WO2011075802A1 - Production of diacylglycerols by lipase - catalyzed hydrolysis of palm oil - Google Patents
Production of diacylglycerols by lipase - catalyzed hydrolysis of palm oil Download PDFInfo
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- WO2011075802A1 WO2011075802A1 PCT/BR2010/000384 BR2010000384W WO2011075802A1 WO 2011075802 A1 WO2011075802 A1 WO 2011075802A1 BR 2010000384 W BR2010000384 W BR 2010000384W WO 2011075802 A1 WO2011075802 A1 WO 2011075802A1
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- WIPO (PCT)
- Prior art keywords
- amano
- lipase
- palm oil
- hydrolysis
- reaction
- Prior art date
Links
- 102000004882 Lipase Human genes 0.000 title claims abstract description 91
- 108090001060 Lipase Proteins 0.000 title claims abstract description 91
- 239000004367 Lipase Substances 0.000 title claims abstract description 91
- 235000019421 lipase Nutrition 0.000 title claims abstract description 91
- 235000019482 Palm oil Nutrition 0.000 title claims abstract description 83
- 239000002540 palm oil Substances 0.000 title claims abstract description 83
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 53
- 150000001982 diacylglycerols Chemical class 0.000 title claims abstract description 35
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 230000007062 hydrolysis Effects 0.000 title abstract description 47
- 238000003756 stirring Methods 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 31
- 230000008569 process Effects 0.000 claims abstract description 29
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 49
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 4
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 3
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 claims description 2
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 2
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 claims description 2
- YCOZIPAWZNQLMR-UHFFFAOYSA-N heptane - octane Natural products CCCCCCCCCCCCCCC YCOZIPAWZNQLMR-UHFFFAOYSA-N 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims 1
- 230000035484 reaction time Effects 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 59
- 239000000463 material Substances 0.000 abstract description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
- 239000003921 oil Substances 0.000 description 15
- 235000019198 oils Nutrition 0.000 description 15
- 150000003626 triacylglycerols Chemical class 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 7
- 235000014113 dietary fatty acids Nutrition 0.000 description 7
- 239000003925 fat Substances 0.000 description 7
- 229930195729 fatty acid Natural products 0.000 description 7
- 239000000194 fatty acid Substances 0.000 description 7
- 150000004665 fatty acids Chemical class 0.000 description 7
- 235000013305 food Nutrition 0.000 description 5
- 150000002759 monoacylglycerols Chemical class 0.000 description 5
- 244000005700 microbiome Species 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 235000014593 oils and fats Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 3
- 102000001708 Protein Isoforms Human genes 0.000 description 3
- 108010029485 Protein Isoforms Proteins 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000005886 esterification reaction Methods 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 3
- 235000015112 vegetable and seed oil Nutrition 0.000 description 3
- 239000008158 vegetable oil Substances 0.000 description 3
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 2
- 235000006008 Brassica napus var napus Nutrition 0.000 description 2
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 2
- 244000188595 Brassica sinapistrum Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- UXDDRFCJKNROTO-UHFFFAOYSA-N Glycerol 1,2-diacetate Chemical compound CC(=O)OCC(CO)OC(C)=O UXDDRFCJKNROTO-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 240000007817 Olea europaea Species 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 244000000231 Sesamum indicum Species 0.000 description 2
- 235000003434 Sesamum indicum Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000008162 cooking oil Substances 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- -1 fatty acid esters Chemical class 0.000 description 2
- 230000037406 food intake Effects 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 238000007210 heterogeneous catalysis Methods 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- 238000010907 mechanical stirring Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000005809 transesterification reaction Methods 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical group 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 244000124209 Crocus sativus Species 0.000 description 1
- 235000015655 Crocus sativus Nutrition 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 101710084373 Lipase 1 Proteins 0.000 description 1
- 102100030659 Lipase member I Human genes 0.000 description 1
- 241001313618 Moritella sp. Species 0.000 description 1
- 241000235395 Mucor Species 0.000 description 1
- 241000498617 Mucor javanicus Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 241000235403 Rhizomucor miehei Species 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 241000303962 Rhizopus delemar Species 0.000 description 1
- 241000235545 Rhizopus niveus Species 0.000 description 1
- 240000005384 Rhizopus oryzae Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 235000004626 essential fatty acids Nutrition 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical class COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 150000002314 glycerols Chemical class 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 229940087559 grape seed Drugs 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229940068065 phytosterols Drugs 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000013974 saffron Nutrition 0.000 description 1
- 239000004248 saffron Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 235000015067 sauces Nutrition 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
Definitions
- the present invention relates to the production of diacylglycerols by heterogeneous catalysis using commercial enzymes to catalyze the production of diacylglycerols by hydrolysis reactions of palm oil.
