WO2011114251A1 - Produits alimentaires - Google Patents

Produits alimentaires Download PDF

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Publication number
WO2011114251A1
WO2011114251A1 PCT/IB2011/050869 IB2011050869W WO2011114251A1 WO 2011114251 A1 WO2011114251 A1 WO 2011114251A1 IB 2011050869 W IB2011050869 W IB 2011050869W WO 2011114251 A1 WO2011114251 A1 WO 2011114251A1
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WO
WIPO (PCT)
Prior art keywords
cyclodextrin
enzyme
foodstuff
present
dough
Prior art date
Application number
PCT/IB2011/050869
Other languages
English (en)
Inventor
Jørn Borch SØE
Jens Frisbæk SØRENSEN
Lene Bojsen Jensen
Original Assignee
Danisco A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Danisco A/S filed Critical Danisco A/S
Publication of WO2011114251A1 publication Critical patent/WO2011114251A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
    • A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/14—Organic oxygen compounds
    • A21D2/18—Carbohydrates
    • A21D2/183—Natural gums
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/14—Organic oxygen compounds
    • A21D2/18—Carbohydrates
    • A21D2/186—Starches; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01004—Phospholipase A2 (3.1.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01026—Galactolipase (3.1.1.26)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01032—Phospholipase A1 (3.1.1.32)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • This invention relates to a foodstuff. It also relates to a method for producing the foodstuff and uses of certain ingredients of the foodstuff to provide novel technical effects.
  • lipolytic enzymes are capable of hydrolysing ester bonds a range of lipids present in wheat flour (in particular, triglycerides, glycolipids and phospholipids), either to liberate free fatty acid molecules or to transfer fatty acyl groups to other acceptor molecules.
  • WO 2005/087918 describes a lipolytic enzyme and its use in baking.
  • the enzyme described therein and having the sequence listing of SEQ !D No. 1 of this specification is commercially available from Danisco A/S under the trade mark GRINDA YL POWERBAKE 4070. Lipolytic enzymes provide a number of advantageous effects in baking applications.
  • lipolytic enzymes can act on triglycerides present in flour to generate in situ mono- and diglycerides, which are useful as emulsifiers.
  • Lipolytic enzymes can also act on glycolipids present in flour to generate in situ mono- and diglycosyl mono- and diglycerides, and on phospholipids present in flour to generate in situ lysophospholipids.
  • the 5-membered macrocycle is not natural.
  • Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring, creating a cone shape.
  • Examples of known cyclodextrins are a-cyclodextrin (6- membered glucose ring molecule), ⁇ -cyclodextrin (7-membered glucose ring molecule) and ⁇ -cyclodextrin (8-membered glucose ring molecule).
  • Cyclodextrins are known to interact with free fatty acids by formation of inclusion compounds, where the fatty acid is included in the hydrophobic torus of the cyclodextrin - see Bru e ai, Colloids and Surfaces A: Physicochemical and Engineering Aspects 97 (1995), 263-269.
  • Cyclodextrins may be generated in situ from starch or other polysaccharides present in a dough mixture, for example by the action of a cyclodextrin glycosyltransferase enzyme (also known as cyclodextrin glucosyltransferase or CGTase) (E.G. 2.4.1.19), the enzyme acting firstly to cleave the glycosidic bonds in the polysaccharide followed by bonding the glucose units together to form the ring. Examples of CGTases and their use in baking are described in WO 02/06508 and EP 0687414A.
  • EP 0 493 045 A1 describes a foodstuff to which a ⁇ -cyclodextrin and a phospholipase D is added. This differs from the present invention in that phospholipase D cleaves phospholipids at the phosphate group to liberate phosphatidic acid and an alkanolamine, rather than a free fatty acid.
  • a foodstuff comprising an effective amount of:
  • a foodstuff comprising an effective amount of:
  • a method of producing the above foodstuff comprising adding the lipolytic enzyme and the cyciodextrin to one or more other ingredients of the foodstuff and, if necessary, treating the ingredients to produce the foodstuff.
  • a cyclodextrin for decreasing the uncompiexed free fatty acid content of a foodstuff incorporating a lipolytic enzyme.
  • a cyclodextrin for decreasing the uncompiexed free fatty acid content of a foodstuff incorporating a lipolytic enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule.
  • a cyclodextrin into a foodstuff (particularly although not exclusively a dough and a baked product prepared from dough) enables the amounts of free fatty acid present in the foodstuff to be reduced.
  • this effect is caused by the free fatty acid molecules forming an inclusion complex with the cyclodextrin in which the free fatty acid molecule is included in the hydrophobic interior of the cyclodextrin: the free fatty acids do not exhibit the deleterious effects described above when in the form of an inclusion complex.
  • Inclusion complex' or 'inclusion compound' is understood in the art as meaning a complex in which one component (the host) forms a cavity in which molecular entities of a second component (the guest) are located. There is no covalent bonding between guest and host, the attraction being generally due to van der VVaals forces.
  • the term 'compiexed' in relation to free fatty acids means that the free fatty acid is present as a component of an inclusion complex (as defined above) in which at least part of the fatty acid molecule is located within the cavity of the cyclodextrin molecule.
  • the term 'uncomplexed' means that the free fatty acid molecule is present in the foodstuff as a separate entity from the cyclodextrin molecule.
  • foodstuff' as used herein means a substance which is suitable for human and/or animal consumption.
  • the term "foodstuff ' as used herein may mean a foodstuff in a form which is ready for consumption.
  • the term foodstuff as used herein may mean one or more food materials which are used in the preparation of a foodstuff.
  • the term foodstuff encompasses both baked goods produced from dough as well as the dough used in the preparation of said baked goods.
  • the foodstuff may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
  • composition of the present invention may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient.
  • the present invention provides a foodstuff as defined above wherein the foodstuff is selected from one or more of the following: eggs, egg-based products, including but not limited to mayonnaise, salad dressings, sauces, ice creams, egg powder, modified egg yolk and products made therefrom; baked goods, including breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers and cookies; confectionery, including chocolate, candies, caramels, halawa, gums, including sugar free and sugar sweetened gums, bubble gum, soft bubble gum, chewing gum and puddings; frozen products including sorbets, preferably frozen dairy products, including ice cream and ice milk; dairy products, including cheese, butter, milk, coffee cream, whipped cream, custard cream, milk drinks and yoghurts; mousses, whipped vegetable creams, meat products, including processed meat products; edible oiis and fats, aerated and non- aerated whipped products, oil-in-water
  • the foodstuff in accordance with the present invention may be a "fine food", including cakes, pastry, confectionery, chocolates, fudge and the like.
  • the foodstuff in accordance with the present invention may be a dough product or a baked product, such as bread, a fried product, a snack, cakes, pies, brownies, cookies, noodles, snack items such as crackers, graham crackers, pretzels, and potato chips, and pasta.
  • the foodstuff in accordance with the present invention may be a convenience food, such as a part-baked or part-cooked product.
  • a convenience food such as a part-baked or part-cooked product.
  • Examples of such part-baked or part-cooked product include part-baked versions of the dough and baked products described above.
  • the foodstuff in accordance with the present invention may be a plant derived food product such as flours, pre-mixes, oils, fats, cocoa butter, coffee whitener, salad dressings, margarine, spreads, peanut butter, shortenings, ice cream, cooking oils.
  • the foodstuff in accordance with the present invention may be a dairy product, including butter, milk, cream, cheese such as natural, processed, and imitation cheeses in a variety of forms (including shredded, block, slices or grated), cream cheese, ice cream, frozen desserts, yoghurt, yoghurt drinks, butter fat, anhydrous milk fat, other dairy products.
  • the enzyme according to the present invention may improve fat stability in dairy products.
  • the foodstuff in accordance with the present invention may be a food product containing animal derived ingredients, such as processed meat products, cooking oils, shortenings.
  • the foodstuff in accordance with the present invention may be a beverage, a fruit, mixed fruit, a vegetable, a marinade or wine.
  • the foodstuff in accordance with the present invention is a plant derived oil (i.e. a vegetable oil), such as olive oil, sunflower oil, peanut oil or rapeseed oil.
  • the oil may be a degummed oil.
  • the maximum free fatty acid content of the foodstuff is 5%, preferably 2%, more preferably 1 %, even more preferably 0.5%, yet more preferably 0.2%, still more preferably 0.1 %, and most preferably 0.05%.
  • 'cyclodextrin' means a cyclic oligosaccharide composed of 5 or more glucose units linked in a 1 ->4 manner so as to form a ring.
  • the cyclodextrin comprises a cyclic oligosaccharide containing 5 to 10, preferably 6 to 8, glucose units.
  • the cyclodextrin may optionally be substituted with a side chain comprising 1 to 3 monosaccharide, preferably glucose, units.
  • suitable cyclodextrins include a-cyclodextrin (6-membered glucose ring molecule), ⁇ - cyciodextrin (7-membered glucose ring molecule) and y -cyciodextrin (8-memoered glucose ring molecule).
  • the cyciodextrin is ⁇ -cyclodextrin.
  • the amount of cyciodextrin present is 0.05 to 50 g per kg of the total weight of the foodstuff.
  • the amount of cyciodextrin present is 0.1 to 20 g per kg of the total weight of the foodsiuff. More preferably, the amount of cyciodextrin present is 0.5 to 10 g per kg of the total weight of the foodstuff.
  • the amount of cyciodextrin present is 0.05 to 50 g per kg of the flour used to form the dough.
