EP2799531A1 - Utilisation de phosphatases pour la démucilagination enzymatique de triglycérides - Google Patents

Utilisation de phosphatases pour la démucilagination enzymatique de triglycérides Download PDF

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Publication number
EP2799531A1
EP2799531A1 EP13166529.1A EP13166529A EP2799531A1 EP 2799531 A1 EP2799531 A1 EP 2799531A1 EP 13166529 A EP13166529 A EP 13166529A EP 2799531 A1 EP2799531 A1 EP 2799531A1
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Prior art keywords
oil
phospholipase
enzymes
phytase
phosphatase
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EP13166529.1A
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German (de)
English (en)
Inventor
Kirstin Dr. Suck
Ulrich Dr. Sohling
Paul Bubenheim
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Clariant Produkte Deutschland GmbH
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Clariant Produkte Deutschland GmbH
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Priority to EP13166529.1A priority Critical patent/EP2799531A1/fr
Priority to PCT/EP2014/001161 priority patent/WO2014177279A1/fr
Priority to ARP140101797A priority patent/AR096185A1/es
Publication of EP2799531A1 publication Critical patent/EP2799531A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
    • 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
    • C11B3/02—Refining fats or fatty oils by chemical reaction
    • C11B3/04—Refining fats or fatty oils by chemical reaction with acids
    • 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

Definitions

  • the invention relates to a method for increasing the oil yield in the degumming of triglycerides by using a phosphatase. Furthermore, the invention relates to the use of at least one phosphatase for degumming crude vegetable oils. Another aspect of the invention relates to the de-oiling of vegetable oil slime, wherein in addition to the increase in oil yield, the recovery and purification of lecithin is in the foreground.
  • Raw vegetable oils contain phosphatides, protein and carbohydrate-containing substances, phytochemicals and colloidal compounds, which greatly reduce the shelf life of the oil. These substances must therefore be removed.
  • the chemical refining consists of the processes 1. Degumming, 2. Neutralization, 3. Bleaching, 4. Deodorization. During degumming, phospholipids ("gums") and metal ions (Ca 2+ , Mg 2+ ) are removed from the oil. The neutralization serves to extract the fatty acids. Bleaching removes the dyes, other metal ions and residual mucilage. Deodorization is a steam distillation process that removes other compounds that affect the smell and taste of the oil. In physical refining, deacidification is performed along with deodorization at the end of the refining process.
  • the degumming of the oils can be carried out by extraction of the phospholipids with water or an aqueous solution of an acid which complexes Ca 2+ and Mg 2+ ions, such as e.g. As citric acid or phosphoric acid. Often one first carries out an aqueous, so-called pre-degumming with which the water-soluble phospholipids are removed. This is called hydratable phospholipids.
  • triglycerides is understood to mean triple esters of glycerol with fatty acids, which are the main constituent of natural fats and oils, be it of vegetable or animal origin. Triglycerides include vegetable or animal fats and oils, and mixtures thereof both with each other and with synthetic or modified fats and oils.
  • vegetable oil is understood here to mean any oil of plant origin.
  • preferred oils are soybean oil, rapeseed oil, canola oil, sunflower oil, olive oil, palm oil, jatropha oil, camelina oil or cottonseed oil.
  • the vegetable oil according to the invention comprises mixtures of different vegetable oils with one another, as well as the mixture of vegetable oil with animal and / or synthetic or modified fats and oils.
  • raw refers to the fact that the oil has not yet undergone degumming, neutralization, bleaching and / or deodorization step. It is also possible in the context of the method according to the invention that a mixture of several crude oils is used or pretreated, eg pre-degummed and / or preconditioned oils are treated with the enzymes.
  • pre- degumming or "wet degumming” is understood to mean a treatment of the crude oil with water or an aqueous acid solution in order to remove water-soluble phospholipids as far as possible from the oil. Also, in the context of a pre- or wet degumming after the addition of acid, if necessary, an addition of alkali to neutralize the acid. Before the enzyme addition, the separation of the aqueous phase takes place. After pre-degumming, the phosphorus content in the crude oil is reduced from approx. 500-1500 ppm, eg for soybean and rapeseed, to below 200 ppm in the pre-degummed oil.
  • lecithin can be obtained from the resulting slime phase or the slime phase can be worked up as feed.
  • the disadvantage of separating off the aqueous phase or lowering the phosphorus content is a yield loss with respect to the oil.
  • the phosphatides passing into the aqueous phase have an emulsifying effect and cause some of the oil to be emulsified in the aqueous phase and separated therewith. Subsequently, the oil can be further treated enzymatically, wherein the enzymes may need to be separated in a further step.
  • preconditioning of the oil in this invention means the addition of water or an aqueous acid solution to the untreated oil. Subsequently, by addition of alkali, for. For example, sodium hydroxide, adjusted to a pH at which the following enzymatic reaction takes place. Ideally, the optimal pH for the enzyme reaction is adjusted from 4 to 7. Subsequently, however, no separation of the aqueous phase, but immediately the addition of the enzymes. The existing mucilages remain so for the time being in the oil or in the emulsion. The separation of the aqueous phase and thus the Enzymes take place only after the action of the enzymes on the (possibly preconditioned) crude oil.
  • alkali for.
  • sodium hydroxide adjusted to a pH at which the following enzymatic reaction takes place.
  • the optimal pH for the enzyme reaction is adjusted from 4 to 7.
  • no separation of the aqueous phase but immediately the addition of the enzymes.
  • the existing mucilages remain so for the time being in the
  • phosphatase is understood to mean a group of enzymes which split off phosphoric acid from phosphoric esters or polyphosphates.