- the use of diacylglycerols instead of triacylglycerols which are found in oils and fats began at the 80' s. Its use was based on two important health benefits.
- the first benefit relates to the suppression of triglycerides postprandial increase in serum and the second benefit is the suppression of body fat accumulation.
- Fats and oils consist of a mixture of triacylglycerols, also called triglycerides, and differ only by the physical state, fats being solid or pasty and oils being liquid at room temperature. Such difference is mainly due to the fact that oils are richer in unsaturated fatty acid residues than fats.
- Triacylglycerols consist of a highly efficient manner of storing metabolic energy, once they are less oxidized than carbohydrates and proteins and do not absorb much water, due to their apolar condition. Thus, triacylglycerols occupy a smaller volume in the organism, contrary to glycogen, which is another energy source that binds to water in an amount almost twice its weight.
- Vegetable oils are mainly composed by glycerides of plant fatty acids and may contain phospholipids, free fatty acids and unsaponifiable constituents. Their physical characteristics vary according to the structure and distribution of fatty acids in the present triacylglycerols .
- the triacylglycerol can be represented by the structure (I) below:
- Rl, R2 and R3 are fatty acid residues.
- Oils have a key role in food since they are important sources of essential fatty acids, carry fat-soluble vitamins and participate in the synthesis of many endogenous substances, among others. However, its excessive consumption is directly related to cardiovascular diseases, obesity and insulin resistance.
- Diacylglycerols or diglycerides are glycerol esters which have two hydroxyl groups esterified by fatty acids, and they can exist in two stereochemical forms, known as sn-l,2-DAG (or 2,3) and sn-l,3-DAG, the natural isomeric relationship being 3:7 as a function of the acyl group migration during the process of oil refining.
- the 1,2-DAG isoform is considered as a metabolic intermediate, which is formed after the ingestion of triacylglycerol.
- the 1,3-DAG isoform is responsible for the beneficial effect, since it is metabolized by a route which is different from that of TAG and 1,2-DAG.
- 1,3-DAG, 2,3-DAG and 1,2-DAG are respectively represented by structures (II), (III) and (IV) below: CH j OCOR
- Diacylglycerol is a natural component in several and fats, comprising about 10% by weight, as demonstrated in Table 1 below.
- the diacylglycerol is broadly used as an emulsifier and stabilizer in food, cosmetic and pharmaceutical industries. It has also been used in Japan and in the United States as cooking oil due to its recently discovered properties of reducing lipid levels, thus reducing weight and fat accumulation.
- the commercial oil was introduced in Japan in 1999 under the trade name Econa. In the United States, said oil was registered as Enova ® oil after 15 years of research, clinical studies and experiments performed by Kao Corporation .
- Enova contains approximately 20% by weight of triacylglycerol and 80% by weight of diacylglycerol, 56% thereof as the sn-l,3-DAG isoform. Its applications include the use as cooking oil, the use for the preparation of sauces and seasonings and products based on oils and fats.
- Japan, weight and fat mass can be reduced by replacing 10 to 20 grams of conventional oils of the diet by diacylglycerol .
- the DAG is produced by glycerolysis, hydrolysis or chemical esterification of oils and fats. These processes often use high temperatures and/or toxic catalysts. Such conditions cause changes in the flavor and in the color of the oil, reduce selectivity and increase energy expenditure, thereby increasing process costs.
- Enzymatic techniques have several advantages over the chemical process, including increased selectivity, increased product purity, the use of milder temperatures and the suppression of toxic catalysts. To this end, the use of lipases has been widely studied.
- diacylglycerol The most used processes to obtain diacylglycerol are the glycerolysis and hydrolysis of fatty acid esters as is shown below.
- Triglyceride Glycerol 1 3-Diacylglycerol 1, 2-Diacylglycerol
- H 2 C I C0 2 R .
- Triglyceride 1 3-Diacylglycerol 1, 2-Diacylglycerol
- a transesterification reaction between a triacylglycerxde and glycerol catalyzed by a lipase occurs in the glycerolysis process.
- the reactions occur without solvent and mechanical stirring is used, since the triacylglycerides have high viscosity.
- water is the agent which, together with the lipase, will hydrolyze the ester linkages of the triacylglyceride .
- the reactions occur in the absence of solvent and mechanical stirring is used.
- the stoichiometry of the reactants is as important as the adjustment of mechanical (stirring) and thermal (heat) factors. Thus, a huge variety of reaction conditions can be used in order to obtain the desired result.
- the products are formed by two types of diacylglycerols that differ only in the position of the fatty acid chains (1,3 and 1,2).
- the enzymatic catalysis provides only one of them due to its high specificity.
- the state of the art includes some patent documents related to the production of diacylglycerols from fats and oils, catalyzed by lipases.
