Classifications

• • 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

Definitions

• the present invention relates to an improved process for enzymatic reducing the content of phosphorus containing components in an edible oil.
• Oils obtained from the usual oil and fat production processes by compressing oil-bearing materials or by extracting oil from the materials and removing the extraction solvent contain impurities such as polar lipids mainly composed of phospholipids, as well as fatty acids, pigments, odor components and the like. Thus it is necessary to remove these impurities by a refining process. Such a process may require a degumming step.
• EP 622446 suggest to use high amount of water in the enzymatic degumming process. See page 3, line 33-44 and claim 4 in said document, which suggest to use more than 30 percent of water by weight of the oil in said process.
• the problem, to be solved, by the present invention is to provide a simplified and economically cheaper process for enzymatic degumming of edible oils.
• the present invention relates to a process for reducing the content of phosphorus containing components in an edible oil, having from 50 to 10.000 part per million (pp ) of phosphorous content, which method comprises contacting said oil at a pH from 1.5 to 8 with an aqueous solution of a -2- phospholipase Al (PLA1) , phospholipase A2 (PLA2) , or phospholipase B (PLB) which is emulsified in the oil until the phosphorous content of the oil is reduced to less than 12 ppm, and then separating the aqueous phase from the treated oil, and wherein said process is characterized by that said emulsified condition is formed using from 0.01 to 1.5 percent of water by weight of the oil, preferably from 0.01 to 1.0 percent of water by weight of the oil, more preferably from 0.01 to 0.75 percent of water by weight of the oil, even more preferably from 0.01 to 0.5 percent of water by
• the lower range above of 0.01 percent of water by weight of the oil may preferably be 0.1 percent of water by weight of the oil.
• An advantage of the process described herein is that costs for water and waste water treatment may be reduced. Furthermore, oil recovery yields may be increased because less amount of oil will be wasted to the aqueous phase. Further, an advantage of the process described herein may be that an oil-mill using this process may skip sludge recycling of the polluted water used in the process.
• Said recycling step may be saved by the low amount of water used in the process described herein.
• enzymatic degumming carried out according to the art (e.g. US 5,264,367) a heat treatment to e.g. 65-75 °C of the water in oil emulsion is usually carried out in order to facilitate separation of the oil and aqueous phases by e.g. centrifugation.
• the thermostable phospholipase LecitaseTM Novo Nordisk A/S, Denmark
• the aqueous phase containing the enzyme can advantageously be reused several times (with or without addition -3-
• Embodiment (s) of the present invention is described below, by way of example (s) only.
• any edible oil may be degummed according to a process of the invention.
• Example of oils are crude oils and water degummed oils.
• a crude oil (also called a non-degummed oil) may be a pressed or extracted oil or a mixture thereof from e.g. rapeseed, soybean, or sunflower.
• the phosphatide content in a crude oil may vary from 0.5-3% w/w corresponding to a phosphorus content in the range of 200-10.000 ppm, more preferably in the range of 250-1200 ppm.
• the crude oil also contains small concentrations of carbohydrates, sugar compounds and metal/phosphatide acid complexes of Ca, Mg and Fe.
• said edible oil is an oil from which mucilage has previously been removed and which has a phosphorus content from 50 to 250 ppm.
• Such an oil is generally obtained by a water-degumming process and termed "a water-degummed oil".
• a water-degummed oil is typically obtained by mixing 1-3% w/w of hot water with warm (60-90°C) crude oil. Usual treatment periods are 30-60 minutes.
• the water-degumming step removes the phosphatides and mucilaginous gums which become insoluble in the oil when hydrated.
• the hydrated phosphatides and gums can be separated from the oil by settling, filtering or centrifuging - centrifuging being the more prevalent practice.
• water-degumming may be called “wet refining to remove mucilage” (see US 5,264,367) . -4-
• an edible is preferably an vegetable oil.
• a phospholipase used in the process of the invention is a phospholipase obtained from a microorganism, preferably a filamentous fungus, a yeast, or a bacterium.