  • Phytase is the name for enzymes that hydrolytically degrade phytic acid and thus release the bound phosphate. Depending on which phosphate group is broken down, a distinction is made between 3-, 4-, 5- and 6-phytase. Phytases are known from animals, plants, fungi and bacteria; the best characterized phytases are from fungi. The phytase formed by many plants and bacteria acts as a 3-phytase; this is used commercially together with 6-phytase. 4-phytase is an acid phosphatase.
  • Acid phosphatases are phosphatases whose pH optimum is in the acidic pH range, such as 4.0 to 5.5.
  • Phospholipases D which split or exchange the head group.
  • Phospholipases B which cleave the fatty acid in both the sn 1 and sn 2 positions to form a 1,2-lysophospholipid.
  • acyltransferase is understood to mean an enzyme that acyl groups, for example fatty acids from a phospholipid, onto one suitable acceptor, eg a sterol, to form an ester transfers.
  • the phosphorus content of the oil when the oils are used, for example, for the production of biodiesel.
  • the phosphorus content of the biodiesel is limited to 5 ppm and it is expedient to carry out the phosphorus reduction already on the oil side.
  • a particularly low phosphorus content ie a phosphorus content which is as low as possible and preferably 0, is necessary if the oils are hydrogenated in further treatment steps either for the use of food, ie converted unsaturated into saturated fatty acids, or if, according to the NxBTL process of Neste, hydrogenation is carried out in such a way that alkanes, that is to say a conventional biodiesel fuel, but produced from vegetable oil, are obtained as the end product.
  • the so-called enzymatic degumming avoids several disadvantages of existing processes or further improves the extraction process.
  • the enzymatic degumming is brought about in the prior art by the use of phospholipases, in particular phospholipase A1 and A2, phospholipase B, phospholipase C or phospholipase D or a combination of phospholipases.
  • An alternative concept for oil degumming represents the systems of the company Danisco, in which a Lipidacyltransferase is used. This enzyme also turns a phospholipid into a lysophospholipid, but transfers the fatty acid residue to a sterol in the oil phase.
  • the corresponding enzymes and methods of using these enzymes are in the WO 2006/008508 and the WO 2009/081094 described.
  • Phytases are marketed primarily as feed additives on the market. About two-thirds of phosphorus in plant feed for livestock are bound to phytic acid or its salts and can be cleaved only by the phytases and thereby made available for the phosphate metabolism of livestock. However, the phytases do not occur in the gastrointestinal tract of pigs and poultry. In order to improve the utilization of phosphorus from vegetable feeds, phytases are added to the animal feed.
  • Such phytases are for example in the patents EP1066373B1 (Novozymes), EP2295553 (Novozymes), US6475762 (Genencor International), WO9855599 (Gist Brocades); WO9949740 (DSM); WO2004071218 (DSM); WO2011117396 (Novozymes), US2009081331 (BASF); WO2009129489 (Danisco US INC.).
  • Typical phytases available on the market are the products Natuphos® from BASF (3-phytase), Ronozyme® from DSM, Axtra® PHY and Phyzyme® from Danisco.
  • phytases are in the modification of plant foods and is for example in the WO2004 / 071218 and the US2004058049 described.
  • the phytic acid especially in the neutral environment of the intestine, forms (sparingly soluble) salts with the divalent ions of the minerals and thus reduces their absorption. This should be the case in particular for the ions of Fe, Zn and Ca.
  • these effects can be reduced. It is discussed to pretreat foods with phytase to make the minerals contained therein more available / absorbable. That's how it describes US2004058049A1 the addition of phytases in soft drinks.
  • the object of the present application is to develop an alternative process for the enzymatic degumming of triglycerides, which reduces the phosphorus content of the oil to be degummed, increases the oil yield and / or increases the reaction rate of the enzymatic degumming. At the same time, this method should enable economic implementation on an industrial scale.
  • a further object of the invention is to provide a process for obtaining lecithin from triglycerides, in particular from crude soybean, sunflower or rapeseed oil, in high yield, wherein the content of oil in the lecithin obtained is as low as possible, or in other words, the De-oiling the lecithin or reducing the oil content in the vegetable oil slime.
  • an (acidic) phosphatase and / or a phytase are particularly suitable for degumming triglycerides or for de-oiling lecithin.
  • the use of acidic phosphatases or phytases in the enzymatic oil degumming was not previously known.
  • only applications of these enzymes in purely aqueous systems have been known, but not in oil-water two-phase systems as used in aqueous oil degumming.
  • those phytases are suitable, which are already used for the treatment of food.
  • vegetable oil slime can be used, which is obtained in the degumming of vegetable oils, either by degumming by conventional or the inventive method.
  • the vegetable oil slime is brought into contact with the at least one phosphatase according to step a) and then after step b2), which is carried out analogously to b1), separated into an aqueous, lecithin-containing phase and an oil-containing phase.
  • the slime phase or the vegetable oil slime is used in particular in the production of lecithin.
  • the at least one phosphatase is an acid phosphatase or a phytase or a combination of both enzymes.
  • the acid phosphatase is preferably one or more enzymes of the enzyme class EC 3.1.3.2.
  • the enzymes of this class are generally substrate-unspecific, cleave from a variety of organic substrates phosphate groups and have a pH optimum of less than or equal to 7, preferably less than or equal to 6.5, more preferably of less than or equal to 6.
  • the lower limit of the pH is preferably 3.0, more preferably 3.5. In this pH range, a fast reaction is possible.
  • acid phosphatases can also be isolated from milk such as mammalian milk.
