US20180368424A1 - Improved enzymatic modification of wheat phospholipids in bakery applications - Google Patents

Improved enzymatic modification of wheat phospholipids in bakery applications Download PDF

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US20180368424A1
US20180368424A1 US16/064,218 US201616064218A US2018368424A1 US 20180368424 A1 US20180368424 A1 US 20180368424A1 US 201616064218 A US201616064218 A US 201616064218A US 2018368424 A1 US2018368424 A1 US 2018368424A1
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dough
enzyme
acts
phospholipase
baked product
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Jorn Borch Soe
Rene Mikkelsen
Tina Lillan Jorgensen
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DuPont Nutrition Biosciences ApS
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    • 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
    • A21D2/16—Fatty acid esters
    • A21D2/165—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/08—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils with fatty acids

Definitions

  • the present invention relates to novel enzyme combinations and their use in the manufacture of dough or baked products.
  • the present invention further relates to methods of making dough or a baked product using novel enzyme combinations.
  • Lipids constitute approximately 2% of wheat flour and these lipids are considered highly important for the baking quality of wheat flour.
  • Wheat flour lipids can be divided into non-polar and polar lipids, and it has been shown that improved baking and bread properties are mainly due to polar lipids.
  • N-acyl phosphatidyl ethanolamine NALPE
  • NALPE N-acyl lysophosphatidyl ethanolamine
  • NAGPE N-acyl glycerophospho-ethanolamine
  • the first aspect of the present invention provides a food enzyme composition
  • a food enzyme composition comprising: a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position; and an enzyme that acts on a polar lipid at the sn1 position.
  • a method of making a dough comprising admixing a dough component, a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position, and an enzyme that acts on a polar lipid at the sn1 position.
  • the invention provides the use of a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position in the manufacture of a dough or a baked product for improving the specific volume of a baked product; dough characteristics (such as dough development; dough extensibility); improving crust crispiness of a baked product; improving the crumb structure (such as improving crumb pore size of a baked product or improving crumb pore homogeneity of a baked product); improving softness (such as improving softness of a baked product); improving the oven spring of a baked product; increasing N-acyl lysophosphatidyl ethanolamine in the dough and/or baked product (preferably increasing N-acyl lysophosphatidyl ethanolamine having a fatty acid moiety containing 14-20 carbon atoms, preferably increasing N-acyl lysophosphatidyl ethanolamine having a saturated
  • kits comprising a phospholipase A2 enzyme which is capable of acting on N-acylphosphatidyl ethanolamine at the sn2 position; an enzyme that acts on a polar lipid at the sn1 position; and a set of instructions for use.
  • the present invention yet further provides a dough obtainable by (preferably obtained by) a method according to the present invention or a baked product obtainable by (preferably obtained by) a method according to the present invention.
  • SEQ ID NO: 1 is the amino acid sequence of an enzyme in POWERBAKE® 4080 and POWERBAKE® 4090 that acts on a polar lipid at the sn1 position (same as SEQ ID NO: 6 from U.S. Pat. No. 8,012,732; hereby incorporated by reference). This enzyme is known to have both galactolipase and phospholipase activity.
  • SEQ ID NO: 2 is the amino acid sequence of a mature lipid acyltransferase (GOAT) derived from Aeromonas salmonicida (See U.S. Pat. No. 9,175,271).
  • GOAT mature lipid acyltransferase
  • SEQ ID NO: 3 is the amino acid sequence of a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position found in MAXAPAL® A2.
  • SEQ ID NO: 4 is the amino acid sequence of a phospholipase A2 enzyme (CRC08335) which acts on NAPE (N-acyl phosphatidyl ethanolamine) at the sn2 position.
  • CRC08335 phospholipase A2 enzyme
  • SEQ ID NO: 5 is the nucleotide sequence of a phospholipase A2 enzyme (CRC08335) which acts on NAPE (N-acyl phosphatidyl ethanolamine) at the sn2 position.
  • CRC08335 phospholipase A2 enzyme
  • SEQ ID NO: 6 is an N-terminal predicted signal peptide sequence of CRC08335.
  • FIG. 1 shows a list of polar and non-polar lipids found in flour (particularly wheat flour) from Pomeranz, Y. in Modern Cereal Science and Technology ((1987) VCH Publishers, New York, N.Y.).
  • FIG. 2 shows the softness effect observed when a phospholipase A2 enzyme (SEQ ID NO: 3) which acts on N-acyl phosphatidyl ethanolamine at the sn2 position (MAXAPAL®); and an enzyme that acts on a polar lipid at the sn1 position (POWERBAKE® 4090) in combination is used in baking white pan bread.
  • SEQ ID NO: 3 which acts on N-acyl phosphatidyl ethanolamine at the sn2 position
  • POWERBAKE® 4090 an enzyme that acts on a polar lipid at the sn1 position
  • FIG. 3 shows the softness effect observed when a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position (MAXAPAL®); and an enzyme that acts on a polar lipid at the sn1 position (POWERBAKE®4090) in combination is used in baking 100% whole wheat bread.
  • FIG. 4 shows the amino acid sequence (SEQ ID NO: 1) of POWERBAKE® 4080 and POWERBAKE® 4090 (both commercially available from DuPont Nutrition Biosciences ApS).
  • FIG. 5 shows the amino acid sequence (SEQ ID NO: 4) of CRC08335.
  • FIG. 6 shows the nucleotide sequence (SEQ ID NO: 5) of CRC08335.
  • FIG. 7 shows the Plasmid map of pZKY512-1 harboring the synthetic gene of CRC08335.
  • a seminal finding of the present invention is that advantageous properties in a foodstuff (e.g. a dough and/or a baked product) can be achieved by using a combination of a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine (NAPE) at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position.
  • a foodstuff e.g. a dough and/or a baked product
  • NAPE N-acyl phosphatidyl ethanolamine
  • the present inventors have shown the synergistic effects provided by the combination of a phospholipase A2 enzyme which is capable of acting on NAPE at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position in a foodstuff, e.g. a dough or a baked product.
  • a phospholipase A2 enzyme which is capable of acting on NAPE at the sn2 position; and an enzyme that acts on a polar lipid at the sn1 position in the preparation of a dough or products obtainable from the dough.
  • the present invention yet further provides a food enzyme composition comprising a phospholipase A2 enzyme which is capable of acting on NAPE at the sn2 position; and an enzyme that acts on a polar lipid at the sn1 position.
