Silica adsorbent for removal of chlorophyll derivatives from triacylglycerol-based oils

Abstract

SOLUTION: 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.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a process for treating oils containing chlorophyll derivatives. In one embodiment, the present disclosure provides a method for removing impurities, including chlorophyll derivatives and / or trace metals, from oil. Improved process for the preparation of silica-based adsorbents and improved silica-based adsorbents for use in such processes. Regarding adhesives. Sequence Listing

[0002] This application has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The above ASCII code was created on March 26, 2019. The file name is "35893-513_SEQ_LIST.txt" and the size is is 36,864 bytes. [Background technology]

[0003] Crude triacylglycerol oil obtained from either the expression or solvent extraction process is Acylglycerol, phospholipid, sterol, tocopherol, diacylglycerol, A complex mixture of free fatty acids, trace metals, chlorophyll, beta-carotene, and other trace compounds It is a high quality fully refined product with a pleasant taste, light color, and long shelf life. In order to produce a refined oil or salad oil, phospholipids, free fatty acids, trace metals, chlorophyll, It is advisable to remove β-carotene and β-carotene.

[0004] Removal of phospholipids results in the largest amount of neutral oils associated with the refinement of triacylglycerol oils. The removal of chlorophyll results in the loss of the fatty acids associated with the refining of triacylglycerol-based oils. The second largest amount of neutral oil is lost.

[0005] The removal of phospholipids can be achieved by water degumming, enzyme-assisted water degumming, acid degumming, caustic refining, and enzyme A number of different techniques can be used, including processing.

[0006] Water degumming is usually applied to crude oils that contain a large amount of hydratable phospholipids. Due to its properties, the resulting phospholipids can be used as lecithin (a natural emulsifier). Although the oil obtained from this technique is generally only partially degummed, Water-degummed oils are referred to in the industry as "degummed". Water-degummed oils still contain large amounts of phospholipids, especially Contains non-hydratable phospholipids, resulting in high quality finish with high stability and light color. To produce oil, other processes such as caustic refining or phospholipase A (PLA) enzymatic degumming are required. The use of process techniques may be required.

[0007] In the water degumming process, water is added to the crude oil with mixing to remove the phospholipids present in the oil. Hydration of the phospholipids or "gums" causes them to swell and flocculate, This is then separated from the remainder of the oil. If the oil loss from the water degumming process is significant This has a negative impact on the overall economic balance of refined oil processing costs.

[0008] Enzyme-assisted water degumming is usually applied to crude oils that contain a large amount of hydratable phospholipids, The purpose of the method is to react all of the hydratable phospholipids and convert them to diacylglycerol to form the diacylglycerols in the oil. The goal of this process is to increase the yield of oil while preserving the non-hydratable phospholipids. The enzymes used are phospholipase C (PLC) and phosphatidylinositol phosphorylase. p53 (PI-PLC).

[0009] In the enzyme-assisted water degumming process, water and PLC are added to the crude oil while mixing. The enzyme reacts with the phospholipids in the oil while shear mixing to form phosphatidylcholine in the oil. Choline (PC), phosphatidylethanolamine (PE), and diacylglycerol (PI) The heavy phase (water, denatured proteins, and phosphorus compounds) is more concentrated than the oil. It has a high specific gravity and can be separated by industrial practices of sedimentation, filtration, or centrifugation The enzyme-assisted water degumming process primarily removes only hydratable phospholipids. Phosphatidic Acid The remaining phospholipids, measured as salts, can be removed in subsequent processing operations. .

[0010] Acid degumming is usually applied to crude oils when the objective is to remove all phospholipids. The resulting oil is usually referred to in the industry as "super degummed" or "fully degummed". Treat with phosphoric or citric acid. The acid improves the hydrophilicity of non-hydratable phospholipids (NHPs), Water is then added to the acid-treated crude oil and the oil is mixed. Hydration of the phospholipids or "gums" causes the gums to swell and become more flexible. The acid degumming process removes most of the phospholipids. However, enough still remains in the degummed oil to require further processing. As with the ethanol process, some of the oil is emulsified and considered a process loss, which adds to the cost of processing the refined oil. It will have a negative impact on the overall economic balance.

[0011] Caustic refining is usually performed when the objective is to remove all phospholipids and free fatty acids. It is applied to crude oil or water degummed oil. Crude oil or water degummed oil is treated with phosphoric acid or citric acid. The acid improves the hydrophilicity of the NHP, thereby aiding in its removal. A caustic solution is added to the acid treated oil. Neutralizes free fatty acids, neutralizes excess acid, and dissolves the remaining phosphates along with the resulting sodium soap. This helps hydrate and emulsify all of the ingredients. Mix the sodium hydroxide solution / oil, then The oil is then separated by settling, filtration, or industrially by centrifugation. The caustic treated oil is then "washed" The oil obtained from the centrifuge is called "Once Refined". For food applications, the water is generally known as "wash water". The "refined" oil is usually subjected to bleaching and deodorization to produce salad oil. Alternatives to water washing The caustic oil is treated with an adsorbent silica gel to remove residual soap and water that is not removed by the first centrifugation. The primary objective of the present invention is to filter out and remove phospholipids and phospholipids.

[0012] "Enzymatic purification" or "enzymatic degumming" refers to the process where the objective is to remove all phospholipids. Generally, the enzymatic degumming processes of the prior art are used in one of several ways, typically This has been carried out on oils that have already been degummed by water degumming. Enzymatically degummed oil is treated with bleaching and degumming, a process known in the industry as "physical refining." Enzymatic degumming gives better oil yields than water, acid, or caustic degumming. and the economic efficiency will be improved.

[0013] The enzyme reaction changes the properties of the phospholipids, and some of the phospholipid moieties are cleaved. This reduces the emulsifying properties of the phospholipids, which reduces the amount of oil lost when the gum is separated from the oil. The enzymes active against phospholipids are generally called "phospholipids". It is called "phosphatase". The types of phospholipases are based on the position in the phospholipid molecule with which the enzyme reacts and are known as PLA1, PLA2, PLC, and PLD. Different types of phospholipases produce different compounds when reacting with phospholipids.

[0014] Commercially available PLA1 enzymes with phospholipase activity are Lecitase® Ultra and QuaraLowP. Commercially available PLA2 enzymes with phospholipase activity are Rohalase Xtra and LysoMax. These products are known to produce polar lysophospholipids and polar fatty acids when mixed with degummed oil together with 1 - 1.5% aqueous citric acid - NaOH buffer at 4.5 < pH < 7.0 and 40°C < T < 55°C. .5 < pH < 7.0 and 40°C < T < 55°C and 1 - 1.5% aqueous citric acid - NaOH buffer. When mixed with degummed oil, they produce polar lysophospholipids and polar fatty acids. PLA1 selectively hydrolyzes fatty acids on the opposite side of the phosphate functional group in the glycerol backbone, and PLA2 selectively hydrolyzes fatty acids at the center of the glycerol backbone of phospholipids. PLA is non - selective about the phospholipids with which it reacts.

[0015] The resulting reaction produces lysophospholipids and fatty acids. Lysophospholipid molecules have lost one hydrophilic functional group, and the remaining alcohol groups at the reaction site are hydrophilic. Since they have two hydrophilic sites, lysophospholipid molecules are water - soluble and have lost their emulsifying properties. Therefore, in the PLA1 or PLA2 degumming process, the purification loss is reduced because the gum no longer removes any neutral oil at all, and the only loss is the original phospholipid molecule.

[0016] The PLC enzyme reacts with phospholipids by selectively hydrolyzing the phosphate functional group. It is known in the art that the reaction produces diacylglycerol. ("DAG") and phosphatidic acid groups. Diacylglycerol molecules are The PLC degumming process removes the phosphate functional groups from the cellulose. Reduces purification losses by preserving the original phospholipid molecule while removing only the functional groups However, PLC does not react with all of the phospholipids present in the oil. Generally, PLC is a phosphatidic acid (PA) or phosphatidylinositol (PI ) does not react with any of the following: PC, P However, non-hydratable phosphatides are not able to react with and remove phosphate, phosphate, and PI after water degumming. Therefore, enzyme-assisted water degummed oils require caustic solubilization to remove the residual gums. The cellulose must be further treated with PLA1 or PLA2.

[0017] Oilseeds such as soybean and canola, and oil fruits such as palm, as well as algae origin The triacylglycerol oil from the oil contains chlorophyll. Seed crushing, oil extraction, defrosting Chlorophyll is removed during many stages of the oil production process, including gumming, caustic treatment, and bleaching. The last of these, the bleaching process, removes residual chlorophyll and makes the water acceptable. This chlorophyll is typically produced by heating the oil and placing it on an adsorbent. Throughout, chlorophyll and other colorants can affect the appearance and / or stability of the finished oil. These are removed from the oil in a bleaching process step which involves removing the compounds.

[0018] High concentrations of chlorophyll pigments impart undesirable color and induce oxidation of the oil during storage. In the edible oil processing industry, the quality of the oil is guaranteed in terms of color and organoleptic properties. To achieve this, a bleaching process is used to reduce the chlorophyll concentration to 0.02 ppm. The whitening process increases processing costs and reduces oil yields due to entrainment in the bleaching clay. The "used" clay must be disposed of in an environmentally friendly manner, and pyrophoric acid-treated materials and Due to the high content of sorbed oil (approximately 30% by weight), it is a hazardous material to transport.

[0019] Chlorophyll is formed by the loss of a magnesium ion from the porphyrin (chlorine) ring. During oil processing, they are modified into derivatives known as pheophytins (see Figure 1). Typically, pheophytins are more abundant in processed oils than chlorophyll. Pheophytin can be further decomposed into pyropheophytin (Behavior of Chlorophyll Derivatives in Canola Oil Processing”, JAOCS, Vol. no. 9 Sept. 1993, p. (See, e.g., 837-841.) Pyropheophytin is formed primarily during the processing of vegetable oils. (See, for example, The lipid handbook' ed. Frank D. Gunn stone,John L.Harwood,Albert J.Dijkstra.2 007-- 3rd ed., p.56). Chlorophyll, phaeophytic Pyropheophytin and pyropheophytin exist in two forms, A and B. The A component is C7 Component B has an aldehyde at the C7 position.

[0020] The use of enzymes to remove pyropheophytin from vegetable oils is described in International Publication No. 2010 It is known from WO 2013 / 160372 and WO 2013 / 143149. No. 2010 / 143149 describes, for example, wheat (Triticum aestivum) and konamidrim. It can hydrolyze pyropheophytin derived from Chlamydomonas reinhardtii. A method for treating a composition containing pyropheophytin using an enzyme that is In WO 2013 / 160372, for example, Arabidopsis thaliana ) and several chlorophyllase enzymes from wheat have been disclosed, which are involved in the production of chlorophyll in oils. Pheophytin and pyropheophytin could be converted. Summary of the Invention [Problem to be solved by the invention]

[0021] Silica has also been used as an adsorbent to remove impurities from triacylglycerol-based oils. Examples of silicas used to purify oils include U.S. Pat. 295,810, 4,781,864, and 9,493,748. However, such silicas are not effective in removing impurities from oils. Even after silica treatment, pigments such as chlorophyll derivatives are not sufficiently effective to Undesirable levels of impurities, including

[0022] Therefore, we have developed an alternative system that can remove impurities from triacylglycerol-based oils. Rika is needed.

[0023] In one aspect, the present disclosure is a process for treating oil containing chlorophyll derivatives. The oil is then contacted with an adsorbent comprising silica treated with an alkaline earth metal oxide. The adsorbent has a pH of about 7 or more, for example, about 7 to about 10, and is about Contains at least 0.1% by weight of an alkaline earth metal oxide such as MgO, preferably at least about 3% by weight. or a water content of about 10% by weight or more, or about 25% by weight to about 75% by weight. The purpose is to:

[0024] In one particular aspect, the present disclosure provides a process for treating oils containing chlorophyll derivatives. contacting the oil with an adsorbent comprising silica treated with an alkaline earth metal oxide; wherein the adsorbent has a pH of about 7 to about 10 and a pH of about 2.5 to about 2.5 on a dry basis. 15% by weight, or about 5 to about 25% by weight, or about 10 to about 20% by weight of MgO, The process is directed to a water content of from 5 to about 75% by weight.

[0025] In another aspect, the present disclosure provides a process for treating oil containing chlorophyll derivatives. The oil is treated with a polypeptide having decolorase activity or a polypeptide having the same. contacting the oil with a composition containing tide to produce a decolorized enzyme-treated oil; contacting the sorbent with an adsorbent comprising silica treated with an alkaline earth metal oxide; The adsorbent has a pH of about 7 or more, for example, about 7 to about 10, and has a weight average molecular weight of about 0.1 on a dry basis. % or more by weight of an alkaline earth metal oxide such as MgO, The process is directed to a water content of 10% by weight or more, or from about 25% to about 75% by weight.

[0026] In another aspect, the present disclosure provides a process for treating oil containing chlorophyll derivatives. The oil is treated with a polypeptide having a decolorizing enzyme activity or a composition containing the polypeptide. contacting the oil with an alkaline earth metal to produce a decolorized enzyme-treated oil; and contacting the silica with an adsorbent comprising a metal oxide-treated silica, the adsorbent comprising has a pH of about 7 to about 10 and is present in an amount of about 2.5 to about 15% by weight, or about 5 to about 25% by weight, on a dry basis. %, or about 10 to about 20% by weight of MgO and has a water content of about 25 to about 75% by weight. The purpose of the process is to

[0027] In another aspect, the present disclosure provides a process for treating oil containing pyropheophytin. The oil is treated with a polypeptide having pyropheophytinase activity or a polypeptide having pyropheophytinase activity. contacting the composition with a pyropheophytin-containing compound, and optionally, pheophytin is converted to a pheophorbide to form a pyrrole phosphatase. producing a pyropheophytinase-treated oil; and treating the pyropheophytinase-treated oil with an alkali. and contacting the adsorbent with an adsorbent comprising silica treated with an alkaline earth metal oxide, The adhesive has a pH of about 7 or more, for example, about 7 to about 10, and is about 0.1% by weight or more on a dry basis. Contains alkaline earth metal oxides such as MgO, at least about 3% by weight, preferably at least about 10% by weight or from about 25 to about 75 weight percent water.

[0028] In another aspect, the present disclosure provides a process for treating oil containing pyropheophytin. The oil is treated with a polypeptide having pyropheophytinase activity or a polypeptide having pyropheophytinase activity. contacting the composition with a pyropheophytin-containing compound, and optionally, pheophytin is converted to a pheophorbide to form a pyrrole phosphatase. producing a pyropheophytinase-treated oil; and treating the pyropheophytinase-treated oil with an alkali. and contacting the adsorbent with an adsorbent comprising silica treated with an alkaline earth metal oxide, The adhesive has a pH of about 7 to about 10 and is present in an amount of about 2.5 to about 15% by weight on a dry basis, or about 5 to about 25% by weight, or about 10 to about 20% by weight MgO, and about 25 to about 75% by weight water content The present invention relates to a process having the following features:

[0029] In another aspect, the present disclosure provides a process for treating oil containing chlorophyll derivatives. The oil is treated with a polypeptide having a decolorizing enzyme activity or a composition containing the polypeptide. contacting the oil with an alkaline earth metal to produce a decolorized enzyme-treated oil; and contacting the silica with an adsorbent comprising a metal oxide-treated silica, the adsorbent comprising has a pH of about 7 or more, for example, about 7 to about 10, and has about 0.1% by weight or more of Mg on a dry basis. 0, and about 3% by weight or more, preferably about 10% by weight or more For example, the water content is about 25 to about 75% by weight, and the chloroform in the composition is reduced by the treatment. The total concentration of chlorophyll derivatives in the composition before contact with the adsorbent is The process is aimed at reducing the total body concentration by at least 5% by weight.

[0030] In another aspect, the present disclosure provides a process for treating oil containing chlorophyll derivatives. The oil is treated with a polypeptide having a decolorizing enzyme activity or a composition containing the polypeptide. contacting the oil with an alkaline earth metal to produce a decolorized enzyme-treated oil; and contacting the silica with an adsorbent comprising a metal oxide-treated silica, the adsorbent comprising has a pH of about 7 to about 10 and is present in an amount of about 2.5 to about 15% by weight, or about 5 to about 25% by weight, on a dry basis. %, or about 10 to about 20% by weight of MgO and has a water content of about 25 to about 75% by weight. The total concentration of the chlorophyll derivative in the composition is increased by the treatment. at least 5% by weight compared to the total concentration of chlorophyll derivatives in the composition before The purpose of the process is to decrease.

[0031] In another aspect, the polypeptide used in the process of the present disclosure is pyropheophytin. A polypeptide having an enzyme activity, a. at least 80%, 85%, 86% to amino acids 1 to 318 of SEQ ID NO:1; 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity to the isolated polypeptide. Do, and b. At least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 11 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, or at least 99%, or 100% identical to Polypeptides is selected from the group consisting of:

[0032] In one embodiment, the polypeptide used in the process of the present disclosure is a chlorophyll substrate. The chlorophyll substrates are chlorophyll, pheophytin, , pyropheophytin, and combinations thereof; The fluoroproducts are chlorophyllides, pheophorbides, pyropheophorbides, and may be selected from the group consisting of a combination of:

[0033] In one embodiment, the oil comprises pyropheophytin and the polypeptide is a polypeptide as disclosed herein. Pyropheophytinase activity is a potent enzyme that converts pyropheophytin into pyropheophorbide. will be converted.

[0034] In another aspect, the present disclosure is directed to the oil produced by the processes disclosed herein. The target.

[0035] In another aspect, the present disclosure provides an improved silica-based adsorbent for use in the processes of the present disclosure. The adsorbent has a pH of about 7 or more and a water content of about 3% by weight or more in the final adsorbent. with an alkaline earth metal oxide, preferably magnesium oxide, in an amount sufficient to provide The present invention includes amorphous porous silica. definition

[0036] The term "control sequence" can be used interchangeably with the term "expression regulating nucleic acid sequence." The term, as used herein, refers to a gene that is operably linked to a particular host organism or in vitro. It refers to a nucleic acid sequence that is necessary and / or influences the expression of a coding sequence expressed by two nucleic acid sequences. When columns are operably linked, they are typically in the same orientation and leading The same goes for frames. They are usually contiguous in nature, although this is not required. Expression control nucleic acid sequences, such as appropriate transcription initiation, termination, promoter, linker, etc. a sequence encoding a sequence of a nucleic acid which is a sequence encoding ... Synthetic-Dalgarno sequences, repressor, or activator sequences; splicing and poly Efficient RNA processing signals, including adenylation signals; stabilizes cytoplasmic mRNA sequences that improve translation efficiency (e.g., ribosome binding sites); and, if desired, a sequence that enhances secretion of the protein. The nucleic acid sequence may be any nucleic acid sequence that exhibits activity in the host organism of interest, and may be used to bind to a host cell. The gene may be derived from a gene encoding a protein that is either endogenous or heterologous. Each control sequence is native to the nucleic acid sequence encoding the polypeptide. If desired, the control sequence may be a foreign (heterologous) sequence encoding the polypeptide. Specific restriction enzyme sites that facilitate ligation of the coding region of the nucleic acid sequence and the regulatory sequences. The control sequence may be provided with a linker for the purpose of introducing a It can be optimized.

[0037] The term "chlorophyll derivative" as used herein refers to a chlorophyll substrate and Chlorophyll substrates include chlorophyll, pheophytin, and picolinic acid. Chlorophyll products include chlorophyllides, pheophorbides, and pyropheophorbide. Chlorophyll derivatives include the so-called a and b compounds. nothing.

[0038] The term "decolorizing enzyme" (as well as those containing the phrase "polypeptide having decolorizing enzyme activity") As used herein, the term "variant of" refers to a polypeptide that binds one or more chlorophyll substrates. This means that the polypeptide can be converted into a chlorophyll product. hydrolyzes pheophytin to chlorophyllide and pheophytin to pheophorbide and / or pyropheophytin can be hydrolyzed to pyropheophorbide. Therefore, the term "decolorizing enzyme activity" includes chlorophyllase activity, pheophytinase activity, and The enzyme may comprise an enzyme activity, a pyropheophytinase activity, or a combination thereof.

[0039] The term "hydrogel" as used herein refers to a hydrogel having a concentration of about 25% by weight or more, preferably about 25 to 40% by weight. It is used to refer to a silica-based adsorbent having a water content of about 75% by weight.

[0040] The term "amorphous" as used herein refers to a material that is amorphous in nature and has no constituent atoms, molecules, or structures. A solid material in which ions are arranged in a random, unordered pattern extending in all three directions. and can be measured by X-ray diffraction or differential scanning calorimetry.

[0041] The term "porous" as used herein refers to the Bar Measured by Rett-Joyner-Halenda (BJH) nitrogen porosimetry This refers to a material that has an internal porosity of 0.1 cc / g or more when compressed.

[0042] The term "treated" is used herein in reference to treatment with an alkaline earth metal oxide. When used in combination with silica, it refers to the intimate mixing of silica and alkaline earth metal oxides under high shear conditions. vinegar.

[0043] The term "particle surface area" refers to the Brunauer Emmet Teller (BET) It is defined to mean the particle surface area as measured by the elementary adsorption method.