  • Purple Acid Phospatases are a distinct class of acid phosphatases that contain dinuclear transition metal centers and show a violet color in solution.
  • a typical representative of this class is the purple acid phosphatase from the sweet potato, as you for example from Kusudo T, Sakaki T, Inouye K. Purification and Characterization of Purple Acid Phosphatase PAP1 from Dry Powder of Sweet Potato, Biosci. Biotechnol. Biochem. 2003 67 (7): 1609-11 is described.
  • PAPs are also widely available and can also be isolated from soybean, rice, onions and red kidney beans. In addition, such enzymes can also be isolated from bacteria or mammals.
  • the acid phosphatase is derived from plants, in particular from sweet potato, from wheat germ or soya, from mammalian milk or from fungi, yeasts or bacteria.
  • the phytase used in the process according to the invention is a single phytase or a mixture of two or more phytases. It is preferably a so-called 3-phytase (enzyme class EC 3.1.3.8), 4-phytase (enzyme class EC 3.1.3.26), 5-phytase (enzyme class EC 3.1.3.72) or 6-phytase (enzyme class EC 3.1.3.26) , (Citation: Enzymes in Industry, Wolfgang Aehle Editor, Wiley VCH, Weinheim, Second Edition, 2004 Chapter 5 Industrial Enzyme, p.
  • the numbers refer to the position of the phosphate groups in the phytic acid, which are the first cleaved by the action of phytases. Thereafter, depending on the reaction time and the reaction conditions, further phosphate groups are split off.
  • Phytases are bulk enzymes that are industrially produced on a large scale.
  • the phytase is derived from the following organisms: plants, bacteria, yeasts and fungi.
  • the phytase is derived from corn, soybean, wheat bran, Bacillus species, Escherichia coli species, Klebsiella species, Pseudomonas species, Aspergillus species, Trichoderma Species, Rhizopus species, Saccharomyces species, Pichia species, Penicillium species, Schwanniomyces species, Arxula adeninivorans.
  • the phytases used in the process according to the invention have a pH optimum at a pH of less than or equal to 8, preferably less than or equal to 7; most preferably less than or equal to 6.
  • the lower limit of the pH is preferably 3.0, more preferably 3.5.
  • a phospholipid-cleaving enzyme is used.
  • the phospholipid-cleaving enzyme is a phospholipase A1, phospholipase A2, phospholipase B, phospholipase C, phospholipase D or acyltransferase. More preferably, the phospholipid-cleaving enzyme is selected from phospholipase A1, phospholipase A2 or phospholipase B.
  • the phospholipase A1 is obtained from the following species: Thermomyces lanuginosus, Fusarium oxysporium, Aspergillus oryzae, Aspergillus niger, Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Listeria monocytogenes, Pseudomonas species, Trichoderma reesei, Pichia pastoris, porcine pancreas or bovine pancreas.
  • the phospholipase A1 is from Thermomyces lanunginosus.
  • Phospholipase A2 is preferably and independently produced from the following sources: porcine pancreas, bovine pancreas, Streptomyces violaceoruber, Naja mossambica, Thermomyces lanuginosus, Fusarium oxysporium, Aspergillus oryzae, Aspergillus niger, Bacillus cereus, Bacillus subtilis, Trichoderma reesei, Clostridium perfringens, Listeria monocytogenes or Pseudomonas species.
  • the phospholipase B is preferably and independently obtained from the following species: Thermomyces lanuginosus, Fusarium oxysporium, Aspergillus oryzae, Aspergillus niger, Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Listeria monocytogenes, Pichia pastoris, Trichoderma reesei, Pseudomonas species, porcine pancreas or bovine pancreas.
  • Phospholipase C is preferably and independently obtained from the following species: Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Listeria monocytogenes, Thermomyces lanuginosus, Fusarium oxysporium, Aspergillus oryzae, Aspergillus niger, Pichia pastoris or Pseudomonas species.
  • the phospholipase D is derived from the following species: porcine pancreas, bovine pancreas, cabbage, peanut, Streptomyces species, Streptomyces chromofuscus, Streptomyces violaceoruber, Naja mossambica, Thermomyces lanuginosus, Fusarium oxysporium, Aspergillus oryzae, Aspergillus niger, Bacillus cereus, Bacillus subtilis, Trichoderma reesei, Clostridium perfringens, Listeria monocytogenes or Pseudomonas species.
  • the phospholipase A1 is selected from Thermomyces lanuginosus or Fusarium oxysporium, and / or independently, the phospholipase A2 is selected from porcine pancreas, bovine pancreas, Streptomyces violaceoruber, Trichoderma reesei, Aspergillus niger or Naja mossambica, and / or independently, the phospholipase C is selected selected from Bacillus cereus, Clostridium perfringens, Pichia pastoris or Listeria monocytogenes.
  • the process according to the invention is preferably carried out in one of the aqueous phases of the oil-water mixture of less than 7, preferably less than 6, particularly preferably less than 5.
  • the lower limit of the pH is preferably 3.0, more preferably 3.5.
  • the method of the present invention is carried out at a pH of 4.0 to 5.5.
  • the ratio of the phosphatase to the phospholipid-cleaving enzyme ranges from 0.01: 6 units / g of oil to 6: 0.01 units / g of oil.
  • the phosphatase and / or the phospholipid-cleaving enzyme are in supported form.
  • soybean oil, rapeseed oil, canola oil, sunflower oil, olive oil, palm oil, jatropha oil, camelina oil or cottonseed oil are used as oils.
  • an aqueous vegetable oil slime which is obtained in the oil degumming of one of the abovementioned oils, is used.
