Red Yellow Refined 0.05 396 3.3 0.9 0.2 b.d. 321 2.19 n.m. n.m. 160 0.03 tr b.d. 0.2 b.d. b.d. 301 1.84 9.1 70 165 n.m. b.d. b.d. 0.2 b.d. b.d. 155 1.73 8.7 70 1610 n.m. n.m. 0.1 0.1 b.d. b.d. 79 n.m. 8.6 70 1620 n.m. n.m. b.d. 0.1 b.d. b.d. 59 1.16 8.3 70 160-Deo 0.02 n.m. n.m. n.m. n.m. n.m. 210 0.94 0.2 3.3 tr = trace b.d. = below detection n.m. = not measured *= HPLC was a measurement of total chlorophyll derivatives

[0340] The results in Table 24 show the chlorophyll substrates and products in the reaction 16 oil following deodorization or silica treatment as described above. The results demonstrate the need for treatment with an adsorbent for final green color removal.

TABLE-US-00025 TABLE 24 Chlorophyll substrate and product composition in oils after enzyme treatment or after enzyme and silica treatment a a′ b CHYL PYN PPYN POB PPOB PYN POB CHYL PYN ppm ppm ppm ppm ppm ppm ppm ppm ppm Refined 0.21 0.36 0.12 b.d. b.d. 0.63 b.d. 0.28 0.12 160 0.17 0.32 0.10 0.04 0.03 0.61 b.d. 0.22 0.11 165 0.17 0.32 0.06 b.d. b.d. 0.61 b.d. 0.22 0.11 1610 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 1620 0.17 0.31 0.02 b.d. b.d. ND b.d. 0.21 0.11 160-Deo 0.11 0.31 0.11 b.d. b.d. ND b.d. 0.17 ND b b′ Decolorase PPYN POB PPOB CHYL PYN POB Total Sub. Prod. ppm ppm ppm ppm ppm ppm ppm ppm ppm Refined 0.26 b.d. b.d. b.d. 0.21 b.d. 2.19 2.19 b.d. 160 0.24 b.d. b.d. b.d. b.d. b.d. 1.84 1.77 0.07 165 0.24 b.d. b.d. b.d. b.d. b.d. 1.73 1.73 b.d. 1610 n.m. n.m. n.m. n.m. n.m. n.m. 1620 0.23 b.d. b.d. 0.11 b.d. b.d. 1.16 1.16 b.d. 160-Deo 0.24 b.d. b.d. b.d. b.d. b.d. 0.94 0.94 b.d. CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = below detection ND = not detected

Example 10

Incubation of Once Refined Canola (ORCAN) Oil with the CHL26 Enzyme, and Treatment with Silica SP-2115 or Commercial Silica or Bleaching Clay

[0341] A five gallon plastic pail of Once Refined Canola (ORCAN) oil was mixed with a high shear mixer to make uniform. Samples were pulled to use in the following experiments.

Reaction 17—CHL26

[0342] 3,000 grams of the once refined canola oil was placed into a 4 liter glass beaker on a hot plate with overhead mixing. The oil was heated to 60° C. under agitation. Once the material reached 60° C., the oil was moved to the shear mixer. 15 g of the decolorase enzyme CHL26 (prepared as described in Example 4) and 150 g of deionized water were added. The material was shear mixed for 1 minute. The glass beaker was moved back to the overhead mixer and covered with plastic wrap. The oil was mixed for 4 hours at 60° C. The oil temperature was increased to 75° C., and the oil was centrifuged utilizing a Gyro-Centrifuge with a bowl with holes closed. Oil and heavy samples were collected.

[0343] The oil and heavy phase remaining in the bowl were poured into a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The oil remaining in the tubes was discarded and the liquid heavy phases were combined. The heavy phase was a dark green.

Reactions 18-20—CHL26

[0344] 3000 grams of the once refined canola oil was placed into a 4 liter jacket glass beaker. The temperature was set at 60° C. Once the material reached 60° C., the oil was moved to a shear mixer. 15 g of the decolorase enzyme CHL26 (prepared as described in Example 4) and 150 g of deionized water were added to the hot oil. The material was shear mixed for 1 minute. The glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 4 hours at 60° C. The oil temperature was increased to 75° C., and the oil was centrifuged utilizing a Gyro-Centrifuge with a bowl with holes closed. The procedure was repeated three times and the oil was collected and combined for reactions 18-20.