- the Japanese document JP 1071495 describes a method for the preparation of diglycerides with high purity and yield, which comprises an esterification reaction between glycerol and saturated or unsaturated fatty acids with 4 to 22 carbon atoms in the presence of an 1 , 3-selective lipase immobilized with an ion exchange resin, and in which water or lower alcohol produced by the reaction are removed to maximum from the reaction system to increase the ester synthesis yield and reduce the amount of monoglycerides .
- 3-selective lipases are selected among the ones originating from microorganisms of Rhizopus, Mucor and Aspergillus species, more specifically from Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Aspergillus niger, Mucor javanicus and Mucor miehei.
- the process for producing diacylglycerols described in the US document 2007/0148745 involves the use of an immobilized 1 , 3-selective lipase in the presence of water to promote the hydrolysis of triacylglycerols found in oils and fats.
- Said document teaches a controlled hydrolysis with dehydration at the end of the process, using various vegetable oils, including palm oil.
- lipases are used as catalysts, preferably lipases immobilized with ion exchange resins. Temperatures between 20 and 90 °C are applied and the amount of water ranges from 20 to 180 parts of water per 100 parts of oil.
- the amount of enzyme used in the reactions is not specified; however, the examples suggest 10% by weight of enzyme to oil.
- the diacylglycerol is purified and 0.5% to 25% by weight of phytosterols and ferulic acid esters are added in relation to the weight of diacylglycerol.
- the reactions are catalyzed by Amano PS and Amano IM enzymes. Nevertheless, the amount of water used in the process of the present invention is four times less than the amount of water added in the process disclosed by document US 2007/0148745, since the minimum percentage used in such document was 20% by weight, while in the present invention only 5% by weight was used.
- Document WO03/094634 Al relates to the beneficial effects of using diacylglycerols in food and beverages. Said document discloses the physical-chemical changes obtained by adding different amounts of diacylglycerols in food, thus leading to new organoleptic properties.
- the object of the present invention relates to the production of diacylglycerols by heterogeneous catalysis.
- Amano PS and Amano IM commercial enzymes are used to catalyze the production of diacylglycerols by the hydrolysis reaction of refined palm oil.
- the object of the present invention comprises a process for the production of diacylglycerols by the hydrolysis of refined palm oil catalyzed by Amano PS and
- Amano PS and Amano IM commercial lipases are used as lipase sources to catalyze the hydrolysis of refined palm oil.
- the reactions should be conducted under optimal conditions for lipase activity, or as near as possible of such conditions.
- the optimal conditions involve temperature, reactant concentration and stirring.
- the enzymatic hydrolysis under reference is carried out in aqueous medium during a period of time within the range of 1 to 24 hours, thus resulting in a mixture of diacylglycerols, fatty acids and glycerol.
- the reaction catalyzed by lipases should be operated in batch.
- an apolar organic solvent is added to the reaction medium (for example, t- butanol, hexane, heptane or isooctane alone or associated with 10 to 90% by weight of water) and then the reaction mixture is filtered and washed with sodium chloride saturated solution. The organic phases are separated, dried with anhydrous sodium sulfate and the solvent is evaporated at reduced pressure.
- Example 1 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 60°C for 24h under stirring (1300 rpm) .
- Example 2 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 60°C for 24h under stirring (1300 rpm) .
- Example 3 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano PS lipase at 60°C for 24h under stirring (1300 rpm) .
- Example 4 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano IM lipase at 60°C for 24h under stirring (1300 rpm) .
- Example 5 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano PS lipase at 60°C for 24h under stirring (1300 rpm) .
- Example 6 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano IM lipase at 60°C for 24h under stirring (1300 rpm) .
- Example 7 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 40°C for 24h under stirring (1300 rpm) .
- Example 8 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 40°C for 24h under stirring (1300 rpm) .
- Example 9 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 50°C for 24h under stirring (1300 rpm) .
- Example 10 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 50°C for 24h under stirring (1300 rpm) .
- Example 11 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 55°C for 24h under stirring (1300 rpm) .
- Example 12 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 55°C for 24h under stirring (1300 rpm) .
- Example 13 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (700 rpm) .
- Example 14 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (700 rpm) .
- Example 15 Hydrolysis of palm oil by Amano PS lipase. The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (700 rpm) .
- Example 16 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (700 rpm) .
- Example 17 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (700 rpm) .
- Example 18 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (700 rpm) .
- Example 19 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 40°C for 24 hours under stirring (700 rpm) .
- Example 20 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 40°C for 24 hours under stirring (700 rpm) .
- Example 21 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 50°C for 24 hours under stirring (700 rpm) .
- Example 22 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 55°C for 24 hours under stirring (700 rpm) .
- Example 24 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 55°C for 24 hours under stirring (700 rpm) .