  • soybean oil is degummed with a phytase, preferably a 3-phytase or 6-phytase, and optionally additionally with a phospholipase A1.
  • rapeseed oil is degummed with an acid phosphatase and optionally additionally with a phospholipase A1.
  • the crude vegetable oil is contacted with water and / or acid.
  • This measure is known as preconditioning if, before carrying out step a), there is no separation into water or oil, or as pre-degumming, if a separation into water or oil takes place before carrying out step a).
  • one of the enzymes as defined above is used to increase the oil yield in a process for aqueous oil degumming, to reduce the emulsifiability of vegetable oils in aqueous phases, or to de-oil vegetable leucite or lecithin.
  • a combination of two or more of the aforementioned phosphatase enzymes can be used, which may originate from the same or different sources.
  • the enzymes can be derived from any organism (eg isolated from a thermophilic organism) or from a synthetic source. The same applies to the phospholipid-cleaving enzymes or their mixtures with phosphatases.
  • the method of the present invention is advantageous because the use of the phosphatase enzymes, in particular by the relatively inexpensive bulk enzyme phytase or by acid phosphatases, the cleavage of components that are present in the mucus phase can take place.
  • the phosphatase enhances the action of the phospholipid-cleaving enzyme when used in addition to the phosphatase. It is believed that by using the phosphatase, in particular by phytase and / or acid phosphatase, a decrease in the viscosity of the mucous phase, an increase in the mobility of the phospholipids or better accessibility of the phospholipids can be achieved. An additional increase of the effects described above results when in addition phospholipid-cleaving enzymes are used.
  • phosphatase In particular, phytase and acid phosphatase are suitable for this purpose.
  • Two or more phytases can be used, which may come from the same or different sources; they can e.g. also be isolated from a thermophilic organism or derived from a synthetic source.
  • acidic phosphatases and their mixture with phytase (n) the same applies mutatis mutandis.
  • the phospholipid-cleaving enzyme may be a phospholipase capable of cleaving either a fatty acid residue or a phosphatidyl residue or a head group from a phospholipid.
  • a so-called acyltransferase in which the cleavage of the fatty acid residue from a phospholipid is associated with a transfer of this residue to a free sterol with ester formation in the oil phase.
  • the present invention relates to a process wherein a phospholipid-cleaving enzyme from the phospholipase A1, phospholipase A2, phospholipase C, phospholipase B, phospholipase D or acyltransferase.
  • Typical enzymes from this group are the Lecitase® Ultra from Novozymes®, a phospholipase A1; Lecitinase® from Novozymes, a phospholipase A2; Rohalase® MPL, a phospholipase A2 from AB Enzymes, Darmstadt (D); Gumzyme TM, a phospholipase A2 from DSM, Purifine®, a phospholipase C from DSM; Lysomax®, an acyltransferase from Danisco.
  • phospholipid-cleaving enzymes within the group of phospholipid-cleaving enzymes, a combination of two or more of the aforementioned phospholipid-cleaving enzymes can be used, which may originate from the same or different sources.
  • the enzymes can be derived from any organism (eg isolated from a thermophilic organism) or from a synthetic source.
  • an addition of water or an aqueous acid solution and optionally of alkali to neutralize the acid to crude oil in the sense of preconditioning done but omits the separation of the aqueous phase before the addition of the enzymes (in the sense of Naughtvorentschleimung).
  • an increase in the oil yield is possible.
  • An increase in the oil yield of one percentage point has enormous economic importance, since this percentage corresponds to about 400,000 t of oil, based on the annual production of soybean oil.
  • the inventive method thus allows the immediate use of crude oils from soybean or rape with phosphorus contents of 500 to 1500 ppm phosphorus. Moreover, it is a simplification of the process, because the separation step before enzyme addition is omitted.
  • the enzyme activity of the phosphatase (s) is selected in the range of 0.01 to 10 units / g of oil, more preferably in the range of 0.1 to 6 units / g of oil, more preferably in the range of 0.2 to 5 units / g of oil.
  • the enzyme activity of the phospholipid-cleaving enzymes in the range of 0.01 to 6 units / g of oil, preferably 0.1 to 3 units / g of oil and more preferably in the range of 0.2 to 2.5 units / g of oil, and most preferably in the range of 0.25 to 1 units / g of oil. (Unit: International unit for enzyme activity, 1 unit corresponds to substrate turnover of 1 ⁇ mol / min).
  • the ratio of the enzyme activity of the phosphatase to the enzyme activity of the phospholipid-cleaving enzymes will be in the range of 0.01: 6 units / g of oil to 6: 0.01 units / g of oil, preferably in the range of 0.1: 4 units / g oil to 4: 0.1 units / g oil. It is also preferred if the proportion of phosphatase (s) and the proportion of phospholipid-cleaving enzymes is the same, for example, if both proportions in the range of 0.1 to 5 units / g of oil, preferably in the range 0.2 to 3 units / g of oil.
  • the ⁇ lentschleimung or de-oiling of the plant mucus in acid pH is carried out.
  • a preferred pH of the aqueous portion of the oil-water mixture is in the range of from 3.0 to 7.5, preferably from 3.5 to 6.5, most preferably from 3.8 to 6.0. If the pH is within this range, rapid reaction can be observed.
  • the phosphatase enzymes and / or phospholipid-cleaving enzymes can be used, for example, freeze-dried and dissolved in an enzyme buffer.
  • an enzyme buffer As examples, citrate buffer 0.01-0.25 M, pH 3.8-7.5, preferably 0.1 M citrate buffer at pH 5.0, or acetate buffer 0.01-0.25 M, pH 3.8 7.5, preferably 0.1 M acetate buffer at pH 4.0, are called.