  • the amount of cyciodextrin present is 0.1 to 20 g per kg flour. More preferably, the amount of cyciodextrin present is 0.5 to 10 g per kg flour.
  • the cyciodextrin is present as an initial component of the foodstuff (i.e. the cyciodextrin is produced separately of the foodstuff and is added as a formed product to the foodstuff during preparation of the same).
  • the cyciodextrin is generated in situ during preparation of the foodstuff, typically by the action of a cyciodextrin glycosyltransferase enzyme (E.G. 2.4.1.19) on polysaccharides present in the foodstuff.
  • 'cyciodextrin glycosyltransferase', 'cyciodextrin glucosyltransferase' and 'CGTase' all mean an enzyme capable of acting on a polysaccharide so as to produce a cyciodextrin (as defined above, either in its broadest aspect or a preferred aspect).
  • the polysaccharide on which the CGTase enzyme (if present) acts is selected from starch, amylose or amylopectin.
  • Suitable CGTase enzymes are described in WO 02/06508 and EP 0687414A.
  • a particular example of a suitable CGTase enzyme is the cyciodextrin glucanotransferase (Bacillus macerans) enzyme available from Amano Enzymes, Japan.
  • the amount of CGTase enzyme present is suitably 0.01 g to 10 g per kg of the total weight of the foodstuff.
  • the amount of CGTase enzyme present is 0.05 g to 1 g per kg of the total weight of the foodstuff.
  • the amount of CGTase enzyme present is suitably 0.01 mg to 2 mg per kg of the total weight of the foodstuff.
  • the amount of CGTase enzyme present is 0.05 mg to 1 mg per kg of the total weight of the foodstuff.
  • the amount of CGTase enzyme present is suitably 6 to 1200 units of enzyme activity (U) per kg of the total weight of the foodstuff.
  • the activity can be measured according to the Tilden-Hudson method described in Tilden E.B. and Hudson C.S. J. Bacterial. (1942), 43, 527-544.
  • the CGTase enzyme may be incorporated into the foodstuff before addition of the lipolytic enzyme.
  • the CGTase enzyme may be incorporated into the foodstuff at the same time as the lipolytic enzyme.
  • the amount of CGTase enzyme, where present is 0.01 to 10 g per kg of the flour used to form the dough.
  • the amount of CGTase enzyme, where present is 0.05 to 1 g per kg flour.
  • the amount of CGTase enzyme, where present is suitably 0.01 mg to 2 mg per kg of the flour used to form the dough.
  • the amount of CGTase enzyme, where present is 0.05 mg to 1 mg per kg of flour.
  • the amount of CGTase enzyme present is suitably 6 to 1200 units of enzyme activity (U) per kg of the flour used to form the dough.
  • the activity can be measured according to the Tilden-Hudson method referred to above.
  • the term lipolytic enzyme' is defined as an enzyme capable of acting on a lipid substrate to liberate a free fatty acid molecule.
  • the lipolytic enzyme is an enzyme capable of hydro!ysing an ester bond in a lipid substrate (particularly although not exclusively a triglyceride, a giycolipid and/or a phospholipid) to liberate a free fatty acid molecule. Examples of possible lipid substrate are described below.
  • the lipolytic enzyme used in the present invention has activity on both non-polar and polar lipids.
  • polar lipids as used herein means phospholipids and/or glycolipids.
  • polar lipids as used herein means both phospholipids and glycolipids.
  • Polar and non-polar lipids are discussed in Eliasson and Larsson, "Cereals in Breadmaking: A Molecular Colloidal Approach", publ. Marcel Dekker, 1993.
  • the lipolytic enzyme used in the present invention has activity on the following classes of lipids: triglycerides; phospholipids, particularly but not exclusively phosphatidylcholine (PC) and/or N-acylphosphatidylethanolamine (APE); and glycolipids, particularly although not exclusively digalactosyl diglyceride (DGDG).
  • lipids particularly but not exclusively phosphatidylcholine (PC) and/or N-acylphosphatidylethanolamine (APE); and glycolipids, particularly although not exclusively digalactosyl diglyceride (DGDG).
  • such an acyl group is an alkanoyl group.
  • such an acyl group comprises an alkenoyl group, which may have, for example, 1 to 5 double bonds, preferably 1 , 2 or 3 double bonds.
  • the lipolytic enzyme for use in the present invention may have one or more of the following activities selected from the group consisting of: phospholipase activity (such as phospholipase A1 activity (E.G. 3.1.1.32) or phospholipase A2 activity (E.G. 3.1 .1.4); glycolipase activity (E.G. 3.1.1.26), triacylglycerol hydrolysing activity (E.G. 3.1.1 ,3), lipid acy!transferase activity (generally classified as E.G. 2.3.1.x in accordance with the Enzyme Nomenclature Recommendations (1992) of the Nomenciature Committee of the International Union of Biochemistry and Molecular Biology), and any combination thereof.
  • phospholipase activity such as phospholipase A1 activity (E.G. 3.1.1.32) or phospholipase A2 activity (E.G. 3.1 .1.4
  • glycolipase activity E.G. 3.1.1.26
  • the lipolytic enzyme for use in the present invention may be a phospholipase (such as a phospholipase A1 (E.G. 3.1.1.32) or phospholipase A2 (E.G. 3.1.1.4)); glycolipase or galactolipase (E.G. 3.1.1 .26), triacylglyceride lipase (E.G. 3.1.1.3).
  • a phospholipase such as a phospholipase A1 (E.G. 3.1.1.32) or phospholipase A2 (E.G. 3.1.1.4)
  • glycolipase or galactolipase E.G. 3.1.1 .26
  • triacylglyceride lipase E.G. 3.1.1.3
  • Such enzyme may exhibit additional side activities such as lipid acyltransferase side activity.
  • the lipolytic enzyme for use in the present invention may have the activity of a phospholipase (such as
  • the lipolytic enzyme may be any commercially available lipolytic enzyme.
  • the lipolytic enzyme may be any one or more of: Lecitase UltraTM, Novozymes, Denmark; Lecitase 10TM; a phospholipase A1 from Fusarium spp e.g. Lipopan FTM, LipopanTM LipopanXtraTM, YieldMaxTM; a phospholipase A2 from Aspergillus niger; a phospholiapse A2 from Streptomyces violaceruber e.g.
  • LysoMax PLA2TM a phospholipase A2 from Tuber borchii; or a phospholipase B from Aspergillus niger, Lipase 3, Grindamyl EXEL 16TM, and GRINDA YL POWERBake 4070, PanamoreTM or GRINDAMYL POWERBake 4100.
  • the lipolytic enzyme is GRINDAMYL POWERBake 4070TM, Lipopan FTM, LipopanTM' LipopanXtraTM, or PanamoreTM.
  • the lipolytic enzyme is obtainable from a microorganism, examples of which include bacteria and fungi (such as yeasts).
  • the lipolytic enzyme is obtainable from a filamentous fungus.
  • the lipolytic enzyme is obtainable from a fungus of the genus Fusarium, Aspergillus or Streptomyces, especially from a fungus of the genus Fusarium.
  • the lipolytic enzyme is obtainable from Fusarium heterosporum. !n a particularly preferred embodiment, the lipolytic enzyme is obtainable from Fusarium heterosporum CBS 782.83.
  • prepro-polypeptides which when post-translationally processed in a host organism produces a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid, wherein the prepropolypeptide comprises an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 and SEQ ID No. 25.
  • the lipolytic enzyme for use in the present invention is a polypeptide which has hydrolytic activity towards an ester bond in a polar lipid, which polypeptide is obtainable from a prepro-polypeptide comprising an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25.
  • prepro-sequences often go through post- translational modification.
  • the present enzymes it is relatively common for the organism to remove the N-terminal region of the prepro sequence, i.e. remove all or part of the amino acids 1 -30 of SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23 or SEQ ID No. 25.
  • the host organism may remove slightly more amino acids than those shown as amino acids 1 -30 of SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No.
  • the host organism may introduce an alternative N-terminal sequence which may encompass all or part of the amino acids shown as amino acids 1 -30 or may comprise a completely different N-terminal sequence (such as EAEA or EA for instance).
  • the mature enzyme produced from the prepro-sequence by the host organism may be a heterogen at its N-terminus end.
  • the post- translational modification may mean modification in the C-terminal region of the prepro sequence. For example, all or part of the amino acids 306-348 may be removed from SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No.
  • the mature enzyme produced from the prepro-sequence by the host organism may be a heterogen at its C-terminus end.
  • the present invention encompasses the use of all mature forms of the protein obtainable from a prepro-polypeptide comprising an amino acid sequence shown as SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ SD No. 23 or SEQ ID No. 25, particularly those obtained from the host organism Trichoderma reesei.
  • the lipolytic enzyme for use in the present invention may comprise one of the following amino acid sequences:
  • n an amino acid sequence as set forth in SEQ ID No. 26;
  • the lipolytic enzyme for use in the present invention may have at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity with any of the sequences shown in a) to n).
  • the lipolytic enzyme for use in the present invention may have an amino acid sequence as set forth in SEQ ID No. 1 or an amino acid sequence having at least 70% identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity thereto.
  • the lipolytic enzyme for use in the present invention may be a lipolytic enzyme encoded by any one of the following nucleic acid sequences:
  • n a nucleic acid sequence encoding a lipolytic enzyme having at least 70% identity to any of the sequences in a) to I);
  • the lipolytic enzyme for use in the present invention may be a lipolytic enzyme encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably af least 95%, preferably at least 98%, preferably at least 99% identity with any of the sequences shown in a) to I).