Reaction 17—Silica Treatment with SP-2115

[0345] 500 grams of once refined canola oil from Reaction 17 was added to a 1000 mL round bottom flask with the equipment configuration of above. The oil was heated to 80° C. and 2.0 g of silica SP-2115 was mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel. This experiment was repeated under the same conditions, except using 4, 6, and 8 grams of silica SP-2115.

Reactions 18-20—Silica Treatment. Comparison of SP-2115 and Commercial Silica and Bleaching Clay

[0346] The once refined canola oil from the combined reactions 18-20 was treated with a commercially available silica (TriSyl® 300), a bleaching clay (Clariant 126FF), or two separate lots of SP-2115, as described below.

[0347] 500 grams of once refined canola oil from the combined reactions 18-20 was added to a 1000 mL round bottom flask with the equipment configuration of above. The oil was heated to 80° C. and 2.0 g of TriSyl® 300 was mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and the oil was mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel.

[0348] 500 grams of once refined canola oil from the combined reactions 18-20 was added to a 1000 mL round bottom flask with the equipment configuration of above. The oil was heated to 80° C. and 2.0 g of the bleaching clay Tonsil® supreme 126 FF(Clariant) was mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and the oil was mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel.

[0349] 500 grams of once refined canola oil from combined reactions 18-20 was added to a 1000 mL round bottom flask with the equipment configuration of above. The oil was heated to 80° C. and 2.0 g of the first lot of silica SP-2115 was mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and the oil was mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel.

[0350] 500 grams of once refined canola oil from combined reactions 18-20 was added to a 1000 mL round bottom flask with the equipment configuration of above. The oil was heated to 80° C. and 2.0 g of the second lot of silica SP-2115 was mixed into the oil and a vacuum of approximately 100 mbar was added. The temperature was increased to 100° C. and the oil was mixed for 30 minutes. The vacuum was broken and the material filter with a Buchner Funnel.

[0351] The results in Table 25 show the contents of free fatty acids (FFA), soap, P, Ca, Mg, Fe, and sodium (Na) in the reaction 17 oil or the combined reaction 18-20 oil following treatment with silica SP-2115, TriSyl® silica, or bleaching clay, as described above.

TABLE-US-00026 TABLE 25 Composition of oils after treatment with CHL26 or after treatment with CHL26 and SP-2115, TriSyl ® 300 silica, or bleaching clay Soap FFA P Ca Mg Fe Na Oil (ppm) (%) (ppm) (ppm) (ppm) (ppm) (ppm) Once Refined Can 171 0.09 4.8 1.1 0.2 0.10 11.4 Rxn 17 - CHL26 Can b.d. 0.05 1.0 1.9 tr 0.05 b.d. Rxn 17 - 2.0 g SP- Can n.m. 0.05 b.d. 0.5 b.d. 0.02 b.d. 2115 Rxn 17 - 4.0 g SP- Can n.m. 0.05 tr 0.2 b.d. 0.03 b.d. 2115 Rxn 17 - 6.0 g SP- Can n.m. 0.04 0.1 0.2 b.d. 0.02 b.d. 2115 Rxn 17 -- 8.0 g SP- Can n.m. 0.04 0.3 0.3 b.d. tr b.d. 2115 Rxn 18-20 -- CHL26 Can b.d. 0.05 0.8 2.0 tr 0.12 b.d  Rxn 18-20 - 2.0 g Can n.m. 0.05 b.d. 0.4 b.d. b.d. b.d. TriSyl ®300 Rxn 18-20 - 2.0 g Can n.m. 0.06 0.2 0.9 b.d. 0.04 b.d. Clariant 126FF Rxn 18-20 - 2.0 g Can n.m. 0.05 0.8 0.9 b.d. b.d. b.d. SP-2115 (2.sup.nd lot) Rxn 18-20 - 2.0 g Can n.m. 0.05 0.7 0.6 b.d. 0.05 b.d. SP-2115 (1.sup.st lot) b.d.—below detection tr—trace n.m.—not measured Once Refined means washed and dried Can—canola

[0352] The oil samples (˜1 gram) were diluted in 100 ml volumetric flask with CHCl.sub.3 (chloroform) and measured for chlorophyll content using the UV-Vis method looking at the peak absorbance at 670 nm. Measurements were also made using the HPLC method. The results are set forth in Table 26.