  • the enzymes are taken up in water or enzyme buffer and added to the crude oil. In order to achieve a better solubility of the enzymes - especially in the mixtures containing phospholipids - also the addition of organic solvents is possible. These find application for. B. in the separation of phospholipids and are described in the literature.
  • non-polar organic solvents such as.
  • hexane, or acetone or mixtures preferably in an amount of 1 to 30 wt .-% (examples of possible solvents are described in the EP 1531182 A2 ).
  • the phosphatase enzyme and / or the phospholipid-cleaving enzyme are used in supported form, wherein the enzymes are adsorptively or covalently bound to a carrier material.
  • Preferred support materials are inorganic support materials, such as. As silica gels, precipitated silicas, silicates or aluminosilicates, and organic support materials such. As methacrylates or ion exchange resins.
  • the support materials facilitate the separability and recyclability of the (relatively expensive) enzymes from the oil-water emulsion in a subsequent process step and contribute to the economics of the process.
  • the phosphatase enzyme comprises at least one enzyme selected from the group consisting of phytase, acid phosphatase, alkaline Phosphatase, and phosphatidic acid-specific phosphatase consists.
  • the phospholipid-cleaving enzyme preferably comprises at least one enzyme selected from the group consisting of phospholipase A1, phospholipase A2, phospholipase B, phospholipase C, phospholipase D, and acyltransferase.
  • freeze-dried enzymes are used which are present in a buffer selected from citrate buffer or acetate buffer. It is also preferred if at least the phosphatase enzyme is present in adsorptive or covalent binding on a carrier.
  • phosphatase enzymes in particular acid phosphatase and / or phytase, are suitable for effectively and effectively reducing the emulsifiability of vegetable oil in aqueous phases.
  • this effect can be enhanced by combining the phosphatase enzymes with phospholipid-cleaving enzymes.
  • the method can be advantageously used for the degumming of crude vegetable oil or for the treatment of mucilage.
  • the mucilages may be obtained, for example, by a conventional degumming process or by the process according to the invention when it is used for degumming crude vegetable oil.
  • Triglycerides preferably crude or untreated triglycerides, can be used as starting material, which are brought into contact with the phosphatase enzyme, optionally in combination with phospholipid-cleaving enzymes (step a) and subsequently separated into mucilages and (degummed) triglycerides become; (Step b1). In this case, the enzyme according to the invention is added before separation step b1).
  • a vegetable oil slime obtained for example, by a conventional degumming process, such as by treatment with water or aqueous acid, optionally followed by a neutralization with brine, may be contacted with the phosphatase enzyme (Step a) and then separated into an aqueous lecithin-containing phase and an oil phase; (Step b2).
  • the phosphatase enzyme is added after separation of the vegetable oil mucus. With this procedure, it is thus possible to obtain from vegetable oil slime both further oil and de-oiled lecithin.
  • the phosphatases optionally in combination with the phospholipases A1, A2 or B, can be used for de-oiling vegetable oil slime.
  • phosphatase enzymes it has been found that it is possible by the use of phosphatase enzymes to increase the oil yield of the reaction. Furthermore, it was surprisingly found that the combination of phosphatase enzymes with phospholipid-cleaving enzymes, the oil yield of the reaction is further increased. Moreover, the addition of the phosphatases further reduces the phospholipid content of the crude oil compared to the pure use of phospholipid-cleaving enzyme (in the prior art), increases the reaction rate during enzymatic degumming, and / or improves the separability of the gum phase formed.
  • phosphatase enzymes in particular by acid phosphatase and / or phytase, it is possible in conjunction with phospholipid-cleaving enzymes, the dosage of phospholipid-cleaving enzymes such.
  • phospholipase A1, A2 or B optionally combined with phospholipase C, and thus to save costs in addition to the above-mentioned advantages for the process.
  • the "bringing into contact” can be carried out in the context of the inventive method in any way that is known to those skilled in the art as suitable for the purpose of the invention.
  • a preferred mode of contacting is to mix the crude oil and the enzymes.
  • the mixture of crude oil and enzyme is preferably stirred, more preferably with a paddle stirrer at 200 to 800 rpm, preferably 250 to 600 rpm and most preferably 300 to 500 U / min.
  • the temperature of the mixture during the contacting is preferably in the range of 15 to 99 ° C, more preferably in the range of 20 to 95 ° C, more preferably 22 to 80 ° C, also preferably 30 to 80 ° C, more preferably from 32 to 80 ° C, and most preferably from 35 to 80 ° C.
  • the temperature of the mixture should always be chosen so that the denaturation temperature of the enzymes is not exceeded.
  • the temperature of the mixture is at least 5 ° C below the denaturation temperature of the enzymes or the lowest denaturation temperature of the enzymes. When using enzymes that have been isolated from thermophilic organisms, generally higher temperatures are to be preferred.
  • the process temperature is preferably in the range from 80 to 120 ° C., more preferably in the range from 85 to 100 ° C.
  • the use of thermostable enzymes has the advantage that thus an elevated process temperature can be selected, whereby the viscosity of the vegetable oil is reduced and the process can be shortened overall - also due to an increased reaction rate of the enzymes.
  • a pretreatment which advantageously also at followed by cooling below a lower denaturation temperature of the enzymes used.
  • the use of thermostable enzymes thus leads to a process shortening and cost reduction.
  • the duration of the contacting is preferably in the range from 1 minute to 12 hours, more preferably from 5 minutes to 10 hours, also preferably from 10 minutes to 6 hours, more preferably from 15 minutes to 4 hours.