- Example 25 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 60 °C for 24 hours under stirring (100 rpm).
- Example 26 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (100 rpm) .
- Example 27 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (100 rpm) .
- Example 28 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (100 rpm) .
- Example 29 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (100 rpm) .
- Example 30 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (100 rpm) .
- Example 31 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 40°C for 24 hours under stirring (100 rpm) .
- Example 32 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 40°C for 24 hours under stirring (100 rpm) .
- Example 33 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 50 °C for 24 hours under stirring (100 rpm) .
- Example 34 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 50°C for 24 hours under stirring (100 rpm) .
- Example 35 Hydrolysis of palm oil by Amano PS lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 55°C for 24 hours under stirring (100 rpm) .
- Example 36 Hydrolysis of palm oil by Amano IM lipase.
- the reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 55°C for 24 hours under stirring (100 rpm) .
- Amano PS and Amano IM enzymes are an alternative to the use of other commercial enzymes, showing that their results depend on the reaction temperature, concentration of the enzyme and stirring speed.
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Abstract
The present invention relates to a process for the production of 1,2 and 1,3-diacylglycerols catalyzed by enzymes from the hydrolysis of lipidic material. In the process under reference, Amano PS and Amano IM commercial lipases are used to catalyze the production of diacylglycerols by hydrolysis of lipidic material from palm oil. Different reaction conditions were employed, wherein variations in temperature, amount of enzyme and stirring speed were studied.
Description
PRODUCTION OF DIACYLGLYCEROLS BY LIPASE - CATALYZED
HYDROLYSIS OF PALM OIL
TECHNICAL FIELD
The present invention relates to the production of diacylglycerols by heterogeneous catalysis using commercial enzymes to catalyze the production of diacylglycerols by hydrolysis reactions of palm oil.
BACKGROUND ART
The use of diacylglycerols instead of triacylglycerols which are found in oils and fats began at the 80' s. Its use was based on two important health benefits. The first benefit relates to the suppression of triglycerides postprandial increase in serum and the second benefit is the suppression of body fat accumulation.
Fats and oils consist of a mixture of triacylglycerols, also called triglycerides, and differ only by the physical state, fats being solid or pasty and oils being liquid at room temperature. Such difference is mainly due to the fact that oils are richer in unsaturated fatty acid residues than fats.
Triacylglycerols consist of a highly efficient manner of storing metabolic energy, once they are less oxidized than carbohydrates and proteins and do not absorb much water, due to their apolar condition. Thus, triacylglycerols occupy a smaller volume in the organism, contrary to glycogen, which is another energy source that binds to water in an amount almost twice its weight.
Vegetable oils are mainly composed by glycerides of plant fatty acids and may contain phospholipids, free fatty acids and unsaponifiable constituents. Their physical
characteristics vary according to the structure and distribution of fatty acids in the present triacylglycerols . The triacylglycerol can be represented by the structure (I) below:
CHjOCOR1
R2OCOCH
CHjOCOR3
(I)
wherein Rl, R2 and R3 are fatty acid residues.
Oils have a key role in food since they are important sources of essential fatty acids, carry fat-soluble vitamins and participate in the synthesis of many endogenous substances, among others. However, its excessive consumption is directly related to cardiovascular diseases, obesity and insulin resistance.
Diacylglycerols or diglycerides are glycerol esters which have two hydroxyl groups esterified by fatty acids, and they can exist in two stereochemical forms, known as sn-l,2-DAG (or 2,3) and sn-l,3-DAG, the natural isomeric relationship being 3:7 as a function of the acyl group migration during the process of oil refining.
The 1,2-DAG isoform is considered as a metabolic intermediate, which is formed after the ingestion of triacylglycerol. The 1,3-DAG isoform is responsible for the beneficial effect, since it is metabolized by a route which is different from that of TAG and 1,2-DAG. 1,3-DAG, 2,3-DAG and 1,2-DAG are respectively represented by structures (II), (III) and (IV) below:
CHjOCOR
HOCH
CH-OCORJ
Diacylglycerol is a natural component in several and fats, comprising about 10% by weight, as demonstrated in Table 1 below.