TABLE-US-00027 TABLE 26 Chlorophyll content of oils after treatment with CHL26 or after treatment with CHL26 and SP-2115, TriSyl ® silica, or bleaching clay UV/Vis HPLC* Oil (ppb) (ppb) Starting Material (ORCO) 32733 36856 Rxn 17 30781 27090 Rxn 17, 2 g SP-2115 16527 16722 Rxn 17, 4 g SP-2115 14490 13680 Rxn 17, 6 g SP-2115 4524 5652 Rxn 17, 8 g SP-2115 3165 4812 Rxn 18-20 Combined 31070 28840 Rxn 18-20, 2 g TriSyl 300 27881 25888 Rxn 18-20, 2 g Clariant 126 FF 11975 6551 Rxn 18-20, 2 g SP-2115 (2.sup.nd lot) 15462 13207 Rxn 18-20, 2 g SP-2115 (1.sup.st lot) 18487 15595 *= HPLC was a measurement of total chlorophyll derivatives

[0353] The results in Tables 27-28 show the chlorophyll substrates and products in the reaction 17 or combined reaction 18-20 oils after treatment as described above. The remaining levels are close to the level of chlorophyll substrates and products needed in an industrial process. Optimizing the reaction conditions for the decolorase enzyme will enable the elimination of bleaching earth for very green canola oils.

TABLE-US-00028 TABLE 27 Chlorophyll substrate and product composition in oils after treatment with CHL26 or after treatment with CHL26 and SP-2115, TriSyl ® 300, or bleaching clay a a′ b b′ CHYL PYN PPYN POB PPOB PYN POB CHYL PYN PPYN POB PPOB CHYL PYN POB Total ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm Starting 0.41 11.83 13.76 b.d. b.d. 2.87 b.d. 0.21 3.21 3.71 b.d. b.d. b.d. 0.86 b.d. 36.86 Material (ORCO) Rxn 17 b.d. 2.95 6.85 4.52 2.76 2.90 0.58 0.19 0.88 3.77 0.97 0.04 b.d. 0.60 0.09 27.09 Rxn 17-2 g 0.12 3.11 5.88 0.19 b.d. 2.16 0.12 0.21 0.81 3.52 b.d. b.d. b.d. 0.60 b.d. 16.72 SP-2115 Rxn 17-4 g 0.11 2.48 5.28 0.07 b.d. 1.78 0.06 0.17 0.60 2.69 b.d. b.d. b.d. 0.44 b.d. 13.68 SP-2115 Rxn 17-6 g 0.13 1.00 0.68 b.d. b.d. 0.94 b.d. 0.18 0.43 1.95 b.d. b.d. b.d. 0.35 b.d. 5.65 SP-2115 Rxn 17-8 g 0.10 0.80 0.48 b.d. b.d. 0.94 b.d. 0.19 0.37 1.62 b.d. b.d. b.d. 0.32 b.d. 4.81 SP-2115 Rxn 18-20 b.d. 2.80 6.94 4.74 2.74 2.44 0.61 0.18 0.83 3.70 1.51 1.61 b.d. 0.52 0.19 28.84 combined Rxn 18-20 - b.d. 2.33 7.09 3.53 1.90 3.42 0.45 0.18 0.67 3.66 0.96 0.88 b.d. 0.69 0.12 25.89 TriSyl ® 300 Rxn 18-20 - b.d. 1.58 1.83 0.57 0.11 b.d. 0.12 0.18 0.63 0.18 0.40 0.37 b.d. 0.47 0.10 6.55 Clariant 126 FF Rxn 18-20 - 0.10 2.74 5.27 0.15 0.11 b.d. b.d. 0.19 0.74 3.43 b.d. b.d. b.d. 0.49 b.d. 13.21 2 g SP-2115 (2nd lot) Rxn 18-20 - 0.10 3.06 7.08 0.10 0.07 b.d. b.d. 0.19 0.74 3.78 b.d. b.d. b.d. 0.47 b.d. 15.60 2 g SP-2115 (1st lot) CHYL = Chlorophyll; PYN = Pheophytin; PPYN = Pyropheophytin; POB = Pheophorbide; PPOB = Pyropheophorbide; Sub = Substrates; Prod = Products b.d. = below detection