  • the present invention relates to the lysis of specific polar lipids in a specific way in dough and food products obtainable from the dough.
  • the polar lipids contained in most cereal flours include phospholipids and galactolipids.
  • NAPE N-acyl phosphatidyl ethanolamine
  • Schafferczyk et al J. of Agricultural and Food Chemistry (2014) 62: 8229-8237 teaches that wheat flour contains on average 0.1% NAPE compared with 0.02% phosphatidylcholine (PC).
  • Flour particularly wheat flour
  • Galactolipids such as digalactosyldiglyceride (DGDG) or monogalactosyldiglyceride (MGDG) are naturally occurring (or endogenous) lipid components in flour, particularly wheat flour.
  • DGDG digalactosyldiglyceride
  • MGDG monogalactosyldiglyceride
  • the phospholipids and/or galactolipids acted on by the enzymes used in the present invention are naturally occurring phospholipids and/or galactolipids within the flour.
  • the phospholipase A2 enzyme which acts on NAPE at the sn2 position according to the present invention is one which has PLA2 activity in the “Assay for the Determination of phospholipase activity and position specificity on NAPE” taught herein.
  • Substrate 0.6% 16:0-18:1 NAPE (N-linoleoyl-(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) (obtained from Avanti on request or produced according to J. L. Newman et al., Chemistry and Physics of Lipids (1986) 42: 240-260),
  • the collected fatty acid fraction (fraction 2) is evaporated to dryness and fatty acid content is analyzed by GLC.
  • Enzyme activity on NAPE is calculated as pmol fatty acid produced per minutes under assay conditions
  • Enzyme ⁇ ⁇ activity 2 ⁇ A ⁇ 1000000 ⁇ D 100 ⁇ MV ⁇ 10 ⁇ 0.1
  • the enzyme specificity is calculated as:
  • Relative ⁇ ⁇ PLA ⁇ ⁇ 1 ⁇ ⁇ activity % ⁇ ⁇ C ⁇ ⁇ 16 ⁇ : ⁇ 0 ⁇ 100 % ⁇ ⁇ C ⁇ ⁇ 16 ⁇ : ⁇ 0 + % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1
  • Relative ⁇ ⁇ PLA ⁇ ⁇ 2 ⁇ ⁇ activity % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1 ⁇ 100 % ⁇ ⁇ C ⁇ ⁇ 16 ⁇ : ⁇ 0 + % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1
  • a phospholipase A2 enzyme which preferentially lyses, e.g. hydrolyses, NAPE at the sn2 position would be one which in the “Assay for the Determination of phospholipase activity and position specificity on NAPE” taught herein has at least 50% more relative PLA2 activity on NAPE.
  • An enzyme with 50% more relative PLA2 activity means that the enzyme has less than 25% sn1 activity and more than 75% sn2 activity.
  • phospholipase A2 enzyme which preferentially lyses e.g.
  • NAPE at the sn2 position would be one which in the “Assay for the Determination of phospholipase activity and position specificity on NAPE” taught herein has at least 10% more relative PLA2 activity compared with relative PLA1 activity.
  • the “Assay for the Determination of phospholipase activity and position specificity on NAPE” taught herein is used. However, in some embodiments this may be determined using the EnzChek Phospholipase A2 Assay Kit from Invitrogen cat. No. E10217, optionally together with a dough test which analyses whether the enzyme reduces NAPE with increased formation of NALPE in a dough.
  • the term “specifically” in relation to the phospholipase A2 enzyme which acts on NAPE at the sn2 position means that the enzyme will catalyse only one particular reaction, e.g. the lysis (or hydrolysis) of NAPE at the sn2 position to produce 1-NALPE.
  • a phospholipase A2 enzyme which specifically lyses, e.g. hydrolyses, NAPE at the sn2 position would be one which in the “Assay for the Determination of phospholipase activity and position specificity on NAPE” taught herein has at least 80% more relative PLA2 activity than PLA1 activity.
  • the phospholipase A2 enzyme which acts on NAPE at the sn2 position according to the present invention has one or more of the following enzyme activities: phospholipase A2 activity (e.g. E.C. 3.1.1.4) or lipid acyltransferase activity (e.g. E.C. 2.3.1.43).
  • phospholipase A2 activity e.g. E.C. 3.1.1.4
  • lipid acyltransferase activity e.g. E.C. 2.3.1.43
  • the phospholipase A2 enzyme which acts on NAPE at the sn2 position according to the present invention is one which is capable of converting NAPE into 1-NALPE under dough conditions.
  • the phospholipase A2 enzyme which acts on NAPE at the sn2 position according to the present invention is one which converts NAPE into 1-NALPE wherein the fatty acid moiety of the produced NALPE contains 14-20 carbon atoms.
  • the phospholipase A2 enzyme which acts on NAPE at the sn2 position according to the present invention is one which converts NAPE into 1-NALPE wherein the fatty acid moiety of the produced NALPE is saturated and contains 14-20 carbon atoms.
  • the phospholipase A2 enzyme which acts on NAPE at the sn2 position according to the present invention is one which converts NAPE into 1-NALPE wherein the fatty acid moiety of the produced NALPE is saturated and contains 16 carbon atoms (C16:0).
  • a phospholipase A2 enzyme which converts NAPE into 1-NALPE wherein the fatty acid moiety of the produced NALPE is saturated and contains 16 carbon atoms can be determined using the “Assay for the Determination of phospholipase activity and position specificity on NAPE” taught herein and/or using “HPLC/MS method for analysis of phospholipids extracted from dough” taught herein.
  • use of the enzyme combination in accordance with the present invention results in the amount of C16:0 NALPE in the dough being increased by a factor of at least 1.5 compared with a dough without enzyme addition.
  • the amount of C16:0 NALPE In the dough may be increased by a factor of at least 2.0, preferably at least 3.0.
  • use of the enzyme combination in accordance with the present invention results in the amount of C16:0 NALPE in the dough being increased by a factor of between about 1.5 and about 4.0 compared with a dough without enzyme addition.
  • Dough conditions are well known to one skilled in the art. These may include the conditions during the mixing of dough components or resting and storage of dough. Suitably dough conditions include dough mixing, dough resting, dough scaling and moulding, and dough fermentation.