[0044] The phrase "median particle size" refers to the amount of particles dissolved in water or in an organic solvent such as acetone or ethanol. The median particle size (D50, 50% of the particles) measured by dynamic light scattering when slurried at The volume percent size is less than this number, and 50 volume percent of the particles have a size of this number. (The volume distribution is greater than the number of

[0045] The term "triacylglycerol-based oil" refers to an oil that contains triacylglycerol.

[0046] The term "expression" includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. This includes, but is not limited to, any step involved in the production of a polypeptide.

[0047] An expression vector is a vector for expressing a polynucleotide in vitro or in a host cell. The gene contains appropriate control sequences for translation (promoter, RBS / Shine-Dalgarno, and transcription and A polypeptide encoding a polypeptide operably linked to a translation stop signal, etc. Contains leotide.

[0048] Expression vectors are useful in recombinant DNA techniques and are used to express polynucleotides. Any vector (e.g., a plasmid or virus) capable of causing expression of The selection of the vector typically depends on the compatibility of the vector and the cell into which the vector is to be introduced. The vector may be a linear or a closed circular plasmid. Autonomous replicating vectors, i.e. extrachromosomal entities whose replication is independent of chromosomal replication, Vectors that exist as organisms, such as plasmids, extrachromosomal elements, minichromosomes, or Alternatively, the vector may be an artificial chromosome. It may be integrated into the genome and replicated together with the chromosome into which it has been integrated. Cloning vectors integrate either randomly or at a predetermined target locus in the host cell chromosome. A vector system may be a single vector or plasmid, or a vector that is integrated into the genome of a host cell. two or more vectors or plasmids which together contain the total DNA to be introduced into the It may be a transposon.

[0049] A host cell as defined herein is an organism suitable for genetic manipulation and is capable of expressing the polypeptide according to the invention. The host cell can be cultured at a cell density useful for industrial production of a target product, such as a polypeptide. The host cell may be a host cell found in nature or a parent cell after genetic manipulation or classical mutagenesis. The host cell may be a host cell derived from a host cell. Advantageously, the host cell is a recombinant host cell. .

[0050] The host cell may be a prokaryotic, archaeal, or eukaryotic host cell. The cell may be, but is not limited to, a bacterial host cell. Eukaryotic host cells include, but are not limited to, yeast. The host cell may be, but is not limited to, a fungal, amoeba, algae, plant, animal, or insect host cell. It won't be done.

[0051] The term "heterologous" as used herein refers to a nucleic acid or nucleic acid that does not naturally occur in the host cell. In other words, the nucleic acid or amino acid sequence is naturally expressed in a host cell. It is not the same as what is seen in nature.

[0052] A nucleic acid or polynucleotide sequence, as used herein, is defined as a sequence of at least 5 nucleotides or is defined as a nucleotide polymer containing nucleic acid units. A nucleotide or nucleic acid is R The terms "nucleic acid" and "polynucleotide sequence" are used herein to refer to NA and DNA. are used interchangeably.

[0053] A "peptide" is a short chain of amino acid residues linked by peptide (amide) bonds. The shortest peptides, dipeptides, are two peptides joined by a single peptide bond. It consists of three amino acids.

[0054] The term "polypeptide" refers to a molecule consisting of more than five amino acid residues linked by peptide bonds. The term "protein" as used herein refers to a molecule that contains amino acid residues that contain At times, the term is synonymous with the term "polypeptide" and may refer to two or more polypeptides. Thus, the terms "protein" and "polypeptide" can be used interchangeably. The polypeptides may optionally be modified to add functionality (e.g., glycosylation, phosphorylation, acylation, farnesylation, prenylation, sulfonation, etc.). A polypeptide that exhibits activity under specific conditions and in the presence of a specific substrate is called an enzyme. As a result of the degeneracy of the genetic code, there are multiple genes that code for a given polypeptide. It will be understood that the nucleotide sequence may be generated.

[0055] An "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state. It is one piece.

[0056] The term "isolated polypeptide" as used herein refers to a polypeptide that is free of at least some of the polypeptides with which it is naturally associated. It means a polypeptide that has been removed from at least one component, e.g., other polypeptide material. An isolated polypeptide may be free of any other impurities. The method may be SDS-PAGE or any other analytical method suitable for this purpose and known to those skilled in the art. At least 50% purity, for example at least 60% purity, At least 70% pure, at least 75% pure, at least 80% pure, at least 8 5% purity, at least 80% purity, at least 90% purity, or at least 95% purity; The purity may be 96%, 97%, 98%, 99%, 99.5%, or 99.9%. The peptide may be produced by a recombinant host cell.

[0057] The term "promoter" as used herein refers to a promoter that binds RNA polymerase and directs the transcription of a nucleic acid sequence. Defined as a DNA sequence that targets polymerase to a specific downstream transcription start site to initiate transcription. The promoter sequence may be native to the nucleic acid sequence encoding the polypeptide. or may be of a different species.

[0058] The term "recombinant" when used in reference to a cell, nucleic acid, protein, or vector. and the cell, nucleic acid, protein, or vector is capable of introducing a heterologous nucleic acid or protein. have been modified by introduction or alteration of the native nucleic acid or protein, or It indicates that the cell is derived from a cell that has been modified in this way. Recombinant cells express genes that are not found within the native (non-recombinant) form of the cell. or that would be aberrantly expressed, under-expressed, or not expressed at all if not recombinant. The term "recombinant" is used interchangeably with "genetically modified" and "transgenic." It is synonymous with "Nick".

[0059] The terms "sequence identity" and "sequence homology" are used interchangeably herein. For purposes of clarity, the term "sequence homology" used herein refers to the degree of sequence identity between two amino acid sequences or between two nucleic acid sequences. Or align the sequences for optimal comparison purposes to determine the percentage of sequence identity. To optimize the alignment between two sequences, the sequence is compared. Gaps may be introduced into either of the two sequences being aligned. Such an alignment may be performed by The alignment may be performed over the entire length of the sequences to be compared. Alternatively, the alignment may be performed over a shorter length. For example, about 20, about 50, about 100, or more nucleic acids / bases or amino acids. Sequence identity may be determined by comparing the sequence identity of two sequences over the reported alignment region. It is the percentage of identical matches between two amino acid sequences or between two nucleotide sequences. Percent sequence identity is calculated using the Needleman and The algorithm can be used to find the sigma-based ... .B. and Wunsch, CD (1970) J.Mol.Biol.48,4 43-453). Both amino acid and nucleotide sequences are generated by algorithms. The Needleman-Wunsch algorithm can be used to align Implemented in the computer program NEEDLE. For the purposes of this invention, EMB We used the NEEDLE program from the OSS package (version 2.8.0 or later). ,EMBOSS:The European Molecular Biology O pen Software Suite (2000) Rice,P.Longden ,I.and Bleasby,A.Trends in Genetics 16,( 6) pp276- 277,http: / / emboss.bioinformati For protein sequences, use EBLOSUM62 as the substitution matrix. For nucleotide sequences, use EDNAFULL. The parameters of choice were Gap Open Penalty 10 and Gap Extension Penalty 10. The null value is 0.5. A person skilled in the art can easily derive a small difference between all these different parameters. Different results are obtained, but the overall similarity of the two sequences is not guaranteed when using different algorithms. It will be appreciated that the percent identity does not change significantly.

[0060] After alignment using the program NEEDLE as described above, the query sequence and the The percentage of sequence identity between the control and the control sequence is calculated as follows: The number of corresponding positions in the alignment that show identical amino acids or nucleotides is called the alignment. The identity is calculated by subtracting the total number of gaps in the alignment and dividing it by the total length of the alignment. When defining in detail, the NOBRIEF option can be used to This is obtained from the above and is labeled as "longest identity" in the program output.

[0061] The nucleic acid and protein sequences of the disclosure can be identified by performing searches against public databases, e.g. For example, as a "query sequence" to identify other family members or related sequences. Such searches can also be used in the literature. 90) NBLAST and XBLAST in J.Mol.Biol.215:403-10 The nucleic acid molecules of the present invention can be produced using the program (version 2.0). To obtain homologous nucleotide sequences, the NBLAST program, score=100, word count=1, was used. BLAST nucleotide searches can be performed using a domain length of 12. To obtain amino acid sequences homologous to the protein molecule, the XBLAST program was used, score = 50. BLAST protein searches can be performed using the following: To obtain a gapped alignment, use the method of Altschul et al. (1997 ) Nucleic Acids Res. 25(17):3389-3402 Gapped BLAST can be used. When using AST programs, the respective programs (e.g., XBLAST and N The default parameters of the BLAST program may be used. National Center for Biotechnology Information homepage, http Please see http: / / www.ncbi.nlm.nih.gov / .

[0062] A "synthetic molecule," such as a synthetic nucleic acid or synthetic polypeptide, is a molecule that is synthesized by in vitro chemical or It is produced by enzymatic synthesis, which involves engineering it with optimal codon usage for the intended host organism. These include, but are not limited to, mutant nucleic acids produced by the method.

[0063] The synthetic nucleic acid is preferably a nucleic acid according to the method of WO 2006 / 023664, which is incorporated herein by reference. According to the method described in WO 2008000632 and / or WO 20077258, WO 2008 / 000632 discloses a method for optimizing the use of the Codon pair optimization deals with optimizing the codon pairs of a nucleotide sequence that encodes a polypeptide. Improve expression of the nucleotide sequence and / or improve production of the encoded polypeptide. In order to achieve this, the polypeptides modified with respect to codon usage, particularly the codon pairs used, are A codon pair is a method of optimizing a nucleotide sequence that is being read. A codon is defined as a set of two subsequent triplets (codons) that form a sequence of two sequences. The donor usage frequency must be adapted depending on the host species, and as a result, significant differences in the sequence of SEQ ID NO:1 are observed. Variants that are less homologous but still encode the polypeptides according to the invention. You will understand that you can get

[0064] As used herein, the terms "mutant," "derivative," "mutation," or "homologue" and can be used interchangeably to refer to either a polypeptide or a nucleic acid. Variants may include substitutions, insertions, deletion ... Mutants include truncations, transversions, and / or inversions. For example, site-saturation mutagenesis, systematic mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, By mutagenesis, directed evolution, and a variety of other recombinant approaches known to those of skill in the art. Mutant genes of nucleic acids can be made by techniques known in the art. It can be artificially synthesized.

[0065] Detailed Description The present disclosure relates to phosphorus compounds such as lingam, soap, trace metals, chlorophyll derivatives, and free fatty acids. For removing impurities, including contaminants, from oils, particularly from triacylglycerol-based oils More specifically, the present disclosure relates to a process for the preparation of alkaline earth metal oxide-treated non-ferrous metals. US20130232633A1 - Adsorbents comprising amorphous porous silica and methods for removing impurities from oil - Google Patents Intended for use.

[0066] Alkaline earth metals such as magnesium, especially alkaline earth metals such as magnesium oxide By treating porous amorphous silica having a specific water content with metal oxides, it is possible to obtain a method for producing a porous amorphous silica having a specific water content. Compared to silica, which is used in oil purification, it removes impurities such as chlorophyll derivatives and trace metals from oil. It has been found that an adsorbent having improved ability to absorb water is obtained. Silica adsorbents treated with alkaline earth metal oxides having a pH of 7 to about 10 are xerophoric. It is preferred over other types of silica-based prior adsorbents such as triacyl sorbents, gels or acid hydrogels. More impurities from glycerol-based oils (e.g., trace metals, chlorophyll derivatives, etc.) It has been found that it is possible to remove

[0067] Thus, in one aspect, the present disclosure provides a method for treating oils containing chlorophyll derivatives. The process includes contacting the oil with a sorbent, the sorbent being MgO or the like. and silica, particularly porous amorphous silica, which is treated with an alkaline earth metal oxide of about 7 or more. and about 3% by weight or more, or preferably about 10% by weight or more, or about 25 to about 75 In one particular embodiment, the adsorbent is hygroscopic. It is a hydrogel having a pH of about 7 to about 10 and a water content of about 25 to about 75% by weight, and is in a dry state. Approximately 2.5 to 15% by weight, or approximately 5 to 25% by weight, or approximately 10 to 20% by weight of M Contains gO.

[0068] In one embodiment, the adsorbent is present at about 0.1% by weight or more on a dry basis, more typically at about 1% by weight. % to about 40% by weight, or about 5% to about 25% by weight, or about 10% to about 20% by weight, or A sufficient amount of aluminum to provide about 2.5% to about 15% by weight of alkaline earth metal oxide. In one particular embodiment, the silica is porous and amorphous and is treated with a potassium earth metal oxide. Silica is treated with magnesium oxide (MgO) and amorphous silica is a gel. In such embodiments, the adsorbent comprises at least about 0.1% MgO by weight on a dry basis, or at least about 1% MgO by weight on a dry basis. about 40% by weight, or about 2.5% by weight to about 15% by weight, or about 5% by weight to about 25% by weight %, or about 5% to about 15% by weight, or about 10% to about 20% by weight, or or about 10% to about 15% by weight MgO. In one particular embodiment, the adsorbent comprises about 1 Contains 0% to about 20% by weight of MgO.

[0069] In some embodiments, the adsorbents of the present disclosure have a ratio of about 1:3.8 to about 1:26, e.g., about 1:12.77 to about 1:3.3, or about 1:12.77 to about 1:4.89, or about 1:8 0.09 to about 1:4.90, or about 1:5.44 to about 1:4.89, of MgO to SiO2 In one particular embodiment, the adsorbent has a molar ratio of about 1:5.44 to about 1:4. It has a MgO to SiO2 molar ratio of 89.

[0070] The adsorbents of the present disclosure comprise at least about 3% by weight, more typically at least about 10% by weight, It may have a moisture content of 20% by weight or more, or even about 55% by weight or more. The adsorbent of the present disclosure is advantageously prepared from a silica hydrogel, and has a concentration of about 25% by weight to about 75% by weight. In other embodiments, the adsorbent has a water content of about 30% to about 65% by weight. or about 40% by weight to about 70% by weight, or about 50% by weight to about 65% by weight, or about 55% by weight to about 67% by weight, or from about 58% to about 65% by weight.

[0071] The adsorbent of the present disclosure has a pH of about 7 or more, for example, about 1:3.8 to about 1:26, about 7 to about 10 , or about 7.5 to about 9.7, or about 8.0 to about 9.5, or about 8.0 to about 9.0, or about As discussed herein and demonstrated in the Examples, As shown, the adsorbents of the present disclosure are more efficient at dissolving oils than prior adsorbents based on acidic hydrogels. It has been found that the method is excellent at removing impurities such as acetic acid, acetic acid, and acetic acid.

[0072] The adsorbents of the present disclosure have a particle size of about 0.1 to about 2000 micrometers, for example about 1 to about 100 0 micrometers, or about 2 to about 500 micrometers, or about 5 to about 50 micrometers In one embodiment, the adsorbent may have a median particle size of about 10 to about 30 micrometers. It has a median particle size of chromium.

[0073] The adsorbent of the present disclosure has a viscosity of about 50 ml 2 / g or more, for example, about 300m 2 / g or more or about 650 m 2 In some embodiments, the adsorbents of the present disclosure may have a BET When measured by surface area measurement, it is approximately 50m 2 / g~about 800m 2 / g or about 300m 2 / g~about 700m 2 / g surface area.

[0074] The adsorbents of the present disclosure have a pore size of about 0.1 cc / g or more, preferably about 0.4 cc / g or more. In some embodiments, the pore volume of the adsorbent may be greater than or equal to the nitrogen porosimetry. When measured by Lee, it is about 0.2cc / g to about 2.0cc / g or about 0.7cc / g ~ about 2.0 cc / g.

[0075] In one particular embodiment, the adsorbent of the present disclosure is treated with magnesium oxide (MgO). and an amorphous silica gel having a pH of about 7 to about 10, It contains 15% by weight of MgO and has a water content of about 25% to about 75% by weight. In one embodiment, the adsorbent of the present disclosure is treated with MgO and has a pH of about 8 to about 9. The amorphous silica gel contains about 10% to about 20% by weight of MgO on a dry basis, and about It has a water content of 50% to about 65% by weight.

[0076] Suitable silicas that can be used to prepare the adsorbents of the present disclosure include amorphous silicas. The precipitate has a moisture content of about 3% by weight or more in the pores of the porous silica gel and silica. In one embodiment, the amorphous silica is about 25% by weight or more, preferably 40% by weight or more. %, most preferably greater than 50% by weight, and even more preferably from about 30 to about 65% by weight. In some embodiments, a suitable silica hydrogel is Logels include TRISYL® silica and TRISYL® 300 (WR Grace & Co.-Conn., Columbia, Md.) Examples of suitable silica gel include, but are not limited to, commercially available silica gel.

[0077] In another embodiment, substantially moisture-free silica is used as the starting silica. "Substantially free of water" means that the silica used to prepare the adsorbent is This means that the silica has less than about 15% water. The gel is then hydrated by contacting it with a sufficient amount of water, preferably alkaline. The desired water content in the pores of the silica prior to combination with the earth metal oxide is about 3 The amount of water added is proportional to the pore volume of the particular silica used. Those skilled in the art can readily determine the appropriate molecular weight for use in the processes of the present disclosure. A non-limiting illustration of the preparation of a suitable silica is provided in the Examples.

[0078] Suitable alkaline earth metal oxides useful in preparing the adsorbents of the present disclosure include magnesium oxide. Sodium oxide, calcium oxide, strontium oxide, barium oxide, beryllium oxide, or The present invention is not limited to these combinations. The metal oxide is magnesium oxide.

[0079] The adsorbent of the present disclosure is preferably prepared by subjecting a desired amount of alkali metal oxide powder to high shear conditions. is prepared by physically blending with an amorphous silica gel having the desired water content. The blending is carried out for a time and at a temperature sufficient to provide a free-flowing powder. Preferably, the blending is carried out at a temperature ranging from about room temperature to about 100° C. until a homogenous mixture is obtained. The final adsorbent of the present invention is prepared in a manner sufficient to achieve the desired results, for example, for about 1 second or more. % by weight or more and a pH of about 7 or more. In one embodiment, the final adsorbent comprises silica gel prepared by W.R. Grace & Co. - A free-flowing powder offered by Conn under the product name SP-2115, The product contains about 10 to about 13 weight percent magnesium oxide on a dry basis and about 50 to about 65 % water content and a pH of about 8.2 to about 9.3.

[0080] Generally, the process of the present disclosure comprises mixing an oil with an effective amount of a soluble sorbent of the present disclosure to remove impurities from the oil. In a non-limiting embodiment, the oil is contacted with an adsorbent of In another embodiment, the oil is contacted with the adsorbent of the present disclosure in an amount of about 10% by weight or less of the oil. In an amount of about 0.01% to about 10% by weight, or about 0.1% to about 8% by weight, based on weight or in an amount of about 0.1% to about 5% by weight, or in an amount of about 0.1%, about 1%, or about In an amount of from about 0.1% to about 0.5% by weight, or in an amount of from about 0.1% to about 0.4% by weight, or Adsorbent in an amount of 0.1% to about 0.3% by weight, or in an amount of about 0.1% to about 0.2% by weight and bring it into contact with.

[0081] In certain embodiments, the oil is heated at a temperature of about 60° C. to less than about 100° C., for example at about 100° C. 30° C., or more typically at a temperature of about 80° C. In some embodiments, the oil is heated to a temperature of less than about 110° C., for example, from about 70° C. to about 130° C. The adsorbent may be contacted with the adsorbent of the present disclosure under vacuum at a temperature of about 100° C. or at a temperature of about 100° C. In one embodiment, the vacuum may be from about 50 to about 700 mbar, more typically about 1 In one embodiment, the oil is heated to about 100 mbar at a temperature of about 100 mbar. In another embodiment, the oil is contacted with the adsorbent of the present disclosure at a temperature of about 60° C. with the adsorbent of the present disclosure at a temperature of from about 50 to about 100° C. or less than about 80° C. A vacuum of about 700 mbar, more typically about 100 mbar, is applied and the temperature is increased to about 70° C. Increase temperature to approximately 130°C. In one particular embodiment, the oil is contacted with the adsorbent of the present disclosure at a temperature of about 80° C., followed by A vacuum of about 100 mbar is applied and the temperature is increased to about 100° C. In one embodiment, the adsorbent is contacted with the oil for about 5 to about 240 minutes, for example, about 15 to about 60 minutes. After treatment, the silica can be removed from the oil using any suitable technique, including filtration. do.

[0082] Impurities that can be removed from oil using the process of the present disclosure include lingam Phosphorus-containing compounds such as soaps, trace metals (sodium, potassium, magnesium, calcium (including but not limited to zinc, iron, aluminum, and lead), chlorophyll Substrates (including chlorophyll, pheophytin, and pyropheophytin), chlorophyll inducers (including chlorophyllides, pheophorbides, and pyropheophorbides), and free These include, but are not limited to, fatty acids (FFA).

[0083] The treatment of oil with the adsorbents of the present disclosure as described above can be used to trade edible oils, including cooking oils. and oils that meet acceptable standards for transportation. Such standards include Nat. onal Institute of Oilseed Products(NIOP) , American Oil Chemists Society, and ISO Some examples include:

[0084] Oils that may be treated using the process of the present disclosure include canola oil, castor oil, cocoa oil, and corn oil. Coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hemp seed oil, linseed oil , mango kernel oil, meadowfoam oil, cow foot oil, olive oil, palm oil, palm kernel oil, Muolein, peanut oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, sesame oil, soybean oil , sunflower seed oil, tall oil, camellia oil, vegetable oil, and oil derived from algae. In one embodiment, the oils include, but are not limited to, triacylglycerol-based oils. The oil is derived from algae.