  • the lipolytic enzyme for use in the present invention may be encoded by a nucleic acid sequence as set forth in SEQ ID No. 3 or a nucleic acid sequence having at least 70% identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99% identity thereto.
  • Lipolytic enzymes have a number of advantageous effects in baking applications.
  • the lipolytic enzyme for use in the present invention may confer one or more of the following effects on dough and/or a baked product prepared from dough: reducing stickiness; improving machinability; reducing blistering during baking; improved volume; improved softness; prolonged shelf life; reduced staling; improved crumb structure; improved pore homogeneity; reducing mean pore size; enhancing the gluten index; improved flavour; improved colour of the crust. These effects remain substantially unchanged by treatment with cyclodextrin.
  • a cyclodextrin in combination with a lipolytic enzyme allows any or all of the above advantageous effects to be exhibited while reducing the content of free fatty acid in the dough and/or baked product prepared from dough and avoiding the deleterious effects associated therewith.
  • the lipolytic enzyme is in a delayed release form.
  • delayed release also known as “slow release” when used in its broadest sense means that the lipolytic enzyme is released into the foodstuff a time later than that immediately following its introduction.
  • the term “delayed release” means that 50% of the lipolytic enzyme is released into the foodstuff at a time at least 5 minutes after its introduction and 100% of the lipolytic enzyme is released into the foodstuff after at a time at least 10 minutes after its introduction. More preferably, the term “delayed release” means that 50% of the lipolytic enzyme is released into the foodstuff at a time between 5 minutes and 3 hours after its introduction and 100% of the lipolytic enzyme is released into the foodstuff after at a time between 10 minutes and 5 hours after its introduction.
  • the provision of the lipolytic enzyme in delayed release form confers the advantage that the rate of generation of free fatty acid by the lipolytic enzyme can be slowed down.
  • the provision of the lipolytic enzyme in delayed release form allows sufficient time for the CGTase enzyme to generate the cyclodextrin before significant quantities of free fatty acid are generated by the lipolytic enzyme.
  • the lipolytic enzyme is encapsulated. Encapsulation is a process of surrounding or coating an ingredient with a substance in order to prevent or delay the release of the ingredient until a certain time or set of conditions is achieved.
  • capsules Various techniques are employed to form the capsules, including spray drying, spray chilling or spray cooling, extrusion coating, fluidized bed coating, liposome entrapment, coacervation, inclusion complexation, centrifugal extrusion and rotational suspension separation.
  • Fats, emulsifiers, starches, dextrins, alginates, protein and lipid materials can be employed as encapsulating materials.
  • Release can be site-specific, stage-specific or signalled by changes in pH, temperature, irradiation or osmotic shock.
  • the lipolytic enzyme used in the present invention is capable of hydrolysing an ester bond in a lipid substrate.
  • the term 'lipid substrate' is defined as meaning a fat-soluble (lipophilic), naturally-occurring or synthetic molecule, having an ester group capable of being hydrolysed to liberate a free fatty acid molecule (as defined above).
  • suitable lipids include triglycerides, mono- and polyglycerolipids (especially monog!ycerides and diglyce rides), giycolipids, phospholipids, and the like.
  • the lipid substrate may comprise a mixture of lipids.
  • lipophilic' means soluble in non-polar organic solvents.
  • such non-polar organic solvents have one or more of the following properties:
  • a low dielectric constant for example, a dielectric constant less than 20, preferably less than 10.
  • a weak or zero dipole moment for example, a dipoie moment of less than 1 D, preferably less than 0.5 D
  • non-polar organic solvents examples include aliphatic hydrocarbons such as pentane, hexane, heptane, aiicylic hydrocarbons such as cyclohexane, aromatic hydrocarbons such as benzene, toluene or xylene, ethers such as diethyl ether, and halogenated hydrocarbons such as dichloromethane, trichloromethane (chloroform) and 1 ,2-dichloroethane.
  • such acyl groups have a total of 4 to 40 carbon atoms, preferably 6 to 40 carbon atoms, such as at least 10 to 40 carbon atoms, for example 12 to 40, such as 14 to 40, 16 to 40, 18 to 40, 20 to 40 or 22 to 40 carbon atoms, more preferably 10 to 24, especially 12 to 22, particularly 14 to 18, for example 16 or 18 carbon atoms.
  • such an acyl group is an alkanoyl group.
  • such an acyl group comprises an alkenoyl group, which may have, for example, 1 to 5 double bonds, preferably 1 , 2 or 3 double bonds.
  • acyl groups include saturated acyl groups such as butanoyl (butyryl), hexanoyl (caproyl), octanoyl (capryl), decanoyl (caprinyl), dodecanoyl (lauroyl), tetradecanoyl, (myristoyl), hexadecanoyl (palmitoyl), octadecanoyl (stearoyl), eicosanoyl (arachidonyl), docosanoyl (behenoyl) and tetracosanoyl (lignoceroyl) groups, and unsaturated acyl groups such as c/s-tetradec-9-enoyl (myristoleyl), c/s- hexadec-9-enoyl (palmitoleyl), c s-octadec-9-enoyl (oleyl), cis c/s-9, 12-
  • lipids suitable as substrates in the present invention are described below.
  • the lipid substrate is a triglyceride.
  • 'triglyceride' also known as triacylglycerol
  • triacylglycerol means a compound comprising three acyl groups (which may be the same or different) covalently bonded to a single glycerol moiety via ester linkages.
  • the lipid substrate is a glycoglycerolipid (also known as a glycosylglyceride).
  • glycoglycerolipid also known as a glycosylglyceride
  • 'glycoglycerolipid' when used to define the lipid substrate molecule, means a lipid comprising a single glyceroi moiety covalently bound via ester linkages to one or more acyl groups (the typical and preferred lengths of which are defined and exemplified above) and having one or more monosaccharide moieties attached to the glycerol moiety via a glycosidic linkage, provided it contains at least one free hydroxy! group to enable transfer to take place.
  • the monosaccharides may be bonded to different oxygen atoms on the glycerol backbone, may be bonded to each other to comprise a di-, oligo- or polysaccharide moiety attached to one oxygen atom on the glycerol moiety, or any combination thereof.
  • Glycerophospholipids also referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells, as well as being involved in metabolism and signaling.
  • Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria or the sn-1 position in the case of archaebacteria.
  • glycerophospholipids found in biological membranes are phosphatidylcholines (also known as PC or GPCho, and lecithin), phosphatidylethanolamines (PE or GPEtn), phosphatidylserine (PS or GPSer), phosphadityl inositol, lysophosphatidylcholines, lysophosphatidylethanolamines, N- acyl phosphatidylethanolamines and N-acyl lysophosphatidylethanolamines.
  • phosphatidylcholines also known as PC or GPCho, and lecithin
  • PE or GPEtn phosphatidylethanolamines
  • PS or GPSer phosphatidylserine
  • phosphadityl inositol lysophosphatidylcholines
  • lysophosphatidylethanolamines lysophosphatidylethanolamines
  • some g!ycerophospholipids in eukaryotic cells such as phosphatidylinositois and phosphatide acids are either precursors of, or are themselves, membrane-derived second messengers.
  • phosphatidylinositois and phosphatide acids are either precursors of, or are themselves, membrane-derived second messengers.
  • these hydroxyl groups are acylated with long-chain fatty acids (the number of carbon atoms in the chains typically as set out above), but there are also alkyl-linked and 1 Z- alkenyl-linked (plasmalogen) glycerophospholipids, as well as dialkylether variants in prokaryotes.
  • Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • a sugar substitutes for the glycerol backbone that is present in glycerolipids and glycerophospholipids.
  • the most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria.
  • Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E.
  • the lipid substrate is a lysophospholipid.
  • a lysophospholipid comprises a glycerol moiety having only one acyl group (as defined and exemplified above) covalent!y bonded to a glycerol oxygen atom via an ester linkage and a phosphate group covalently bonded to another glycerol oxygen atom to form a phosphate ester: the said lysophospholipids therefore possess a free OH group on the remaining glycerol carbon atom.
  • Suitable lysophospholipids include lysophosphatidylcholines (also known as lyso-PC), lysophosphatidylethanolamines (PE or GPEtn), phosphatidylserine (PS or GPSer), phosphadityl inositol, , N-acyl phosphatidyl-ethanolamines and N-acyl lysophosphatidyl-ethanolamines.
  • the lysophospholipid may be formed in situ by hydrolysis of one of the ester linkages on the corresponding phospholipid.
  • the amount of lipolytic enzyme present is 0.001 g to 2 g per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 0.005 g to 1 g per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 0.001 g to 2 g per kg of the flour used to form the dough.
  • the amount of lipolytic enzyme present is 0.005 g to 0.5 g per kg of the flour.
  • the amount of lipolytic enzyme present is 0.001 mg to 2 mg per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 0.005 mg to 1 mg per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 0.001 mg to 2 g per kg of the flour used to form the dough.
  • the amount of lipolytic enzyme present is 0.005 to 0.5 mg per kg of the flour.
  • the amount of lipolytic enzyme present is 50 to 15,000 TIPU per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 100 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 TIPU per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 50 to 15,000 TIPU per kg of the flour used to form the dough.
  • the amount of lipolytic enzyme present is 100 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 TIPU per kg of the flour.
  • the TIPU unit of activity and the assay used to determine it is described in more detail below.