TABLE 1
Acylglycerol content in edible oils from different sources
(g/100g)
Total
Oil TAG 1,2-DAG 1,3-DAG MAG Others
DAG
Soybean 97.9 1.0 ND1 ND1 0.0 1.1
Palm 93.1 5.8 ND1 ND1 <LD2 1.1
Cotton 87.0 9.5 ND1 ND1 0.2 3.3
Corn 95.8 2.8 1.5 2.9 <LD2 1.4
Saffron 96.0 2.1 1.2 2.7 <LD2 1.9
Olive 93.3 5.5 ND1 ND1 0.2 2.3
Olive (mol%) 18-41 8-20 - - - -
Rapeseed 96.8 0.8 ND1 ND1 0.1 2.3
Vegetal (canola +
98.3 1.7 0.6 1.1 <LD2 ND1 soybean)
Canola 97.1 2.9 1.0 1.9 <LD2 ND1
Sesame 95.2 4.1 1.2 2.9 0.8 ND1
Pure sesame 95.5 3.9 1.2 2.7 0.6 ND1
Rice 92.4 7.6 2.4 5.2 <LD2 ND1
Rice germ 91.2 8.8 2.7 6.1 <LD2 ND1
Corn germ 95.5 4.5 1.5 2.9 <LD2 ND1
Grape seed 94.2 5.8 2.1 3.7 <LD2 ND1
DAG 17.3 81.4 28.4 53.0 1.2 ND1
Not detected
Below the detection limit
The diacylglycerol is broadly used as an emulsifier and stabilizer in food, cosmetic and pharmaceutical industries. It has also been used in Japan and in the United States as cooking oil due to its recently discovered properties of reducing lipid levels, thus reducing weight and fat accumulation.
The commercial oil was introduced in Japan in 1999 under the trade name Econa. In the United States, said oil was registered as Enova® oil after 15 years of research, clinical studies and experiments performed by Kao Corporation .
Enova contains approximately 20% by weight of triacylglycerol and 80% by weight of diacylglycerol, 56% thereof as the sn-l,3-DAG isoform. Its applications include the use as cooking oil, the use for the preparation of sauces and seasonings and products based on oils and fats.
According to clinical studies in the United States and
Japan, weight and fat mass can be reduced by replacing 10 to 20 grams of conventional oils of the diet by diacylglycerol .
The DAG is produced by glycerolysis, hydrolysis or chemical esterification of oils and fats. These processes often use high temperatures and/or toxic catalysts. Such conditions cause changes in the flavor and in the color of the oil, reduce selectivity and increase energy expenditure, thereby increasing process costs.
Enzymatic techniques have several advantages over the chemical process, including increased selectivity,
increased product purity, the use of milder temperatures and the suppression of toxic catalysts. To this end, the use of lipases has been widely studied.
The most used processes to obtain diacylglycerol are the glycerolysis and hydrolysis of fatty acid esters as is shown below.
GLYCEROLYSIS REACTIONS
H2C I C02 H2CI OH H2C C02R H2C C02R
Lipase I J
CH C02R + CH OH →■ CH OH CH C02R
¾C I C02R H2CI OH H2CI C02R H2CI OH
Triglyceride Glycerol 1 , 3-Diacylglycerol 1, 2-Diacylglycerol
HYDROLYSIS REACTIONS
H2C I C02R . H2C C02R h½C C02R
Lipase
CH CO2R + water ^ CH—0H or CH COiR
H2C C02R H2C C02R Hfi OH
Triglyceride 1 , 3-Diacylglycerol 1, 2-Diacylglycerol
These reactions do not necessarily lead to the formation of diacylglycerol, since the reaction conditions must be controlled so that it does not lead to the total breakdown . of the fatty acid ester. In this case, reaction byproducts like fatty acids, monoacylglycerols and glycerol can be obtained.
A transesterification reaction between a triacylglycerxde and glycerol catalyzed by a lipase occurs in the glycerolysis process. Typically, the reactions occur without solvent and mechanical stirring is used, since the triacylglycerides have high viscosity. In hydrolysis reactions, water is the agent which, together with the lipase, will hydrolyze the ester linkages of the triacylglyceride . As in glycerolysis reaction, the reactions occur in the absence of solvent and mechanical stirring is used.
The stoichiometry of the reactants is as important as the adjustment of mechanical (stirring) and thermal (heat) factors. Thus, a huge variety of reaction conditions can be used in order to obtain the desired result.
After the hydrolysis and glycerolysis reactions, the products are formed by two types of diacylglycerols that differ only in the position of the fatty acid chains (1,3 and 1,2). However, the enzymatic catalysis provides only one of them due to its high specificity.
The state of the art includes some patent documents related to the production of diacylglycerols from fats and oils, catalyzed by lipases.
The Japanese document JP 1071495 describes a method for the preparation of diglycerides with high purity and yield, which comprises an esterification reaction between glycerol and saturated or unsaturated fatty acids with 4 to 22 carbon atoms in the presence of an 1 , 3-selective lipase immobilized with an ion exchange resin, and in which water or lower alcohol produced by the reaction are removed to maximum from the reaction system to increase the ester synthesis yield and reduce the amount of monoglycerides . 1, 3-selective lipases are selected among the ones originating from microorganisms of Rhizopus, Mucor and Aspergillus species, more specifically from Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Aspergillus niger, Mucor javanicus and Mucor miehei.