[0085] The adsorbents of the present disclosure can be used to remove impurities from oil at various stages during oil processing. For example, the oil being treated may be crude undegummed oil, degummed oil, caustic refined oil, caustic refined and In one embodiment, the oil is a crude oil. The process comprises contacting the crude oil with the silica of the present disclosure.

[0086] In another embodiment, the oil is first subjected to a decolorizing enzyme treatment before contacting it with the adsorbent. For example, in one embodiment, the process comprises treating an oil containing chlorophyll derivatives with a decolorizing enzyme having decolorizing enzyme activity. and contacting the oil with a polypeptide that exhibits the above-mentioned effect or a composition that contains the above-mentioned polypeptide to produce a decolorized enzyme-treated oil. and contacting the decolorized enzyme-treated oil with the adsorbent of the present disclosure. Examples of suitable polypeptides having decolorizing enzyme activity that can be used in the process of Examples of polypeptides include, but are not limited to, those discussed in detail below. A suitable process for producing colored enzyme-treated oils, as well as various oil processing methods, are also described below. The details are described in.

[0087] Treatment of oil with the adsorbents of the present disclosure advantageously reduces impurities in the oil prior to contact with the adsorbent. For example, the oil (including the decolorized enzyme-treated oil) is treated with the By contacting the oil with the silica shown in the table, the chlorophyll present in the oil before contacting with the silica is reduced. The concentration of chlorophyll derivatives (chlorophyll 1, chlorophyll 2, chlorophyll 3, chlorophyll 4, chlorophyll 5, chlorophyll 6, chlorophyll 7, chlorophyll 8, chlorophyll 9, chlorophyll 10, chlorophyll 11, chlorophyll 12, chlorophyll 13, chlorophyll 14, chlorophyll 15, chlorophyll 16 The total concentration of the substrate and / or chlorophyll product is at least 5% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, At least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight %, at least 90% by weight, at least 95% by weight, at least 99% by weight, or 10% by weight. It can be reduced to 0% by weight.

[0088] In another embodiment, the chlorophyll derivatives in the oil include pyropheophytin, and the oil ( by contacting the oil (including the decolorized enzyme-treated oil) with the adsorbent of the present disclosure. The pyropheophytin concentration in the oil was compared to the total concentration (by weight) of pyropheophytin present in the previous oil. The total concentration of pheophytin is at least 5% by weight, at least 10% by weight, at least 20% by weight, %, at least 30%, at least 40%, at least 50%, At least 60% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight , at least 95%, at least 99%, or 100% by weight. Cut.

[0089] In another embodiment, the chlorophyll derivatives in the oil include pheophytin, and the oil (bleaching By contacting the oil (including enzyme-treated oil) with the adsorbent of the present disclosure, the oil before contacting with silica can be The concentration of pheophytins in the oil compared to the total concentration (by weight) of pheophytins present in the oil The total concentration of the amines should be at least 5% by weight, at least 10% by weight, at least 20% by weight, or At least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight %, at least 70% by weight, at least 80% by weight, at least 90% by weight, at least It may be reduced by 95%, at least 99%, or 100% by weight.

[0090] In another embodiment, the chlorophyll derivatives in the oil include chlorophyll and the oil (decolorizing enzymes). By contacting a raw oil (including unprocessed oil) with the silica of the present disclosure, the oil before contacting with the silica can be The total chlorophyll content in the oil is compared to the total concentration (by weight) of chlorophyll present in the oil. At least 5% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, At least 70% by weight, at least 80% by weight, at least 90% by weight, at least 95% by weight %, at least 99% by weight, or 100% by weight.

[0091] In another embodiment, oil (including decolorized enzyme-treated oil) is contacted with the silica-based adsorbent of the present disclosure. By soaking the oil in water, the amount of trace metal impurities (sodium, These include potassium, magnesium, calcium, iron, aluminum, and lead. The total concentration of trace metal impurities in the oil compared to the total concentration (by weight) of At least 5% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight %, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least at least 70% by weight, at least 80% by weight, at least 90% by weight, at least 95% by weight, It may be reduced by at least 99% by weight, or even 100% by weight.

[0092] In another embodiment, the oil containing the chlorophyll derivative, particularly the chlorophyll substrate, is treated with a decolorizing enzyme. A decolorizing enzyme is produced by contacting the polypeptide having decolorizing activity or a composition containing the polypeptide. In one such embodiment, the chlorophyll substrate is Pyropheophytic The decolorizing enzyme treatment removes at least a portion of the pyropheophytin. The decolorized enzyme-treated oil is converted into olbide and contacted with the adsorbent of the present disclosure to form the adsorbent. The total concentration (by weight) of pyropheophorbide present in the decolorized enzyme-treated oil before contact with the The total concentration of pyropheophorbide in the decolorized enzyme-treated oil is at least 5% by weight compared to the %, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, At least 80% by weight, at least 90% by weight, at least 95% by weight, at least 99% by weight In another embodiment, the chlorophyll substrate is reduced by phaeophytic phosphate esters (PPS) or by 100% by weight. The decolorizing enzyme treatment converts at least a portion of the pheophytin into pheophorbi. The decolorized enzyme-treated oil is then contacted with the adsorbent of the present disclosure to form a decolorized enzyme-treated oil. The total concentration (by weight) of pheophorbides present in the decolorized enzyme-treated oil before and after the treatment was compared. and the total concentration of pheophorbide in the oil is at least 5% by weight, at least 10% by weight, At least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight %, at least 60%, at least 70%, at least 80%, At least 90% by weight, at least 95% by weight, at least 99% by weight, or 100% by weight reduction In another embodiment, the chlorophyll substrate comprises chlorophyll and is decolorized by the decolorizing enzyme treatment. As a result, at least a portion of the chlorophyll is converted to chlorophyllide, and the decolorized enzyme-treated oil is By contacting the oil with the disclosed adsorbent, the decolorized enzyme-treated oil before contacting with the adsorbent can be The chlorophyllides in the decolorized enzyme-treated oils were found to be higher than the total concentration (by weight) of chlorophyllides present. A total concentration of phyllides of at least 5% by weight, at least 10% by weight, at least 20% by weight , at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 6 0% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, The weight loss is reduced by at least 95%, at least 99%, or 100% by weight.

[0093] In one embodiment, the process of the present disclosure comprises treating an oil or a decolorized enzyme treated oil with a phospholipase, Pheophytinase, pyropheophytinase, pheophorbidase, chlorophyll and combinations thereof. Included.

[0094] In another embodiment, the present disclosure provides a process for treating oils containing pyropheophytin. The oil is treated with a polypeptide having pyropheophytinase activity or a polypeptide having pyropheophytinase activity. contacting the peptide with a composition comprising the peptide, and optionally, pheophytin is converted to a pheophorbide and pyrrolidone. producing a pyropheophytinase-treated oil; and treating the pyropheophytinase-treated oil. and contacting the adsorbent of the present disclosure. Polypeptides

[0095] Any suitable polypeptide having decolorizing enzyme activity can be used in the process of the present disclosure. In one particular embodiment, the polypeptide has pyropheophytinase activity. is a polypeptide having the properties a. at least 80%, 85%, 86% to amino acids 1 to 318 of SEQ ID NO:1; 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity to the isolated polypeptide. Do, and b. At least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 11 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, or at least 99%, or 100% identical to Polypeptides is selected from the group consisting of:

[0096] A polypeptide having pyropheophytinase activity is a polypeptide having amino acids 1 to 3 of SEQ ID NO:1. 18. The disclosed polypeptides comprise at least 85% amino acid sequence identical to amino acids 1-318 of SEQ ID NO:1. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or at least 99% identity to the sequence disclosed herein. The polypeptide to be tested has at least 85%, 80%, or more amino acids 1 to 318 of SEQ ID NO:1. 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The nucleic acid sequence disclosed herein may have 6%, 97%, 98%, or at least 99% identity. The polypeptide having pyropheophytinase activity is selected from the group consisting of amino acids 1 to 31 of SEQ ID NO: 1. It may comprise, contain, or consist of 8. The polypeptide having enzyme activity may contain amino acids 2 to 318 of SEQ ID NO:1. Surprisingly, the amino acid sequence of SEQ ID NO: 1 may be A polypeptide having at least 80% identity to amino acids 1 to 318 or amino acids 2 to 318. The peptide was found to contain pyropheophytinase activity.

[0097] The polypeptide having pyropheophytinase activity is capable of converting pyropheophytin to pyrofumab. The pyropheophytic acid disclosed herein is hydrolyzed to a pyrophorbide (see also FIG. 1). The polypeptide having enzyme activity is preferably pyropheophytin a and pyropheophytin b. Phytin b is hydrolyzed into these pyropheophorbide a and b compounds. Pyropheophytinase activity can be determined by the formation of pyropheophorbide. can.

[0098] The polypeptides disclosed herein may further comprise pheophytinase activity. Polypeptides with pheophytinase activity convert pheophytin to pheophorbide. Preferably, the polypeptides disclosed herein are pheophytin a and and / or hydrolyze pheophytin b to their respective pheophorbide compounds. Therefore, pheophytinase activity can be determined by the formation of pheophorbide. can be done.

[0099] The polypeptides disclosed herein that have pyropheophytinase activity include chlorophyll, Polypeptides having chlorophyllase activity may also contain chlorophyllase activity. Preferably, the polypeptides disclosed herein are hydrolyzed to chlorophyllides. The chitosan converts chlorophyll a and / or chlorophyll b into their respective chlorophyllides. Hydrolyze to compound.

[0100] In one embodiment, the polypeptides disclosed herein have pyropheophytinase activity. It has phytochemical, pheophytinase, and chlorophyllase activities.

[0101] Pyropheophytin, pheophytin, chlorophyll, and the reaction product pyrof The determination of aeolides, pheophorbides, and chlorophyllides is as disclosed in the Examples. can be carried out by HPLC.

[0102] The polypeptide may be derived from any suitable source, for example, from a plant, algae, or cyanobacteria. The polypeptides disclosed herein may be derived from plants, such as Hordeum sp. .) species or Triticum, for example, barley (Hordeum vulgare) or wheat (Triticum The polypeptides disclosed herein may be derived from the standard They can also be produced using conventional molecular techniques, for example, de novo synthesis.

[0103] The present invention relates to a method for producing a polysaccharide having decolorizing enzyme activity, such as pyropheophytinase activity, as disclosed herein. A peptide may be an isolated, pure, recombinant, synthetic, or mutant polypeptide. The polypeptides disclosed herein may be purified. Protein Purification can be carried out by a number of methods known to those skilled in the art.

[0104] The mutant polypeptides having pyropheophytinase activity disclosed herein The peptide is selected from amino acids 1 to 318 of SEQ ID NO: 1 or amino acids 2 to 318 of SEQ ID NO: 1. At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical The polypeptide may be a polypeptide that

[0105] The polypeptides having pyropheophytinase activity disclosed herein are those having SEQ ID NO: When aligned with the amino acid sequence according to SEQ ID NO:1, one or more Polypeptides that contain substitutions, deletions, and / or insertions at amino acid positions, e.g., mutant polypeptides. For example, the polypeptides disclosed herein may be the polypeptide of SEQ ID NO:1. When aligned with the peptide, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more amino acid substitutions, deletions, and / or insertions, but A polypeptide that still has the activity or function of the polypeptides disclosed herein. Those skilled in the art will appreciate that these minor modifications may be present in the polypeptides disclosed herein. Minor amino acid changes can occur without loss of function or activity of the protein (e.g., It is important to understand that mutations can be naturally occurring or engineered (e.g., using rDNA techniques). These mutations may affect the binding domain, active site, or other functional domain of the polypeptide. When present in a polypeptide, the properties of the polypeptide may be altered, but the polypeptide may retain its activity. In close proximity to active sites, binding domains, or other functional domains, If an absent mutation is present, the effect may be predicted to be less.

[0106] The polypeptides disclosed herein are intended to be illustrative, not limiting, and are not intended to be limiting unless otherwise specified. The polypeptide may be encoded by any suitable polynucleotide sequence, so long as it exhibits phytopheophytinase activity. Typically, the enzyme has pyropheophytinase activity as disclosed herein. The polynucleotide sequence encoding the polypeptide may be a codon-optimized sequence, or a sequence that is specifically It is a codon pair optimized sequence for expression of a polypeptide in a given host cell. composition

[0107] The process of the present disclosure comprises treating an oil with a decolorizing enzyme activity, such as those disclosed herein. The method may further comprise contacting the subject with a composition comprising the polypeptide.

[0108] Such compositions may include a carrier, excipient, or other compound. Typically The composition or formulation has decolorizing enzyme activity (including pyropheophytinase activity). The polypeptides can be formulated into compounds that are suitable for use in formulations. Liquid formulations such as emulsions, suspensions, and solutions, pastes, gels, granules, and lyophilized or sprayed formulations. Mist-dried powders are included.

[0109] As used herein, an excipient is a compound that is formulated with the polypeptides disclosed herein. Inactive substances such as sucrose or lactose, glycerol, sorbitol, or is sodium chloride. The compositions comprising the polypeptides disclosed herein are liquid compositions. The composition may be a liquid or solid composition. Liquid compositions usually contain water. When used, the composition typically contains glycerol, sorbitol, or sodium chloride (Na The polypeptides disclosed herein include a component that reduces water activity, such as HCl. The solid composition may comprise granules containing the polypeptide, or the composition may comprise a liposome. in a liquid matrix such as gel or in a gel such as alginate or carrageenan. The present invention relates to a method for encapsulating or granulating a polypeptide or an enzyme. There are many techniques known in the art (e.g., GMH Meesters, “Encapsulation of Enzymes and Peptides”, Chapter 9, in NJZuidam and VANedovic (eds.)”Encapsulation Technologies for Ac tive Food Ingredients and food processing g” 2010).

[0110] The compositions disclosed herein also include a carrier comprising a polypeptide disclosed herein. For example, the polypeptides disclosed herein may be immobilized on silica. The polypeptides disclosed herein can be any polypeptide known in the art. It may be bound or immobilized to a carrier by techniques.

[0111] Having decolorizing enzyme activity (including pyropheophytinase activity) as disclosed herein The composition comprising the polypeptide may further comprise one or more additional enzymes, e.g., a lipase, e.g., a phosphoenzyme, Phospholipases such as lipase A, B and / or C, chlorophyllase, phaeophytic enzymes, Further enzymes may include phosphatase, pyropheophytinase, and / or pyropheophytinase. Phospholipase C (PLC), phosphatidylinositol PLC, and / or phospholipase The enzyme may be phospholipase A, such as phospholipase A1 or phospholipase A2.

[0112] Having decolorizing enzyme activity (including pyropheophytinase activity) as disclosed herein The composition comprising the polypeptide may be a cell in which the polypeptide having decolorizing enzyme activity is produced. Fractions may include, for example, cellular fractions from host cells. Cell fractions may be, for example, cellular fractions from host cells. After disruption of the host cells by ultrasonication and / or the use of glass beads, the cells are cultured by a variety of methods. It can be generated by:

[0113] The present disclosure also provides methods for preparing compositions comprising the polypeptides disclosed herein. The process comprises spray drying the fermentation medium containing the polypeptide or a method according to the invention as disclosed herein. and preparing the composition. Regarding the process. Nucleic acids, expression vectors, and recombinant host cells

[0114] The present disclosure also provides nucleic acid sequences encoding the polypeptides disclosed herein. At least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical The nucleic acids disclosed herein are identical to or have 100% identity to the nucleic acids disclosed herein. The acid is at least 80%, 85%, 86%, 87%, 88%, 89% relative to SEQ ID NO:2. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or less The nucleic acids disclosed herein may be nucleic acids having at least 99% identity to the nucleic acid. The nucleic acids disclosed herein may include or contain sequence number 2. It may further comprise a promoter sequence and / or other control sequences.

[0115] Having decolorizing enzyme activity (including pyropheophytinase activity) as disclosed herein The nucleic acid encoding the polypeptide may be codon optimized or may be modified to suit the particular host. Codon pair optimized sequences for expressing the polypeptides disclosed herein in cells The host cell may be, for example, a Pseudomonas spp., e.g., It may be Pseudomonas fluorescens.

[0116] In another embodiment of the invention, an isolated, pure, recombinant nucleic acid of SEQ ID NO:2 is Disclosed herein are nucleic acids that are synthetic or mutant nucleic acids. Mutant nucleic acid sequences include, for example, the sequence At least 80%, 85%, 86%, 87%, 88%, 89%, 90% for number 2 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 9 9% sequence identity.

[0117] The present invention also relates to an expression vector comprising a nucleic acid as disclosed herein, the nucleic acid comprising operably linked to one or more control sequences that direct expression of the polypeptide in a host cell. The present invention relates to an expression vector.

[0118] There are several methods for inserting a nucleic acid into a nucleic acid construct or expression vector known to those of skill in the art. For example, Sambrook & Russell, Molecular C loning:A Laboratory Manual,3rd Ed.,CSHL Press, Cold Spring Harbor, NY, 2001. Control sequences, such as promoter and terminator sequences, are used to encode the polypeptide of the present invention. In some cases, it may be desirable to manipulate the nucleic acid being encoded.

[0119] The promoters include any transcriptionally active promoter, including mutant, truncated, and hybrid promoters. The promoter sequence may be any suitable promoter sequence suitable for a eukaryotic or prokaryotic host cell. The introduction of extracellular or intracellular polypeptides, either endogenous (native) or heterologous (foreign) to the cell. The promoter can be derived from a polynucleotide encoding a constitutive peptide. Alternatively, the promoter may be an inducible promoter. Preferably, the promoter is an inducible promoter, e.g. For example, a starch-inducible promoter.

[0120] In filamentous fungi, preferred promoters include the Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, aspartic acid proteinase Aspergillus gpdA promoter, A. niger neutral alpha α-amylase, Aspergillus niger acid stable alpha amylase, Aspergillus niger or Awamo A. awamori glucoamylase (glaA), Aspergillus niger or Awamo Aspergillus oryzae endoxylanase (xlnA) or beta-xylosidase (xlnD), Trichoderma reesei (T. reesei) cellobiohydrolase I (CBHI), Rhizomuco Le Miehei Lipase, Aspergillus oryzae Alkaline Protease, Aspergillus oryzae Triose Phosphate Isomerase, A. nidulans acetamidase, Fusarium venenata Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Dania (International Publication No. 00 / 569 00), Fusarium venenatum Quinn (International Publication No. 0 / 56900), Fusarium oxysporum trypsin-like Protease (International Publication No. 96 / 00787), Trichoderma reesei beta glucoside Cellobiohydrolase I, Trichoderma reesei lobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III , Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase Canase V, Trichoderma reesei xylanase I, Trichoderma reesei xylana Polynucleotides encoding Trichoderma reesei beta-xylosidase II and Trichoderma reesei beta-xylosidase The promoter obtained from the tpi promoter and the NA2-tpi promoter (Aspergillus niger neutral Polynucleotides encoding sulpha amylase and Aspergillus oryzae triose phosphate isomerase (hybrids of promoters from nucleotides), as well as their mutants, truncations, and hybrids. The promoter may be selected from the group including, but not limited to, promoters of It is data.

[0121] In bacterial hosts, preferred promoters include the E. coli lac promoter, the aro H promoter, araBAD promoter, 77 promoter, trc promoter, tac promoter The promoter may be selected from the group consisting of the trp promoter, the trp promoter, and the trp promoter. Another example is the agarase gene from Streptomyces coelicolor. Promoter of gene (dagA), Bacillus lentus alkaline phosphatase Promoter of the enzyme gene (aprH) from Bacillus licheniformis heniformis alkaline protease gene (subtilisin Carlsberg gene) The promoter of the Bacillus subtilis levansucrase gene (sacB) α, Bacillus subtilis alpha-amylase gene (amyE) promoter, Bacillus lichenifera Promoter of the ruminant alpha-amylase gene (amyL), Bacillus stearothermolyticus Bacillus stearothermophilus maltogenic amylase gene (amyM ), or the promoter of Bacillus amyloliquefaciens ) the promoter of the alpha amylase gene (amyQ). Another example is the "-35" region. A “codon” having the sequence TTGACA for the “−10” region and TATAAT for the “−10” region is a promoter of the "Consensus".

[0122] The invention also relates to a composition comprising a nucleic acid as disclosed herein, or an expression vector as disclosed herein. The present disclosure relates to a recombinant host cell, wherein the nucleic acid is heterologous to the host cell. The recombinant host cells disclosed herein can express the decolorizing enzyme (pyropheophytin) disclosed herein. The nucleic acid having the activity (including the enzyme) and the encoded polypeptide are heterologous to the host cell. It may be a host cell.

[0123] The host cells disclosed herein can be any suitable microbial, plant, or insect cell. Suitable host cells include, for example, Acremonium, Aspergillus Aspergillus, Chrysosporium, Fusarium , Penicillium, Rasamsonia, Trich oderma, Saccharomyces, Kluyveromyces ), Pichia, e.g., Aspergillus niger, Aspergillus awamorii, Aspergillus Aspergillus foetidus, Aspergillus oryzae, Aspergillus schonoskii jae), Rasamsonia emersonii, Chrysosporium la Chrysosporium lucknowense, Fusarium oxysporum, Trichoderma Luma reesei, Saccharomyces cerevisiae, Kluyveromyces lactis It is derived from Kluyveromyces lactis or Pichia pastoris. It may be a fungal cell.