  • the amount of lipolytic enzyme present is 35 to 10,000 LIPU-NEFA per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 50 to 5,000, preferably 200 to 1 ,000 LIPU-NEFA per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 35 to 10,000 LIPU-NEFA per kg of the f!our used to form the dough.
  • the amount of lipolytic enzyme present is 50 to 5,000, preferably 200 to 1 ,000 LIPU-NEFA per kg of the flour.
  • the LIPU-NEFA unit of activity and the assay used to determine it is described in more detail below.
  • the amount of lipolytic enzyme present is 15 to 15,000 GLU per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 50 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 GLU per kg of the total weight of the foodstuff.
  • the amount of lipolytic enzyme present is 15 to 15,000 GLU per kg of the flour used to form the dough.
  • the amount of lipolytic enzyme present is 50 to 10,000, preferably 100 to 5,000, preferably 200 to 1 ,000 GLU per kg of the flour.
  • the GLU unit of activity and the assay used to determine it is described in more detail below.
  • glycolipase activity, phospholipase activity and triacyiglyceride lipase activity of the lipolytic enzyme as used in the present invention can be determined using the assays presented hereinbelow.
  • Phospholipase activity was measured using two different methods which give comparable results. Either of these methods can be used to determine phospholipase activity in accordance with the present invention. Preferably, the PLU assay is used for determining the phospholipase activity of any enzyme.
  • Free fatty acid in the samples was analyzed by using the NEFA HR(2) kit from WAKO GmbH. Enzyme activity PLU-7 at pH 7 was calculated as micromoles of fatty acid produced per minute under assay conditions.
  • TIPU Tiration Phospholipase Unit
  • Phospholipase A1 and A2 catalyse the conversion of lecithin to lyso-lecithin with release of the free fatty acid from position 1 and 2, respectively.
  • Phospholipase activity can be determined by continuous titration of the fatty acids liberated from lecithin during enzymation, since the consumption of alkali equals the amount of fatty acid liberated.
  • An enzyme solution was prepared to give a slope on the titration curve between 0.06 and 0.18 ml/min with an addition of 300 ⁇ _ enzyme.
  • a control sample of known activity is included. The samples were dissolved in demineralised water and stirred for 15 min. at 300 rpm. 25.00 ml substrate was thermostatted to 37.0°C for 10-15 minutes before pH was adjusted to 7.0 with 0.05 M NaOH. 300 ⁇ _ enzyme solution was added to the substrate and the continuous titration with 0.05 M NaOH was carried out using a pH-Stat titrator (Phm 290, Mettler Toledo). Two activity determinations are made on each scaling. After 8 minutes the titration is stopped and the slope of the titration curve is calculated between 5 and 7 minutes. The detection limit is 3 TIPU/ml enzyme solution.
  • TIPU/g enzyme The phospholipase activity (TIPU/g enzyme) was calculated in the following way:
  • a is the slope of the titration curve between 5 and 7 minutes of reaction time (ml/min); N is the normality of the NaOH used (mol/l);
  • ⁇ / ⁇ is the volume in which the enzyme is dissolved (ml);
  • Free fatty acid in the samples was analyzed by using the NEFA HR(2) kit from WAKO GmbH. Enzyme activity LSPU-NEFA at pH 5.5 was calculated as micromoles of fatty aid produced per minute under assays conditions.
  • one or more further food grade enzymes may be used, for example added to the food, dough preparation, or foodstuff.
  • At least one further enzyme may be added to the baked product and/or the dough.
  • Such further enzymes include starch degrading enzymes such as endo- or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g.
  • glucose oxidase pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidises maltose, for example hexose oxidase (HOX), lipases, phospholipases and hexose oxidase, proteases, and acyltransferases (such as those described in WO 2004/064987 for instance).
  • HOX hexose oxidase
  • lipases phospholipases and hexose oxidase
  • proteases and acyltransferases
  • the amylase may be a non-maltogenic amylase, such as a polypeptide having non- maltogenic exoamylase activity, in particular, giucan 1 ,4-alpha-maltotetrahydrolase (EC 3.2.1.60) activity (as disclosed in WO 2005/003339).
  • a suitable non-maltogenic amylase is commercially available as PowersoftTM (available from Danisco A/S, Denmark).
  • the combined use of alpha amylases and the lipolytic enzyme of the invention may be used in a dough, and/or the production of a baked product, such as bread, cakes, doughnuts, cake doughnuts or bagels.
  • the combination of alpha amylases and the lipolytic enzyme of the invention is also considered as preferable for use in methods of production of tortillas, such as wheat and/or maize tortillas.
  • the lipolytic enzyme according to the present invention may be used in combination with a xylanase in producing food products.
  • GR!NDAMYLTM and POWERBake 7000 are examples of commercially available xylanase enzymes available from Danisco A/S. Other examples of xylanase enzymes may be found in WO 03/020923 and WO 01/42433.
  • the lipolytic enzyme according to the present invention may be used in combination with a xylanase and an alpha amylase.
  • the alpha amylase may be a maltogenic, or a non-maltogenic alpha amylase (such as GRINDAMYLTM or POWERSoft, commercially available from Danisco A/S), or a combination thereof.
  • the lipolytic enzyme of the invention can also preferably be used in combination with an oxidising enzyme, such as a maltose oxidising enzyme (MOX), for example hexose oxidase (HOX). Suitable methods are described in WO 03/099016.
  • MOX maltose oxidising enzyme
  • HOX hexose oxidase
  • Suitable methods are described in WO 03/099016.
  • Commercially available maltose oxidising enzymes GRINDAMYLTM and SUREBake are available from Danisco A/S.
  • an alpha-amylase such as a non-maltogenic exoamylase and/or a maltogenic amylases, and/or a maltose oxidising enzyme (MOX) in combination with the enzyme according to the present invention may be used in methods of preparing a dough, a baked product, tortilla, cake, instant noodle/fried snack food, or a dairy product such as cheese.
  • a non-maltogenic exoamylase and/or a maltogenic amylases and/or a maltose oxidising enzyme (MOX) in combination with the enzyme according to the present invention
  • MOX maltose oxidising enzyme
  • the invention further comprises methods for including the lipolytic enzyme in the foodstuff or other composition.
  • the invention provides in a further aspect a method of producing a foodstuff according to the invention, comprising adding the lipolytic enzyme and the cyclodextrin to one or more other ingredients of the foodstuff or other composition and, if necessary, treating the ingredients to produce the foodstuff.
  • the method further comprises adding a cyclodextrin glycosyltransferase enzyme and, if necessary, a polysaccharide substrate to generate cyclodextrin in situ.
  • the cyclodextrin glycosyltransferase enzyme may be added to the foodstuff before addition of the lipolytic enzyme.
  • the cyclodextrin glycosyltransferase enzyme is added to the foodstuff or other composition at least 10 minutes (more preferably between 10 minutes and 3 hours) before the lipolytic enzyme.
  • the cyclodextrin glycosyltransferase enzyme may be added to the foodstuff or other composition after addition of the lipolytic enzyme.
  • the cyclodextrin glycosyltransferase enzyme is added to the foodstuff or other composition at least 10 minutes (more preferably between 10 minutes and 3 hours) after the lipolytic enzyme.
  • the cyclodextrin glycosyltransferase enzyme is added at the same time as the lipolytic enzyme.
  • Such methods are generally known to the skilled person, and include adding the lipolytic enzyme directly to the foodstuff or composition, addition of the lipolytic enzyme in combination with a stabilizer and/or carrier, and addition of a mixture comprising the lipolytic enzyme and a stabilizer and/or carrier.
  • Suitable stabilizers for use with the present invention include but is not limited to inorganic salts (such as NaCL ammonium sulphate), sorbitol, emulsifiers and detergents (such as Tween 20, Tween 80, Panodan AB100 without triglycerides, polyglycerol ester, sorbitan monoo!eate), oil (such as rape seed oil, sunflower seed oil and soy oil), pectin, trehalose and glycerol.
  • inorganic salts such as NaCL ammonium sulphate
  • sorbitol such as sorbitol
  • emulsifiers and detergents such as Tween 20, Tween 80, Panodan AB100 without triglycerides, polyglycerol ester, sorbitan monoo!eate
  • oil such as rape seed oil, sunflower seed oil and soy oil
  • pectin such as rape seed oil, sunflower seed oil and soy oil
  • Suitable carriers for use with the present invention include but is not limited to starch, ground wheat, wheat flour, NaCS and citrate.
  • the bread and/or dough improving composition may further comprise another enzyme, such as one or more other suitable food grade enzymes, including starch degrading enzymes such as endo- or exoamylases, pullulanases, debranching enzymes, hemicellulases including xylanases, cellulases, oxidoreductases, e.g.
  • glucose oxidase pyranose oxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidises maltose, for example hexose oxidase (HOX), lipases, phosphoiipases and hexose oxidase, proteases and acyltransferases (such as those described in WO 2004/064987 for instance).
  • HOX hexose oxidase
  • lipases phosphoiipases and hexose oxidase
  • proteases acyltransferases
  • Oxidoreductases such as for example glucose oxidase and hexose oxidase, can be used for dough strengthening and control of volume of the baked products and xylanases and other hemicellulases may be added to improve dough handling properties, crumb softness and bread volume.
  • Lipases are useful as dough strengtheners and crumb softeners and a-amylases and other amylolytic enzymes may be incorporated into the dough to control bread volume.
  • Further enzymes may be selected from the group consisting of a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase.