The process for producing diacylglycerols described in the US document 2007/0148745 involves the use of an immobilized 1 , 3-selective lipase in the presence of water to promote the hydrolysis of triacylglycerols found in oils and fats.
Said document teaches a controlled hydrolysis with dehydration at the end of the process, using various vegetable oils, including palm oil. Furthermore, lipases are used as catalysts, preferably lipases immobilized with ion exchange resins. Temperatures between 20 and 90 °C are applied and the amount of water ranges from 20 to 180 parts of water per 100 parts of oil. The amount of enzyme used in the reactions is not specified; however, the examples suggest 10% by weight of enzyme to oil. At the end of the process, the diacylglycerol is purified and 0.5% to 25% by weight of phytosterols and ferulic acid esters are added in relation to the weight of diacylglycerol.
In the present invention, the reactions are catalyzed by Amano PS and Amano IM enzymes. Nevertheless, the amount of water used in the process of the present invention is four times less than the amount of water added in the process disclosed by document US 2007/0148745, since the minimum percentage used in such document was 20% by weight, while in the present invention only 5% by weight was used.
Additionally, interesting results were obtained with the teachings of the present invention with only 1% by weight of enzyme to palm oil and with 0.5% by weight to palm olein, whereas in said US document the percentage of enzyme is not mentioned, but suggesting 10% by weight in its examples, as was previously mentioned.
Document O2008/018147 Al teaches the composition of certain oils and identifies the relationship that should exist among free fatty acids, 1,2 and 1, 3-diacylglycerols and triacylglycerols .
The process described in document WO2008/003314 Al involves the formation of glycosides derived from mono- and
diacylglycerols of plant material. Said document describes in details the process of extracting glycosides derived from mono and diacylglycerols without any enzymatic process involved.
The process described in document WO2007/097160 Al involves the use of microorganisms of Moritella sp. species in the production of monoacylglycerols.
The process disclosed in the Japanese document JP 330289/1992 describes glycerolysis reactions wherein stoichiometric amounts of glycerol are used for the formation of diacylglycerol . In this process, a number of phospholipases for the production of diacylglycerol by transesterification reaction of vegetable oils with glycerol is used.
Document WO03/094634 Al relates to the beneficial effects of using diacylglycerols in food and beverages. Said document discloses the physical-chemical changes obtained by adding different amounts of diacylglycerols in food, thus leading to new organoleptic properties.
Document WO2005/048722 Al relates to the beneficial effects of regular ingestion of diacylglycerols. In the same manner as the process described above, said document shows the influence of using diacylglycerols as additives in food and supplements.
The process disclosed in document WO2006/022356 Al describes the formation of diacylglycerols catalyzed by microorganisms. In this process, the microorganisms lead to the formation of a mixture of diacylglycerols, monoacylglycerols and free fatty acids.
SUMMARY OF THE INVENTION
The object of the present invention relates to the
production of diacylglycerols by heterogeneous catalysis. In the process of the present invention, Amano PS and Amano IM commercial enzymes are used to catalyze the production of diacylglycerols by the hydrolysis reaction of refined palm oil.
DETAILED DESCRIPTION OF THE INVENTION
The object of the present invention comprises a process for the production of diacylglycerols by the hydrolysis of refined palm oil catalyzed by Amano PS and
Amano IM commercial lipases.
In the present invention, Amano PS and Amano IM commercial lipases are used as lipase sources to catalyze the hydrolysis of refined palm oil.
In order to obtain diacylglycerol conversions from refined palm oil, the reactions should be conducted under optimal conditions for lipase activity, or as near as possible of such conditions. The optimal conditions involve temperature, reactant concentration and stirring.
The enzymatic hydrolysis under reference is carried out in aqueous medium during a period of time within the range of 1 to 24 hours, thus resulting in a mixture of diacylglycerols, fatty acids and glycerol.
The reaction catalyzed by lipases should be operated in batch.
After the end of the reaction, an apolar organic solvent is added to the reaction medium (for example, t- butanol, hexane, heptane or isooctane alone or associated with 10 to 90% by weight of water) and then the reaction mixture is filtered and washed with sodium chloride saturated solution. The organic phases are separated, dried with anhydrous sodium sulfate and the solvent is evaporated
at reduced pressure.
The examples presented below are only intended to illustrate the invention and facilitate its understanding, and are not intended to limit it in any manner.
EXAMPLES
Example 1 : Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 60°C for 24h under stirring (1300 rpm) .
Example 2 : Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 60°C for 24h under stirring (1300 rpm) .
Example 3 : Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano PS lipase at 60°C for 24h under stirring (1300 rpm) .
Example 4 : Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano IM lipase at 60°C for 24h under stirring (1300 rpm) .