• the term "obtained from”, as used herein in connection with a specific microbial source, means that the enzyme and consequently the DNA sequence encoding said enzyme is produced by the specific source.
• the enzyme is then obtained from said specific source by standard known methods enabling the skilled person to obtain a sample comprising the enzyme and capable of being used in a process of the invention.
• Said standard methods may be direct purification from said specific source or cloning of a DNA sequence encoding the enzyme followed by recombinant expression either in the same source (homologous recombinant expression) or in a different source (heterologous recombinant expression) .
• a phospholipase used in a process of the invention is obtained from a filamentous fungal species within the genus Fusarium, such as a strain of F. cul orum, F. heterosporum, F . solani , or in particular a strain of F. oxysporum; or a filamentous fungal species within the genus Aspergillus, such as a strain of Aspergillus awamori , Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or in particular Aspergillus oryzae .
• VOD 5 Degomma VOD (Roehm, Germany) , which is believed to comprise an Aspergillus phospholipase is suitable to be used in a process of the invention.
• a phospholipase used in a process of the invention exhibits certain properties.
• embodiment of the invention relates to i) a process according to the invention, wherein the phospholipase is a phospholipase which is substantively independent of Ca 2+ concentration measured as, relative phospholipase activity at 5 mM EDTA and 5mM Ca 2+ in 5 a phospholipase activity assay measuring release of free fatty acids from lecithin in a buffer comprising 2% lecithin, 2% Triton X-100, 20 mM citrate, pH 5; incubated for 10 min. at 37°C followed by stop of reaction at 95°C for 5 min.
• the ratio of relative phospholipase activity at 5mM 0 EDTA/5 mM Ca 2+ is greater than 0.25, more preferably greater than 0.5; and/or ii ) a process according to the invention, wherein the phospholipase is a phospholipase which has a phospholipase activity which is capable of releasing at least 7 ⁇ mol of free 25 fatty acid/min./mg enzyme; more preferably at least 15 ⁇ mol of free fatty acid/min./mg enzyme; measured as, phospholipase activity is measured in an assay measuring release of free fatty acids from lecithin in a buffer comprising
• the invention relates to a process according to the invention, wherein the phospholipase is a phospholipase having an polypeptide sequence selected from the group comprising of:
• a phospholipase obtained from F . oxysporum and having the polypeptide sequence shown in (b) above exhibits both of the above mentioned functional characteristic. Accordingly, this phospholipase is the most preferred phospholipase to be used in a process of the invention. A working example herein demonstrates the use of this phospholipase (vide infra) . Finally an example of a suitable non-microbial phospholipase is the commercial available PL (LecitaseTM, Novo Nordisk A/S, Denmark) obtained from porcine pancreas.
• the enzymatic treatment is conducted by dispersing an aqueous solution of the phospholipase, preferably as droplets with an average diameter below 10 ⁇ (micro) . -7-
• the amount of water is from 0.01 to 1.5% by weight in relation to the oil.
• An emulsifier may optionally be added. Mechanical agitation may be applied to maintain the emulsion.
• the enzymatic treatment can be conducted at any pH in the range 1.5-8, preferably from pH 3-6.
• the pH may be adjusted by adding citric acid, a citrate buffer, NaOH or HC1.
• a suitable temperature is generally 30-75°C (particularly 40-60°C) .
• the reaction time will typically be 0.5-12 hours (e.g. 2-6 hours) , and a suitable enzyme dosage will usually be 100- 5000 IU per liter of oil, particularly 200-2000 IU/1.
• the enzymatic treatment may be conducted batchwise, e.g. in a tank with stirring, or it may be continuous, e.g. a series of stirred tank reactors.
• the enzymatic treatment is followed by separation of an aqueous phase and an oil phase. This separation may be performed by conventional means, e.g. centrifugation.
• the process of the invention can reduce this value to below 12 ppm, more preferably below 10, and even more preferably below 5 ppm.
• the equipment consists of a 1 1 jacketed steel reactor fitted with a steel lid, a propeller (about 600 rpm) , baffles, a temperature sensor, an inlet tube at the top, a reflux condenser (about 4°C) at the top, and an outlet tube at the bottom.
• the reactor jacket is connected to a thermostat bath.
• the outlet tube is connected via silicone tubing to a Silverson in-line mixer head equipped with a "square hole high shear screen", driven by a Silverson L4RT high shear lab mixer (about 8500 rpm, flow ca. 1.1 1/minute) .
