Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01014—Chlorophyllase (3.1.1.14)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/40—Colouring or decolouring of foods
  • A23L5/49—Removing colour by chemical reaction, e.g. bleaching
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
  • C11B3/00—Refining fats or fatty oils
  • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
  • C11B3/00—Refining fats or fatty oils
  • C11B3/16—Refining fats or fatty oils by mechanical means
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

• Chlorophyll may be removed during many stages of the oil production process, including the seed crushing, oil extraction, degumming, caustic treatment and bleaching steps.
• the bleaching step is usually the most significant for reducing chlorophyll residues to an acceptable level.
• the adsorbent used in the bleaching step is typically clay.
• the use of such steps typically reduces chlorophyll levels in processed oil to between 0.02 to 0.05 ppm.
• the bleaching step increases processing cost and reduces oil yield due to entrainment in the bleaching clay.
• the use of clay may remove many desirable compounds such as carotenoids and tocopherol from the oil.
• the use of clay is expensive, this is particularly due to the treatment of the used clay (i.e. the waste) which can be difficult, dangerous (prone to self-ignition) and thus costly to handle.
• attempts have been made to remove chlorophyll from oil by other means, for instance using the enzyme chlorophyllase.
• chlorophyllase In plants, chlorophyllase (chlase) is thought to be involved in chlorophyll degradation and catalyzes the hydrolysis of an ester bond in chlorophyll to yield chlorophyllide and phytol.
• WO 2006009676 describes an industrial process in which chlorophyll contamination can be reduced in a composition such as a plant oil by treatment with chlorophyllase.
• the water- soluble chlorophyllide which is produced in this process is also green in colour but can be removed by an aqueous extraction or silica treatment.
• Chlorophyll is often partly degraded in the seeds used for oil production as well as during extraction of the oil from the seeds.
• One common modification is the loss of the magnesium ion from the porphyrin (chlorin) ring to form the derivative known as pheophytin (see Figure 32).
• the loss of the highly polar magnesium ion from the porphyrin ring results in significantly different physico-chemical properties of pheophytin compared to chlorophyll.
• pheophytin is more abundant in the oil during processing than chlorophyll.
• pheophorbidase A 28-29 kDa enzyme from Chenopodium album named pheophorbidase is reportedly capable of catalyzing an analogous reaction on pheophorbide, to produce the phytol-free derivative of pyropheophytin known as pyropheophorbide (see Figure 32). Pyropheophorbide is less polar than pheophorbide resulting in the pyropheophoribe having a decreased water solubility and an increased oil solubility compared with pheophorbide.
• the enzyme is contacted with the oil in the presence of 1 to 5% by weight water.
• the degumming step comprises water degumming.
• the enzyme may comprise, for example, a chlorophyllase, pheophytinase, pyropheophytinase or pheophytin pheophorbide hydrolase.
• the enzyme comprises a polypeptide sequence as defined in any one of SEQ ID NOs: 1 to 31, or a functional fragment or variant thereof.
• the enzyme comprises a polypeptide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1 to 31 over at least 50 amino acid residues.
• the activity of chlorophyllases in crude oils having a relatively low phospholipid content can be improved by recycling the gum phase back into the crude oil in a continuous refining process.
• gum recycling increases the phospholipid content of the oil during the enzymatic treatment step, thereby improving the reaction kinetics and favouring rapid hydrolysis of chlorophyll and chlorophyll derivatives to phytol-free compounds.
• Figure 4 shows the amino acid sequence of a Triticum aestivum chlorophyllase (SEQ ID NO:4).
• Figure 18 shows the amino acid sequence of a Sorghum bicolor chlorophyllase (SEQ ID NO: 18).
• Figure 23 shows the amino acid sequence of a Brachypodium distachyon chlorophyllase (SEQ ID NO:23).
• Figure 28 shows the amino acid sequence of a Oryza sativa Japonica chlorophyllase (SEQ ID NO:28).
• Figure 29 shows the amino acid sequence of a Oryza sativa Japonica chlorophyllase (SEQ ID NO:29).
• the chlorophyll and/or chlorophyll derivatives may be present in the oil naturally, as a contaminant, or as an undesired component in a processed product.
• the chlorophyll and/or chlorophyll derivatives e.g. chlorophyll, pheophytin and/or pyropheophytin
• the process of the present invention comprises a step of contacting the oil with an enzyme which is capable of hydrolysing chlorophyll or a chlorophyll derivative.
• hydro lyzing chlorophyll or a chlorophyll derivative means hydrolysing an ester bond in chlorophyll or a (phytol-containing) chlorophyll derivative, e.g. to cleave a phytol group from the chlorin ring in the chlorophyll or chlorophyll derivative.
• the enzyme typically has an esterase or hydrolase activity.
• the enzyme has esterase or hydrolase activity in an oil phase, and optionally also in an aqueous phase.
• chlorophyllase pheophytinase and/or pyropheophytinase activity suitable for use in the process may be identified by determining the presence of conserved sequence motifs present e.g. in known chlorophyllase, pheophytinase or pyropheophytinase sequences.
• conserved sequence motif GHSRG SEQ ID NO: 32
• amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”.
• amino acid sequence is synonymous with the term “peptide”.
• the amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques. Suitably, the amino acid sequences may be obtained from the isolated polypeptides taught herein by standard techniques.
• sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
• Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
• the method of the invention can be practiced with immobilized enzymes, e.g. an immobilized chlorophyllase, pheophytinase and/or pyropheophytinase.
• the enzyme can be immobilized on any organic or inorganic support.
• Exemplary inorganic supports include alumina, celite, Dowex-1 -chloride, glass beads and silica gel.
• Exemplary organic supports include DEAE- cellulose, alginate hydrogels or alginate beads or equivalents.
• immobilization of the enzyme can be optimized by physical adsorption on to the inorganic support.
• E. coli BL21(DE3) (Novagen).
• the cells were cultured at 37°C in LB containing carbenicillin (50mg/ml) until OD 6 oo 0.6-0.8.
• For induction the culture was added 1 mM IPTG and incubated at 25°C for another 20-24 h before harvesting the cells by centrifugation.
• the recombinant chlorophyllases were released from the cell pellet by sonication and cellular debris removed by centrifugation.

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Citations (27)

• 2013
  • 2013-04-24 WO PCT/EP2013/058542 patent/WO2013160374A1/fr not_active Ceased
  • 2013-04-26 AR ARP130101441A patent/AR090870A1/es unknown

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