  • oxidases such as a glucose oxidase (EC 1.1 .3.4), carbohydrate oxidase, glycerol oxidase, pyranose oxidase, galactose oxidase (EC 1 .1.3.10), a maltose oxidising enzyme such as hexose oxidase (EC 1.1 .3.5).
  • the further enzyme is at least a xyianase and/or at least an antistaling amylase.
  • xyianase refers to xylanases (EC 3.2.1 .32) which hydrolyse xylosidic linkages.
  • amylase refers to amylases such as a-amylases (EC 3.2.1.1 ), ⁇ -amylases (EC 3.2.1.2) and ⁇ -amylases (EC 3.2.1.3).
  • the further enzyme can be added together with any dough ingredient including the flour, water or optional other ingredients or additives, or a dough improving composition.
  • the further enzyme can be added before the flour, water, and optionally other ingredients and additives or the dough improving composition.
  • the further enzyme can be added after the flour, water, and optionally other ingredients and additives or the dough improving composition.
  • the further enzyme may conveniently be a liquid preparation. However, the composition may be conveniently in the form of a dry composition.
  • host cell in relation to the present invention includes any cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein.
  • a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the enzyme of the present invention.
  • the cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
  • the host cells are not human cells. Examples of suitable bacterial host organisms are gram positive or gram negative bacterial species.
  • eukaryotic hosts such as yeasts or other fungi may be preferred.
  • some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast).
  • a different fungal host organism should be selected.
  • suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
  • the host cell may be a protease deficient or protease minus strain.
  • the genotype of the host cell may be modified to improve expression.
  • host cell modifications include protease deficiency, supplementation of rare tRNA's, and modification of the reductive potential in the cytoplasm to enhance disulphide bond formation.
  • the host cell may be deficient in a number of reducing enzymes thus favouring formation of stable disulphide bonds as exemplified/described in Bessette (Proc Natl Acad Sci USA (1999), 96, 13703-13708 "Efficient folding of proteins with multiple disulphide bonds in the Escherichia coli cytoplasm").
  • the enzymes for use in the present invention may be in an isolated form.
  • isolated means that the sequence or protein is at least substantially free from at least one other component with which the sequence or protein is naturally associated in nature and as found in nature.
  • the enzymes for use in the present invention may be used in a purified form.
  • purified means that the sequence is in a relatively pure state - e.g. at least about 51 % pure, or at least about 75%, or at least about 80%, or at least about 90% pure, or at least about 95% pure or at least about 98% pure.
  • a nucleotide sequence encoding either a polypeptide which has the specific properties as defined herein or a polypeptide which is suitable for modification may be isolated from any cell or organism producing said polypeptide.
  • Various methods are well known within the art for the isolation of nucleotide sequences.
  • a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the polypeptide.
  • labelled oligonucleotide probes may be synthesised and used to identify polypeptide-encoding clones from the genomic library prepared from the organism.
  • a labelled oligonucleotide probe containing sequences homologous to another known polypeptide gene could be used to identify polypeptide-encoding clones. Sn the latter case, hybridisation and washing conditions of lower stringency are used.
  • polypeptide-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing an enzyme inhibited by the polypeptide, thereby allowing clones expressing the polypeptide to be identified.
  • an expression vector such as a plasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library
  • the nucleotide sequence encoding the polypeptide may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al (1981 ) Tetrahedron Letters 22, 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, 801 -805.
  • the phosphoroamidite method oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
  • the nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence.
  • the DNA sequence may also be prepared by polymerase chain reaction (PGR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al (Science (1988) 239, 487-491 ).
  • nucleotide sequences encoding polypeptides having the specific properties as defined herein.
  • nucleotide sequence refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homoiogues, fragments and derivatives thereof (such as portions thereof).
  • the nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be doubie-stranded or single-stranded whether representing the sense or antisense strand.
  • nucleotide sequence in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence.
  • the nucleotide sequence per se encoding a polypeptide having the specific properties as defined herein does not cover the native nucleotide sequence in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment.
  • the term "non-native nucleotide sequence” means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment.
  • amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism.
  • amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
  • the polypeptide is not a native polypeptide.
  • native polypeptide means an entire polypeptide that is in its native environment and when it has been expressed by its native nucleotide sequence.
  • nucleotide sequence encoding polypeptides having the specific properties as defined herein is prepared using recombinant DNA techniques (i.e. recombinant DNA).
  • recombinant DNA i.e. recombinant DNA
  • the nucleotide sequence could be synthesised, in whole or in part, using chemical methods wel! known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et a/ (1980) Nuc Acids Res Symp Ser 225-232).
  • an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme-encoding nucleotide sequence has been identified, it may be desirable to modify the selected nucleotide sequence, for example it may be desirable to mutate the sequence in order to prepare an enzyme in accordance with the present invention.
  • Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
  • EP 0 583 265 refers to methods of optimising PGR based mutagenesis, which can also be combined with the use of mutagenic DNA analogues such as those described in EP 0 866 796.
  • Error prone PGR technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics.
  • WO 02/06457 refers to molecular evolution of lipases.
  • a third method to obtain novel sequences is to fragment non-identical nucleotide sequences, either by using any number of restriction enzymes or an enzyme such as Dnase I, and reassembling full nucleotide sequences coding for functional proteins. Alternatively one can use one or multiple non-identical nucleotide sequences and introduce mutations during the reassembly of the full nucleotide sequence.
  • DNA shuffling and family shuffling technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics. Suitable methods for performing 'shuffling' can be found in EP 0 752 008, EP 1 138 763, EP 1 103 606. Shuffling can a!so be combined with other forms of DNA mutagenesis as described in US 6, 180,406 and WO 01/34835.
  • mutations or natural variants of a polynucleotide sequence can be recombined with either the wild type or other mutations or natural variants to produce new variants.
  • Such new variants can also be screened for improved functionality of the encoded polypeptide.
  • the nucleotide sequence encoding a lipolytic enzyme used in the invention may encode a variant, i.e. the lipolytic enzyme may contain at least one amino acid substitution, deletion or addition, when compared to a parental enzyme.
  • Variant enzymes retain at least 70%, 80%, 90%, 95%, 97%, 99% homology with the parent enzyme.
  • Variant lipolytic enzymes may have decreased activity on triglycerides, and/or monoglycerides and/or digiycerides compared with the parent enzyme.
  • the variant enzyme may have no activity on triglycerides and/or monoglycerides and/or digiycerides. Alternatively, the variant enzyme may have increased thermostability.
  • the variant enzyme may have increased activity on one or more of the following, polar lipids, phospholipids, lecithin, phosphatidylcholine, glycolipids, digalactosyl monoglyceride, monogalactosyl monoglyceride.
  • variants of lipid acyltransferases are known, and one or more of such variants may be suitable for use in the methods and uses according to the present invention and/or in the enzyme compositions according to the present invention.
  • variants of lipid acyltransferases are described in the following references may be used in accordance with the present invention: Hilton & Buckley J Biol. Chem. 1991 Jan 15: 266 (2): 997-1000; Robertson et al J. Biol. Chem. 1994 Jan 21 ; 269(3):2146-50; Brumlik et al J. Bacteriol 1996 Apr; 178 (7): 2060-4; Peelman et al Protein Sci. 1998 Mar; 7(3):587-99.
  • AMINO ACID SEQUENCES AMINO ACID SEQUENCES
  • the present invention also encompasses the use of amino acid sequences encoded by a nucleotide sequence which encodes an enzyme for use in any one of the methods and/or uses of the present invention.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”.
  • amino acid sequence is synonymous with the term “peptide”.
  • amino acid sequence is synonymous with "enzyme”.
  • the amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
  • amino acid sequences may be obtained from the isolated polypeptides taught herein by standard techniques.
  • One suitable method for determining amino acid sequences from isolated polypeptides is as follows: Purified polypeptide may be freeze-dried and 100 pg of the freeze-dried material may be dissolved in 50 ⁇ of a mixture of 8 M urea and 0.4 M ammonium hydrogen carbonate, pH 8.4. The dissolved protein may be denatured and reduced for 15 minutes at 50°C following overlay with nitrogen and addition of 5 ⁇ of 45 mM dithiothreitol. After cooling to room temperature, 5 pi of 100 mM iodoacetamide may be added for the cysteine residues to be derivatized for 15 minutes at room temperature in the dark under nitrogen.
  • the resulting peptides may be separated by reverse phase HPLC on a VYDAC C18 column (0.46x15cm; 10pm; The Separation Group, California, USA) using solvent A: 0.1 % TFA in water and solvent B: 0.1 % TFA in acetonitrile.
  • Selected peptides may be re-chromatographed on a Develosil C18 column using the same solvent system, prior to N-terminal sequencing. Sequencing may be done using an Applied Biosystems 476A sequencer using pulsed liquid fast cycles according to the manufacturer's instructions (Applied Biosystems, California, USA).
  • homologue means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences.
  • the term “homology” can be equated with “identity”.
  • the homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the enzyme.
  • a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or 98% identical, preferably at least 95 or 98% identical to the subject sequence.
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence).
  • the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.
  • a suitable computer program for carrying out such an alignment is the Vector NTI (Invitrogen Corp.).
  • Other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4 th Ed - Chapter 18), and FASTA (Altschul et al 1990 J. Mol. Biol. 403-410). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60). However, for some applications, it is preferred to use the Vector NTI program.
  • BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1 ): 187-8 and tafiana@ncbi.nlm.nih.gov).
  • % homology can be measured in terms of identity
  • the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI Advance ' 10 package.