  • the claimed phospholipase A2 enzyme which acts on NAPE at the sn2 position is incapable or substantially incapable of acting on N-acyl lysophosphatidylethanolamine (NALPE).
  • NALPE N-acyl lysophosphatidylethanolamine
  • substantially incapable of acting on N-acyl lysophosphatidylethanolamine means that the enzyme which in the same dosage tested in both the “Assay for the Determination of phospholipase activity and position specificity on NAPE” and in the “Assay for the Determination Lysophospholipase activity on N-acyl lysophosphatidylethanolamine (NALPE)” has less than 20% activity on NALPE compared to activity on NAPE.
  • an enzyme which is substantially incapable of acting on N-acyl lysophosphatidylethanolamine has less than 10% activity on NALPE compared with activity on NAPE, more suitably less than 5% activity on NALPE, even more preferably less than 1% NALPE activity.
  • the determination of fatty acid moiety saturation and length can be performed by methods known in the art. As a non-limiting example gas chromatography (GC) or liquid chromatography-mass spectrometry (HPLC/MS) as taught herein.
  • NALPE N-acyl lysophosphatidylethanolamine
  • NALPE N-linoleoyl-(1-oleoyl-glycero-3-phosphoethanolamine) (obtained from Avanti on request or produced according to “Synthesis of N-acyl lysophosphatidylethanolamine (NALPE)”), 0.4% TRITONTM-X 100 (Sigma, X-100), and 5 mM CaCl 2 ) were dissolved in 0.05 M HEPES buffer pH 7.0. For pancreatic enzyme 0.003 M Deoxy-cholate was also added.
  • the sample was centrifuged at 1520 g for 10 min.
  • One 500 mg amine (NH2)—Bond Elut SPE column (Agilent) was placed on a Bond Elut Vacuum System.
  • the column was conditioned with 8 mL Petroleum-ether.
  • the MTBE phase from the extraction was applied onto the column and eluted with:
  • the collected fatty acid fraction (fraction 2) was evaporated to dryness and fatty acid content was analyzed by GLC.
  • Enzyme ⁇ ⁇ activity 2 ⁇ A ⁇ 1000000 ⁇ D 100 ⁇ MV ⁇ 10 ⁇ 0.1
  • NALPE N-acyl lysophosphatidylethanolamine
  • the phospholipase A2 enzyme which acts on NAPE at the sn2 position according to the present invention may be MAXAPAL®, LYSOMAX® Oil, Pancreatic PLA2, Lipomod 699L from Biocatalysts.
  • the enzyme that acts on a polar lipid at the sn1 position is one which acts on a polar lipid at the sn1 position as determined using one or both of the following assays: “Assay for determining an enzyme that acts on a polar lipid (a phospholipase) at the sn1 position” and/or “Assay for determining an enzyme that acts on a polar lipid (a galactolipid; MGDG) at the sn1 position”.
  • Phospholipase A1 activity was measured using PED-A1 (N-((6-(2,4-DNP)Amino)Hexanoyl)-1-(BODIPY® FL C5)-2-Hexyl-Sn-Glycero-3-hosphoethanolamine (A10070 from ThermoFisher Scientific) as a substrate.
  • the substrate is specific for PLA1 and is a dye labeled glycerophosphoethanolamine with BODIPY® FL dye-labeled acyl chain at the sn1 position, and dinitrophenyl quencher-modified head group. Quenching efficiency is decreased by cleavage of the BODIPY® FL pentanoic acid substituent at the sn1 position and with an enzyme resistant ether linkage in the sn2 position. The result is a PLA1 dependent increase in fluorescence emission detected at 515 nm.
  • a “lipid mix” was prepared by mixing 30 ⁇ L 10 mM dioleoylphosphatidylcholine in ethanol, 30 ⁇ L 10 mM Dioleylphosphatidylglycerol in ethanol and 30 ⁇ L 2 mM PED-A1 in DMSO.
  • micro titer plate well To a micro titer plate well add 50 ⁇ L enzyme sample (or standard or control) and 50 ⁇ L substrate liposome mix. Incubate at room temperature for 30 minutes, protected from light. Measure the fluorescence using a micro titer plate reader with excitation at 470 nm and emission at 515 nm.
  • a calibration curve is constructed based on a number of standard PLA1 solutions of different enzyme concentration from 0 to 10 U/mL.
  • the enzyme standard is a PLA1 (L3295 from Sigma) of known activity.
  • a calibration curve of fluorescence intensity as a function of enzyme concentration U/mL was constructed. Based on the standard curve the activity of the unknown sample was measured (U/mL).
  • Substrate 0.6% 1-linoleyl-2-oleyl-3-O-(-D-galactopyranosyl)-sn glycerol (C18:2, C18:1 MGDG) 0.4% TRITONTM-X 100 (Sigma, X-100), and 5 mM CaCl 2 were dissolved in 0.05 M HEPES buffer pH 7. For pancreatic enzyme 0.003 M Deoxy-cholate was also added.
  • the sample was centrifuged at 1520 g for 10 min.
  • One 500 mg amine (NH2)—Bond Elut SPE column (Agilent) is placed on a Bond Elut Vacuum System.
  • the column is conditioned with 8 mL Petroleum-ether.
  • the MTBE phase from the extraction is applied onto the column and eluted with:
  • the collected fatty acid fraction (fract. 2) is evaporated to dryness and fatty acids are analyzed by GLC. Based on the internal standard C17:0 fatty acid the amount of C18:2 and C18:1 fatty acid is determined.
  • Enzyme activity is calculated as pmol fatty acid produced per minutes under assay conditions
  • Enzyme ⁇ ⁇ activity 2 ⁇ A ⁇ 1000000 ⁇ D 100 ⁇ MV ⁇ 10 ⁇ 0.1
  • the enzyme specificity is calculated as
  • Relative ⁇ ⁇ sn ⁇ ⁇ 1 ⁇ ⁇ activity % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 2 ⁇ 100 % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 2 + % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1
  • Relative ⁇ ⁇ sn ⁇ ⁇ 2 ⁇ ⁇ activity % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1 ⁇ 100 % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 2 + % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1
  • the enzyme that acts on a polar lipid at the sn1 position is one which in the assay entitled “Assay for determining an enzyme that acts on a polar lipid (a phospholipase) at the sn1 position” has at least 20% more relative sn1 activity than relative sn2 activity.