  • the pH of the aqueous portion of the oil / enzyme mixture during contacting is preferably in the range of pH 3 to pH 7.5, more preferably in the range of pH 3.5 to pH 6.5, and most preferably in the range of Range from pH 3.8 to pH 6.0.
  • the separation of the mucilages according to step b) of the method according to the invention can be carried out in any manner which is known to those skilled in the art as suitable for the purpose of the invention, but preferably the separation is carried out by centrifugation or filtration, centrifugation is preferred.
  • the centrifugation phase separates the mixture so that the treated vegetable oil, mucilages and enzyme composition are in separate phases that are easily separated.
  • the separate products differ only in their quantity, but not in their chemical nature.
  • the phase containing the mucilages and the phase containing the enzyme is separated from the treated oil. It is particularly preferred if all enzymes are separated simultaneously with the mucilage.
  • the enzymes can be regenerated or purified after the separation and, for example, in a new purification process be used. In this case, too, it is likewise favorable to work again with the enzymes according to the invention, either by using the phosphatase enzymes directly in combination with supported phospholipases or lipid acyltransferases, or by using the enzyme according to the invention to prepare the vegetable oil mucus, for example supported phospholipases liable to remove in order to better reuse the supported enzymes in a new process can.
  • the bringing into contact is preferably carried out under the same conditions as described above for step a) of the process according to the invention.
  • the phosphatase enzymes and / or the phospholipid-cleaving enzymes are subjected to regeneration or purification before re-contacting.
  • the crude vegetable oil is brought into contact with water and / or acid before contacting in step a) of the process according to the invention.
  • Preferred acids are calcium and magnesium-complexing acids alone or in combination, such as. As citric acid and phosphoric acid. This is called a so-called "pre-degumming".
  • bringing into contact with water at a temperature between 30 ° C to 90 ° C for 15 to 60 minutes, preferably 30 to 60 minutes, wherein a temperature of 35 to 85 ° C is preferred and a temperature of 40 to 80 ° C is particularly preferred.
  • the bringing into contact with acid takes place in the context of the method according to the invention preferably at a temperature between 30 ° C to 90 ° C for 5 to 60 minutes, preferably 15 to 60 minutes, with a temperature of 35 to 85 ° C is preferred and a temperature of 40 to 80 ° C is particularly preferred.
  • the acidic, aqueous phase is then z. B.
  • a neutralization step will be carried out with a corresponding base to achieve a pH of from 3.5 to 8.0, preferably from 4 to 7.
  • the oil of the mucins obtained by z As centrifugation or filtration are separated. It is also possible and preferred according to the method of the present invention if the enzyme (s) are added directly to the vegetable oil without previously undergoing a separation step.
  • the phosphatase (s) and optionally the phospholipase (s) should preferably be taken to ensure that the reaction temperature does not exceed the optimum temperature range of the enzyme to prevent denaturation of the enzyme.
  • Temperatures between 35 to 75 ° C, better between 45 to 70 ° C, are suitable, by use of enzymes from thermophilic organisms, ie particularly thermally stable enzymes, use at 80 to 100 ° C is possible, so that between the in- Contacting the crude vegetable oil with water and / or acid and bringing into contact with the enzyme or enzymes no temperature reduction must be made.
  • An increase in temperature stability can also be achieved by immobilizing the enzymes. Since many enzymes have a certain tolerance to organic solvents (Faber, K., Biotransformations in Organic Chemistry (2001), Springer-Verlag, Heidelberg), accordingly pretreated oils or mucus can be treated with the enzymes in the context of the present invention.
  • the method of the present invention comprises the steps:
  • a so-called preconditioning is carried out before step a) of the process by mixing the crude oil in a separate process step with an amount of 300-1,000 ppm of organic acid, preferably citric acid.
  • the temperature of the mixture is in this case preferably set to 45 to 85 ° C, more preferably 48-80 ° C.
  • the mixture After a reaction time of preferably 10 minutes to 2 hours, more preferably 15 minutes - 1 hour, the mixture by addition of a stoichiometric amount of alkali, preferably sodium hydroxide, in an amount of preferably 0.5 to 2 mol / l, more preferably 1 mol / l, adjusted to a pH of 4-5. Only then is the process according to the invention proceeded according to step a).
  • This preferred embodiment B) is combined in a preferred embodiment with a post-degumming by the addition of an organic acid and / or alkali (after step b) or b1)).
  • the temperature of the mixture is in this case preferably set to 35 to 60 ° C, more preferably 50 ° C.
  • an alkali preferably sodium hydroxide, in a concentration of preferably 0.5 to 2 mol / l, more preferably 1 mol / l, to a pH of 4-5 set.
  • a slime phase which has been separated by a conventional or the degumming method according to the invention is brought into contact with the enzyme (s).
  • the method is preferably carried out according to the embodiment A), B) or D).
  • this process allows the recovery of degummed oil that has been separated with the slime phase. It thus allows a recovery of the oil and leads to an indirect increase in the oil yield; It also allows de-oiling of the lecithin.
  • any phosphatidic acids still dissolved in the vegetable oil and not split by the phospholipases can be further reduced by reducing the Ca and / or Mg content of the oil treated according to the invention. Therefore, in general embodiments, the abovementioned embodiments of the process A) to D) according to the invention are supplemented by a subsequent step in which, by adding again complexing agents, such as e.g. Citric acid or phosphoric acid, the content of bivalent ions and parallel to the content of P in the oil is further reduced.
  • complexing agents such as e.g. Citric acid or phosphoric acid
  • the method according to the invention it is possible to greatly lower the phosphorus value in the crude vegetable oil. It will the phosphorus value is reduced to below 20 ppm, more preferably below 10 ppm.