• the mixer head is fitted with a cooling coil (5-10 °C) and an outlet tube, which is connected to the -8-
• the reactor/mixer system is emptied and rinsed with 2x500 ml 10% Deconex/DI water solution followed by minimum 3x500 ml of DI water.
• Table 2 is a presentation of the various additions and samplings during the reaction.
• Phosphorus analysis Take 10 ml of water in oil emulsion in a glass centrifuge tube. Heat the emulsion in a boiling water bath for 30 minutes. Centrifuge at 5000 rpm for 10 minutes. Transfer about 8 ml of upper (oil) phase to a 12 ml polystyrene tube and leave it (to settle) for 12-24 hours. After settling draw about 1-2 g from the upper clear phase for P-analysis.
• a calibration curve was obtained by dissolving 0.6 g of citric acid monohydrate in 27 g of DI water; pH of this solution was measured by pH electrode Orion (pH real ) . 100 ⁇ l were mixed with 2 ml MilliQ water, and pH of this solution was measured by pH electrode Orion (pH measured ) . pH of the citric acid solution was changed gradually by adding NaOH solution, and for each adjustment dilution and pH measurements were carried out as described above.)
• the enzyme was recombinantly expressed and purified as described in EP Patent application number 97610056.0.
• Phospholipase activity was measured as the release of free fatty acids from lecithin. 50 ⁇ l 4% L-alpha- phosphatidylcholine (plant lecithin from Avanti, USA) , 4% Triton X-100, 5 mM CaCl 2 in 50 mM HEPES, pH 7 was added, 50 ⁇ l enzyme solution diluted to an appropriate concentration in 50 mM HEPES, pH 7. The samples were incubated for 10 min at 30°C and the reaction stopped at 95 °C for 5 min prior to centrifugation (5 min at 7000 rpm) .
• Free fatty acids were determined using the NEFA C kit from Wako Chemicals GmbH; 25 ⁇ l reaction mixture was added to 250 ⁇ l reagent A and incubated for 10 min at 37°C. Then 500 ⁇ l Reagent B was added and the sample was incubated again, 10 min at 37 °C. The absorption at 550 nm was measured using an HP 8452A diode array spectrophotometer. Samples were run at least in duplicates. Substrate and enzyme blinds (preheated enzyme samples (10 min at 95°C) + substrate) were included. Oleic acid was used as a fatty acid standard. 1 PHLU equals the amount of enzyme capable of releasing 1 ⁇ mol of free fatty acid/min under these conditions.
• the assay was run at 37°C in 20 mM citrate buffer, pH 5 (Ca 2+ -dependence) or 20 mM Britton-Robinson buffer (pH-profile/temperature-profile/stability) .
• Phospholipase Al activity was measured using 1-(S- decanoyl) -2-decanoyl-l-thio-sn-glycero-3-phosphocholine (D3761
• HEPES, 0.3 mM DTNB, 2 mM CaCl 2 , pH 7) in a 200 ⁇ l cuvette were added to 10 ⁇ l enzyme, and the absorption at 410 nm was measured as a function of time on the HP 8452A diode array spectrophotometer at room temperature. Activity was calculated as the slope of the curve in the linear range.
• PLA1 equals the amount of enzyme capable of releasing 1 ⁇ mol of free fatty acid (thiol)/min at these conditions. -17-
• Phospholipase A2 activity was measured at 40°C using 1- hexadecanoyl-2- (1-pyrenedecanoyl) -sn-glycero-3-phosphocholine (H361 Molecular Probes) .
• 2 ml substrate 50 ⁇ l 1% Triton X-100 + 25 ⁇ l 0.1% H361 in methanol + 10 ml 50mM HEPES, pH 7) in a 2 ml cuvette with stirring was added to 10 ⁇ l enzyme, and the pyrene fluorescence emission was measured at 376 nm (excitation at 340 nm) as a function of time (1 sec. intervals) using the Perkin Elmer LS50 apparatus.
• Triton X-100/phospholipid micelles the concentration of phospholipid was adjusted to have excimer formation (emits at 480 nm) . Upon cleavage the fatty acid in the 2-position containing the pyrene group is released into the aqueous phase resulting in an increase in the monomer emission. PLA2 was taken as the slope of the curve in the linear range at equal conditions.

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• Preparation Of Compounds By Using Micro-Organisms (AREA)
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• 1999
  • 1999-04-07 AU AU30263/99A patent/AU3026399A/en not_active Abandoned
  • 1999-04-07 CN CNB998040053A patent/CN1139652C/zh not_active Expired - Fee Related
  • 1999-04-07 CA CA002324653A patent/CA2324653A1/en not_active Abandoned
  • 1999-04-07 BR BR9909502-5A patent/BR9909502A/pt not_active IP Right Cessation
  • 1999-04-07 EP EP99911648A patent/EP1071734A1/de not_active Withdrawn
  • 1999-04-07 AR ARP990101569 patent/AR017484A1/es unknown
  • 1999-04-07 WO PCT/DK1999/000202 patent/WO1999053001A1/en not_active Application Discontinuation

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