  • the enzyme that acts on a polar lipid at the sn1 position is one which in the assay entitled “Assay for determining an enzyme that acts on a polar lipid (a phospholipase) at the sn1 position” has at least 50% more relative sn1 activity than relative sn2 activity.
  • the enzyme that acts on a polar lipid at the sn1 position according to the present invention is one which in the assay entitled “Assay for determining an enzyme that acts on a polar lipid (a MGDG) as the sn1 position” has at least 20% more relative sn1 activity than relative sn2 activity. In one embodiment, the enzyme that acts on a polar lipid at the sn1 position according to the present invention is one which in the assay entitled “Assay for determining an enzyme that acts on a polar lipid (a MGDG) at the sn1 position” has at least 50% more relative sn1 activity than relative sn2 activity
  • the enzyme that acts on a polar lipid at the sn1 position according to the present invention is one which in a dough can hydrolyse at least 10% of the DGDG using HPTLC analysis of dough lipids.
  • polar lipid means a polar lipid found in flour (preferably wheat flour). Polar lipids found in wheat flour are defined by Pomeranz, Y. (supra; see FIG. 1 ). In one embodiment the term “polar lipid” means one or more of the group consisting of: a phospholipid, a galactolipid, or a combination thereof.
  • the phospholipid may be one or more of phosphatidyl choline, N-acyl phosphatidyl ethanolamines, phosphatidyl ethanolamines, phosphatidyl serines or phosphatidyl inositol. In one embodiment preferably the phospholipid may be phosphatidyl choline.
  • the galactolipid may be one or more of digalactosyl diglyceride, ceramide diglycerides, 6-o-acetylsteryl glucosides or ceramide diglucosides. In one embodiment preferably the galactolipid may be digalactosyl diglyceride.
  • the enzyme that acts on a polar lipid at the sn1 position is a phospholipase A1, e.g. has phospholipase A1 activity and may be classified as E.C. 3.1.1.32.
  • the enzyme that acts on a polar lipid at the sn1 position may act on a galactolipid (e.g. digalactosyldiglyceride (DGDG) or monogalactosyldiglyceride (MGDG). This may be in addition to its phospholipase A1 activity.
  • DGDG digalactosyldiglyceride
  • MGDG monogalactosyldiglyceride
  • the enzyme that acts on a polar lipid at the sn1 position is a galactolipase, e.g. and may be classified as E.C. 3.1.1.26.
  • the enzyme that acts on a polar lipid at the sn1 position acts on DGDG at the sn1 position.
  • the term “acts on” in relation to the enzyme that acts on a polar lipid at the sn1 position as used herein means that the enzyme removes the fatty acid from the sn1 position of a polar lipid (e.g. by hydrolysis) e.g. thus releasing free fatty acid.
  • the term “preferentially” in relation to the enzyme that acts on a polar lipid at the sn1 position means that the enzyme prefers to catalyse the hydrolysis of a polar lipid at the sn1 position, e.g. compared with catalysing the lysis of the polar lipid at the sn2 position.
  • An enzyme which acts on a polar lipid at the sn1 position can be determined using the assay(s): “Assay for determining an enzyme that acts on a polar lipid (a phospholipase) at the sn1 position” and/or “Assay for the Determination of phospholipase activity and sn1 and sn2 position specificity on PC (phosphatidylcholine)” and/or “Assay for determining an enzyme that acts on a polar lipid (a galactolipid; MGDG) at the sn1 position”.
  • assay(s) “Assay for determining an enzyme that acts on a polar lipid (a phospholipase) at the sn1 position” and/or “Assay for the Determination of phospholipase activity and sn1 and sn2 position specificity on PC (phosphatidylcholine)” and/or “Assay for determining an enzyme that acts on a
  • Substrate 0.6% 16:0-18:1 PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids Inc., Alabaster, Ala.; cat. 850457)
  • the collected fatty acid fraction (fract. 2) is evaporated to dryness and fatty acids are analyzed by GLC. Based on the internal standard C17:0 fatty acid the amount of C16:0 and C18:1 fatty acid is determined.
  • Enzyme activity is calculated as pmol fatty acid produced per minutes under assay conditions
  • Enzyme ⁇ ⁇ activity 2 ⁇ A ⁇ 1000000 ⁇ D 100 ⁇ MV ⁇ 10 ⁇ 0.1
  • the enzyme specificity is calculated as
  • Relative ⁇ ⁇ PLA ⁇ ⁇ 1 ⁇ ⁇ activity % ⁇ ⁇ C ⁇ ⁇ 16 ⁇ : ⁇ 0 ⁇ 100 % ⁇ ⁇ C ⁇ ⁇ 16 ⁇ : ⁇ 0 + % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1
  • Relative ⁇ ⁇ PLA ⁇ ⁇ 2 ⁇ ⁇ activity % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1 ⁇ 100 % ⁇ ⁇ C ⁇ ⁇ 16 ⁇ : ⁇ 0 + % ⁇ ⁇ C ⁇ ⁇ 18 ⁇ : ⁇ 1
  • the term “preferentially” in relation to the enzyme that acts on a polar lipid at the sn1 position means that the enzyme prefers to catalyse the hydrolysis of a polar lipid at the sn1 position, e.g. compared with catalysing the lysis of the polar lipid at the sn2 position.
  • An enzyme which acts on a polar lipid at the sn1 position can be determined using the assay(s): “Assay for determining an enzyme that acts on a polar lipid (a phospholipase) at the sn1 position” and/or “Assay for the Determination of phospholipase activity and sn1 and sn2 position specificity on PC (phosphatidylcholine)” and/or “Assay for determining an enzyme that acts on a polar lipid (a galactolipid; MGDG) at the sn1 position”.
  • assay(s) “Assay for determining an enzyme that acts on a polar lipid (a phospholipase) at the sn1 position” and/or “Assay for the Determination of phospholipase activity and sn1 and sn2 position specificity on PC (phosphatidylcholine)” and/or “Assay for determining an enzyme that acts on a
  • An enzyme that preferentially acts on polar lipids at the sn1 position means that the relative PLA1/sn1 activity when determined using the “Assay for the Determination of phospholipase activity and sn1 and sn2 position specificity on PC (phosphatidylcholine)” and/or “Assay for determining an enzyme that acts on a polar lipid (a galactolipid; MGDG) at the sn1 position” would be at least 60%.