  • percentage homologies may be calculated using the multiple alignment feature in Vector NT! AdvanceTM 10 (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1 ), 237-244).
  • the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides.
  • the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
  • the default parameters for the programme are used for pairwise alignment.
  • the following parameters are the current default parameters for pairwise alignment for BLAST 2:
  • the sequence identity for the nucleotide sequences and/or amino acid sequences may be determined using BLAST2 (blastn) with the scoring parameters set as defined above.
  • the degree of identity is based on the number of sequence elements which are the same.
  • the degree of identity in accordance with the present invention for amino acid sequences may be suitably determined by means of computer programs known in the art such as Vector NTI AdvanceTM 1 1 (Invitrogen Corp.).
  • the scoring parameters used are preferably BLOSUM62 with Gap existence penalty of 1 1 and Gap extension penalty of 1.
  • the degree of identity with regard to an amino acid sequence is determined over at least 20 contiguous amino acids, preferably over at least 30 contiguous amino acids, preferably over at least 40 contiguous amino acids, preferably over at least 50 contiguous amino acids, preferably over at least 60 contiguous amino acids, preferably over at least 100 contiguous amino acids.
  • the degree of identity with regard to an amino acid sequence may be determined over the whole sequence.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention aiso encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • Non-homologous substitution may also occur i.e.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine
  • pyridylalanine thieny!alanine
  • naphthylalanine phenylglycine
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including a Iky I groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ -alanine residues.
  • Iky I groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ -alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue's nitrogen atom rather than the ⁇ -carbon.
  • Nucleotide sequences for use in the present invention or encoding a polypeptide having the specific properties defined herein may include within them synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences.
  • the present invention also encompasses the use of nucleotide sequences that are complementary to the sequences discussed herein, or any derivative, fragment or derivative thereof, !f the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
  • Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways.
  • Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • other viral/bacterial, or cellular homoiogues particularly cellular homoiogues found in mammalian cells (e.g.
  • rat, mouse, bovine and primate cells may be obtained and such homoiogues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homoiogues and allelic variants of the polypeptide or nucleotide sequences of the invention.
  • Variants and strain/species homoiogues may also be obtained using degenerate PGR which will use primers designed to target sequences within the variants and homoiogues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PGR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction polypeptide recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PGR primer, a primer for an alternative amplification reaction, a probe e.g.
  • primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
  • Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PGR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mPxNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
  • the present invention also encompasses the use of sequences that are complementary to the sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
  • hybridisation shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PGR) technologies.
  • the present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the subject sequences discussed herein, or any derivative, fragment or derivative thereof.
  • the present invention also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences discussed herein.
  • Hybridisation conditions are based on the melting temperature (Tm) of the nucleotide binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • a maximum stringency hybridisation can be used to identify or detect identical nucleotide sequences while an intermediate (or low/) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences.
  • the present invention also relates to the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).
  • the present invention also relates to the use of nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).
  • polynucleotide sequences that are capable of hybridising to the nucleotide sequences discussed herein under conditions of intermediate to maximal stringency.
  • the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under stringent conditions (e.g. 50°C and 0.2 x SSC).
  • stringent conditions e.g. 50°C and 0.2 x SSC.
  • the present invention covers the use of nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under high stringency conditions (e.g. 65°C and 0.1 x SSC).
  • the variant sequences etc. are at least as biologically active as the sequences presented herein.
  • biologically active refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.
  • sequence for use in the present invention is a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.
  • sequence for use in the present invention is a synthetic sequence - i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms - such as the methylotrophic yeasts Pichia and Hansenula.
  • a nucleotide sequence for use in the present invention or for encoding a polypeptide having the specific properties as defined herein can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate and express the nucleotide sequence, in polypeptide form, in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the polypeptide produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intrace!lularly depending on the sequence and/or the vector used.
  • the coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • the expression vector is incorporated into the genome of a suitable host organism.
  • the term "incorporated” preferably covers stable incorporation into the genome.
  • the nucleotide sequence encoding an enzyme for use in the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism.
  • the vectors for use in the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide of the present invention.
  • vector e.g. a plasmid, cosmid, or phage vector
  • the choice of vector e.g. a plasmid, cosmid, or phage vector will often depend on the host cell into which it is to be introduced.
  • the vectors for use in the present invention may contain one or more selectable marker genes such as a gene which confers antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Alternatively, the selection may be accomplished by co-transformation (as described in W091/17243).
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect, transform, transduce or infect a host cell.
  • the vector may further comprise a nucleotide sequence enabling the vector to replicate in the host eel! in question.
  • a nucleotide sequence enabling the vector to replicate in the host eel! in question.
  • sequences are the origins of replication of plasmids pUC19, pACYC177, pUB1 10, pE194, pAMB1 and plJ702.
  • the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell.
  • the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • regulatory sequences includes promoters and enhancers and other expression regulation signals.
  • promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site.
  • Enhanced expression of the nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions.
  • the nucleotide sequence according to the present invention is operably linked to at least a promoter.
  • Suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.
  • construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes a nucleotide sequence encoding a polypeptide having the specific properties as defined herein for use according to the present invention directly or indirectly attached to a promoter.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Sh1-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention.
  • fused in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wiid type gene promoter and when they are both in their natural environment.
  • the construct may even contain or express a marker which allows for the selection of the genetic construct.
  • the construct comprises at least a nucleotide sequence of the present invention or a nucleotide sequence encoding a polypeptide having the specific properties as defined herein operably linked to a promoter.
  • organism in relation to the present invention includes any organism that could comprise a nucleotide sequence according to the present invention or a nucleotide sequence encoding for a polypeptide having the specific properties as defined herein and/or products obtained therefrom.
  • transgenic organism in relation to the present invention includes any organism that comprises a nucleotide sequence coding for a polypeptide having the specific properties as defined herein and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence coding for a polypeptide having the specific properties as defined herein within the organism.
  • a promoter can allow expression of the nucleotide sequence coding for a polypeptide having the specific properties as defined herein within the organism.
  • the nucleotide sequence is incorporated in the genome of the organism.
  • Suitable organisms include a prokaryote, fungus yeast or a plant.
  • transgenic organism does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment.
  • the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, a nucleotide sequence coding for a polypeptide having the specific properties as defined herein, constructs as defined herein, vectors as defined herein, plasmids as defined herein, cells as defined herein, or the products thereof.
  • the transgenic organism can also comprise a nucleotide sequence coding for a polypeptide having the specific properties as defined herein under the control of a promoter not associated with a sequence encoding a lipid acyltransferase in nature.
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • suitable prokaryotic hosts include bacteria such as E. coli and Bacillus licheniformis, preferably B. licheniformis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et a! (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • the transgenic organism can be a yeast.
  • Filamentous fungi cells may be transformed using various methods known in the art - such as a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the ceil wall in a manner known.
  • Aspergillus as a host microorganism is described in EP 0 238 023.
  • T. reesei is the host organism.
  • Another host organism can be a plant.
  • a review of the general techniques used for transforming plants may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol (1991 ) 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
  • a host organism may be a fungus - such as a filamentous fungus.
  • suitable such hosts include any member belonging to the genera Fusarium, Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like.
  • Trichoderma is the host organism, preferably T. reesei.
  • the host organism can be of the genus Aspergillus, such as Aspergillus niger.
  • a transgenic Aspergillus according to the present invention can also be prepared by following, for example, the teachings of Turner G. 1994 (Vectors for genetic manipulation, in: Martinelli S.D., Kinghorn J R. (Editors) "Aspergillus: 50 years on”; Progress in industrial microbiology vol 29; Elsevier Amsterdam 1994; pp. 641 -666).
  • the transgenic organism can be a yeast.
  • yeast - such as the species Saccharomyces cerevisi or Pichia pastoris or Hansenula polymorpha (see FEMS Microbiol Rev (2000 24(1 ):45-66), may be used as a vehicle for heterologous gene expression.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et a/., (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H ef al (1983, J Bacteriology 153, 163-168).
  • the transformed yeast cells may be selected using various selective markers - such as auxotrophic markers dominant antibiotic resistance markers.
  • a suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as, but not limited to, yeast species selected from Pichia spp., Hansenula spp., Kluyveromyces, Yarrowinia spp., Saccharomyces spp., including S. cerevisiae, or Schizosaccharomyce spp. including Schizosaccharomyce pom be.
  • a strain of the methylotrophic yeast species Pichia pastoris may be used as the host organism.
  • the host organism may be a Hansenula species, such as H. polymorpha (as described in WO 01/39544).
  • a host organism suitable for the present invention may be a plant.
  • a review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol ( 991 ) 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27), or in WO 01/16308.
  • the transgenic plant may produce enhanced levels of phytosterol esters and phytostanol esters, for example.
  • a host organism suitable for the present invention may be a plant.
  • a review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994
  • Host cells transformed with the nucleotide sequence of the present invention may be cultured under conditions conducive to the production of the encoded enzyme and which facilitate recovery of the enzyme from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the enzyme.
  • the protein produced by a recombinant cell may be displayed on the surface of the cell
  • the enzyme may be secreted from the host cells and may conveniently be recovered from the culture medium using well-known procedures.
  • the polypeptide may be secreted from the expression host into the culture medium from where the enzyme may be more easily recovered.
  • the secretion leader sequence may be selected on the basis of the desired expression host.
  • Hybrid signal sequences may also be used with the context of the present invention.