Example 5: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano PS lipase at 60°C for 24h under stirring (1300 rpm) .
Example 6: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano IM lipase at 60°C for 24h under stirring (1300 rpm) .
Example 7 : Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of
palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 40°C for 24h under stirring (1300 rpm) .
Example 8 : Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 40°C for 24h under stirring (1300 rpm) .
Example 9: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 50°C for 24h under stirring (1300 rpm) .
Example 10: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 50°C for 24h under stirring (1300 rpm) .
Example 11: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 55°C for 24h under stirring (1300 rpm) .
Example 12 : Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 55°C for 24h under stirring (1300 rpm) .
Example 13: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (700 rpm) .
Example 14: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (700 rpm) .
Example 15: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (700 rpm) .
Example 16: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (700 rpm) .
Example 17: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (700 rpm) .
Example 18: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (700 rpm) .
Example 19: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 40°C for 24 hours under stirring (700 rpm) .
Example 20: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 40°C for 24 hours under stirring (700 rpm) .
Example 21: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 50°C for 24 hours under stirring (700 rpm) .
Example 22: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 50°C for 24 hours under stirring (700 rpm) .
Example 23: Hydrolysis of palm oil by 7Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 55°C for 24 hours under stirring (700 rpm) .
Example 24: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 55°C for 24 hours under stirring (700 rpm) .
Example 25: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 60 °C for 24 hours under stirring (100 rpm).
Example 26: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (100 rpm) .
Example 27: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (100 rpm) .
Example 28: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 2.0% w/w of Amano IM lipase at 60°C for 24 hours under stirring (100 rpm) .
Example 29: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano PS lipase at 60°C for 24 hours under stirring (100 rpm) .
Example 30: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 3.0% w/w of Amano IM lipase
at 60°C for 24 hours under stirring (100 rpm) .
Example 31: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 40°C for 24 hours under stirring (100 rpm) .
Example 32 : Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 40°C for 24 hours under stirring (100 rpm) .
Example 33: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 50 °C for 24 hours under stirring (100 rpm) .
Example 34 : Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 50°C for 24 hours under stirring (100 rpm) .
Example 35: Hydrolysis of palm oil by Amano PS lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano PS lipase at 55°C for 24 hours under stirring (100 rpm) .
Example 36: Hydrolysis of palm oil by Amano IM lipase.
The reaction was carried out by incubating 2.0 g of palm oil, 0.1 mL of water and 1.0% w/w of Amano IM lipase at 55°C for 24 hours under stirring (100 rpm) .
Table 2 below shows the results of the hydrolysis reactions of the examples above.
TABLE 2
Water % by weight of Temp. Yield
Example rpm Enzyme
(mL) enzyme (°C) (%)
1 1300 0.1 1 Amano PS 60 36
1300 0.1 1 Amano IM 60 52
1300 0.1 2 Amano PS 60 30
1300 0.1 2 Amano IM 60 45
1300 0.1 3 Amano PS 60 32
1300 0.1 3 Amano IM 60 42
1300 0.1 1 Amano PS 40 24
1300 0.1 1 Amano IM 40 31
1300 0.1 1 Amano PS 50 34
1300 0.1 1 Amano IM 50 42
1300 0.1 1 Amano PS 55 30
1300 0.1 1 Amano IM 55 32
700 0.1 1 Amano PS 60 32
700 0.1 1 Amano IM 60 55
700 0.1 2 Amano PS 60 27
700 0.1 2 Amano IM 60 41
700 0.1 3 Amano PS 60 29
700 0.1 3 Amano IM 60 45
700 0.1 1 Amano PS 40 20
700 0.1 1 Amano IM 40 21
700 0.1 1 Amano PS 50 36
700 0.1 1 Amano IM 50 48
700 0.1 1 Amano PS 55 34
700 0.1 1 Amano IM 55 43
100 0.1 1 Amano PS 60 29
100 0.1 1 Amano IM 60 37
100 0.1 2 Amano PS 60 29
100 0.1 2 Amano IM 60 33
100 0.1 3 Amano PS 60 26
100 0.1 3 Amano IM 60 36
100 0.1 1 Amano PS 40 21
32 100 0.1 1 Amano IM 40 23
33 100 0.1 1 Amano PS 50 24
34 100 0.1 1 Amano IM 50 33
35 100 0.1 1 Amano PS 55 25
36 100 0.1 1 Amano IM 55 33
The results shown in the comparative table above summarize the reaction conditions studied for this process as well as the product yields (diacylglycerol) found in different reaction conditions. The obtained results show that Amano IM enzyme presents superior results in comparison with Amano PS enzyme, regardless of reaction conditions used. However, when temperatures in the range of 60°C are used, better yields are obtained. Such result was not expected for these enzymes, since they are not known for their hydrolytic capacity but by their ability to perform esterification reactions.