[0124] The host cell may be a prokaryotic cell, such as a bacterial cell. The term "bacterial cell" refers to any cell that is a Gram-positive cell. Suitable bacteria include those of the genus Escherichia, both gram-negative and gram-positive. ichia, Pseudomonas, Bacillus, Enterobacter Enterobacter, Lactobacillus, Lactococcus The bacterial cell may be from the genus Streptomyces. , Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis (B. iformis, B.puntis, B.megaterium, Bacillus haematobacterium rodurans (B.halodurans), Bacillus pumilus (B.pumilus), Pseudomonas Zeaxanthinifaciens (Pseudomonas zeaxanthinifaciens), Pseudomonas f The bacteria may be derived from a species of Pseudomonas fluorescens, or Escherichia coli.

[0125] Suitable bacterial host cells include, for example, pseudomonas, such as Pseudomonas fluorescens. It may also be a Monas species. Methods for Producing Polypeptides

[0126] Polypeptides suitable for use in the processes of the present disclosure (e.g., pyropheoside, The polypeptide having decolorizing enzyme activity, including the polypeptide having decolorizing enzyme activity, is The host cell culture disclosed herein is cultured in a suitable fermentation medium under conditions that allow expression of the polypeptide. The polypeptide can be produced by culturing host cells and allowing them to produce the polypeptide. Manufacturers can provide detailed information on the pH, temperature, and composition of the fermentation medium depending on the host cell used. Understand how to carry out the processes for producing the polypeptides disclosed herein. The main cells are grown in shake flasks or in volumes of 0.5 or 1 liter or more, up to 10-100 cubic meters. The culture may be carried out in a fermentor having a volume of up to 1000 liters or more. Depending on the requirements of the culture medium, the culture may be carried out aerobically or anaerobically. For example, in the case of Pseudomonas fluorescens, the host cell culture is carried out under aerobic conditions. It can be done.

[0127] Advantageously, the polypeptides disclosed herein can be purified by, for example, centrifugation techniques known to those skilled in the art. The enzymes having pyropheophytinase activity are recovered or isolated from the fermentation medium by extraction or filtration. Recovery of the polypeptide may also involve the destruction of the cells in which the polypeptide is produced. Disruption of the cells can be accomplished using glass beads and / or sonication as known to those skilled in the art. The polypeptide having decolorizing enzyme activity can be used to decolorize oil containing chlorophyll derivatives. Process for handling

[0128] In one embodiment, the process of the present disclosure also comprises a process for producing a dye having a decolorizing enzyme activity as disclosed herein. A polypeptide that inhibits the chloroform reaction or a composition comprising said polypeptide as disclosed herein, In one embodiment, the method further comprises treating oils containing pyrophytin derivatives (e.g., pyropheophytin). In the present invention, the polypeptides disclosed herein have pyropheophytinase activity. In one embodiment, the polypeptide has pheophytinase activity. The polypeptide has pyropheophytinase activity and pheophytinase activity. In another embodiment, the polypeptide has pyropheophytinase activity, pheophytinase activity, and chlorophyllase activity.

[0129] As discussed herein, a polypeptide having pyropheophytinase activity is It can be hydrolyzed to pyropheophorbide, and pheophytin can be hydrolyzed to pyropheophorbide. The polypeptide having the enzyme activity can hydrolyze pheophytin to pheophorbide. The polypeptides with chlorophyllase activity can convert chlorophyll to chlorophyllide. Thus, in one embodiment, the decolorizing enzyme treatment can hydrolyze 1 In various embodiments, the concentration of one or more chlorophyll substrates can be reduced. The chlorophyll substrates were chlorophyll, pheophytin, and / or pyropheophytin. For example, the treatment with a decolorizing enzyme can remove chlorophyll substrates present in the oil before treatment. The total concentration of chlorophyll substrates in the oil was increased by at least 5 times compared to the total concentration (by weight) of %, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, At least 80% by weight, at least 90% by weight, at least 95% by weight, at least 99% by weight The total concentration of chlorophyll substrates can be reduced by 100% or 100% by weight. , pyropheophytin becomes pyropheophorbide, and pheophytin becomes pheophorbide , and / or may be the result of conversion of chlorophyll to chlorophyllide.

[0130] In another embodiment, the chlorophyll substrate in the oil comprises pyropheophytin, At least a portion of pheophytin is converted to pyropheophorbide as a result of the decolorizing enzyme treatment. For example, the decolorization enzyme treatment converts pyropheoside, which is present in the oil before the decolorization enzyme treatment, into pyropheoside, which is present in the oil before the decolorization enzyme treatment. The total concentration of pyropheophytin in the oil is low compared to the total concentration of pyropheophytin (by weight). At least 5% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, At least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight %, at least 80%, at least 90%, at least 95%, The total concentration of pyropheophytin can be reduced by 99% or 100% by weight. The decrease in intensity may be the result of conversion of pyropheophytin to pyropheophorbide. In another embodiment, the chlorophyll substrate in the oil comprises pheophytin, At least a portion of the pheophorbide is converted to pheophorbide as a result of the treatment. Total concentration (by weight) of pheophytins present in oil before and after decolorization enzymatic treatment The total concentration of pheophytin in the oil is at least 5% by weight, at least 10% by weight, %, at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, at least 90%, at least 95%, at least 99%, or 100% by weight The decrease in the total concentration of pheophytin is due to the fact that pheophytin is In such an embodiment, the polypeptide may be the result of conversion to a phenylide. Shows ophitinase activity.

[0131] In another embodiment, the chlorophyll substrate in the oil comprises chlorophyll, At least a portion of the chlorophyllide is converted to chlorophyllide as a result of the decolorizing enzyme treatment. For example, The total concentration (by weight) of chlorophyll present in the oil before the decolorization enzyme treatment was The total concentration of chlorophyll in the oil is at least 5% by weight and at least 10% by weight. %, at least 20% by weight, at least 30% by weight, at least 40% by weight, at least at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, At least 90% by weight, at least 95% by weight, at least 99% by weight, or 100% by weight The decrease in the total concentration of chlorophyll is due to the fact that chlorophyll is In such an embodiment, the polypeptide may be the result of being converted to a chloroform. It shows enzyme activity.

[0132] Oils containing pyropheophytin, pheophytin, and / or chlorophyll are phospholipids. Optionally, the oils disclosed herein may be processed in a manner similar to that disclosed herein, but which may further comprise other substrates such as fats and oils. The process for obtaining phospholipids further comprises removal of phospholipids, as described below.

[0133] To remove one or more undesirable chlorophyll derivatives from the oil, chlorophyll Any oil containing chlorophyll derivatives, including substrates, may be treated according to the present process. , triacylglycerol-based oils, including various plant-based or algae-based oils. In terms of form, suitable oils that can be used in connection with the present process include canola oil, hiyashi Mashi oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hemp seed oil , linseed oil, mango kernel oil, meadowfoam oil, cow foot oil, olive oil, palm oil, palm Kernel oil, palm olein, peanut oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, sesame oil oil, soybean oil, sunflower or sunflower seed oil, tall oil, camellia oil, vegetable oil, and oil derived from algae In one embodiment, the processing according to the present disclosure includes, but is not limited to, The oils that can be used are canola oil, corn oil, olive oil, palm oil, palm kernel oil, and peanut oil. oil, canola oil, rapeseed oil, rice bran oil, sesame oil, soybean oil, and sunflower seed oil. In one embodiment, the oil is an algae-derived oil.

[0134] The contact of an oil containing one or more chlorophyll substrates with a polypeptide having decolorizing enzyme activity. The contacting can be carried out for any suitable time and at any suitable pH and temperature. The contacting is carried out at a pH and temperature that is applied during the degumming of triacylglycerol oils. The preferred pH range is pH 2 to pH 10, for example, pH 3 to pH 9, pH 4 to pH 8. , pH 5 to pH 7, pH 5 to 8, or pH 6.5 to 7.5. , 4.0 to 7.5, or 4.5 to 8.0, or 4.5 to 7.0, In one embodiment, the oil is contacted with a pH of 4.0 to 5.0, or more specifically, 4.5. In another embodiment, the polypeptide is contacted with oil at a pH of 7.0. Let them touch it.

[0135] The oil containing one or more chlorophyll substrates is treated with a decolorizing enzyme having the decolorizing enzyme activity disclosed herein. The temperature suitable for contacting the polypeptide is 10° C. to 90° C., for example, 20° C. to 80° C. ℃, 30℃ to 70℃, 45℃ to 70℃, 40℃ to 60℃, or 50℃ to 65℃. stomach.

[0136] For example, a polypeptide having decolorizing enzyme activity for oils containing one or more chlorophyll substrates can be prepared. Contact with the solvent is performed at pH 5-8 and temperature 40°C-60°C, or pH 4.5-7.0 and temperature 4 0℃~60℃, or pH 7.0 and temperature 45℃~70℃, or pH 7.0 and temperature 50℃ It can be carried out at ~65°C.

[0137] The polypeptide having decolorizing enzyme activity can be prepared by the addition of any suitable amount of oil containing chlorophyll substrate. For example, the polypeptide may be administered at 1 to 50 U / gram (processing oil), e.g., 1 .4~50U / g (processing oil) or 5~50U / g (processing oil) One unit is defined according to the enzyme activity taught in the Examples below.

[0138] Surprisingly, decolorizing enzymes, such as pyropheophytinase activity, disclosed herein A polypeptide having the formula (I) is more resistant to acidic and caustic conditions than a reference polypeptide. It was found that the chlorophyll substrate was converted to the chlorophyll product in a larger amount than that of the chlorophyll substrate. A peptide is a polypeptide comprising an amino acid sequence according to SEQ ID NO: 12. The present study identified Chlamydomonas reinhardtii ( ) Contains chlorophyllase.

[0139] The contact of an oil containing one or more chlorophyll substrates with a polypeptide having decolorizing enzyme activity. The contacting can be carried out during degumming. Degumming of the oil can be carried out by pressing and / or hexane extraction, For example, those disclosed in International Publication No. 2005 / 086900 or International Publication No. 2011 / 046812 Degumming, refining, bleaching and degumming in the presence of degumming enzymes such as phospholipases. It involves several processing steps such as flavoring. The pyrophenols of oils containing one or more chlorophyll substrates are Possessing ophitinase activity, pheophytinase activity, and / or chlorophyllase activity Contact with a polypeptide having the structure described in more detail below may be used to treat the drift of oil during degumming. It may be carried out during the white stage.

[0140] A polypeptide having pyropheophytinase activity is added to a triacylglycerol oil or The contact with oil, such as algal oil, may involve dissolving an aqueous solution comprising a polypeptide disclosed herein in the oil. The method may include dispersing the treated polypeptide having pyropheophytinase activity. The oil to be used is typically 0.5 to 10 w / w% water, for example 1 to 10 w / w% water, 1 ~5w / w% water, 2~8w / w% water, 2~4w / w% water, 3~6w / w% water, 0 0.5-5w / w% water, 1-3w / w%, 1.5-2w / w% water, or 5w / w% water Includes.

[0141] Polypeptides were incubated for 5 min to 24 h, 10 min to 12 h, 15 min to 10 h, and 0.5 to May be contacted with oil for periods of 24 hours, 1 to 12 hours, 1.5 to 6 hours, or 2 to 4 hours. In one embodiment, the polypeptide may be contacted with the oil for 2 hours. After this contact, the aqueous phase is typically and the oil phase separates.

[0142] The oils treated in the processes disclosed herein may be crude non-degummed, degummed (water degummed, Enzyme degumming, or acid degumming), caustic refining, or caustic refining and water-washed oil, or water-degummed oil In one particular embodiment, the oil comprises a non-degummed crude oil. The oil is usually extracted by pressing or solvent extraction and is generally low in free fatty acids (FFA) and phospholipids. Degummed oil is oil from which most of the phospholipids have been removed. The gum oil contains 0.5 to 200 ppm atomic phosphorus, e.g., 1 to 100 ppm atomic phosphorus, e.g. Refined oil contains 5 to 50 ppm of atomic phosphorus. FFAs are neutralized by caustic treatment and removed. Caustic treatment of oil usually involves treating the oil with sodium hydroxide. .

[0143] For example, the use of decolorizing enzymes disclosed herein (e.g., pyropheophylline) in oil degumming. The contact of the polypeptide having tRNAse activity with the oil may be carried out at any suitable temperature, for example, at 45 It can be carried out at a temperature of up to 70°C, for example 50 to 65°C.

[0144] For example, the use of decolorizing enzymes disclosed herein (e.g., pyropheophylline) in oil degumming. The contact of the polypeptide having tRNAse activity with the oil may be performed at any suitable pH, for example at pH 3. The reaction may be carried out at a pH of 0.5 to 8.0, for example at a pH of 4 to 7.5, for example at a pH of 4.5 to 7.0. do.

[0145] In one embodiment, the treatment process disclosed herein comprises subjecting the oil to water degumming. As discussed herein, water degumming typically contains a large amount of hydratable phospholipids. Due to its mild properties, the resulting phospholipids are called lecithin (natural milk fat). The oil obtained from this process is generally partially Although the gum has only been removed once, it is referred to as "demugmed" in the industry.

[0146] Thus, in one aspect, the treatment process of the present disclosure is a process for the production of chlorophyll derivatives (chlorophyll derivatives). Contacting an oil (e.g., undegummed crude oil) containing a cellulose substrate (e.g., a gel substrate) with water and a polypeptide of the present disclosure. Typically, the temperature of the oil is between 45 and 70°C, for example between 50 and 65°C. Water may be added in an amount of 1 to 5 w / w%, for example 2 to 4 w / w%. ~50U / gram (processing oil), for example, 1.4~50U / gram (processing oil), or 5~50 The polypeptides and water disclosed herein may be administered in an amount of 100 U / gram of processed oil. The polypeptide may be added as a single composition or may be added separately from the water. Typically, no acid or base is added to the resulting mixture and the process is carried out at a neutral pH (e.g. After contact with the polypeptide and water, the mixture is optionally subjected to shear mixing. The oil may then be mixed using a stirrer (e.g., a continuous stirred reactor). Use), 0.5 to 24 hours, or 1 to 12 hours, or 1.5 to 6 hours, or 2 to 4 hours Incubate the oil for a period of time, which aids in the hydration of the phospholipids present in the oil. After incubation and stirring, the oil is heated to a temperature of 70-85°C. The resulting oil is then separated by settling, filtration, and or by the industrial practice of centrifugation. Flow, water degummed oil and wet gum result.

[0147] In another embodiment, the treatment process disclosed herein comprises subjecting oil to enzyme-assisted water degumming. As discussed herein, enzyme-assisted water degumming typically involves the addition of a large amount of It is applied to crude oils containing hydratable phospholipids, the purpose of which is to react all of the hydratable phospholipids. and converting it to diacylglycerol, which improves oil yield while maintaining the non-hydratable phospholipids in the oil. The enzymes used in this process are phospholipase C (PLC) and phosphatidylinositol-phospholipase (PI-PLC). can be.

[0148] Thus, in one aspect, the treatment process of the present disclosure is a process for the production of chlorophyll derivatives (chlorophyll derivatives). The oil (e.g., undegummed crude oil) containing the oil (e.g., a crude oil matrix) is mixed with water, a polypeptide of the present disclosure, and The additional enzymes include PLC, PI-PLC, and the like. In one embodiment, the additional enzyme may be selected from the group consisting of PLC and P Typically, the oil temperature is between 45 and 70°C, for example between 50 and 65°C. Water may be added in an amount of 1 to 5 w / w%, for example 2 to 4 w / w%. is 1 to 50 U / gram (processing oil), for example 1.4 to 50 U / gram (processing oil), or It can be administered in an amount of ~50 U / gram of processed oil. PLC (e.g., Purifine PLC) is 50 to 500 ppm, for example 100 to 400 ppm or 150 to 250 ppm The amount of PI-PLC may be 50 to 500 ppm, for example 100 to 400 ppm. In one embodiment, the additional enzyme may be added in an amount of 150-250 ppm or 150-250 ppm. Purifine 4G contains both LC and PI-PLC. Purifine 4G is 50 to 500 ppm, for example 100 to 400 ppm or The polypeptides disclosed herein may be added in an amount of 150 to 250 ppm. The enzyme and water may be added as a single composition or may be added in the form of a polysaccharide as disclosed herein. The peptide, additional enzyme, and water may be added separately. Typically, the resulting mixture is No acid or base is added and the process proceeds at neutral pH (e.g., around pH 7.0). After contact with the lipid, additional enzymes, and water, the composition may be optionally mixed using a shear mixer. The materials may be mixed. A suitable shear mixer is the continuous shear mixer IKA DispaxR reactor. Then, with stirring (e.g., using a continuous stirred reactor), The composition is then incubated for 0.5 to 24 hours, or 1 to 12 hours, or 1.5 to 6 hours, or 2 to 4 hours. This resulted in the conversion of PC, PE, and PI in the oil to diacylglycerol. After incubation and stirring, the composition is heated to a temperature of 70 to 85°C, e.g., 85°C. The resulting composition is separated by industrial practice of settling, filtration, or centrifugation. By centrifugation, two streams are separated: water-degummed oil and a heavy phase (water, denatured tannic acid). The resulting product contains proteins and phosphorus compounds.

[0149] In another embodiment, the treatment process disclosed herein comprises subjecting the oil to enzymatic degumming. Enzymatic degumming may be performed on crude oil or on water degumming, enzyme assisted water degumming, or acid degumming. It can be applied to oils that have already been degummed by various methods such as degumming. Processors wishing to produce lecithin for the commercial or industrial market must first sterilize the lecithin before further processing. The oil may then be water degummed. Destruction of the phospholipids is unacceptable in lecithin applications.

[0150] Thus, in one aspect, the treatment process of the present disclosure is a process for the production of chlorophyll derivatives (chlorophyll derivatives). Compositions including oils (e.g., crude oils or oils that have already been degummed), For example, 100 to 1000 ppm, e.g., For example, by adding an acid (e.g., citric acid or phosphoric acid) in an amount of 500 ppm, The pH of the oil may be adjusted before contacting it with the polypeptide. Typically, the pH is between 4.5 and 5. The pH of the oil is adjusted to 8.0, for example 4.5 to 7.0. Typically, the temperature of the oil is adjusted After the addition of the acid, the resulting oil is mixed with the mixer (e.g., high shear, The mixing time may range from 5 minutes to 24 hours depending on the type of mixer (mixer, stirrer, etc.). When used, less mixing time is required but lower shear may be applied. Understand that longer mixing times may be required if the mixing is carried out in a non-linear manner (e.g., if a simple stirrer is used). After adjusting the pH, the composition (e.g., oil) is heated to 45-70°C, e.g., 50-65°C. Cool to °C and add water, a polypeptide of the disclosure, and optionally additional enzyme. The enzyme (if used) is selected from the group consisting of PLC, PI-PLC, and combinations thereof. In one embodiment, the additional enzyme includes both PLC and PI-PLC. Water may be added in an amount of 1 to 5 w / w%, for example 2 to 4 w / w%. 1 to 50 U / gram (processing oil), for example, 1.4 to 50 U / gram (processing oil), or 5 to 5 PLC (e.g., Purifine PL) can be added in an amount of 0 U / gram of processing oil. C) is 50 to 500 ppm, for example 100 to 400 ppm or 150 to 250 ppm. PI-PLC may be added in an amount of 50 to 500 ppm, for example 100 to 400 ppm. Alternatively, the additional enzyme may be added in an amount of 150-250 ppm. and Purifine 4G, which contains both PI-PLC. Purifine 4G is 50 to 500 ppm, for example 100 to 400 ppm or 15 The polypeptide, additional enzymes (if present), and The polypeptide, additional enzyme, and water may be added as a single composition, or the polypeptide, additional enzyme, and water may be added in a single composition. may be added separately.

[0151] After contact with the polypeptide, additional enzymes (if present), and water, the mixture is mixed in a high shear mixer. The composition may be mixed using a continuous shear mixer such as the IKA Dis The shear mixing is particularly useful when the composition being treated is crude undegummed oil. If the reaction is or includes, then it is optional. , 0.5 to 24 hours, or 1 to 12 hours, or 1.5 to 6 hours, or 2 to 4 hours, composition Stir.

[0152] After mixing, phospholipase A (PLA) enzyme is added to the oil. In one embodiment, the enzyme is a PLA1 enzyme and / or a PLA2 enzyme. Phospholipase activity was observed in the presence of phospholipids with activity in triacylglycerol oil. The amino acid sequences having phospholipase activity have been widely reported in the art. Commercially available PLA1 enzymes with this property include Lecitase® Ultra and Commercially available PLA2 enzymes with phospholipase activity include QuaraLowP. Examples include Rohalase Xtra and LysoMax. The enzyme added may vary depending on the manufacturer and the type of continuous stirred tank reactor used. After adding the PLA enzyme, the oil is mixed using a shear mixer to A suitable shear mixer is the continuous shear mixer IKA DispaxReactor. The oil is then incubated with stirring (e.g., using a continuous stirring reactor). The oil is incubated with the PLA1 enzyme for 1-8 hours, or 2-7 hours, or The reaction may be allowed to proceed for 3-6 hours. After incubation and stirring, the oil is heated to 70-85°C, e.g. The reaction time is determined by the amount of PLA and the amount of non-hydratable phospholipid (NHP) ( For example, the concentration of phosphatidic acid (Ca and Mg salts) can vary. The separation can be achieved by the industrial practice of sedimentation, filtration, or centrifugation.