  • Typical examples of secretion leader sequences not associated with a nucleotide sequence encoding a lipid acyltransferase in nature are those originating from the fungal amyloglucosidase (AG) gene (g/aA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenula) or the a-amylase gene (Bacillus).
  • AG fungal amyloglucosidase
  • a-factor gene e.g. Saccharomyces, Kluyveromyces and Hansenula
  • Bacillus e.g. Saccharomyces, Kluyveromyces and Hansenula
  • a variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiiuminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3,817,837; US-A-3,850,752; US-A-3,939,350; US-A-3,996,345; US-A-4,277,437; US-A-4,275,149 and US-A-4,366,241.
  • recombinant immunoglobulins may be produced as shown in US-A-4, 816,567.
  • An enzyme for use in the present invention may be produced as a fusion protein, for example to aid in extraction and purification thereof.
  • fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the protein sequence.
  • amino acid sequence of a polypeptide having the specific properties as defined herein may be ligated to a non-native sequence to encode a fusion protein.
  • a non-native sequence For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a non- native epitope that is recognised by a commercially available antibody.
  • sequences for use according to the present invention may also be used in conjunction with one or more additional proteins of interest (POIs) or nucleotide sequences of interest (NO!s).
  • POIs proteins of interest
  • NO!s nucleotide sequences of interest
  • Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, a-galactosidases, ⁇ -ga!actosidases, a-glucanases, glucan lysases, endo- ⁇ - glucanases, glucoamylases, glucose oxidases, a-glucosidases, ⁇ -glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases
  • the POI may even be a fusion protein, for example to aid in extraction and purification.
  • the POI may even be fused to a secretion sequence.
  • sequences can also facilitate secretion or increase the yield of secreted POI.
  • sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821 198.0.
  • the NOI may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations which modify the processing and/or expression of the expression product thereof.
  • the NOI may also be modified to optimise expression in a particular host cell.
  • Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
  • the NO! may include within it synthetic or modified nucleotides- such as methylphosphonate and phosphorothi emerge backbones.
  • the NOI may be modified to increase intracellular stability and half-life.
  • composition of the present invention may be used as a food ingredient.
  • the term "food ingredient” includes a formulation which is or can be added to functional foods or foodstuffs as a nutritional supplement and/or fiber supplement.
  • the term food ingredient as used here also refers to formulations which can be used at low levels in a wide variety of products that require gelling, texturising, stabilising, suspending, film-forming and structuring, retention of juiciness and improved mouthfeel, without adding viscosity.
  • the food ingredient may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
  • the amino acid sequence is used for large scale applications.
  • the amino acid sequence is produced in a quantity of from 1g per litre to about 25g/litre, preferably from above 2.5g/litres to about 18 g/litre, preferably above 8 g per litre of the total cell culture volume after cultivation of the host organism.
  • Figure 1 illustrates mini breads made in Baking Trial 1 to demonstrate the effect of generating increasing levels of free fatty acids in situ using the recipe described in Table 2;
  • Figure 2 illustrates the relative volume (as compared with blank), % of triglyceride and % of free fatty acids as a function of lipase (Exel 16) dose on top of fixed GRINDAMYL POWERBAKE 4070 dose (1000 TIPU/kg flour) from Baking Trial 1 using the recipe described in Table 2;
  • Figures 3A and 3B illustrate mini breads made in Baking Trial 2 to illustrate the effect of adding increasing levels of ⁇ -cyclodextrins together with a high dose of the lipolytic enzyme Grindamyl POWERBAKE 4070 using the recipe described in Table 3 ( Figure 3A showing bread in tins and Figure 3B showing bread made without tins);
  • Figure 4 illustrates the relative volume (as compared with blank) as a function of added amount of ⁇ -cyclodextrin on top of fixed high Grindamyl POWERBAKE 4070 dose (5000 TIPU/kg flour) made in Baking Trial 2 using the recipe described in Table 3B;
  • Figure 5 illustrates a scheme for extraction of free fatty acids from lyophilized dough
  • Figure 7 illustrates mini breads made in Baking Trial 3 to compare the baking effect of ⁇ -cyclodextrin and pectin using the recipe described in Table 4 (no tins);
  • Figure 8 illustrates the relative volume (as compared with blank) as a function of added amount of p-cyclodextrin and pectin on top of increasing dosages of Exel 16 in Baking Trial 3;
  • Figure 9 illustrates mini breads made in Baking Trial 4 to compare the performance of a combination of Grindamyl POWERBAKE 4070 and CGTase compared with a combination of Grindamyl POWERBAKE 4070 and ⁇ -cyclodextrin using the recipe described in Table 5 (in tins);
  • Figure 10 illustrates mini breads with added starch made in Baking Trial 5 to compare the performance of a combination of Grindamyl POWERBAKE 4070 and CGTase compared with a combination of Grindamyl POWERBAKE 4070 and ⁇ -
  • Figure 13 shows an amino acid sequence of a fungal lipolytic enzyme derived from Fusarium heterosporum comprising an N terminal signal sequence (underlined) (SEQ ID No. 2);
  • Figure 14 shows a nucleotide sequence (SEQ ID No. 3) encoding a fungal lipolytic enzyme derived from Fusarium heterosporum in accordance with the present invention
  • Figure 15 shows an amino acid sequence (SEQ ID No. 4) of a lipolytic enzyme derived from Fusarium semitectum;
  • Figure 16 shows a nucleotide sequence (SEQ ID NO. 5) encoding a lipolytic enzyme derived from Fusarium se itectum;
  • Figure 17 shows SEQ ID No. 6 a Humicola lanuginosa lipase produced by Humicola lanuginosa DSM 4109, sold commercially as GrindamyS Exel 16;
  • Figure 18 shows a nucleotide sequence (SEQ ID No.7) of a lipolytic enzyme derived from Fusarium heterosporum which includes a a- factor signal sequence;
  • Figure 19 shows SEQ ID No. 8 which is the DNA sequence for the polypeptide variant designated "mut 3";
  • FIG. 9 shows SEQ ID No. 9 which is the protein preprosequence for the polypeptide variant designated "mut 3";
  • FIG. 21 shows SEQ ID No. 10 which is the DNA sequence for the polypeptide variant designated "mut 4";
  • FIG. 22 shows SEQ ID No. 1 1 which is the protein preprosequence for the polypeptide variant designated "mut 4";
  • FIG. 23 shows SEQ ID No. 12 which is the DNA sequence for the polypeptide variant designated "mut 5";
  • Figure 24 shows SEQ ID No. 13 which is the protein preprosequence for the polypeptide variant designated "mut 5";
  • FIG. 14 shows SEQ ID No. 14 which is the DNA sequence for the polypeptide variant designated "mut 345";
  • FIG. 26 shows SEQ ID No. 15 which is the protein preprosequence for the polypeptide variant designated "mut 345";
  • FIG. 27 shows SEQ ID No. 16 which is the DNA sequence for the polypeptide variant designated "mut 3459”;
  • Figure 28 shows SEQ ID No. 17 which is the protein preprosequence for the polypeptide variant designated "mut 3459”;
  • Figure 29 shows SEQ ID No. 18 which is the DNA sequence for the polypeptide variant designated "mut 9";
  • FIG. 30 shows SEQ ID No. 19 which is the protein preprosequence for the polypeptide variant designated "mut 9";
  • FIG. 31 shows SEQ ID No. 20 which is the DNA sequence for the polypeptide variant designated "mut 10";
  • FIG. 32 shows SEQ ID No. 21 which is the protein preprosequence for the polypeptide variant designated "mut 10";
  • Figure 33 shows SEQ ID No. 22 which is the DNA sequence for the polypeptide variant designated "mut 1 1 ";
  • Figure 34 shows SEQ ID No. 23 which is the protein preprosequence for the polypeptide variant designated "mut 1 1";
  • Figure 35 shows SEQ ID No. 24 which is the DNA sequence for the polypeptide variant designated "mut 12 " ;
  • FIG. 36 shows SEQ ID No. 25 which is the protein preprosequence for the polypeptide variant designated "mut 12".
  • Figure 37 shows SEQ ID No. 26 which is a lipolytic enzyme sold commercially as Lipopan FTM.
  • Lipopan F the enzyme having SEQ ID No. 26 as described herein
  • Novozymes A/S, Denmark 1204468, lot 4010629964 holding 12900 TIPU/g was used as a positive control.
  • Phospholipase Grindamyi POWERBAKE 4070 (the enzyme having SEQ ID No. 1 as described herein) (available from Danisco A S, Denmark) (EDS 129, 5830 TIPU/g) was used in combination with either Lipase Exel 16, ⁇ -cyclodextrin, CGTase or CGTase and starch.
  • Lipase Exel 16 (available from Danisco A/S, Denmark) (Control sample for protocol A770, 37350 LIPU/g) was used in combination with Grindamyi POWERBAKE 4070, ⁇ -cyclodextrin and pectin.
  • CGTase (Amano) was used in combination with either Grindamyi POWERBAKE 4070 or Grindamyi POWERBAKE 4070 and starch
  • ⁇ -cyclodextrin (Sigma C4767) was used in combination with Grindamyi POWERBAKE 4070 or Exel 16.
  • the water absorption of the flour and composite flours was determined using a Farinograph (Brabender, Germany) according to AACC 54-21 .
  • Baking performance was evaluated in small scale baking trials (50 gram mixer and 10 gram loaves) using the recipe shown in Table 1 below.