  • the inventive method to reduce the calcium and magnesium content of the crude vegetable oil to below 20 ppm, more preferably below 15 ppm, most preferably below 10 ppm, also preferably below 8 ppm, and most preferably below 4 ppm.
  • the calcium and magnesium content is reduced to below 3 ppm.
  • the content of free fatty acids is determined by the consumption of sodium hydroxide or potassium hydroxide via a saponification reaction. The percentage of free fatty acids in the examined oil is obtained. The determination was carried out in accordance with DIN 53402 (method DGF C-V 2).
  • the slime phase contained in the oil is measured by enzymatically untreated and enzymatically treated slime.
  • a 10 mL sling glass is heated to the working temperature of the reaction mixture, the samples (2 x 2 mL) are filled and tempered for at least 4 minutes at 3000 rpm centrifuged to separate the slime from the oil. From the upper oil phases samples are taken for analysis. For documentation purposes, the result of the phase formation is additionally photographed.
  • the determination of the oil yield, the (residual) oil content in the slime phase and the slime volume are determined by the determination of the slime volume according to the standardized method as described in PCT / EP 2013/053 199 detectable. Furthermore, by Soxhlet extraction of the isolated mucus of the oil content of the mucus can be determined separately according to DIN ISO 659.
  • the amount of crude to be treated 400 to 600 g, is poured into a Duran reactor DN120 1000 mL and samples for analysis are taken.
  • the oil in the Duran reactor is heated by means of a hot plate to a temperature of 35 to 90 ° C, in particular 48 ° C or 80 ° C, while maintaining a temperature at which the enzyme does not denature. After the temperature is reached, the preconditioning is started.
  • a defined amount of dilute citric acid for example 450-1000 ppm
  • the mixture can be mixed with an Ultraturrax for 1 minute. Alternatively, incubate with stirring at about 600 rpm for 15 minutes to 1 hour to await the reaction of the acid.
  • a defined amount of sodium hydroxide solution (1-4 mol / L, remaining amount to 2-3% v / v, minus water from acid addition and enzyme addition) is added and it is incubated for a further 10 minutes with stirring.
  • the addition of the enzyme, the enzyme mixture or the immobilizate, preferably dissolved in buffer is stirred in, for which the stirrer speed can be briefly increased (1 minute to 900 rpm), then stirring is continued at a lower speed.
  • Sampling takes place at defined time intervals. The sample is taken with the help of a pipette, filled into a tempered glass (temperature of the reaction mixture) and tempered for at least 4 minutes at 3000 rpm to separate the slime phase from the oil. For documentation purposes, the result of phase formation is photographed. From the supernatant samples are taken to determine the phosphorus, calcium and magnesium content.
  • phosphatases and optionally additional enzymes in a suitable combination as free enzymes or immobilized enzymes together with an aqueous phase (enzyme buffer, pH 4-5) 0.05 to 5% w / v, added to the crude oil.
  • the emulsion consisting of water, enzyme, possibly enzyme carriers and oil, is mixed.
  • the Reaction tempered between 20 to 70 ° C, better carried out between 40 to 65 ° C.
  • the phase separation is awaited, the solids settle or can be prepared by a standard method known in the art, for. B. be removed by centrifugation or filtration.
  • the oil can be degummed with dilute acid (eg citric acid) or lye according to a procedure known to the skilled person as degumming.
  • oil slime is treated with enzymes.
  • further enzymes are added to the oil slime, which is obtained by a process known to the expert as degumming. These may be dissolved in an aqueous phase or suspended in an organic solvent. The mixture is ideally tempered to a temperature between 20 to 70 ° C, better to a temperature between 35 to 65 ° C. The batch is mixed until the process is complete. This can be checked by viscosity measurements or visually, by dissolving the otherwise solid slime phase. By centrifugation, a phase separation can be achieved, the individual phases can be separated.
  • the upper phase consists of the recovered oil
  • the middle phase of the phospholipids and the lower phase is an aqueous phase and contains the enzymes.
  • the enzymes can be recycled and reused.
  • the oil or the water phase containing the enzyme must be purified by the addition of complexing agents before further use of the ions.
  • the crude oil is brought to a high temperature, especially 70 to 100 ° C, more precisely 75 to 85 ° C.
  • the crude oil is conditioned with acid and brine according to the procedure described above, the temperature is maintained and thermostable enzymes are added.
  • the enzyme is stirred in, for which the stirrer speed can be briefly increased (eg 1 minute to 900 rpm), then stirring is continued at 600 rpm until the reaction is complete.
  • the separation of the oil slime can be carried out as previously described.
  • reaction variant 1 a soybean oil with the following starting contents was used: phosphorus 700 ppm, calcium 65.6 ppm, magnesium 62.6 ppm and a free fatty acid content of 1%.
  • the crude oil was preconditioned using aqueous citric acid (450 ppm) and aqueous sodium hydroxide solution (1 mol / L). Regular samples were taken (see Table 1). At the end of the reaction, the slime phase was centrifuged off and the residual oil content determined according to Soxhlet.
  • this preconditioning was carried out with the addition of an enzyme, phospholipase A1 from the organism Thermomyces lanuginosus (Sigma-Aldrich) (see FIG. 2 , Table 2).
  • Table 3 shows results of preconditioning with addition of the enzyme PLA1 from Thermomyces lanuginosus and another enzyme, a phytase, ie a 6-phytase from Aspergillus niger (from ASA special enzymes).