[0153] In another embodiment, the treatment process of the present disclosure comprises the step of: The method includes contacting an oil, particularly a single refined oil, containing a refined or unrefined oil, with a polypeptide of the present disclosure. Typically, the temperature of the oil is between 45 and 70° C., for example between 50 and 65° C. 1 to 50 U / gram (processing oil), for example, 1.4 to 50 U / gram (processing oil), or 5 to 5 Water may be added in an amount of 1 to 10 w / w%, for example 2 to 8 w / w. The polypeptides and the like can be added to the oil in an amount of 0.1 w / w%, 3-6 w / w%, or 5 w / w%. The polypeptide and water may be added as a single composition, or the polypeptide may be added separately from the water. Typically, no acid or base is added to the resulting mixture and the process is carried out at a neutral pH ( After contact with the polypeptide and water, a shear mixer is used. The oil may be mixed using a continuous shear mixer such as the IKA Dispax continuous shear mixer. Incubate the oil with stirring for 1.5 to 3 hours, e.g., 2 hours. After incubation and stirring, the oil is heated to a temperature of 70-85°C. The oil can be separated by the industrial practices of settling, filtration, or centrifugation.

[0154] In another embodiment, the present invention provides a method for treating oils containing chlorophyll derivatives. The process for the preparation of the chlorophyll further comprises removing pyropheophorbides and / or pheophorbides. In one embodiment, the oil containing the chlorophyll derivatives disclosed herein may be treated. The process for producing the chlorophyllides, pyropheophorbides, and / or pheophorbides is The method may further include removing rubido. and / or chlorophyllide is added during the water washing of oils, as part of the chemical refining process (to remove excess soap). water addition) or by using the adsorbent of the present disclosure in a deodorization process. can.

[0155] In one embodiment, the oil containing chlorophyll derivatives, e.g. pyropheophytin, is treated with The processes involved in the synthesis of phospholipases, chlorophyllases, pheophytinases, and pyrofumab and combinations thereof. Suitable phospholipases include phospholipase A, phospholipase B, phospholipase C, phospholipase D, phospholipase E, phospholipase F, phospholipase H, phospholipase I ... Phospholipase B, and / or phospholipase C, or any suitable combination of these enzymes. The oil can be treated with phospholipase, so-called enzymatic degumming. This reduces the phospholipid content in the oil, which in turn reduces the atomic phosphorus content in the oil. .

[0156] The present disclosure also relates to oils (e.g., triglycerides, sorbitol, sorbitol derivatives ... The present invention also relates to the use of glycerol oils, vegetable oils, algae-derived oils, etc. The triacylglycerol oil obtainable by the process may be any of the oils disclosed herein. The present invention may include a polypeptide having decolorizing enzyme activity, such as pyropheophytinase activity, which is . [Brief description of the drawings]

[0157] [Figure 1]FIG. 1 is an overview of the conversion of chlorophyll to pheophytin and pyropheophytin and to their respective reaction products chlorophyllide, pheophorbide, and pyropheophorbide. Compound A is shown with a methyl group at the C7 position. Compound B has an aldehyde at the C7 group instead of a methyl group. Structures are taken from PubChem, NCBI.

[0158] [Diagram 2] Figure 2 shows the HPLC results of incubation of pheophytins a and b and pyropheophytins a and b with various putative chlorophyllases for 24 hours at pH 7 and 50°C. The amounts of substrates pheophytins a and b and pyropheophytins a and b, and reaction products pheophorbides a and b and pyropheophorbides a and b, are shown as peak surface areas. The first two columns show the sum of reaction products and substrates, and "nd" means not detectable. [Figure 2A] Figure 2A shows the HPLC results of incubation of pheophytins a and b and pyropheophytins a and b with various putative chlorophyllases for 24 hours at pH 7 and 50°C. The amounts of substrates pheophytins a and b and pyropheophytins a and b, and reaction products pheophorbides a and b and pyropheophorbides a and b, are shown as peak surface areas. The first two columns show the sum of reaction products and substrates, and "nd" means not detectable.

[0159] [Diagram 3] Figure 3 shows the HPLC results of incubation of pheophytins a and b and pyropheophytins a and b with various putative chlorophyllases at pH 5 and 50°C for 24 hours. The amounts of substrates pheophytins a and b and pyropheophytins a and b, and reaction products pheophorbides a and b and pyropheophorbides a and b, are shown as peak surface areas. The first two columns show the sum of reaction products and substrates, and "nd" means not detectable.

[0160] [Figure 4A] FIG. 4A: Chlorophyll derivatives in canola oil after 24 hours of incubation with the barley-derived CHL26 enzyme or the reference enzyme ELDC94 from Chlamydomonas reinhardtii. [Figure 4B] FIG. 4B: Phosphorus compounds in canola oil after 24 hours of incubation with the barley-derived CHL26 enzyme or the reference enzyme ELDC94 from Chlamydomonas reinhardtii.

[0161] [Figure 5A] FIG. 5A: Chlorophyll derivatives in canola oil and soybean oil after several incubations with the CHL26 enzyme from barley and / or the reference enzyme ELDC94 from Chlamydomonas reinhardtii under various reaction conditions. [Figure 5B] FIG. 5B: Phosphorus compounds in canola oil and soybean oil after several incubations with the CHL26 enzyme from barley and / or the reference enzyme ELDC94 from Chlamydomonas reinhardtii under various reaction conditions. [Figure 5C] FIG. 5C shows chlorophyll derivatives in the resulting gums in canola oil and soybean oil after several incubations with the CHL26 enzyme from barley and / or the reference enzyme ELDC94 from Chlamydomonas reinhardtii under various reaction conditions.

[0162] [Figure 6] FIG. 6: Chlorophyll derivatives in canola and soybean oils after caustic refining and incubation with the CHL26 enzyme from barley or the reference enzyme ELDC94 from Chlamydomonas reinhardtii. [Figure 6A] FIG. 6A: Chlorophyll derivatives in canola and soybean oils after caustic refining and incubation with the CHL26 enzyme from barley or the reference enzyme ELDC94 from Chlamydomonas reinhardtii.

[0163] [Figure 7]FIG. 7 is a schematic diagram of a typical chemical refining process for triacylglycerol-based oils. Oilseeds (rapeseed or soybean), oil fruit plants (palm), or single cell sources (algae) are solvent extracted and / or pressed to obtain crude oil. The crude oil is then treated with citric or phosphoric acid to react with non-hydratable phospholipids, and then sodium hydroxide is added to neutralize the free fatty acids and form sodium soaps. The sodium soaps form an emulsion with the water present, allowing the non-hydratable phospholipids to be removed 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. Industrial acidic silica has no ability to remove chlorophyll or chlorophyll derivatives. The oil is then treated with bleaching earth to remove soaps, phospholipids, and chlorophyll and chlorophyll derivatives present in the oil. It is the final step in the deodorization process, steam distillation at high temperature and a vacuum of less than 5 mbar. Distillation primarily removes peroxides, aldehydes, ketones, and other aroma compounds. Distillation also destroys beta-carotene and removes the remaining free fatty acids (0.1 percent) to reach a final free fatty acid (FFA) concentration of 0.02 to 0.05 percent.

[0164] [Figure 8]Figure 8 is a schematic diagram of a typical enzymatic degumming / physical refining process. Crude oil is treated with phosphoric acid or citric acid to allow non-hydratable phospholipids to lose calcium or magnesium bound to it at a pH of approximately 2. Sodium hydroxide is then added to bring the pH above 4 for citric acid or above 6 for phosphoric acid to allow phospholipases to act and leave very low residual phosphorus (<5 ppm) after the enzymatic reaction with PLA. Alternatively, PLA may be reacted with PLC and / or PI-PLC to maximize oil yield and still leave very low residual phosphorus to allow physical refining. The oil is then washed or treated with acidic silica followed or combined with bleaching earth. After the bleaching process, which results in a chlorophyll concentration of less than 50 ppb, the oil is physically refined in a deodorizer. High temperature steam distillation removes all of the compounds mentioned above in Figure 7, but its main purpose is to remove FFAs. The FFAs are distilled and collected in a scrubber. Very limited neutral oil losses occur in the deodorization process, compared to the losses associated with emulsions formed in water degumming or chemical refining.

[0165] [Figure 9] 9 is a schematic diagram of the use of decolorizing enzymes in a water degumming process or an enzyme-assisted water degumming process. The decolorizing enzymes may be added to water at 60° C., or PLC, or a combination of PLC and PI-PLC. After 2 hours of incubation, the oil is heated to 70-85° C. and centrifuged to remove reacted gums and reacted chlorophyll derivatives.

[0166] [Figure 10]FIG. 10 is a schematic diagram of an enzymatic degumming process modified to include treatment with the disclosed decolorizing enzymes and silica adsorbent (such as MgO treated adsorbent). First, the crude oil is treated with citric acid to a pH of approximately 2 to dissociate bound calcium and magnesium ions and raise the pH to above 4, a pH at which the PLC and decolorizing enzymes can operate efficiently. One to five percent water is added for the hydrolysis reaction. After completion of the PLC and decolorizing enzyme incubation, PLA1 or PLA2 may be added to react with the non-hydratable phospholipids present in the oil. After an additional 2 to 6 hours of incubation, the oil is heated to 70 to 85° C. and centrifuged to remove reacted gums and chlorophyll derivatives to produce an oil with less than 5 ppm residual phosphorus in the oil. The enzymatically degummed oil may then be contacted with the disclosed silica adsorbent, optionally under vacuum, to further remove chlorophyll derivatives and trace metals as described herein.

[0167] [Figure 11] FIG. 11 is a schematic diagram of a chemical refining process using decolorizing enzymes followed by treatment with the silica sorbent of the present disclosure. The pH in the early steps of the process is very low (approximately 2) and very high (approximately 14), so decolorizing enzymes do not need to be added in the acid or caustic addition step. The decolorizing enzymes need to be added in the refined oil after the initial centrifugation step. It is advantageous to add the decolorizing enzymes in the washing step at a temperature suitable for the enzymes (50-65° C.). Incubate for at least 2 hours, followed by heating to 70-85° C. before centrifugation. The oil is then further processed by contacting with the silica sorbent of the present disclosure, optionally under vacuum, to further remove chlorophyll derivatives and trace metals as described herein.

[0168] [Figure 12]12 is a schematic diagram of an enzymatic degumming / physical refining process modified to include treatment with a silica adsorbent of the present disclosure but without the decolorizing enzyme treatment. The enzymatically degummed oil or the enzymatically degummed and washed oil is contacted with a silica adsorbent of the present disclosure, optionally under vacuum, and optionally with a bleaching earth to remove chlorophyll derivatives and trace metals as described herein.

[0169] [Figure 13] 13 is a schematic diagram of a chemical refining process for triacylglycerol-based oils modified to include treatment with a silica-based adsorbent of the present disclosure but without a decolorizing enzyme treatment. Refined or first refined oils are contacted with a silica-based adsorbent of the present disclosure, optionally under vacuum, and optionally with a bleaching earth to remove chlorophyll derivatives and trace metals as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0170] array SEQ ID NO:1 = C having a decolorizing enzyme containing pyropheophytinase activity from barley HL26 polypeptide. MASAGDVFDHGRHGTSLARVEQAKNTRCSAASRVDADA QAQQSPPKPLLVAAPCDAGEYPVVVFLHGYLCNNYFYSQL IQHVASHGFIVVCPQLYTVSGPDTTSEINSAAAVIDWLAA GLSSKLAPGIRPNLAAVSISGHSRGGKVAFALGLGHAKTS LPLAALIAVDPVDGTGMGNQTPPPILAYKPNAIRVPAPVM VIGTGLGELPRNALFPPCAPLGVSHAAFYDECAAPACHLV ARDYGHTDMMDDVTTGAKGLATRALCKSGGARAPMRRFVA GAMVAFLNKWVEGKPEWLDAVREQTVAAPVVLSAVEFRDE SEQ ID NO:2: Barley CHL for expression in Pseudomonas fluorescens 26 encoding a polypeptide having a decolorizing enzyme containing pyropheophytinase activity. A codon-optimized nucleic acid sequence. SEQ ID NO:3; CHL25 from Gossypium raimondii Putative chlorophyllase SEQ ID NO: 4; CHL27 putative chlorophyll from date palm (Phoenix dactylifera) Rase SEQ ID NO:5; CHL28 putative chlorogenic acid sequence from Wollemi pine (Wollemia nobilis) Phyllase SEQ ID NO:6; CHL29 putative chlorophyllase from cucumber (Cucumis sativus) SEQ ID NO: 7: CHL30 primer from Tarenaya hassleriana Constant Chlorophyllase SEQ ID NO:8; CHL31 putative chlorophyll from potato (Solanum tuberosum) -ze SEQ ID NO: 9; CHL32 putative chlorophyll from poplar (Populus trichocarpa) Z SEQ ID NO:10; CHL33 putative chlorophyll from mungbean (Vigna radiata) Z SEQ ID NO:11; N1 negative control, green fluorescent protein (GFP) SEQ ID NO: 12; P2, konamidrin cyclopropane having pyropheophytinase activity SEQ ID NO:12 is also referred to herein as ELDC94. SEQ ID NO:13; Spel site and ribosome binding site SEQ ID NO:14; stop codon and Xhol site. EXAMPLES

[0171] Materials and Methods overview Overexpression of the enzyme in the host cell, genetic modification of the host cell, or hybridization Standard genetic techniques, such as the chromosomal replication technique, were developed by Sambrook and Russell (2 001) “Molecular Cloning: A Laboratory Manual al (3 rd edition),Cold Spring Harbor Labo ratory,Cold Spring Harbor Laboratory Pre ss,or F.Ausubel et al,eds.,”Current prot. ocols in molecular biology”,Green Publis hing and Wiley Interscience, New York (19 87) and other methods known in the art. If not specified, Milli-Q water is used. Analysis method:

[0172] pH - the stoichiometric addition of acid and base relative to the percentage of water added to the oil. The reaction of 2% water in 2000 grams gives 40 grams, which is 50 percent of 2.0 grams. Add the citric acid solution, plus 1.6 mL of 4 M sodium hydroxide. An aqueous solution with a pH of 4.5 is obtained. The pH of the oil always remains at 7.

[0173] Soap-American Oil Chemists' Society Office cial Method Cc 13a-43, revised 2017. Free fatty acids - Amer ican Oil Chemists' Society Official Meth od Ca 5a-40, revised 2017.

[0174] Colors - American Oil Chemists' Society Offic ial Method Ce 13e-92, revised 2017. 51 / 4” cell colorimeter PFX-950.

[0175] Phosphorus and Trace Metals - American Oil Chemists' Society y Official Method Ca 17-01-43, revised 2017.

[0176] A phospholipid composition, 31 In the case of P NMR (also called 31-P NMR), % heavy water (D2O, Cambridge Isotope Laboratories, D LM-4), 25 mg / mL deoxycholic acid (Sigma D2510), 5.84 m Disodium EDTA at 100 µg / mL (Titriplex III, Merck 1084 18), and 5.45 mg / mL Tris base (tris(hydroxymethyl)aminomethane 1 mL of aqueous medium containing demineralized water containing 10% DOL Disperse 10 μL of the dispersion, adjust the pH to pH 9 with 4N KOH, and add 2 mg / mL of TIP internal standard (tri-isopropyl phosphate, Aldrich 5546 69) (accurately weighed) was added.

[0177] All samples were analyzed using a Bruker 400 with a Prodigy BBO probe. The measurements were performed using a 1000-mHz Avancell NMR spectrometer. The probe head temperature was , set it to 300K.

[0178] Measurements for quantification were performed with semi-quantitative parameters: 128 scans, 90° pulse, D1 = The time was 5 seconds. Sample dry weight (DOL) values ​​are reported in μmol / g.

[0179] Analysis of Green Content by UV / Vis - Using the AOCS UV / Vis method, Measures the green content of oil. AOCS UV / Vis method is CC 13d-55, As stated in the 2017 reauthorization.

[0180] Analysis of chlorophyll derivatives by HPLC-FLU

[0181] λex 410nm / λem 666nm for A compounds, λem 666nm for B compounds Hwa ng et al J.Food Hyg.Soc.Japan Vol.46,No. 2,45-48 Pheophytin A and B, pyropheophytin A and B, and their phosphatidylcholine In addition to rubido, chlorophyll and chlorophyllide analyses were performed.

[0182] Analysis of the moisture content of the adsorbent

[0183] The moisture content (wt%) was determined by heating the adsorbent to 1750 F until a constant weight was observed. The moisture content is equal to the mass loss divided by the original mass of the material, It is expressed as a percentage. Sample preparation

[0184] The oil sample was diluted in acetone (9 mL of acetone for 1 g of oil) and diluted with 1400 The clear supernatant was transferred to an injection vial and a 10 μL sample was added. The chlorophyll concentration was very low in all real oil samples. Therefore, it was not considered in the analysis. Data analysis

[0185] The peak surface area (arbitrary units) of the chromatogram represents the pheochromic acid present in the oil sample. The amounts of pyropheophytin, pyropheophytin, pheophorbide, and pyropheophorbide are shown. Figures 2 and 3 show the results of incubation with the putative chlorophyllase at pH 5 and pH 7. Pheophytin, pyropheophytin, pheophorbide, and pyropheophytin in oil samples after The peak surface area of ​​phytin, phorbide, and phytate is shown. The sum of the peak surface areas of the pheophorbide and the peak surface areas of the individual compounds are shown. The formation of pyropheobide and pyropheobide is a function of pheophytinase activity and pyropheophytinase activity, respectively. It is a measure of the presence of enzyme activity. enzyme

[0186] Purifine® Phospholipase C (PLC) and Purifine® PI-PLC® and fungal PLA1 were obtained from DSM.

[0187] Purifine® Phospholipase C is a phospholipase that is SEQ ID NO: 1 with the amino acid substitutions 63D, 131S, and 134D disclosed in US Pat. No. 6,233,363; 2, containing amino acids 38 to 282.

[0188] Purifine® PI-PLC is a compound disclosed in WO 2011 / 046812 The present invention includes the mature polypeptide according to SEQ ID NO:8 disclosed in US Pat. No. 6,399,362.

[0189] The fungal PLA1 is represented by the sequence number SEQ ID NO: 1 contains the mature amino acid sequence of equipment

[0190] The overhead mixer is an IKA RW 20 with a flat blade paddle. It was digital.

[0191] The centrifuge is a Delphi centrifuge equipped with a "Bowl Unit" for continuous separation. It was an aval Gyro-Tester. With the plug screws installed, The separator bowl was closed. Shear mixing was performed using an Ultra 1000rpm G450 rotor-stator. - This was done using a Turrax homogenizer SD-45. Silica Adsorbent

[0192] Test adsorbents SP-2113, SP-2114, SP-2115, SP-2116, S P-2117 and SP-2119 are from W.R. Grace & Co.-Conn. TRISYL® silica and TRI SYL® 300 (WR Grace & Co.-Conn., Colum (Philadelphia, Md.) is commercially available. Characteristics of Various Silicas and Adsorbents Used in the Examples are listed below. [Table 1] Example 1. Expression of putative chlorophyllases in Pseudomonas sp.

[0193] The estimated chlorophyllase (CHL) enzymes provided in the tables of Figures 2 and 3 were synthesized by Dow Glo The plasmid was expressed in a Pseudomonas system obtained from Bal Technologies Inc. (U.S. Patent Application Publication Nos. 20050130160, 20050186666, and and No. 20060110747). DNA2.0 (GeneGPS (registered trademark) technology) Optimizing gene codon usage for Pseudomonas according to the algorithm of 2-2A and 3 show the deduced chlorophyllase protein sequence. We designed 12 synthetic genes based on the sequence. For cloning purposes, the DNA sequence was , and a Spel site and a ribosome binding site (ACTAGTAGGAGGTAAC TAATG) (SEQ ID NO: 13), and a stop codon and Xhol site (T GAT GACT CG AG) (SEQ ID NO: 14).

[0194] SEQ ID NO:2 is the codon encoding the putative barley chlorophyllase SEQ ID NO:1. The optimized nucleic acid sequence is shown.

[0195] Using Spel and Xhol restriction enzyme cloning, the DNA sequence was cloned into pDOW11 69 Vector (Dow Global Technologies Inc., U.S. Patent The modified tac promoter was then inserted into the ribosome-specific ribosome-specific tac promoter (Publication No. 20080058262). The pDOW1169 vector contains genes encoding the CHL and PPH enzymes under the control of The vector was introduced into the uracil auxotrophic strain DPfl0 of Pseudomonas fluorescens. The cells were grown in M9 minimal medium (Dow Global Technology) without uracil for 48 h at 30°C. (U.S. Patent Application Publication No. 20050186666) After incubation, transformed cells were selected (Schneider et al. 200 5).