TABLE-US-00029 TABLE 28 Chlorophyll substrate and product composition in oils after treatment with CHL26 or after treatment with CHL26 and SP-2115, TriSyl ® 300, or bleaching clay. Decolorase Substrates Products (ppm) (ppm) Starting Material (ORCO) 36.86 b.d. Rxn 17 18.14 8.95 Rxn 17, 2 g SP-2115 16.41 0.31 Rxn 17, 4 g SP-2115 13.55 0.13 Rxn 17, 6 g SP-2115 5.65 b.d. Rxn 17, 8 g SP-2115 4.81 b.d. Rxn 18-20 combined 17.41 11.43 Rxn 18-20, TriSyl ® 300 18.03 7.85 Rxn 18-20, Clariant 126 FF 4.87 1.68 Rxn 18-20, 2 g SP-2115 (2.sup.nd 12.96 0.25 lot) Rxn 18-20, 2 g SP-2115 (1.sup.st 15.42 0.17 lot)

[0354] The commercial silica “Trisyl® 300 has a limited ability to remove the products generated in the decolorase reactions, and actually appears to convert some of the chlorophyll products back into substrates. The bleaching earth has a greater ability to remove the chlorophyll substrates found in unreacted decolorase oils, but does not remove the products of the decolorase treated oils as well as the silicas of the present disclosure.

Example 11

Preparation of Silica Adsorbents

[0355] This example describes the preparation of the adsorbents in Reactions 21-30 below:

Reaction 21—Preparation of SP-2113

[0356] 600 grams of a TRISYL® silica, was dried at 60° C. to remove 173 grams of water. The silica was then impregnated with a sodium hydroxide solution containing 18.6 grams of NaOH and 81.9 grams of water. The material was blended in a Waring blender for 5 minutes.

Reaction 22—Preparation of SP-2114

[0357] 600 grams of TRISYL® silica, was blended in a Waring blender for 5 minutes with 12.2 g of MgO powder.

Reaction 23—Preparation of SP-2115

[0358] 600 grams of TRISYL® silica was blended in a Waring blender for 5 minutes with 31.6 g of MgO powder.

Reaction 24—Preparation of SP-2116

[0359] 600 grams of TRISYL® 300 silica, was dried at 60° C. to remove 173 grams of water. The silica was then impregnated with a sodium hydroxide solution containing 11.4 grams of NaOH and 92 grams of water. The material was blended in a Waring blender for 5 minutes.

Reaction 25—Preparation of SP-2117

[0360] 600 grams of TRISYL® 300 silica was dried at 60° C. to remove 173 grams of water. The silica was then impregnated with a sodium hydroxide solution containing 17.8 grams of NaOH and 92 grams of water. The material was blended in a Waring blender for 5 minutes.

Reaction 26—Preparation of SP-2119

[0361] 600 grams of a silica xerogel containing less than 10 wt % water, a surface area of 707 m.sup.2/g and a median particle size of 19 microns, was blended in a Waring blender with 30 grams of MgO for 5 minutes.

Reaction 27—Preparation of Adsorbent A

[0362] 4.8 grams of SP-2115 was dried in an oven at 110° C. for 3 hours.

Reaction 28—Preparation of Adsorbent B

[0363] 5 grams of MgO powder and 20 grams TRISYL® silica were blended into a container, sealed, then mixed by shaking for 1 hour.