  • an enzyme that preferentially acts on polar lipids at the sn1 position means that the relative PLA1/sn1 activity when determined using the “Assay for the Determination of phospholipase activity and sn1 and sn2 position specificity on PC (phosphatidylcholine)” and/or “Assay for determining an enzyme that acts on a polar lipid (a galactolipid; MGDG) at the sn1 position” would be at least 70%.
  • the term “specifically” in relation to the enzyme that acts on a polar lipid at the sn1 position means that the enzyme will catalyse only the hydrolysis of a polar lipid at the sn1 position.
  • An enzyme that specifically acts on polar lipids at the sn1 position means that the enzyme has at least 60% (suitably at least 70%) relative PLA1/sn1 activity when determined using the “Assay for the Determination of phospholipase activity and sn1 and sn2 position specificity on PC (phosphatidylcholine)” and/or “Assay for determining an enzyme that acts on a polar lipid (a galactolipid; MGDG) at the sn1 position”.
  • the enzyme that acts on a polar lipid at the sn1 position may include the enzyme as taught in WO02/03805 (which is incorporated herein by reference).
  • the enzyme that acts on a polar lipid at the sn1 position includes POWERBAKE® 4080, POWERBAKE® 4090, PANAMORE®, LIPOPAN FTM, and LIPOPAN EXTRATM.
  • the enzyme that acts on a polar lipid at the sn1 position may be a phospholipase A1 from Fusarium oxysporum (e.g. LIPOPAN FTM).
  • the phospholipase A1 from Fusarium oxysporum may be the enzyme taught in WO98/26057—which is incorporated herein by reference.
  • the enzyme that acts on a polar lipid at the sn1 position according to the present invention is one which has at least 60% sequence identity, more preferably at least 70%, at least 80%, at least 90%, at least 95% or 100% identity to SEQ ID NO: 1.
  • the enzyme that acts on polar lipid at the sn1 position has low activity on NAPE.
  • One advantage of the present invention is the use of a combination of a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine (NAPE) at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position.
  • NAPE N-acyl phosphatidyl ethanolamine
  • the fatty acid composition of phospholipids and galactolipids at the sn1 and sn2 positions differs significantly, both in fatty acid length and saturation levels.
  • a phospholipase A1 may hydrolyse NAPE at the sn-1 position to produce 2-NALPE, e.g. with the fatty acid in the sn-2 position (see Structural Analysis of Wheat Flour Glycerolipids. Lipids, Vol. 6, No. 10 p. 768-776). Therefore, the invention relates to the impact of lysing (e.g. hydrolysing) NAPE at the sn2 position in combination with modifying a polar lipid (e.g. further polar lipid) with an enzyme that acts at the sn1 position.
  • lysing e.g. hydrolysing
  • the phospholipase A2 enzyme and the enzyme that acts on polar lipids are admixed to the dough components in effective amounts that result in an increase of the specific volume of the baked product that is at least 10%, relative to a baked product made under identical conditions except for the addition of the claimed enzymes.
  • a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position are admixed to the dough components in effective amounts that result in an increased softness of the baked product that is at least 5%, preferably at least 10%, more preferably at least 20%, most preferably at least 30% relative to a baked product made under identical conditions except for the addition of the claimed enzymes.
  • a food enzyme composition is considered to increase monogalactosylmonoglyceride content in a dough or baked product when the lipid components are extracted from the dough or baked product (e.g.
  • GC gas chromatography
  • HPLC/MS liquid chromatography-mass spectrometry
  • a food enzyme composition is considered to increase monogalactosylmonoglyceride content in a dough or baked product when the lipid components are extracted from the dough or baked product (e.g. and subject to gas chromatography (GC) or liquid chromatography-mass spectrometry (HPLC/MS) analysis or HPTLC analysis), showing between about 0.005 and 0.1% w/w increase (based on dough dry dough) in monogalactosylmonoglyceride content in comparison to an identical dough or baked product where the enzyme has not been added.
  • a food enzyme composition is considered to decrease digalactosyldiglyceride content in a dough or baked product when the lipid components are extracted from the dough or baked product (e.g.
  • a food enzyme composition is considered to decrease digalactosyldiglyceride content in a dough or baked product. It is analyzed when the lipid components are extracted from the fully proofed dough or baked product (e.g.
  • GC gas chromatography
  • HPLC/MS liquid chromatography-mass spectrometry
  • the phospholipase A2 enzyme of the present invention is present at a concentration of between 100-7500 ePLU/kg flour. In one embodiment the phospholipase A2 enzyme is dosed at 150-2000 ePLU/kg flour.
  • the enzyme that acts on a polar lipid at the sn1 position of the present invention is present at a concentration of between 50-2000 TIPU/kg flour. In one embodiment the enzyme that acts on a polar lipid at the sn1 position of the present invention is dosed at 200-800 TIPU/kg flour.
  • the phospholipase A2 enzyme activity may be determined using the following assay using egg yolk as substrate.
  • the assay is conducted according to Food Chemical Codex (FCC, 8ed., Appendix 5 p. 1328)
  • Phospholipase activity may be determined using the following assay:
  • Substrate 0.6% L- ⁇ Phosphatidylcholine 95% Plant (Avanti, cat. #441601), 0.4% TRITONTM-X 100 (Sigma X-100), and 5 mM CaCl 2 ) were dissolved in 0.05 M HEPES buffer pH 7.
  • Samples, calibration sample, and control sample were diluted in 10 mM HEPES pH 7.0 containing 0.1% TRITONTM X-100. Analysis was carried out using a Konelab Autoanalyzer (Thermo, Finland). The assay was run at 30° C. 34 ⁇ L substrate was thermostatted for 180 seconds at 30° C., before 4 ⁇ L of enzyme sample was added. Enzymation lasted 600 sec. The amount of free fatty acid liberated during enzymation was measured using the NEFA kit obtained from WakoChemicals GmbH, Germany).
  • This assay kit is composed of two reagents
  • CoA coenzyme A
  • ATP adenosine 5-triphosphate disodium salt
  • 4-amino-antipyrine (4-AA) 2.6 U/mL Ascorbate oxidase (AOD) 0.062% Sodium azide
  • NEFA-HR(1) was added and the mixture was incubated for 300 sec. Afterwards 56 ⁇ L NEFA-HR(2) was added and the mixture was incubated for 300 sec. OD 520 nm was then measured. Enzyme activity ( ⁇ mol FFA/min ⁇ mL) was calculated based on a calibration curve made form oleic acid. Enzyme activity TIPU pH 7 was calculated as micromole fatty acid produced per minute under assay conditions.