[0196] Correct transformants were cultured in 24-well sterile deep wells containing 3 mL of M9 medium. The plates were preincubated with Breaths eal(Greiner bio-one,Frickenhausen,German y) and incubated in a Microton incubator at 30°C, 550 rpm, and 80% humidity for 16 hours. Incubator shaker (Infers AG, Bottmingen, Switzerland From these cultures, the cells were incubated at 30°C, 550 rpm for 24 hours. In the meantime, place a second 24-well sterile deep well plate (Ax) containing 3 mL of M9 medium. After 8 h, the culture was incubated at 4°C for 1 h at 4°C for 30 min at 2°C for 1 h. The cells were induced with PTG (final concentration 0.3 mM) and centrifuged at 2750 rpm for 10 min. The cultures were harvested by centrifugation and the supernatant was removed. The cell pellets were stored overnight at -20°C. Cell pellets from 1 mL cultures were suspended in 1 mL lysis buffer and incubated at 37 °C for 1 h. The mixture was incubated in lysis buffer (1 mM EDTA, 50 mM Tris, pH 8, 0.2 5mg / mL lysozyme, 10mg / mL Dnasel, 25mM MgS04, and 0.03% Triton). The lysate was centrifuged at 2750 rpm for 10 min, the supernatant was removed, and It was kept. Example 2. Measurement of pyropheophytinase activity in cell-free extracts of crude canola oil judgement incubation

[0197] Crude canola of North American origin with high pheophytin and pyropheophytin contents. The enzymes in the supernatants produced in Example 1 for pyropheophytins A and B were detected using oil. Activity was determined by the following method: Oil was mixed under high shear mixing using a Silverson mixer. For pH 5, 20 mM citrate buffer was added (5% (v / v)). For pH 7, 20 mM phosphate buffer was used. Fill each well of a clotting plate with 1.425 mL of the buffer-in-oil dispersion. To each well, add 75 μL of 5% (v / v) cell-free extract (supernatant) prepared in Example 1. The list of samples tested is shown in Figures 2-2A and the table in Figure 3, which shows the results of the A positive reference containing pyropheophytinase from Rhizobium and a negative control, green fluorescent protein The microtiter plate was wrapped in plastic foil [Fasson S Each well was stirred using an individual magnetic stir bar. Incubation was performed at 50°C using an iterplate shaker. After 24 hours, samples were The samples were collected and analyzed by HPLC-FLU as described above for pheophytins A and B, and pheophytins B and pheophytins C. The presence of lopheophytins A and B and their phorbides was analyzed.

[0198] The results in Figures 2 and 3 show that only CHL26, a putative chlorophyllase derived from barley, However, at pH 7 and pH 5, all pheophytins and pyropheophytins were It was shown that each of the (pyro)pheophorbides could be hydrolyzed. Example 3. Incubation of crude canola oil with CHL26 versus time

[0199] 5% of the barley putative chlorophyllase CHL26 produced as described in Example 1 Incubation of the cell extract with crude canola oil was performed at pH 7 as described in Example 2. The same procedure was repeated. Samples were removed after 30 minutes, 2 hours, 5 hours, and 24 hours. Pyropheophytin a and b, and pheophytin a and b, pyropheophytin Pheophorbides a and b, and pheophorbides a and b were measured by HPLC as described above. It was determined.

[0200] The reaction products in Table 1, pyropheophorbide a and b, and pheophorbide a The formation of a and b is expressed as a percentage of the amount of reaction product (each phorbidide molecule) after 24 h. It is expressed as:

[0201] Table 2 shows the results after 0.5, 2, and 5 hours, expressed as a percentage of the value at t=0. The relative amounts of pheophytin and pyropheophytin as a function of time are shown (four determinations). (average of 100). [Table 2] [Table 3]

[0202] The results in Tables 1 and 2 show that the barley-derived enzyme CHL26 inhibits pheophytin and pyrrolidin. It was found that the compound was capable of hydrolyzing both pheophytin and both a and b compounds. After 2 hours, all pheophytin was converted (below the detection limit), whereas after 5 hours Almost all of the pyropheophytin was converted. Example 4. Production of CHL26 and ELDC94 by 10 L bioreactor fermentation Strains and inocula

[0203] CHL26 (SEQ ID NO: 1) and Chlamydomonas reinhardtii (ELDC94; SEQ ID NO: 1) described in Example 1 Column No. 12) A preliminary culture of a P. fluorescens strain containing chlorophyllase was added to yeast Extract, slat, and natural medium containing glycerol as a C source. This was prepared in a single-phase shake flask and used as inoculum for a 10 L fermentation at a 10% inoculation ratio as follows: and used it. 10L fermentation

[0204] The fermentation process is an industrial Pseudomonas fluorescens fermentation (fed-batch process, sugar The fermentation process was based on the use of glucose as the C source. Biomass production under exponential feeding, followed by a production phase under IPTG induction. After 23 hours of fermentation (end of biomass production phase), the enzyme production was induced IPTG was added to the culture medium to a final concentration of 0.125 mM. The feeding rate was reduced to approximately 70% of the maximum rate, and the fermentation was extended from 48 to 55 hours after inoculation.

[0205] At the end of the fermentation, the broth is killed and the enzymes are released via benzoate treatment, followed by , increasing the pH of the fermentation broth. collect

[0206] The intracellular enzymes were released by homogenization. A 12-hour cooling period was then allowed to proceed. The homogenized broth was then diluted with 30% water. Dilute it with 15% DBF (Dicalite BF), calcium chloride (20g / kg) broth), and Flocculent C577 (0.1% of the original broth) were added. The pH was adjusted to 8 and the material was clarified and ultrafiltered. The UF was diluted with 50% glycerol. The bacteria was stabilized with MEP (propanediol) to ensure complete destruction of the remaining bacteria. (methyl / ethylparaben in a solution containing 1,2-dichlorophenyl) was added and the product was diluted to approximately 15% v / v. . Activity Activity against p-NP substrate

[0207] The enzyme was assayed using the chromogenic substrate 4-nitrophenyl butyrate (Sigma N9874). Activity was determined. Substrate stock solution: 50 mM pNP-butyrate in acetonitrile. Substrate solution: Before use, the substrate stock solution was diluted with 0.2% BSA and 2.0% Triton X-100. The mixture was mixed with 0.1 M phosphate buffer (pH 7.0) containing 1:4.

[0208] In a microtiter plate, add 120 μL of phosphate buffer (same as above). Mix with 15 μL of substrate solution and equilibrate at 37°C. Add 15 μL of sample. After initiating the reaction with 50 mL of 10 ... A blank measurement was performed by adding 15 μL of buffer instead of the curve. The slope of the linear portion is used as a measure of activity. The increase in absorbance after 5 minutes should be consistently less than 1.0. The sample was diluted to

[0209] The activity is calculated as follows: U / mL = (ΔAbs / min sample - ΔAbs / min blank) / (ε ρNP NP× 5) x 1000 x 150 / 15 x DfW ε ρNP = Molar extinction coefficient of para-nitro-phenol [L.mol-1.cm-1] 5=Incubation time [min] 1000 = mmol to μmol factor 150 = Assay volume [μL] 15 = sample volume μ [L] Df = dilution factor W = weight of sample (g)

[0210] Activity is measured as the liberation of 1 μmole of p-nitrophenol per minute under the test conditions. The enzyme was expressed as the amount of enzyme reacting with the 4-nitrophenol standard solution diluted in the above phosphate buffer. Calibration is performed using a solution (Sigma N7660).

[0211] The CHL26 activity of the final formulation was 1.4 U / g (0.5 w / w%), and ELDC The activity of 94 was 87 U / g (0.04 w / w%). Example 5. Crude canola oil and reference enzyme (E) from Chlamydomonas reinhardtii under various conditions LDC94) derived from crude canola oil and barley Incubation with an enzyme (CHL26) with pyropheophytinase activity

[0212] Crude canola oil was added to barley to obtain 0.5 w / w% pure chlorophyllase CHL26. The cells were incubated with 0.04 w / w% chloroform extract and treated with 0.04 w / w% chloroform extract. The results were compared with cell-free extracts (coded ELDC94=Ref). The enzyme was prepared as described in. The incubation was carried out on a 10 g scale (15 mL of 10 g of oil in a glass reaction vessel was placed on a hotplate aluminum reaction block under temperature control. The plates were incubated on a rack. The contents were kept vigorously stirred by a magnetic bar) at three different temperatures (40, 50, and 60 °C) and under four regimes of varying aqueous phase acidity: Acidity: Pretreatment with 400 ppm citric acid; Weak acid: Pretreatment with 500 ppm citric acid and 138 ppm caustic (NaOH) ; Neutral: water only; Weakly alkaline: Pretreatment with 150 ppm NaOH.

[0213] The total water concentration during incubation was 3% w / w, which was the same as for the enzyme formulation and pretreatment. Prior to the experiment, the pretreated oil was diluted 1:1 with water to prepare an aqueous environment. The acidity was evaluated and then the pH was measured by a pH meter. This allowed the The following pH values ​​were obtained, indicating the acidity of the aqueous environment in the dispersion: acidic: pH 3.4; Weakly acidic: pH 4.5; neutral: pH 5.9, and alkaline pH 7.9.

[0214] To pretreat with citric acid, citric acid (as a 50% w / w solution) was added to the oil at 70°C. After adding and stirring at 70°C for 30 minutes, the temperature was lowered to the incubation temperature and For acidic conditions, NaOH (as a 2.0% w / w solution) was added. Only when necessary, the oil was stirred for 30 minutes at the incubation temperature. During the incubation, samples were taken at 0.5, 2, 4, and 24 hours and The concentration of each of the standards was analyzed by HPLC-Flu as described in Example 2. All substrates (chlorophyll, pheophytin, pyropheophytin-a and -b) and All reaction products (chlorophyllide, pheophorbide, pyropheophorbide-a and b) The concentrations of the substrate and reaction product were calculated by adding them up to obtain the total substrate and reaction product (mg / kg oil). All results are presented in the table below as percentages of substrate and reaction product.

[0215] The results in Tables 3, 4 and 5 show that the barley enzyme CHL26 according to SEQ ID NO: 1 inhibits acid and caustic It has a broader range of activity in the presence of chlorophyll and is more potent than the reference chlorophyllase from Chlamydomonas reinhardtii. These results indicate that the α-aminobutyric acid is also active at high temperatures. [Table 4] [Table 5] [Table 6] Example 6. Inking of crude canola oil with Chlamydomonas reinhardtii enzyme (ELDC94) hubet followed by treatment with various silicas.

[0216] 1,500 grams of crude canola oil was mixed in an overhead mixer with a square paddle. Place the flask in a 2-liter jacketed glass beaker equipped with a stirrer and rotate at 90 revolutions per minute (rpm). The jacket temperature was set at 65°C. After the oil temperature reached the set point, 20 mL of ELDC94 Chlamydomonas (algae) (prepared as described in Example 4) and 10 100 grams of deionized water was added to the oil. Covered with plastic wrap, the material was left to sit for 1 minute. The jacketed glass beaker was placed back into the overhead mixer and the plastic The material was mixed covered at 250 rpm for 24 hours.

[0217] 1.5 grams of 50% (wt%) citric acid was added to the oil mixture. The set point of the oil mixer was lowered to 55°C. Once the material reached 55°C, the oil was transferred to the shear mixer. 1.2 mL of 4N NaOH was added to the oil and sheared for 30 seconds. Purifine® Phospholipase C (PLC) and 30 grams of deionized water The oil was sheared for 1 minute while covered with plastic wrap. Place the glass beaker back on the overhead mixer and cover it again with plastic wrap. The oil was mixed at 250 rpm for 2 hours at 55°C.

[0218] Place the beaker back into the high shear mixer and add 0.1 grams of phospholipase A1 (PLA1). Add the enzyme (Lecitase Ultra) to the oil and shear for 1 minute while covered with plastic wrap. The jacketed glass beaker was placed back into the overhead mixer and again The oil was mixed at 250 rpm for 2 hours at 55°C. The water bath set point was After the oil reached 75°C, the Gyr o The oil was centrifuged using a centrifuge. A sample of the oil was collected. The gum was discarded.

[0219] The above reaction was repeated 11 times and the oils were combined and labeled "Control."

[0220] Six 500 gram samples of the enzyme-treated canola oil "control" were The oil was heated to 80°C and diluted with 0.25, 1.0, and 2 0.0, 4.0, 6.0, and 8.0 grams of Silica SP-2115 were mixed into the oil, and A vacuum of 100 mbar was then applied. The temperature was increased to 100° C. and mixed for 30 minutes. It was unexpected that the oil turned a dark green color during the adsorption process with silica. Silica (TRISYL® (Grace Davison, Columbia, Md.) or SORBSIL® silica (INEOS Silicas, Jol In previous experiments using iet,III), the color of the oil did not change. The material was filtered through a Büchner funnel. The filter discs were dark green in color, but were not stained with commercial silica. When the filter discs and cakes were used, they were always yellow in colour. The cake was a deep green color.

[0221] Two 500 gram portions of the enzyme-treated canola oil "control" obtained above were The sample was divided and added to two 1,000 mL round bottom flasks with the sorbent procedure configured. The oil was heated to 80°C and 1.0 and 2.0 grams of Silica SP-2116 were mixed into the oil. The temperature was increased to 100° C. and mixed for 30 minutes. The color of the oil did not change from the original color during the test. The vacuum was released and the material was filtered through a Buchner filter. The mixture was filtered through a funnel and the filter disc and cake were yellow in color.

[0222] Two 500 gram portions of the enzyme-treated canola oil "control" obtained above were The sample was divided and added to two 1,000 mL round bottom flasks with the sorbent procedure configured. The oil was heated to 80°C and 1.0 and 2.0 grams of Silica SP-2117 were mixed into the oil. The temperature was increased to 100° C. and mixed for 30 minutes. The color of the oil did not change from the original color during the test. The vacuum was released and the material was filtered through a Buchner filter. The mixture was filtered through a funnel and the filter disc and cake were yellow in color.

[0223] The oil contents are listed in Tables 6 and 7. [Table 7] [Table 8] CHYL = chlorophyll; PYN = pheophytin; PPYN = pyropheophytin ;POB=pheophorbides;PPOB=pyropheophorbides bd = below detection limit nd=untested

[0224] The ELDC94 Chlamydomonas (algae) enzyme produced chlorophyll and chlorophyll after 24 hours. The amount of fluorophyl derivatives was reduced from 19.86 ppm to 8.06 ppm (AOCS UV / VIS However, the industrial process The amount of chlorophyll in the chlorophyll a is not large enough for this process to be significantly effective. The enzymes that are more capable of hydrolyzing chlorophyll derivatives and chlorophyll derivatives, as well as the enzymes that hydrolyze these produced derivatives, A process is needed to remove more of the conductor.

[0225] The data above shows that silica SP-2115 has a lower resistance to metals, chlorine, and mercury than the other two silicas. The UV / Vis spectrophotometer shows the highest ability to remove chlorophyll and chlorophyll derivatives. The method was to determine whether treatment with low doses (i.e., 0.25 g and 1 g) of SP-2115 significantly reduced the (i.e., 38.49 ppm) of chlorophyll, The results show that the concentration of 100 ppm of 100% or less of the control is reduced by 2.20 and 12.84 ppm. After treatment with the lowest dose (i.e., 0.25 g) of SP-2116 and SP-2117 The amount of chlorophyll increased by 0.54 ppm and 0.17 ppm, respectively, while the maximum of chlorophyll after treatment with 1 g of SP-2116 and SP2117. The amount of EDTA decreased by 1.98 ppm and 1.83 ppm, respectively. The doses of SP-2116 and SP-2117 show the same pattern of limited reduction. The data also included additional data to more closely resemble the AOCS method for measuring green color in vegetable oils. For chlorophyll and chlorophyll derivatives, by finding the standard and response factors of It also indicates that the HPLC method for HPLC needs to be improved. Additional work on the method has been completed and are included in the examples. Example 7. Barley-derived enzymes compared with a reference enzyme (ELDC94) from Chlamydomonas reinhardtii The enzyme (CHL26) with pyropheophytinase activity and the enzyme activity of solvent-extracted crude canola. Incubation with La oil

[0226] 35 lbs of solvent extracted crude canola oil was poured into a large stainless steel container and cooled. The mixture was homogenized using a KA mixer.

[0227] After mixing, approximately 1.5 kg of crude canola was fed into an overhead chuck with a square paddle. The mixture was placed in a 2-liter jacketed glass beaker equipped with a 300-rpm mixer and rotated at 90 rpm ( The oil was mixed at 100 rpm. The jacket temperature was set at 65°C. When the oil temperature reached the set point, Then, 0.7 grams of enzyme ELDC94 (reaction 1) produced as described in Example 4 or 7.5 grams of CHL26 (reaction 2) was added to the oil along with 100 grams of deionized water. The material was shear mixed for 1 minute while covered with plastic wrap. The beaker was placed back into the overhead mixer and covered with plastic wrap. Incubated with enzyme for 24 hours at 250 rpm.

[0228] 1.5 grams of 50% (wt%) citric acid was added to the oil mixture. The set point of the oil mixer was lowered to 55°C. Once the material reached 55°C, the oil was transferred to the shear mixer. 1.2 mL of 4N NaOH was added to the oil and sheared for 30 seconds. Purifine® Phospholipase C (PLC) and 30 grams of deionized water The oil was sheared for 1 minute while covered with plastic wrap. Place the glass beaker back on the overhead mixer and cover it again with plastic wrap. The oil was mixed at 250 rpm for 2 hours at 55°C.

[0229] Place the beaker back into the high shear mixer and add 0.1 gram of fungal phospholipase Ai (PLA). 1) The enzyme was added to the oil and mixed under shear for 1 minute while covered with plastic wrap. Place the glass beaker with the nozzle back on the overhead mixer and wrap it again with plastic wrap. The oil was mixed at 250 rpm for 2 hours at 55°C. The water bath set point was increased to 75°C. After the oil reached 75°C, a Gyro centrifuge with a bowl with the holes closed was used. The oil was centrifuged using a centrifuge. Oil and gum samples were collected and analyzed as described above (using HPLC). The soil was analyzed for the presence of P, Ca, Mg and Fe, as well as chlorophyll derivatives.

[0230] Pour the oil and heavy phase mixture remaining in the centrifuge bowl into a 400 mL beaker. The oil was decanted from it. The remaining oil and heavy phase was placed in a 50 mL centrifuge tube and spun. The oil from the decanted bowl and the oil in the centrifuge tube were discarded and the heavy liquid phases were combined.

[0231] The results in Table 8 and FIG. 4a) show that pyropheophytinase activity according to SEQ ID NO:1 CHL26 enzyme reduces chlorophyll derivatives in solvent-extracted crude canola oil The chlorophyll substrates are chlorophyll, pheophytin, and picolinic acid. The chlorophyll products are chlorophyllide, pheophorbide, and pyropheophorbide. [Table 9] * Starting material (crude canola oil)

[0232] The results in Figure 4b) show that unreacted phospholipids still exist in the recovered heavy phase. , indicating that the phospholipase reaction was too short to go to completion. Example 8. Interaction of CHL26 enzyme with pressed crude canola oil under various conditions Incubation and treatment with Silica SP-2115

[0233] A 35 pound container of pressed crude canola oil was poured into a large stainless steel container and poured into the I The mixture was homogenized using a KA mixer. Reaction 3-CHL26 was incubated with PLC and PI-PLC at pH 4.5 for 2 h. After incubation, the cells were incubated with PLA1 for 2 hours.

[0234] Approximately 1.5 kg of crude canola was mixed in an overhead mixer with a square paddle. The oil was mixed at 90 rpm in a 2 L jacketed glass beaker equipped with a The jacket temperature was set at 70° C. 1.5 grams of 50% (wt%) citric acid was added to The oil was added to the mix and shear mixed for 1 minute. The jacket set point was reduced to 60°C. After the material reached 60°C, the oil was transferred to a shear mixer. 1.2 mL of 4N NaO H was added to the oil and sheared for 30 seconds. 0.3 grams of Purifine PLC (L R79.14 Feb 2018), 0.02 grams of Purifine PI-PLC 7.5 grams of CHL26 enzyme (two-rowed barley (Hor deum vulgare var.distichum (barley, plant)], and 10 0 grams of deionized water. The material was shear mixed for 1 minute while covered with plastic wrap. Place the jacketed glass beaker back into the overhead mixer and reinsert the plastic Cover with plastic wrap. The oil was mixed at 250 rpm for 2 hours at 60°C.

[0235] The jacketed glass beaker was again transferred to the hear mixer and 0. Add 0.75 grams of PLA1 (notebook, 0743B2) and shear the oil for 1 minute. Mix. Place the jacketed glass beaker back into the overhead compartment and cover with a plastic wrap. The oil was mixed and the reaction was allowed to continue for 2 hours at 250 rpm. The water bath set point was set to 75 After the oil reached 75°C, the Gyro centrifuge was used with a bowl with the hole closed. The oil was centrifuged using a centrifuge. Oil and gum samples were collected.

[0236] Pour the oil and heavy phase mixture remaining in the centrifuge bowl into a 400 mL beaker. The oil was decanted from it. The remaining oil and heavy phase was placed in a 50 mL centrifuge tube and spun. The oil from the decanted bowl and the oil in the centrifuge tube were discarded and the heavy liquid phases were combined. Reaction 4 - ELDC94 was incubated with PLC and PI-PLC at pH 4.5 for 2 h. After baiting, the cells were incubated with PLA1 for 2 hours.

[0237] 0.61 grams of the formulated enzyme solution (prepared as described in Example 4) was used to The same procedure as in reaction 1 above was used for crude ELDC94.

[0238] Split two 450 gram samples of the canola oil treated with the enzyme from reaction 4, The oil was added to two 1000 mL round-bottom flasks containing the adsorbent procedure. Heat and mix 1.0 and 2.0 grams of Silica SP-2115 into the oil and add approximately 100 ml of A vacuum of 1 bar was applied. The temperature was increased to 100° C. and mixed for 30 minutes. During the adsorption process used, the oil turned a dark green color. The vacuum was released and the material was placed in a Büchner funnel. The filter disc and cake were deep green in color. Reaction 5-CHL26 was incubated with PLC and PI-PLC at pH 4.5 for 2 h. After incubation, the cells were incubated with PLA1 for 4 hours.