  • FIG. 1 shows how out FIG. 1 it can be seen that the use of acid and lye on the crude oil as preconditioning leads to a not inconsiderable mucus volume, which does not decrease significantly in the sequence despite the use of a stirrer at 600 rpm.
  • the single photo corresponds to a sampling. The samples are taken at times 10, 60, 120, 180 and 240 minutes. Table 1 lists the associated analytical data: the phosphorus content dropped from 33 ppm to 21 ppm after 240 minutes; the concentration of divalent ions calcium and magnesium increases slightly in the case of calcium from 7.8 ppm to 9.8 ppm; the concentration of magnesium decreases from 4.1 ppm to 3.2 ppm during the course of the reaction. The content free Fatty acids remain almost unchanged.
  • the preconditioning serves as a preparatory reaction for the oil degumming and at the same time as a reference treatment.
  • FIG. 2 When using the enzyme Phospholipase A1 from Thermomyces lanuginosus (Sigma-Aldrich) a decrease in the mucus volume in the course of the reaction can be seen (one photo per measurement / sampling). The associated data and times of sampling are shown in Table 2.
  • Tab. 2 shows a decrease in the calcium concentration from 9.6 ppm to 8.1 ppm, a decrease in the magnesium concentration from 4.4 ppm to 3 ppm and a decrease in the phosphorus content from 23 ppm to 15 ppm; the content of free fatty acids increases from 0.79% to 1.24%, in each case after 180 min reaction time.
  • the increase in free fatty acid is a sign of the activity of PLA1, which splits off the fatty acids from the phospholipid molecules and also the mucus volume decreases continuously.
  • FIG. 3 the volume of the mucus phase of a pre-conditioned crude oil treated with PLA1 and additionally with phytase, ie a 6-phytase from Aspergillus niger (ASA Spezialenzyme), is shown. It is evident from the associated analytical data from Table 3 that, surprisingly, a reduced mucus volume of 2.0% is already reached after 120 minutes, compared to the mucus volume of 2.8% when using PLA1 alone (Table 2). In addition, the ion values (Table 3) are reduced more rapidly compared to the values in Table 2 to 4.7 ppm Ca, 1.7 ppm Mg and 11 ppm P z. B. after 180 min reaction.
  • phytase ie a 6-phytase from Aspergillus niger
  • reaction variant 1 a rapeseed oil with the following starting contents was used: phosphorus 1200 ppm, calcium 365 ppm, magnesium 145 ppm and a free fatty acid content of 1.65%.
  • the crude oil was preconditioned using aqueous citric acid (1000 ppm) and aqueous sodium hydroxide solution (4 mol / L). Samples were taken regularly (see Table 4). At the end of the reaction, the slime phase was centrifuged off and the residual oil content determined according to Soxhlet.
  • this preconditioning was carried out with the addition of an enzyme, phospholipase A1 from the organism Thermomyces lanuginosus (Sigma-Aldrich) (see FIG. 5 , Table 5).
  • Table 6 shows results of preconditioning with the addition of the enzyme PLA1 (from Thermomyces lanuginosus) and another enzyme, a sweet-potato acid phosphatase (Sigma-Aldrich).
  • FIG. 4 shows the use of acid and lye on the crude oil as preconditioning leads to a not inconsiderable mucus volume, which does not decrease significantly in the sequence despite the use of a stirrer at 600 rpm.
  • the single photo corresponds to a sampling; the samples are taken at times 10, 60, 120, 180 and 240 minutes.
  • Table 4 shows the associated analytical data, the phosphorus content decreased from 247 ppm to 14 ppm after 240 minutes, the concentration of divalent ions calcium and magnesium in the case of calcium decreased from 76 ppm to 9.5 ppm, the concentration of magnesium decreases from 31 ppm to 1.7 ppm during the course of the reaction. The content of free fatty acids remains almost unchanged.
  • the preconditioning serves as a preparatory reaction for the oil degumming and at the same time as a reference treatment.
  • FIG. 5 If the enzyme Phospholipase A1 from Thermomyces lanuginosus (Sigma-Aldrich) is used, a decrease in the mucus volume of 1% can be detected in the course of the reaction (one photograph per measurement / sampling). The associated data and times of sampling are shown in Table 5.
  • Tab. 5 shows a decrease of the calcium concentration from 26 ppm to 7.9 ppm, a decrease of the magnesium concentration from 9.7 ppm to 1.5 ppm and a decrease in the phosphorus content from 82 ppm to 12 ppm, the free fatty acid content increases from 1.76% to 2.14%.
  • the increase in free fatty acid is indicative of the activity of PLA1, which cleaves the fatty acids from the phospholipid molecules, and also the mucus volume decreases.
  • FIG. 6 the volume of the mucus phase of a pre-conditioned crude oil treated with PLA1 (from Thermomyces lanuginosus ) and additionally with sweet potato acid phosphatase is shown. It is evident from the associated analytical data from Table 6 that, surprisingly, the additional use of acid phosphatase compared to the sole use of phospholipase ( Fig. 5 Tab.5) the mucus volume of the reaction is significantly reduced. This is synonymous with an increase in oil yield (cf. Fig. 5 and 6 , Table 5 + 6) and thus a larger profit margin of the process.
  • soybean oil About 43.5 million tonnes of soybean oil are produced each year worldwide (USDA FAS - 2012). With a 97.1% oil yield increase to 97.7% in the standard process through the phytase and PLA1 enzymatic process, 260,000 tonnes more soybean oil would be produced each year.

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EP13166529.1A 2013-05-03 2013-05-03 Utilisation de phosphatases pour la démucilagination enzymatique de triglycérides Withdrawn EP2799531A1 (fr)

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