  • a flour dough may not contain sufficient amounts of all of the lipid substrates for the composition of the invention. It is therefore within the scope of the invention to supplement the dough with at least one of a galactolipid, a phospholipid or a combination thereof to provide sufficient substrates for the enzyme(s). It will be appreciated that the expression “sufficient substrate” implies that none of the lipid substrates is limiting for obtaining a dough improving or baked product improving effect as described above.
  • the supplementary lipid substrate for the enzyme of the invention may be a polar lipid.
  • a particularly useful lipid is an oil or a fat derived from cereals such as oat oil.
  • Oat oil typically contains, in addition to triglycerides, 5-25% phospholipids and 5-12% glycolipids.
  • Oat oil can be fractionated to yield fractions having a high content of polar lipids.
  • phospholipids can be added to the dough.
  • useful phospholipids include phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidylcholine (PC).
  • composition of the present invention further comprises lecithin.
  • composition for use according to the present invention further comprises lecithin.
  • the method of the present invention further comprises admixing lecithin.
  • the lecithin of the present invention is soya-derived lecithin.
  • the lecithin of the present invention has been enzymatically modified.
  • the lecithin of the present invention has been enzymatically modified by an enzyme with phospholipase A2 activity.
  • the lecithin of the present invention has been modified by a phospholipase A2 that is capable of acting at the sn2 position of N-acetyl phosphatidylethanolamine.
  • the present invention yet further provides the use of a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position in the manufacture of a dough or a baked product for improving the specific volume of a baked product; dough characteristics (such as dough development; dough extensibility); improving crust crispiness of a baked product; improving the crumb structure (such as improving crumb pore size of a baked product or improving crumb pore homogeneity of a baked product); improving softness (such as improving softness of a baked product); improving the oven spring of a baked product; increasing N-acyl lysophosphatidyl ethanolamine in the dough and/or baked product (preferably increasing N-acyl lysophosphatidyl ethanolamine with a fatty acid moiety containing 14-20 carbon atoms, preferably increasing N-acyl lysophosphatidyl ethanolamine with a saturated fatty
  • the present invention may further advantageously provide a method for obtaining a baked product having improved quality characteristics (such as improved specific volume, improved crust crispiness of a baked product; improved crumb structure (such as improved crumb pore size of a baked product or improved crumb pore homogeneity of a baked product); improved softness (such as improved softness of a baked product); improved capping of a baked product; improved oven spring of a baked product).
  • improved quality characteristics such as improved specific volume, improved crust crispiness of a baked product; improved crumb structure (such as improved crumb pore size of a baked product or improved crumb pore homogeneity of a baked product); improved softness (such as improved softness of a baked product); improved capping of a baked product; improved oven spring of a baked product).
  • the method of the present invention comprises as a further step that the dough is baked to obtain a baked product.
  • One particularly desired property of baked bread products is a high specific volume as defined in the examples.
  • the addition of the enzymes of the invention preferably results in an increase of the specific volume of the baked product that is at least 10%, relative to a baked product made under identical conditions except that the enzyme is not added. More preferably, the increase of the specific volume is at least 20% such as at least 30%, e.g. at least 40%.
  • enzymes other than lipases may contribute to improved dough properties and quality of baked products. It is within the scope of the invention that, in addition to the composition of the invention, at least one further enzyme may be added.
  • Such further enzymes include a lipase, starch degrading enzyme (e.g. an amylase or an amyloglucosidase), a hemicellulase (e.g. xylanase), a cellulase, an oxidoreductase (e.g. a glucose oxidase, such as hexose oxidase), a lipid acyltransferase, a debranching enzyme (e.g. a pullulanase), a lactase and a protease.
  • starch degrading enzyme e.g. an amylase or an amyloglucosidase
  • hemicellulase e.g. xylanas
  • the claimed method comprises a further step wherein a further enzyme is admixed to the dough components.
  • Specific volume in baked products can be defined as the volume of the product divided by its weight. (g/mL or g/ccm)
  • the present invention relates to improving the specific volume of a baked product.
  • the present invention may relate to improving dough characteristic, such as dough development; dough extensibility.
  • the present invention does not negatively impact dough stickiness.
  • the present invention may relate to improving crust crispiness of a baked product.
  • baked products e.g. bread or bread rolls as follows:
  • the present invention may relate to improving the crumb structure (such as improving crumb pore size of a baked product or improving crumb pore homogeneity of a baked product). These may be measured in baked products, e.g. bread or bread rolls as follows:
  • the present invention may relate to improving softness (such as improving softness of a baked product).
  • Softness may also be measured by any method known in the art.
  • baked products e.g. bread or bread rolls as follows:
  • the softness (or hardness) of bread slices was determined from a texture profile analysis (TPA) using a Texture analyser TAXTplus from Stable Microsystems.
  • TPA texture profile analysis
  • TAXTplus from Stable Microsystems.
  • a 35 mm metal probe may be used to measure softness on days 1 (D1) and 3 (D3).
  • the present invention does not negatively affect capping of a baked product
  • capping occurs when the top has set (i.e., hardened), and then this top is pushed up, allowing batter from the interior of the baked product, e.g. muffin or roll, to ooze out to the side. The result is an undesirable baked product, e.g. muffin or roll.
  • Capping was subjectively evaluated by examining the baked product and the amount of capping observed was assigned a qualitative number.
  • the present invention may relate to improving the oven spring of a baked product
  • organ spring means the rapid increase in volume (rising) of baked products, e.g. bread when they are placed into a hot oven.
  • increasing or improving means increasing or improving compared with the same dough or product obtainable from said dough (e.g. a baked product) but without addition of the enzymes in accordance with the present invention.
  • Additional technical effects of the present invention include increasing N-acyl lysophosphatidyl ethanolamine in the dough and/or baked product (preferably increasing N-acyl lysophosphatidyl ethanolamine with a fatty acid moiety containing 14-20 carbon atoms, preferably increasing N-acyl lysophosphatidyl ethanolamine with a saturated fatty acid moiety containing 14-20 carbon atoms); increasing a lyso-phospholipid in the dough and/or baked product; increasing a digalactosylmonoglyceride and/or monogalactosylmonoglyceride in the dough and/or baked product.