[0239] The procedure was the same as reaction 1, except that the PLA1 reaction was allowed to react for 4 hours instead of just 2 hours. I followed the order.

[0240] Split two 450 gram samples of the canola oil treated with the enzyme from reaction 5, The oil was added to two 1000 mL round-bottom flasks containing the adsorbent procedure. Heat and mix 2.0 and 3.0 grams of Silica SP-2115 into the oil and add approximately 100 ml of A vacuum of 1 bar was applied. The temperature was increased to 100°C and mixed for 30 minutes. The vacuum was released. The material was filtered through a Buchner funnel. The filter disc and cake were a deep green color. Reaction 6-CHL26 was incubated with PLC and PI-PLC at pH 4.5 for 2 h. After incubation, the cells were incubated with PLA1 for 4 hours.

[0241] Reaction 3 was repeated except that twice the amount of CHL26 (15 grams total) was added to the reaction. The same procedure was followed.

[0242] Split two 450 gram samples of the canola oil treated with the enzyme from reaction 6, The oil was added to two 1000 mL round-bottom flasks containing the adsorbent procedure. Heat and mix 1.0 and 2.0 grams of Silica SP-2115 into the oil and add approximately 100 ml of A vacuum of 1 bar was applied. The temperature was increased to 100°C and mixed for 30 minutes. The vacuum was released. The material was filtered through a Buchner funnel. The filter disc and cake were a deep green color. . Reaction 7-CHL26 was incubated with PLC and PI-PLC at neutral pH for 2 h. After that, the cells were incubated with PLA1 for 4 hours.

[0243] The same procedure was followed as for reaction 1, except no pH adjustment was performed. Reaction 8-SBO CHL26 was incubated with PLC and PI-PLC at pH 4.5 for 2 h. After incubation, the cells were incubated with PLA1 for 2 hours.

[0244] The same procedure as reaction 1 was followed, except that the oil was solvent extracted crude soybean oil (SBO). I obeyed.

[0245] Divide three 450 g samples of the soybean oil treated with the enzyme from reaction 8 into two samples of the adsorbent. The oil was heated to 80° C. and added to three 1000 mL round-bottom flasks having the following composition: 0.25, 0.5, and 1.0 grams of Silica SP-2115 were mixed into the oil to obtain approximately 1 A vacuum of 1000 mbar was applied. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was The mixture was then removed and the material was filtered through a Buchner funnel. The filter disc and cake were dark green in color. there were.

[0246] Table 9 and Figure 5b show the results of the enzyme treatment of oil and each gum before and after reactions 1 to 6. The phosphorus (P), calcium (Ca), magnesium (Mg), and iron (Fe) contents are shown. At neutral pH, more P remained in the oil compared to the reaction at pH 4.5. Table 9 also showed the P, Ca, Mg, and Fe contents of the oil after treatment with silica SP-2115. This data shows that silica treatment meets industry standards for oil bleached without bleaching earth. These demonstrate the removal of trace amounts of phosphorus and metals to concentrations sufficient to satisfy the It did not lose its ability to adsorb these impurities when O was added.

[0247] The results in Table 10 and FIG. 5a show that when the enzymes were incubated under the same conditions (reaction 1 and 2) the CHL26 enzyme was found to be in higher abundance in crude canola oil compared to ELDC94. Table 10 also shows that the enzyme-treated oil converts chlorophyll derivatives of silica. By contacting with SP-2115, the cleavage of the enzyme-treated oil without contacting with silica was improved. The amount of chlorophyll substrates and products in the oil was compared to the amount of chlorophyll substrates and products. It has also been shown to reduce both the amount of

[0248] In this example, the CHL26 enzyme was found to be more efficient than the acidic condition (pH 4.5) (reaction 7). 3, 5, and 6), and higher amounts of chlorophyll in canola oil under neutral conditions. The substrates were converted to their respective chlorophyll products.

[0249] The CHL26 enzyme also converts chlorophyll substrates in soybean oil into the respective chlorophyll-forming products. Convert into something (reaction 8).

[0250] The results in Table 10 also show that when oil was reacted with the CHL26 enzyme, it was significantly lower than that with the ELDC94 enzyme. It was also shown that a larger amount of chlorophyll product was found in the gum (heavy phase) compared to the reaction vinegar. [Table 10] bd-Below detection limit Tr-trace [Table 11] bd-below detection limit; Example 9. CHL26 enzyme and silico in caustic refining applications for canola and soybean oils Use of Ka Processing

[0251] The following experiments were performed to determine whether the oil was processed as it is in the industrial process for canola and soybean oil. Evaluation of CHL26 in phosphoric acid and sodium hydroxide treated caustic refining applications. The "single purified" product was treated with phosphoric acid and then with sodium hydroxide to release The fatty acids (FFA) are water-soluble and are removed in the water or "heavy" phase of the "purifying" centrifuge. The oil was then converted into thorium soap. The oil was then washed with water (2-10 percent w / w). This was then performed to remove any remaining soaps and residual phospholipids present in the oil. Optionally, a water wash step was performed. The enzyme was evaluated after purification centrifugation at 37°C, but at a much lower temperature.

[0252] A 5-gallon plastic bucket of once-refined canola (ORCAN) oil was mixed in a high shear mixer. The mixture was mixed in a stirrer to make it homogenous. A sample of 2-3 kg was taken out for use in the following experiments. Got it. Reaction 9 - ELDC94 - Comparison

[0253] 2 kg of once-refined canola was mixed in a hot The oil was heated to 60°C under stirring. After the mixture reached 60°C, the beaker was transferred to a shear mixer. 0.8 grams of enzyme ELD C94 (prepared as described in Example 4) and 100 grams of deionized water were added to the oil. Shear the material for 1 minute while covered with plastic wrap to minimize loss of protein. The glass beaker was placed back into the overhead mixer and covered with plastic wrap. The oil was mixed at 250 rpm for 4 hours at 60°C. The temperature was increased to 75°C. Gyr The oil was separated using a centrifuge and the separated oil was collected.

[0254] Pour the oil and heavy phase mixture remaining in the centrifuge bowl into a 400 mL beaker. The oil was decanted from it. The remaining oil and heavy phase was placed in a 50 mL centrifuge tube and spun. The oil from the decanted bowl and the oil in the centrifuge tube were discarded and the liquid heavy phase was combined. The color was a deep green. Reaction 10 - ELDC94 - Comparison

[0255] Reaction 10 consisted of 3 kg of oil and 2.0 grams of ELDC94 (prepared as described in Example 4). This was a repeat of reaction 9, except that reaction mixture (formula 1) was used.

[0256] After the oils from reactions 9 and 10 were analyzed, the oils were mixed together and analyzed again. Reaction 11-CHL26

[0257] Reaction 11 used 10.1 grams of CHL26 (described in Example 4) instead of ELDC94. The heavy phase was a repeat of reaction 9, except that 1,2-dichlorophenyl ether (prepared as in reaction 9) was used. It was a lighter green than 10. Reaction 12-CHL26

[0258] Reaction 12 is a repeat of reaction 10, except that 20 grams of CHL26 was utilized. It was.

[0259] After analysis, the oils from reactions 11 and 12 were combined, mixed, and re-analyzed after mixing. Reaction 13 - ELDC94 - Comparison

[0260] 3kg of once refined soybean oil (ORSBO) is washed with water and centrifuged in a caustic refinery production line. The oil was poured into a 4-liter glass beaker and mixed with a square mixing paddle. Place on hot plate with overhead mixing (90 rpm). After cooling, the beaker was transferred to a shear mixer. E was produced as described in Example 4. 1.0 gram of LDC94 enzyme and 150 grams of deionized water were added to the oil. The material was mixed for 1 minute while covered with plastic wrap to minimize shear. The glass beaker was placed back into the overhead mixer and re-covered with plastic wrap. The oil was mixed at 250 rpm for 4 hours at 60°C. The temperature was increased to 75°C and then Gy The oil was centrifuged using a ro centrifuge.

[0261] Oil and heavy samples were collected for further analysis.

[0262] The oil and heavy phase remaining in the centrifuge bowl were poured into a 400 mL beaker and then The oil was decanted. The remaining oil and heavy phase were placed in a 50 mL centrifuge tube and spun. The remaining oil was discarded and the liquid heavy phases were combined. The heavy phase was colorless with no discernible color pigments. It was. Reaction 14-CHL26

[0263] Reaction 14 used 15 grams of CHL26 (as described in Example 4) instead of ELDC94. This was a repeat of reaction 13, except that a 2-methylphenyl ether was used (formed by reaction with 2-methylphenyl ether). Reaction 15 - EDLC94 - Comparison

[0264] 3kg of once refined soybean oil (ORSBO) was produced by water washing and centrifuging followed by caustic purification. The oil was drawn from line number 1. The oil was placed in a 4 liter glass beaker and placed in a square mixing bowl. The materials were placed on a hot plate with overhead mixing (90 rpm) at 600 rpm. After cooling to 0° C., the beaker was transferred to a shear mixer. 1.2 grams of ELDC94 enzyme and 150 grams of deionized water were added to the oil. Shear mix the ingredients for 1 minute while covered with plastic wrap to minimize loss. The glass beaker was placed back into the overhead mixer and re-covered with plastic wrap. The oil was mixed at 250 rpm for 4 hours at 60°C. The temperature was increased to 75°C and then The oil was centrifuged using a Gyro centrifuge.

[0265] Oil and heavy phase (gum) samples were collected for further analysis.

[0266] The oil and heavy phase remaining in the centrifuge bowl were poured into a 400 mL beaker and then The oil was decanted. The remaining oil and heavy phase were placed in a 50 mL centrifuge tube and spun. The remaining oil was discarded and the liquid heavy phases were combined. The heavy phase was colorless with no discernible color pigments. It was. Reaction 16-CHL26

[0267] Reaction 16 is a repeat of reaction 15, except that 15 grams of CHL26 was utilized. It was.

[0268] The results of reactions 9 to 16, as well as reactions 9 and 10 and reactions 11 and 12, were combined and mixed. The results for the combined oils are shown in Table 11 and Figures 6 to 6A.

[0269] The results in Table 9 and Figures 6 to 6A show that the enzyme CHL26 having pyropheophytinase is It produces a larger amount of chlorophyll substrate (chlorophyll, Pheophytin and pyropheophytin) are converted to their chlorophyll products (chlorophyllides). , pheophorbide, pyropheophorbide). [Table 12] bd = below detection limit nm=not measurable;

[0270] The results in Table 12 show the results of the enzyme treatment of once refined canola oil and once refined soybean oil. Free fatty acids (FFA), soaps, and phosphorus, as well as Ca, Mg, Fe, and / or chloroform. The content of the film is shown. [Table 13] tr=trace amount bd = below detection limit nm=not measurable; td = too dense to measure * = HPLC was a measure of total chlorophyll derivatives. Reactions 9 and 10 - Silica Treatment

[0271] The oils from reactions 9 and 10 were combined and then the three 5% of the enzyme treated ORCAN oil was Divide the sample into three 1,000 mL round-bottom flasks with the sorbent procedure configuration. The oil was heated to 80°C and 1.0, 2.0, and 3.0 grams of Silica SP were added. -2115 was mixed into the oil and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100°C. During the adsorption process with the test silica, the oil turned dark green. The vacuum was released and the material was filtered through a Büchner funnel. The filter discs were dark green in color. The oils were labeled 9101, 9102, and 9103, respectively. The oil extracted was a combined sample of reactions 9 and 10.

[0272] Processed canola oil, 9102 (436 g) and 9103 (448 g) were combined. The oil was sparged with nitrogen for approximately 2 minutes. The vacuum was released. At this time, the nitrogen sparge was discontinued and the deodorization process was started with steam from the steam generator. The vacuum achieved was 0.82-0.98mBar during the deodorization process. Heated under water sparge (3 wt%) to 230° C. The sparge and temperature were maintained for 2 hours. The heat was discontinued and the oil was allowed to cool under vacuum and sparged with water. The vacuum was released with nitrogen at approximately 100°C. The oil was removed and allowed to cool further to 70°C before being opened to air. The oil had a dark greenish / grey colour. The oil was labeled 91023-DEO.

[0273] The results in Table 13 show the results of the combined reaction 9 and 10 in oil after silica treatment as described above. The contents of free fatty acids (FFA), soaps, P, Ca, Mg, Fe, and chlorophyll are shown. [Table 14] tr=trace amount bd = below detection limit nm=not measurable; td = too dense to measure * = HPLC was a measure of total chlorophyll derivatives.

[0274] The results in Table 14 show the results of the chlorine content in oils obtained by combining reactions 9 and 10 after silica treatment as described above. The rolophile substrate and product are shown. [Table 15] CHYL = chlorophyll; PYN = pheophytin; PPYN = pyropheophytin Chin; POB = pheophorbide; PPOB = pyropheophorbide; Sub = substrate; P rod = product bd = below detection limit

[0275] Reaction using ELDC94 at 18.71 ppm as reported by HPLC method The combined feedstock from the samples was 1 in the matrix after treatment with SP-2115. A dramatic reduction in S from 0.39 ppm in the combined sample to 4.04 ppm. Showing complete removal of products below detection limits after treatment with P-2115. Reactions 11 and 12 - Silica Treatment

[0276] The oils from reactions 11 and 12 were combined and then the three fractions of enzyme treated ORCAN oil were Divide the 500 g sample into three 1000 mL round-bottom flasks with the sorbent procedure. The oil was heated to 80° C. and 1.0, 2.0, and 3.0 grams of silica S were added. P-2115 was mixed into the oil and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100°C. The mixture was heated to 350° C. and mixed for 30 minutes. The vacuum was released and the material was filtered through a Buchner funnel. The turdisk and cake were dark green in color. The oils were 11121, 11122, and 11123, respectively. The oil labeled 11120 was from reactions 11 and 12. The oils were combined in the sample.

[0277] Processed canola oil, 11122 (449 g) and 11123 (451 g) combined The oil was mixed and placed in a 3L Claisen flask and assembled according to the deodorization procedure. The vacuum was started, the nitrogen sparge was discontinued, and steam from the steam generator was removed. The deodorization process was started with a vacuum of 0.28 to 0.56 during the deodorization process. The oil was heated to 230° C. under vacuum and water sparge (3 wt%). The pressure and temperature were maintained for 2 hours. The heat was discontinued and the oil was allowed to cool under vacuum and sparged with water. The vacuum was released with nitrogen at approximately 100°C and the oil was allowed to cool further to 70°C before being opened to air. The oil was labeled 111223-DEO.

[0278] The results in Table 15 show the catalytic activity of the combined reaction 11 and 12 oils after silica treatment as described above. The contents of free fatty acids (FFA), soaps, P, Ca, Mg, Fe, and chlorophyll are shown. [Table 16] tr=trace amount bd = below detection limit nm=not measurable; td = too dense to measure * = HPLC was a measure of total chlorophyll derivatives.

[0279] The results in Table 16 show the Cr content in the combined Reaction 11 and 12 oils after silica treatment as described above. The rolophile substrate and product are shown. [Table 17] CHYL = chlorophyll; PYN = pheophytin; PPYN = pyropheophytin Chin; POB = pheophorbide; PPOB = pyropheophorbide; Sub = substrate; P rod = product bd = below detection limit

[0280] As reported by HPLC, the reaction was carried out using 18.19 ppm CHL26. The combined feedstock from the samples was 12% in the matrix after treatment with SP-2115. The product showed a dramatic reduction from 6.19 ppm to 0.1 The reduction in the substrate and product of the enzyme reaction of CHL26 was shown to be It is clear that it has the ability to remove both in an adsorption process. Reactions 5 and 13 - Silica treatment. Comparison of SP-2114 and SP-2115

[0281] Divide three 500 gram samples of the enzyme-treated refined soybean oil from reaction 13 into The oil was added to three 1000 mL round bottom flasks containing the adsorbent procedure. The oil was heated to 80 °C. Heat and mix 0.5, 1.0, and 2.0 grams of Silica SP-2115 into the oil. A vacuum of approximately 100 mbar was applied. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was released and the material was filtered through a Buchner funnel. The filter disc and cake were thick. The oils were labeled 135, 1310, and 1320, respectively. The collected oil was the sample from reaction 13.

[0282] A 500 gram sample of the enzyme treated refined soybean oil from reaction 5 was subjected to the sorbent procedure. The oil was heated to 80° C. and 2.0 grams of ethanol was added to a 1000 mL round bottom flask containing 1.0 mL of ethanol. of silica SP-2114 was mixed into the oil and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100°C and mixed for 30 minutes. The color change in the oil was greenish / brown. The vacuum was released and the material was filtered through a Büchner funnel. The filter disc and cake were light green in color. Sample was labeled 132-2114. did.

[0283] The results in Table 17 show the free fat in reaction 5 and reaction 13 oils after silica treatment as described above. The contents of acids (FFA), soaps, P, Ca, Mg, Fe, and chlorophyll are shown. [Table 18] bd = below detection limit nm=not measurable; HPLC was a measure of total chlorophyll derivatives.

[0284] The results in Table 18 show the chlorophyll content in reaction 5 and 13 oils after silica treatment as described above. Substrates and products are shown. [Table 19] CHYL = chlorophyll; PYN = pheophytin; PPYN = pyropheophytin Chin; POB = pheophorbide; PPOB = pyropheophorbide; Sub = substrate; P rod = product bd = below detection limit ND = Not detectable

[0285] In a direct comparison between SP-2114 and SP-2115, SP-2114 is faster than SP-211 5, but as reported by the UV / Vis method, SP-2115 was used. The results showed that the chlorophyll in reaction 13 oil was reduced to 298 ppb compared to 41 ppb achieved using We were able to reduce the concentration of ions to 104 ppb. Reaction 14 - Silica Treatment. Ratio of SP-2113, SP-2115, and SP-2119 comparison

[0286] Divide three 500 gram samples of the enzyme-treated refined soybean oil from reaction 14 into The adsorbent was added to three 1000 mL round-bottom flasks having the composition of the procedure. The oil was heated to 80°C and 0.5, 1.0, and 2.0 grams of Silica SP-2115 were added. The mixture was mixed into oil and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100° C. and the mixture was stirred for 3 The mixture was mixed for 10 minutes. The vacuum was released and the material was filtered through a Buchner funnel. The oils were labeled 145, 1410, and 1420, respectively. The oil labeled 140 was the sample from reaction 14.

[0287] A 500 gram sample of the enzyme-treated refined soybean oil from reaction 14 was subjected to the adsorbent technique. The oil was heated to 80°C and 2.0 grams of Silica SP-2113 from the company was mixed into the oil and a vacuum of approximately 100 mbar was applied. 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 released and the material was filtered through a Buchner funnel. The color was yellow. The sample was labeled 142-2113.

[0288] A 500 gram sample of the enzyme-treated refined soybean oil from reaction 14 was subjected to the adsorbent technique. The oil was heated to 80°C and 2.0 grams of Silica SP-2119 from the company was mixed into the oil and a vacuum of approximately 100 mbar was applied. 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 released and the material was filtered through a Buchner funnel. The color was yellow. The sample was labeled 142-2119.

[0289] Enzyme-treated and silica-treated samples of once-refined soybean oil, 1420 (471 g) and Combine 1410 (461 g) and place in a 3 L Claisen flask and deodorize according to the deodorization procedure. Assemble. Sparge the oil with nitrogen for approximately 2 minutes. Start vacuum and discontinue nitrogen sparging. The deodorization process was started with steam from a steam generator. The achieved vacuum was The pressure in the oil was 0.28-0.56 mBar during the process. The mixture was heated to 230° C. with a sparge and temperature maintained for 2 hours. Heating was discontinued and the oil was removed under vacuum. The mixture was cooled to 35° C. and sparged with water. The vacuum was released with nitrogen at approximately 100° C. before being opened to air. It was further allowed to cool to 70° C. The oil was colorless with no green tint. It was labeled 20-DEO.

[0290] The results in Table 19 show the free fatty acids (FFA) in the reaction 14 oil after silica treatment as described above. ), soap, P, Ca, Mg, Fe, and chlorophyll contents. [Table 20] bd = below detection limit nm=not measurable; * = HPLC was a measure of total chlorophyll derivatives.

[0291] The results in Table 20 show the chlorophyll substrate and chlorophyll concentration in reaction 14 oil after silica treatment as described above. The product is shown. [Table 21] CHYL = chlorophyll; PYN = pheophytin; PPYN = pyropheophytin Chin; POB = pheophorbide; PPOB = pyropheophorbide; Sub = substrate; P rod = product bd = below detection limit

[0292] Unlike the samples treated with SP-2115, the samples treated with SP-2113 or SP-2119 The samples treated with chlorophyll showed no activity to remove chlorophyll as measured by UV / Vis. This method is accepted in the industry. Reaction 15 - Silica Treatment and / or Deodorization

[0293] Three 500 g samples of the enzyme-treated refined soybean oil from reaction 15 were divided and absorbed. The oil was heated to 80° C. in three 1000 mL round-bottom flasks containing the composition of the adhesive. Then, 0.5, 1.0, and 2.0 grams of silica SP-2115 were mixed into the oil, and approximately A vacuum of 100 mbar was applied. The temperature was increased to 100° C. and mixed for 30 minutes. The temperature was then released and the material was filtered through a Buchner funnel. The filter disc and cake were dark green in color. The oils were labeled 155, 1510, and 1520, respectively. The oil extracted was the sample from reaction 15.