Reaction 29—Preparation of Adsorbent C

[0364] 24.5 grams of a silica xerogel containing less than 10 wt % water, and having a surface area of 707 m.sup.2/g and a median particle size of 19 microns, was impregnated with 23.6 grams of DI water then added to a container containing 2.5 grams of MgO powder. The contents were sealed and mixed by shaking for 1 hour.

Reaction 30—Preparation of Adsorbent D

[0365] 264 grams of silica xerogel with 4 wt % water, a surface area of 330 m.sup.2/g, and a particle size between 88 and 210 microns was impregnated with 303 grams of DI water. The material was divided into six different containers, each containing 6 grams of MgO powder. Each container was mixed by shaking for 1 hour, then the contents were all combined into a larger container and blended by shaking for 1 hour.

Example 12

Enzymatic and Adsorbent Treatment of Oils

[0366] Adsorbents prepared in Example 11 were used to further treat two batches of oil previously subjected to decolorase treatment (i.e., oil 11120, prepared as described in Example 9).

Reaction 31—Treatment of Decolorase Treated Oil with Adsorbent A

[0367] 100 grams of enzymatically treated oil 11120 was heated to 80° C. then 0.21 grams of Adsorbent A was mixed into the oil. A 100 mbar vacuum was applied then the temperature was set to 100° C. and mixed for 30 minutes. The vacuum was broken and the material was filtered with a Buchner Funnel. The filter disc and cake were dark green.

Reaction 32—Treatment of Decolorase Treated Oil with Adsorbent B

[0368] 100 grams of enzymatically treated oil 11120 was heated to 80° C. then 0.4 grams of Adsorbent B was mixed into the oil. A 100 mbar vacuum was applied then the temperature was set to 100° C. and mixed for 30 minutes. The vacuum was broken and the material was filtered with a Buchner Funnel. The filter disc and cake were dark green.

Reaction 33—Treatment of Decolorase Treated Oil with Adsorbent C

[0369] 100 grams of enzymatically treated oil 11120 was heated to 80° C. then 0.4 grams of Adsorbent C was mixed into the oil. A 100 mbar vacuum was applied then the temperature was set to 100° C. and mixed for 30 minutes. The vacuum was broken and the material was filtered with a Buchner Funnel. The filter disc and cake were dark green.

Reaction 34—Treatment of Decolorase Treated Oil with Adsorbent D

[0370] 100 grams of enzymatically treated oil 11120 was heated to 80° C. then 0.4 grams of Adsorbent D was mixed into the oil. A 100 mbar vacuum was applied then the temperature was set to 100° C. and mixed for 30 minutes. The vacuum was broken and the material was filtered with a Buchner Funnel. The filter disc and cake were dark green.

Reaction 35—Treatment of Decolorase Treated Oil with TRISYL® Silica

[0371] 100 grams of enzymatically treated oil 11120 was heated to 80° C. then 0.4 grams of TRISYL® silica was mixed into the oil. A 100 mbar vacuum was applied then the temperature was set to 100° C. and mixed for 30 minutes. The vacuum was broken and the material was filtered with a Buchner Funnel. The filter disc and cake were green.

Reaction 36—Treatment of Decolorase Treated Oil with TRISYL® 300 Silica

[0372] 100 grams of enzymatically treated oil 11120 was heated to 80° C. then 0.4 grams of TRISYL® 300 silica was mixed into the oil. A 100 mbar vacuum was applied then the temperature was set to 100° C. and mixed for 30 minutes. The vacuum was broken and the material was filtered with a Buchner Funnel. The filter disc and cake were yellow.

[0373] The green color concentrations from the oils produced in Reactions 31-36 were determined using the AOCS UV/Vis method. The results are set forth in Table 29.

TABLE-US-00030 TABLE 29 Green color of decolorase treated oil after further treatment with various adsorbents. Reaction Green color (ppm) First batch of oil 11120 11.4 31 0.2% of Adsorbent A 6.5 32 0.4% of Adsorbent B 5.7 33 0.4% of Adsorbent C 5.6 34 0.4% of Adsorbent D 7.0 Second batch of oil 11120 18.9 35 0.4% of TRISYL ® silica 18.6 36 0.4% TRISYL ® 300 silica 18.8