  • the present invention relates to increasing N-acyl lysophosphatidyl ethanolamine (NALPE) (preferably 1-NALPE) together with increasing a lyso-phospholipid and/or a digalactosylmonoglyceride and/or monogalactosylmonoglyceride in the dough and/or baked product.
  • NALPE N-acyl lysophosphatidyl ethanolamine
  • the present inventors have shown the synergistic effects provided by the combination of a phospholipase A2 enzyme which is capable of acting on NAPE at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position in a foodstuff, e.g. a dough or a baked product.
  • “synergy” or “synergistic effect” means an increase in the effect (e.g. bread volume) which is more than the increase obtained from each enzyme when used individually or separately, in the same dosage.
  • the method, uses or compositions of the present invention may be used in the preparation of a foodstuff.
  • the term “foodstuff” is used in a broad sense—and covers foodstuff for humans as well as foodstuffs for animals (i.e. a feedstuffs).
  • the foodstuff is for human consumption.
  • the term “dough component” means any one of flour (e.g. cereal flour, preferably wheat flour), water or yeast or any composition comprising one or more of flour, water and/or yeast.
  • the enzyme(s) of the present invention are admixed with a dough component.
  • the enzyme(s) of the present invention are admixed with flour or with a composition comprising flour.
  • the foodstuff is a dough or a product produced from the dough, e.g. by cooking, such as by baking or steaming, boiling or frying.
  • the baked product is obtainable (preferably obtained) from a dough.
  • the steamed product is obtainable (preferably obtained) from a dough.
  • the boiled product is obtainable (preferably obtained) from a dough.
  • the fried product is obtainable (preferably obtained) from a dough.
  • the foodstuff is a baked product.
  • the foodstuff is a steamed product.
  • the foodstuff is a boiled product.
  • the foodstuff is a fried product.
  • the method, uses or compositions of the present invention can be used in the preparation of a dough or a product produced from the dough, e.g. by cooking, such as by baking, steaming, boiling or frying.
  • the baked product is produced by baking a dough produced in accordance with the present invention.
  • the boiled product is produced by boiling a dough produced in accordance with the present invention.
  • the steamed product is produced by steaming a dough produced in accordance with the present invention.
  • the fried product is produced by frying a dough produced in accordance with the present invention.
  • the foodstuff is a baked product, such as bread (e.g. white, whole-meal or rye bread; typically in the form of loaves or rolls, French baguette-type bread, pita bread, flatbreads, crisp bread or pizza bread), tortillas, pancakes, muffins, pie crusts, pastry, Danish pastry, cakes, biscuits, or cookies.
  • bread e.g. white, whole-meal or rye bread; typically in the form of loaves or rolls, French baguette-type bread, pita bread, flatbreads, crisp bread or pizza bread
  • tortillas e.g. white, whole-meal or rye bread
  • pancakes e.g., muffins, pie crusts
  • pastry e.g. white, whole-meal or rye bread
  • loaves or rolls French baguette-type bread
  • Pizzas e.g., French baguette-type bread, pita bread, flatbreads, crisp bread or pizza bread
  • tortillas e.g. white, whole-meal or
  • the foodstuff is a steamed product, such as a steamed bread, dumplings.
  • the foodstuff is a boiled product, such as noodles or pasta.
  • the foodstuff is a fried product, such as a doughnut.
  • a “food enzyme composition” as defined herein may be any composition suitable for addition to a dough or suitable for admixing with a dough component.
  • food products of the present invention include baked products and dough products.
  • the term “dough” as used herein means a thick, malleable mixture of flour and liquid (e.g. water).
  • the dough may include yeast or other leavening agents.
  • the dough may further comprise other dough components such as a fat or a flavouring(s) or salt or sugar.
  • the dough according to the present invention may be made from one or more of the flours selected from: wheat flour, maize flour, rice flour, rye flour, legume flour, almond flour or other cereal flours.
  • the dough is made from wheat flour.
  • the method and uses of the present invention may be part of any bread making process.
  • the composition of the present invention may be used in any bread making process.
  • the bread making process may be one or more processes selected from the group consisting of: sponge-and-dough; straight; no-time and continuous bread making.
  • the sponge-and-dough mixing method may consist of two distinct stages, a sponge stage and a dough stage.
  • a sponge stage In the first stage (sponge stage) a sponge is made and allowed to ferment for a period of time; and in the second stage (dough stage) the sponge is added to the final dough ingredients creating a total formula.
  • Other terms for “sponge” include yeast starter or yeast pre-ferment.
  • French baking the sponge and dough method may be known as levain-levure.
  • the mixture in the first stage, may contain about one-third to three-quarters of the flour, the yeast, yeast food (e.g. sugar), and malt, and enough water to make a stiff dough or a more liquid brew. Shortening may be added at this stage, although it is usually added later, and one-third to three-quarters of the salt may be added to control fermentation.
  • the sponge may be mixed in any suitable mixing device, suitably with temperature control.
  • suitable mixing device suitably with temperature control.
  • this may be a large, horizontal dough mixer, processing about one ton per batch, and is optionally constructed with heat-exchange jackets, allowing temperature control.
  • the objectives of mixing are a nearly homogeneous blend of the ingredients and “developing” of the dough by formation of the gluten into elongated and interlaced protein network that will form the basic structure of the loaf. Because intense shearing actions must be avoided, the usual dough mixer has several horizontal bars, oriented parallel to the body of the mixer, rotating slowly at 35 to 75 revolutions per minute, stretching and kneading the dough by their action. A typical mixing cycle would be about 12 minutes.
  • the mixed sponge is dumped into a trough, a shallow rectangular metal tank on wheels, and placed in an area of controlled temperature and humidity (e.g., 27° C. and 75% relative humidity), where it is fermented until it begins to decline in volume.
  • controlled temperature and humidity e.g., 27° C. and 75% relative humidity
  • the time required for this process, called the drop or break depends on such variables as temperature, type of flour, amount of yeast, absorption, and amount of malt, which are frequently adjusted to produce a drop in about three to five hours.
  • the sponge is returned to the mixer, and the remaining ingredients are added.
  • the dough is developed to an optimum consistency then either returned to the fermentation room or allowed “floor time” for further maturation.
  • a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position may be added at either the sponge stage or the dough stage, preferably the sponge stage. These may be added simultaneously or sequentially.