Thus, Amano PS and Amano IM enzymes are an alternative to the use of other commercial enzymes, showing that their results depend on the reaction temperature, concentration of the enzyme and stirring speed.
The above description of the present invention was presented for illustration and description purposes. Thus, the description does not intend to limit the invention to the form revealed herein. Therefore, variations and modifications consistent with the teachings above which are based on the skill or knowledge of the relevant art are within the scope of the present invention. The present invention intends to include all modifications and variations thereof which are within the scope described in the present specification, as well as in the attached claims .
Claims
1. A process for the production of diacylglycerols by hydrolysis reaction of palm oil characterized by being catalyzed by 0.1 to 5.0% by weight of lipases at temperatures ranging from 25 to 80°C in aqueous solution, under stirring and in the presence of an apolar organic solvent .
2. The process according to claim 1, wherein the lipase is selected from Amano PS and Amano IM enzymes.
3. The process according to claim 1, wherein the solvent can be selected from t-butanol, hexane, heptane or isooctane, either alone or associated with 10 to 90% by weight of water.
4. The process according to claim 1, wherein the reaction time ranges from 1 to 24 hours.
5. The process according to claim 1, wherein the stirring speed ranges from 100 to 1300 rpm.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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BRPI0905607-6A BRPI0905607A2 (en) | 2009-12-24 | 2009-12-24 | process for the production of diacylglycerols through palm oil hydrolysis reactions catalyzed by the enzymes amano ps and amano im |
BRPI0905607-6 | 2009-12-24 |
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WO2011075802A1 true WO2011075802A1 (en) | 2011-06-30 |
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PCT/BR2010/000384 WO2011075802A1 (en) | 2009-12-24 | 2010-11-24 | Production of diacylglycerols by lipase - catalyzed hydrolysis of palm oil |
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BR (1) | BRPI0905607A2 (en) |
WO (1) | WO2011075802A1 (en) |
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JPS6471495A (en) | 1987-09-09 | 1989-03-16 | Kao Corp | Production of diglyceride |
JPH04330289A (en) | 1991-01-24 | 1992-11-18 | Kao Corp | Production of diglyceride |
WO2003094634A1 (en) | 2002-05-06 | 2003-11-20 | Archer-Daniels-Midland Company | Foods and drinks containing diacylglycerol |
WO2005048722A1 (en) | 2003-11-18 | 2005-06-02 | Archer-Daniels-Midland Company | Foods and drinks containing diacylglycerol |
WO2006022356A1 (en) | 2004-08-24 | 2006-03-02 | Suntory Limited | Process for producing microorganism fat containing diacylglycerol in any amount and the fat |
US20070148745A1 (en) | 2005-12-28 | 2007-06-28 | Lai Oi Ming | Process for the production of diacylglycerol |
WO2007097160A1 (en) | 2006-02-27 | 2007-08-30 | Tokyo University Of Marine Science And Technology | Novel microbe, lipid modifying agent, process for producing 2-acyl-lysophospholipid, process for producing diacylglycerol, process for producing ceramide, and method of degumming oil or fat |
WO2008003314A1 (en) | 2006-07-03 | 2008-01-10 | Hyben Vital Licens Aps | A method of preparing a glycoside of a mono- or diacylglycerol product from a plant material |
WO2008018147A1 (en) | 2006-08-11 | 2008-02-14 | Kao Corporation | Fat or oil composition |
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2009
- 2009-12-24 BR BRPI0905607-6A patent/BRPI0905607A2/en not_active IP Right Cessation
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2010
- 2010-11-24 WO PCT/BR2010/000384 patent/WO2011075802A1/en active Application Filing
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JPS6471495A (en) | 1987-09-09 | 1989-03-16 | Kao Corp | Production of diglyceride |
JPH04330289A (en) | 1991-01-24 | 1992-11-18 | Kao Corp | Production of diglyceride |
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WO2006022356A1 (en) | 2004-08-24 | 2006-03-02 | Suntory Limited | Process for producing microorganism fat containing diacylglycerol in any amount and the fat |
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EP1803819A2 (en) * | 2005-12-28 | 2007-07-04 | Universiti Putra Malaysia | Process for the production of diacylglycerol |
WO2007097160A1 (en) | 2006-02-27 | 2007-08-30 | Tokyo University Of Marine Science And Technology | Novel microbe, lipid modifying agent, process for producing 2-acyl-lysophospholipid, process for producing diacylglycerol, process for producing ceramide, and method of degumming oil or fat |
WO2008003314A1 (en) | 2006-07-03 | 2008-01-10 | Hyben Vital Licens Aps | A method of preparing a glycoside of a mono- or diacylglycerol product from a plant material |
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