  • the amount of flour was adjusted according to the amount of ⁇ -cyclodextrin or pectin added to give a total of 50 g.
  • the same was the case for the trial described in Table 1 , where the amount of flour was adjusted so the total of added starch and flour summed up to 50 g.
  • Table 1 shows the recipe used for evaluation of baking performance.
  • Salt/sugar is a 1 :1 (w/w) mixture.
  • 'Water' is the water absorption determined by Farinograph analysis.
  • the flour and dry ingredients were mixed for one minute, water (with or without enzyme) was added and mixing was continued for another five minutes.
  • the aim of Baking Trial 1 was to test the effect of increasing levels of in situ generated free fatty acid (FFA).
  • the aim of Baking Trial 2 was to test the effect of adding increasing amounts of ⁇ -cyclodextrin for complexing of in situ generated FFA. In both cases mini bread in tins as well as without tins were made.
  • the setup of Baking Trial 2 the high amount of ⁇ -cyclodextrin added to the dough was evaluated by including a ⁇ -cyclodextrin blank.
  • the experimental set-up is listed in Tables 2 and 3.
  • Baking Trial 3 tested the baking effect of ⁇ -cyclodextrin versus the baking effect of the hydrocolloid pectin, which was used as a control. Only mini breads without tins were made. In this trial setup both a ⁇ -cyclodextrin blank and pectin blank was included. The experimental setup is listed in Table 4. Cyclodextrin glycosyltransferase (CGTase) is closely related to a-amylases but has the unique ability to produce cyclodextrins from linear a(1 ⁇ 4)-linked glucans via an intramolecular transg!ycosyiation reaction known as cyclization.
  • CCTase Cyclodextrin glycosyltransferase
  • Baking Trials 4 and 5 The aim of Baking Trials 4 and 5 was to test whether the same baking effect could be obtained by combining Grindamyl POWERBAKE 4070 and CGTase as with combining Grindamyl POWERBAKE 4070 and added ⁇ -cyclodextrin. In Baking Trial 5 additional substrate (starch) for the CGTase was added. In Baking Trial 5 a starch blank was included. In both trials only mini breads without tins were made. The experimental setup is listed in Tables 5 and 6.
  • the aim of Baking Trial 6 was to evaluate the effect of accumulating cyclodextrins in the dough before generating free fatty acids.
  • Table 2 Baking Trial 1 : Trial to test the effect of increasing dosage of Exei 16 in combination with a fixed dose of Grindamyl POWERBAKE 4070 (1000 TIPU/kg flour).
  • Baking Trial 2 Trial to test the effect of increasing amount of ⁇ -cyclodextrin in combination with a fixed dose of Grindamyl POWERbake 4070 (5000 TIPU/kg flour).
  • Baking Trial 4 Trial to test if combination of Grindamyl POWERbake 4070 and CGTase performed on level with combination of Grindamyl POWERbake 4070 and added ⁇ -cyclodextrin.
  • Lyophilized dough samples were milled in a coffee mill before extraction of the lipid fraction applying one of the following procedures.
  • Oven temperature °C. 80 200 240 360
  • Sample preparation Evaporated sample was dissolved in 1 ml pyridine containing internal standard heptadecane, 0.5 mg/ml. 500 ⁇ sample solution was transferred to a crimp vial, 100 pi BSTFA ( ⁇ /, ⁇ -bis-trimethylsilyl-trifluoroacetamide) + TMCS (trimethylchlorosilane) (99+1 ) was added and reacted for 15 minutes at 60°C.
  • the amount of free fatty acid was determined on Konelab Autoanaiyser (Thermo, Finland) using the NEFA HR(2) kit (WAKO GmbH, Germany). Assay was run at 30°C. 150 ⁇ _ solution A and 15 ⁇ _ redispersed extracted lipids were incubated for 3 minutes. 75 ⁇ solution B was added and incubated for 4.5 minutes. The absorbance at 520 nm was measured. The amount of free fatty acid was determined, using the read absorbance and a standard curve based on oleic acid (0.05 mM to 1.0 mM).
  • Table 7 Water absorption relative to fiour at 400 BLJ determined for each p- cyclodextrin concentration tested in Baking Thai 2.
  • This baking trial was conducted to evaluate the effect of adding increasing levels of ⁇ - cyclodextrin together with a high dose of Grindamyl POWERbake 4070 enzyme.
  • the addition of ⁇ -cyclodextrin should serve to lower the concentration of free fatty acids by formation of inclusion compounds and thereby eliminate/minimize the negative baking effect of increasing levels of free fatty acid in terms of reduced bread voiume.
  • Figure 5 shows the flow of the dough extraction made to differentiate between fatty acids incorporated in ⁇ -cyclodextrin inclusion compounds and free fatty acid.
  • extract 1 a small amount of free fatty acid will be extracted.
  • butanohethanol (85:15 (v/v)) to the watery extract will cause the fatty acid-p- cyclodextrin inclusion compounds to break down into free fatty acid and ⁇ - cyclodextrin.
  • the free fatty acid will be in the organic phase (extract 3) and the ⁇ - cyclodextrin will be in the water phase (extract 2).
  • the precipitate from the watery extract will contain the fatty acid not incorporated in inclusion compounds and by adding butanohethanol (85: 15 (v/v)) to the precipitate this fatty acid will be extracted (extract 4).
  • butanohethanol 85: 15 (v/v)
  • the results of the dough extractions depicted in Figure 5 are summarized in Table 8.
  • Table 8 For dough with no enzyme added (b!ank and ⁇ -cyclodextrin) the total amount of free fatty acid was in the proximity of 6 ⁇ /g dough, and for dough with enzyme added the total !evel of free fatty acid was in the range 1 1 -13 rnmol/ g dough.
  • Extract 3 was to be a number for the amount of free fatty acid released from ⁇ - cyclodextrin inclusion compounds upon addition of organic solvent.
  • Extract 2 As expected only a very small amount of free fatty acid was found in Extract 2: see Table 8. Presumably the rather varying free fatty acid numbers determined for mini bread made in tins was caused by inadequate centrifugation of the sample resulting in poor phase separation.
  • the amount of free fatty acid in extract 2, 3 and 4 should add up to the amount determined for the total extract. But as is evident from Table 8 this was not the case as the recovery (sum extract 2, 3 and 4 relative to the total extract) was in the range 40-60%, meaning only close to half the free fatty acid was extracted. The low recovery could be due to gluten agglumeration during water extraction of the iyophilized dough. Agglumerated gluten could trap free fatty acids and render them inaccessible for analysis. In fact a gum-like lump was observed in the precipitate of the water extracted Iyophilized dough indicating gluten agglumeration taking place. To verify if this was the case papain (a plant protease) was added during water extraction of selected Iyophilized doughs.
  • papain a plant protease
  • Table 9 shows the levels of free fatty acids in papain treated Iyophilized dough from baking trial conducted to illustrate the effect of increasing levels of added ⁇ - cyclodextrin (no tins).
  • the Iyophilized doughs were extracted as depicted in Figure 5 with the exception that 500 ppm papain was added during the water extraction. The amount of free fatty acid was determined applying the NEFA method.
  • Extract 1 Extract 2 Extract 3 Extract 4 (Extract (2+3+4) extraction / total)
  • Baking Trial 1 indicated increasing amounts of in situ generated free fatty acids had a negative baking effect in terms of reduced bread volume. Complexing free fatty acids with ⁇ -cyclodextrin had a significant positive effect on bread volume, 40% increase compared to blank, !n combination with Grindamyl POWERbake 4070, ⁇ -cyciodextrin had an additive effect, as an additional 20% volume increase was obtained, giving a total of 60% volume increase compared to blank.
  • Baking Trials 4 and 5 comparing the baking performance of Grindamyl POWERbake 4070 in combination with either ⁇ -cyclodextrin or CGTase showed the two combinations to perform on level. Further results observed in relation to the use of lipases include improved crumb structure, improved pore homogeneity and reduced mean pore size. These effects remain substantially unchanged by the inclusion of cyclodextrin.
  • CGTase was added to a fraction of the flour (20% of flour comprising the dough), together with the water needed to form the dough. Brews were stirred gently and left at 30°C for 1 hour. Afterwards the brew was added the residual amount of flour (80% of flour comprising the dough), mixed to form a dough, proofed, scaled and baked using the below baking procedure. 20% of flour is added to water required for dough (with or without CGTase) and the mixture is stirred gently at 30 °C for 1 hour. At this point, mixing of the dry ingredients (residual flour, yeast, salt, sugar) begins.

Abstract

L'invention porte sur un produit alimentaire contenant une quantité efficace de : (a) une enzyme lipolytique apte à agir sur un substrat lipidique en vue de libérer une molécule d'acide gras libre ; et (b) une cyclodextrine. L'invention porte également sur des procédés de production du produit alimentaire, ainsi que sur des utilisations d'une cyclodextrine pour la diminution de la teneur en acide gras libre d'un produit alimentaire comportant une enzyme lipolytique.
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Publication number Priority date Publication date Assignee Title
WO2019121585A1 (fr) * 2017-12-19 2019-06-27 Dupont Nutrition Biosciences Aps Modification enzymatique améliorée de phospholipides dans des aliments

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US11744254B2 (en) 2017-12-19 2023-09-05 Dupont Nutrition Biosciences Aps Enzymatic modification of phospholipids in food
EP4248757A3 (fr) * 2017-12-19 2023-12-06 DuPont Nutrition Biosciences ApS Modification enzymatique améliorée de phospholipides dans des aliments

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