[0294] Enzyme-treated and silica-treated samples of once-refined soybean oil, 1520 (457 g) and Combine 1510 (461 g) and place in a 3 L Claisen flask and deodorize according to the deodorization procedure. Assemble. Sparge the oil with nitrogen for approximately 2 minutes. Start vacuum and discontinue nitrogen sparging. The deodorization process was started with steam from a steam generator. The achieved vacuum was The pressure in the oil was 0.57-0.97 mBar during the process. The mixture was heated to 230° C. with a sparge and temperature maintained for 2 hours. Heating was discontinued and the oil was removed under vacuum. The mixture was cooled to 35° C. and sparged with water. The vacuum was released with nitrogen at approximately 100° C. before being opened to air. It was further allowed to cool to 70° C. The oil was colorless with no green tint. It was labeled 20-DEO.

[0295] 618 grams of enzyme-treated oil from reaction 15, which did not undergo any adsorbent treatment, was added to a 3 L flask. The oil was placed in an Ezen flask and assembled according to the deodorizing procedure. The oil was sparged with nitrogen for approximately 2 minutes. The vacuum was started, the nitrogen sparge was discontinued, and the deodorization process was started with steam from the steam generator. The vacuum achieved was 1.15-1.40 mBar during the deodorization process. The oil was heated to 230° C. under vacuum and water sparge (3 wt %). The sparge and temperature were maintained for 2 hours. The heat was discontinued and the oil was allowed to cool under vacuum and water sparged. The vacuum was released with nitrogen at 100°C and the flask was allowed to cool to 70°C before being opened to air. The oil was colorless with no green tint. The oil was labeled 150-DEO.

[0296] The results in Table 21 show the free EDTA concentration in Reaction 15 oil after deodorization and / or silica treatment as described above. The contents of fatty acids (FFA), soaps, P, Ca, Mg, Fe, and chlorophyll are shown. [Table 22] tr=trace bd = below detection limit nm=not measurable; * = HPLC was a measure of total chlorophyll derivatives.

[0297] The results in Table 22 show the chloroform content in reaction 15 oil after deodorization and / or silica treatment as described above. The results show that the final adsorbent is more effective in removing less color. Indicates that is required. [Table 23] CHYL = chlorophyll; PYN = pheophytin; PPYN = pyropheophytin Chin; POB = pheophorbide; PPOB = pyropheophorbide; Sub = substrate; P rod = product bd = below detection limit Reaction 16 - Silica treatment or deodorization

[0298] Divide three 500 gram samples of the enzyme-treated refined soybean oil from reaction 16 into The oil was added to three 1000 mL round bottom flasks containing the adsorbent procedure. The oil was heated to 80 °C. Heat and mix 0.5, 1.0, and 2.0 grams of Silica SP-2115 into the oil. A vacuum of approximately 100 mbar was applied. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was released and the material was filtered through a Buchner funnel. The filter disc and cake were thick. The oils were labeled 165, 1610, and 1620. The oil labeled 160 was green in color. The oil used was the sample from reaction 16.

[0299] 825.3 g of enzyme-treated oil that had not been treated with any adsorbent was added to 3 L of Claisenflurane. The oil was placed in a vacuum oven and assembled according to the deodorization procedure. The oil was sparged with nitrogen for approximately 2 minutes. The nitrogen sparge was then turned off and the deodorization process was started with steam from the steam generator. The vacuum achieved was 1.15-1.40 mBar during the deodorization process. and heated to 230° C. under water sparge (3 wt %). The sparge and temperature were maintained for 2 hours. The heat was discontinued and the oil was allowed to cool under vacuum and water sparged. The vacuum was released with nitrogen at 100°C and the flask was allowed to cool to 70°C before being opened to air. The color was predicted to be green, but it was very slightly red and had a greenish tint. The sample was labeled 160-Deo.

[0300] The results in Table 23 show the free fatty acids in reaction 16 oil after deodorization or silica treatment as described above. The contents of (FFA), soap, P, Ca, Mg, Fe, and chlorophyll are shown. [Table 24] tr=trace bd = below detection limit nm=not measurable; HPLC was a measure of total chlorophyll derivatives.

[0301] The results in Table 24 show the chlorophyll content in reaction 16 oil after deodorization or silica treatment as described above. The results show that treatment with an adsorbent is necessary to remove the final green color. Indicates that it is necessary. [Table 25] CHYL = chlorophyll; PYN = pheophytin; PPYN = pyropheophytin Chin; POB = pheophorbide; PPOB = pyropheophorbide; Sub = substrate; P rod = product bd = below detection limit ND = Not detectable Example 10: Incubation of once refined canola (ORCAN) oil with CHL26 enzyme ion, and treatment with Silica SP-2115 or commercial silica or bleaching clay.

[0302] A 5-gallon plastic bucket of once-refined canola (ORCAN) oil was mixed in a high shear mixer. The mixture was mixed in a stirrer until homogenous, and a sample was withdrawn for use in the following experiments. Reaction 17-CHL26

[0303] 3,000 grams of single-refined canola oil was poured into a hot press with overhead mixing. The oil was heated to 60°C under stirring. After the temperature reached 60°C, the oil was transferred to a shear mixer. 150 g of DI water was added (prepared as described in Example 4). The material was shear mixed for 1 minute. The glass beaker was placed back into the overhead mixer and covered with plastic wrap. The oil was mixed for 4 hours at 60°C. The temperature of the oil was increased to 75°C and the mixture was mixed in a bowl with the hole closed. The oil was centrifuged using a Gyro centrifuge equipped with a .

[0304] The oil and heavy phase in the bowl were poured into a 400 mL beaker from which the oil was decanted. The remaining oil and heavy phase were placed in a 50 mL centrifuge tube and spun. The remaining oil in the tube was discarded. The heavy liquid phases were combined and the heavy liquid phase was a deep green color. Reaction 18~20-CHL26

[0305] Add 3,000 grams of once-refined canola oil to a 4-liter jacketed glass beaker. The temperature was set at 60°C. After the material reached 60°C, the oil was transferred to a shear mixer. 15 g of the decolorizing enzyme CHL26 (prepared as described in Example 4) and 150 g of deionized The water was added to the hot oil. The materials were shear mixed for 1 minute. Place the glass beaker back on the overhead mixer and cover it again with plastic wrap. Ta. The oil was mixed at 60°C for 4 hours. The temperature of the oil was increased to 75°C and the bottle with the hole closed was The oil was centrifuged using a Gyro centrifuge equipped with a filter. The procedure was repeated three times to separate the oil. Reactions 18-20 were collected and combined. Silica treatment with reaction 17-SP-2115

[0306] Five hundred grams of the once refined canola oil from reaction 17 was mixed in a 10-well flask with the above-described equipment configuration. The oil was heated to 80°C and 2.0 g of silica SP-211 was added to a 0.00 mL round bottom flask. 5 was mixed into the oil and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100° C. and mixed for 30 minutes. The vacuum was released and the material was transferred to a Buchner funnel. The same procedure was repeated except that 4, 6, and 8 grams of silica SP-2115 were used. The experiment was repeated under the same conditions. Reactions 18-20 - Silica Treatment. Ratios of SP-2115 and Commercial Silica and Bleaching Clay comparison

[0307] The combined single refined canola oil from reactions 18-20 was purified using commercially available Silica (TriSyl® 300), bleaching clay (Clariant 126FF ), or two separate lots of SP-2115.

[0308] Five hundred grams of the once-refined canola oil from the combined reactions 18-20 was then added to the apparatus configured as described above. The oil was heated to 80° C. and 2.0 g of Tr The iSyl® 300 was mixed into the oil and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100°C and the oil was mixed for 30 minutes. The vacuum was released and the material was placed in a Buchner Filter through a funnel.

[0309] Five hundred grams of the once-refined canola oil from the combined reactions 18-20 was then added to the apparatus configured as described above. The oil was heated to 80° C. and 2.0 g of bleach was added to a 1000 mL round bottom flask containing 1.0 g of ethyl acetate. Clay Tonsil® supreme 126 FF (Clariant) oil The mixture was mixed in and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100° C. and the oil was stirred for 3 The mixture was mixed for 10 minutes. The vacuum was released and the material was filtered through a Buchner funnel.

[0310] Five hundred grams of the once-refined canola oil from the combined reactions 18-20 was then added to the apparatus configured as described above. The oil was heated to 80° C. and 2.0 g of the first A lot of silica SP-2115 was mixed into the oil and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100°C and the oil was mixed for 30 minutes. The vacuum was released and the material was separated using a Buchner filter. Filtered through a funnel.

[0311] Five hundred grams of the once-refined canola oil from the combined reactions 18-20 was then added to the apparatus configured as described above. The oil was heated to 80° C. and 2.0 g of 2.0 mL of 1,2-dichlorophenyl ether was added to a 1000 mL round bottom flask containing 1.0 mL of 1,2-dichlorophenyl ether. A lot of silica SP-2115 was mixed into the oil and a vacuum of approximately 100 mbar was applied. The temperature was increased to 100°C and the oil was mixed for 30 minutes. The vacuum was released and the material was separated using a Buchner filter. Filtered through a funnel.

[0312] The results in Table 25 are shown above for silica SP-2115, TriSyl (registered trademark) silica, and Free fat in reaction 17 oil or combined reaction 18-20 oil after treatment with ricin or bleaching clay Indicates the content of fatty acids (FFA), soap, P, Ca, Mg, Fe, and sodium (Na) . [Table 26] bd--Below detection limit tr-trace nm - not measurable First purification means washed and dried Can - Canola

[0313] An oil sample (approximately 1 gram) was placed in a 100 mL measuring flask and dissolved in CHCl3 ( The chloroform was diluted with chloroform and the peak absorbance at 670 nm was measured using the UV-Vis method. The fill content was measured. It was also measured using HPLC. The results are shown in Table 26. . [Table 27] * = HPLC was a measure of total chlorophyll derivatives.

[0314] The results in Tables 27-28 are for reaction 17 or combined reactions 18-20 after treatment as described above. The residual concentrations are based on the amount of chlorophyll substrates and products required for industrial processes. The concentrations of chlorophyll substrate and product are close to those expected for the decolorization enzyme. This allows for the elimination of bleaching earth for very green canola oil. [Table 28] CHYL = chlorophyll; PYN = pheophytin; PPYN = pyropheophytin Chin; POB = pheophorbide; PPOB = pyropheophorbide; Sub = substrate; P rod = product bd = below detection limit [Table 29]

[0315] Commercially available silica "Trisyl (registered trademark) 300" is a product generated by decolorization enzyme reaction. They have limited ability to remove chlorophyll products and actually convert some of the chlorophyll products into substrates. The bleaching earth is believed to remove the chlorophyll substrates found in the unreacted decolorizing enzyme oil. Although it has a higher ability to remove the products of decolorized enzyme treated oil and silica of the present disclosure. Example 11: Preparation of silica adsorbent

[0316] This example describes the preparation of the adsorbent in the following reactions 21-30: Reaction 21 - Preparation of SP-2113

[0317] 600 grams of TRISYL® silica was dried at 60° C. to obtain 173 grams The water was removed. The silica was then mixed with 18.6 grams of NaOH and 81.9 grams of The material was infused with a sodium hydroxide solution containing water. Blended for minutes. Reaction 22 - Preparation of SP-2114

[0318] 600 grams of TRISYL® silica in a Waring blender for 5 minutes. The mixture was blended with 12.2 g of MgO powder for 2 h. Reaction 23 - Preparation of SP-2115

[0319] 600 grams of TRISYL® silica in a Waring blender for 5 minutes. During this time, it was blended with 31.6 g of MgO powder. Reaction 24 - Preparation of SP-2116

[0320] 600 grams of TRISYL® 300 silica was dried at 60° C. for 17 3 grams of water was removed. The silica was then treated with 11.4 grams of NaOH and 92 grams of The material was infiltrated with a sodium hydroxide solution containing 100 ml of water in a Waring blender. Blended for 5 minutes. Reaction 25 - Preparation of SP-2117

[0321] 600 grams of TRISYL® 300 silica was dried at 60° C. for 17 3 grams of water was removed. The silica was then mixed with 17.8 grams of NaOH and 92 grams of The material was infiltrated with a sodium hydroxide solution containing 100 ml of water in a Waring blender. Blended for 5 minutes. Reaction 26 - Preparation of SP-2119

[0322] Contains less than 10% water by weight, 707m 2 / g surface area, and 19 microns 600 grams of silica xerogel having a median particle size of 1.0 mm was blended in a Waring blender for 5 minutes. , and blended with 30 grams of MgO. Reaction 27 - Preparation of Adsorbent A

[0323] 4.8 grams of SP-2115 was dried in a 110° C. oven for 3 hours. Reaction 28 - Preparation of Adsorbent B

[0324] Add 5 grams of MgO powder and 20 grams of TRISYL® silica to a container. The mixture was blended, sealed, and then mixed by shaking for 1 hour. Reaction 29 - Preparation of Adsorbent C

[0325] Contains less than 10% water by weight, 707m 2 / g and a surface area of ​​19 microns 24.5 grams of silica xerogel having a median particle size was infiltrated with 23.6 grams of Dl water. The contents were then sealed and shaken for 1 hour. Mix by shaking. Reaction 30 - Preparation of Adsorbent D

[0326] 4% water by weight, 330m 2 / g surface area and particle size of 88-210 micrometers 264 grams of silica xerogel having the above formula was infiltrated with 303 grams of Dl water. The mixture was divided into two containers, each containing 6 grams of MgO powder. Each container was left for 1 hour. Mix by shaking, then combine all contents into a larger container and shake for 1 hour. Blend by shaking. Example 12: Enzyme and adsorbent treatment of oil.

[0327] The adsorbent prepared in Example 11 was used to decolorize oil (i.e., That is, two batches of oil 11 (120) prepared as described in Example 9 were further processed. Reaction 31 - Treatment of decolorized enzyme-treated oil with adsorbent A

[0328] 100 grams of enzyme treated oil 11120 was heated to 80°C, and then 0.21 grams of Adsorbent A was mixed into the oil. A vacuum of 100 mbar was applied and then the temperature was increased to 100° C. The vacuum was set and mixed for 30 minutes. The vacuum was released and the material was filtered through a Buchner funnel. Filter The discs and cakes were deep green in color. Reaction 32 - Treatment of decolorized enzyme-treated oil with adsorbent B

[0329] 100 grams of enzyme-treated oil 11120 was heated to 80°C, and then 0.4 grams of adsorbent were added. Adhesive B was mixed into the oil. A vacuum of 100 mbar was applied and then the temperature was set to 100°C. The mixture was mixed for 30 minutes. The vacuum was released and the material was filtered through a Buchner funnel. The discs and cake were a deep green color. Reaction 33 - Treatment of decolorized enzyme-treated oil with adsorbent C

[0330] 100 grams of enzyme-treated oil 11120 was heated to 80°C, and then 0.4 grams of adsorbent were added. Adhesive C was mixed into the oil. A vacuum of 100 mbar was applied and then the temperature was set to 100°C. The mixture was mixed for 30 minutes. The vacuum was released and the material was filtered through a Buchner funnel. The discs and cake were a deep green color. Reaction 34 - Treatment of decolorized enzyme-treated oil with adsorbent D

[0331] 100 grams of enzyme-treated oil 11120 was heated to 80°C, and then 0.4 grams of adsorbent were added. Adhesive D was mixed into the oil. A vacuum of 100 mbar was applied and then the temperature was set to 100°C. The mixture was mixed for 30 minutes. The vacuum was released and the material was filtered through a Buchner funnel. The discs and cake were a deep green color. Reaction 3: Treatment of decolorized enzyme-treated oil with 5-TRISYL® silica

[0332] 100 grams of enzyme treated oil 11120 was heated to 80° C., and then 0.4 grams of T RISYL® silica was mixed into the oil. A vacuum of 100 mbar was applied, followed by The temperature was set to 100°C and mixed for 30 minutes. The vacuum was released and the material was transferred to a Buchner funnel. Filtered. The filter disc and cake were green in color. Reaction 36-Treatment of decolorized enzyme treated oil with TRISYL® 300 silica

[0333] 100 grams of enzyme treated oil 11120 was heated to 80° C., and then 0.4 grams of T RISYL® 300 silica was mixed into the oil. A vacuum of 100 mbar was applied. The temperature was then set to 100° C. and mixed for 30 minutes. The vacuum was released and the material was placed in a Buchner Filtered through a funnel. The filter disc and cake were yellow in color.

[0334] The green concentration from the oils produced in reactions 31-36 was measured using the AOCS UV / Vis method. The results are shown in Table 29. [Table 30]

Claims

1. An adsorbent for removing chlorophyll and chlorophyll derivatives from oil, comprising: The amorphous porous silica gel is treated with potassium earth metal oxide, and has a pH of about 7 or more. An alkaline earth metal oxide content of at least 0.1% by weight based on the A sorbent having a water content.

2. 10. The absorbent of claim 1 comprising from 1 to about 40 weight percent alkaline earth metal oxide on a dry basis. Adhesive.

3. The alkaline earth metal oxide is magnesium oxide, calcium oxide, barium oxide. , beryllium oxide, or a combination thereof. Adsorbent.

4. 2. The adsorbent of claim 1, wherein the alkaline earth metal oxide is magnesium oxide. 。

5. 4. The method of claim 1, wherein the amount of magnesium oxide ranges from about 5% to about 25% MgO by weight.

5. The adsorbent according to claim 4.

6. 6. The magnesium oxide of claim 5, wherein the magnesium oxide comprises about 10 to about 20 wt. % MgO. Adsorbent.

7. 10. The adsorbent of claim 1 having a pH of 7.0 to 10.

0.

8. The adsorbent of claim 7 having a pH of from 7.5 to 9.

7.

9. The adsorbent of claim 8 having a pH of 8.0 to 9.

5.

10. 5. The adsorbent of claim 1 or 4 having a water content of 10% by weight or more.

11. The adsorbent of claim 10 having a water content of about 25 to about 75 weight percent.

12. The adsorbent of claim 11 having a water content of about 40 to about 70 weight percent.

13. 13. The adsorbent of claim 12 having a water content of about 55 to about 65 weight percent.

14. 5. The method of claim 1 or 4, having a median particle size of about 0.1 to about 2,000 micrometers. Adsorbent on board.

15. 15. The adsorbent of claim 14 having a median particle size of about 2 to about 500 micrometers.

16. 16. The adsorbent of claim 15 having a median particle size of about 5 to about 50 micrometers.

17. At least 50 meters ２ The adsorbent of claim 1 or 4 having a surface area of ​​1 / g.

18. Approximately 50 to approximately 800m ２ 20. The adsorbent of claim 17 having a surface area of ​​1 / g.

19. 10. The adsorbent of claim 1 or 4 having a pore volume of at least about 0.1 cc / g.

20. 20. The adsorbent of claim 19 having a pore volume of at least about 0.4 cc / g.

21. 21. The adsorbent of claim 20 having a pore volume of about 0.7 to about 2.0 cc / g.

22. a pH of about 7 to about 10, a magnesium oxide amount of about 5 to 25% by weight MgO on a dry basis; and a moisture content of about 25% to about 65% by weight.

23. Magnesium oxide having a pH of about 8 to about 9.5 and about 10 to 20% by weight MgO on a dry basis.

23. The adsorbent of claim 22 having a water content of about 55% to about 65% by weight.

24. 5. The method according to claim 1, 4, which has the ability to remove chlorophyll and chlorophyll derivatives from oil.

24. The adsorbent according to claim 22, 23 or 24.

25. 25. The adsorbent of claim 24, wherein the oil comprises a triacylglycerol-based oil.

26. The triacylglycerol oil is canola oil, castor oil, coconut oil, Under oil, corn oil, cottonseed oil, hazelnut oil, hemp seed oil, linseed oil, mango kernel oil, Meadowfoam oil, beef foot oil, olive oil, palm oil, palm kernel oil, palm olein, pea Nut oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, sesame oil, soybean oil, sunflower seed oil 24 or 25. The oil of claim 23, wherein the oil is selected from the group consisting of oils derived from tall oil, camellia oil, vegetable oil, and algae.

26. The adsorbent according to claim 25.

27. 26. The adsorption method according to claim 24 or 25, wherein the oil is a decolorase-treated oil. Agent.

28. 28. The method of claim 1 or 27, wherein the silica gel is a hydrogel or a hydrated xerogel. Adsorbent.

29. The adsorbent is a compound that reduces the total concentration of chlorophyll and chlorophyll derivatives in the decolorized enzyme-treated oil. The degree of chlorophyll and chlorophyll in the decolorized enzyme-treated oil before contact with the adsorbent was The concentration of the aryl derivatives is reduced by at least 5% by weight compared to the total concentration of the aryl derivatives.

29. The adsorbent according to claim 28.

30. The adsorbent is a compound that reduces the total concentration of chlorophyll and chlorophyll derivatives in the decolorized enzyme-treated oil. The degree of chlorophyll and chlorophyll in the decolorized enzyme-treated oil before contact with the adsorbent was 30. The composition according to claim 29, wherein the concentration of the aryl derivatives is reduced by at least 50% by weight compared to the total concentration of the aryl derivatives. Adsorbent.

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