  • the phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position may be added at the sponge stage (e.g. during admixing the flour and other dough components).
  • the enzyme that acts on a polar lipid at the sn1 position may be added to the dough stage (e.g. during mixing).
  • the phospholipase A2 enzyme which is capable of acting on NAPE is added to a sponge and the enzyme that acts on a polar lipid at the sn1 position is added to the dough.
  • a lecithin may additionally be added, preferably soya-based lecithin, at the sponge stage (e.g. during admixing the flour and other dough components).
  • the lecithin may be added together with at least a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position.
  • the lecithin may be an enzymatically modified lecithin.
  • lecithin may be enzymatically modified by an enzyme with phospholipase A2 activity (preferably the lecithin may be enzymatically modified by a phospholipase A2 that acts on N-acyl phosphatidyl ethanolamine at the sn2 position).
  • the phospholipase A2 or a portion thereof is added during sponge stage.
  • the straight dough method may be a single-mix process of making bread. All components (e.g. ingredients) for making the dough are all placed together and combined in one kneading or mixing session. After mixing, a bulk fermentation rest occurs before division.
  • a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position may be admixed with the dough components. These may be added simultaneously or sequentially.
  • a lecithin may additionally be added, preferably soya-based lecithin.
  • the lecithin may be an enzymatically modified lecithin.
  • lecithin may be enzymatically modified by an enzyme with phospholipase A2 activity (preferably the lecithin may be enzymatically modified by a phospholipase A2 that acts on N-acyl phosphatidyl ethanolamine at the sn2 position).
  • the “no-time” method is a special subset of the straight dough method.
  • increased amounts of yeast and fast-acting oxidants such as ascorbic acid and azodicarbonamide enable the elimination of most of the straight dough bulk fermentation period.
  • the brew After the brew has finished fermenting, it is fed along with the dry ingredients into a mixing device, which mixes all ingredients into a homogeneous mass.
  • the batter like material passes through a dough pump regulating the flow and delivering the mixture to a developing apparatus, where kneading work is applied.
  • the developer is the key equipment in the continuous line. Processing of about 50 kilograms (100 pounds) can occur each 90 seconds, it changes the batter from a fluid mass having no organized structure, little extensibility, and inadequate gas retention to a smooth, elastic, film-forming dough.
  • the dough then moves out of the developer into a metering device that constantly extrudes the dough and intermittently severs a loaf-size piece, which falls into a pan passing beneath.
  • a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position may be added to the liquid pre-ferment or to the dough, e.g. after fermentation and during mixing of the dough. These may be added simultaneously or sequentially.
  • the phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position may be added to the liquid pre-ferment.
  • the enzyme that acts on a polar lipid at the sn1 position may be added to the dough, e.g. after fermentation and during mixing of the dough.
  • a lecithin may additionally be added, preferably soya-based lecithin, at either the pre-ferment stage or to the dough, e.g. after fermentation and during mixing of the dough.
  • the lecithin may be added together with at least a phospholipase A2 enzyme which acts on N-acyl phosphatidyl ethanolamine at the sn2 position.
  • the lecithin may be an enzymatically modified lecithin.
  • lecithin may be enzymatically modified by an enzyme with phospholipase A2 activity (preferably the lecithin may be enzymatically modified by a phospholipase A2 that acts on N-acyl phosphatidyl ethanolamine at the sn2 position).
  • Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.
  • protein includes proteins, polypeptides, and peptides.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”.
  • polypeptide proteins and “polypeptide” are used interchangeably herein.
  • the conventional one-letter and three-letter codes for amino acid residues may be used.
  • the 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
  • Lecithin samples (40 ⁇ 5 mg) were dissolved in 1 mL 4:2:3 CDCl 3 :MeOH:CsCDTA (aq) (deuterochloroform:methanol:caesium-1,2-diaminocyclohexanetetraacetic acid, v/v).
  • the CDTA solution was prepared with a concentration of 1 M in milli-Q-water.
  • CsOH.H 2 O Caesium hydroxide ⁇ water was then added to adjust the pH to 10.5.
  • NMR spectra were acquired under automation at 14.1T using a Bruker Advance III spectrometer (Fällandern, Switzerland), a SampleJet sample changer (Bruker, Desillanden, Switzerland) and a 5 mm BBO (Broadband Observe) probe tuned to phosphorous (Bruker, Desillanden, Switzerland). Spectra were acquired under quantitative conditions.
  • the dough lipid samples were analyzed by liquid chromatography coupled on-line with a triple quadrupole mass spectrometer in full scan m/z 50-1500 with heated electro spray in positive and negative mode.
  • NALPE formed deprotonated ions, [M-H] ⁇ in negative mode.
  • the column was a normal phase column (DIOL) and the mobile phase was acetonitrile/acetone 80/20 with addition of 20 mL water in 1 L.
  • the water contained 5 mM ammonium formate.
  • Samples were solved in 2 mL acetonitrile/acetone (80:20). The traces of selected NALPE were extracted and the areas were compared.
  • the dough is proofed for 45 minutes at 34° C., 85% RH and baked for 13 minutes at 200° C./2 l steam+5 minutes damper open (MIWE oven prog. 1). After baking the rolls are cooled for 25 minutes at ambient temperature before weighing and measuring of volume.
  • the dough sticks to your resting open the dough, touch the slips your fingers fingers cut dough surface with fingers Crust Crispiness of Fracture crust using Leathery crust Crisp crust crust several fingers Crumb Crumb pore size Visual evaluation of sliced Open crumb, big gas Fine crumb, small gas bread, bubbles bubbles size of gas bubbles in crumb Crumb pore Visual evaluation of sliced Big variation in sizes of Constant gas bubble size homogeneity bread, homogeneity of gas gas bubbles bubbles Product shape Capping/ Visual evaluation of A very large hole directly No separation between Hole under the vertical cut surface under the crust. crust and crumb. crust Oven Visual evaluation amount No energy High level of energy spring/Energy of energy in the product
  • DGDG digalactosyldiglyceride
  • DGMG digalactosylmonoglyceride
  • MGMG monogalactosylmonoglyceride
  • NAPE N-acyl phosphatidylethanolamine
  • NALPE N-acyl lysophosphatidylethanolamine
  • NAGPE N-acyl glycerophosphatidylelthanolamine
  • the synergistic baking performance between POWERBAKE® 4080 and LYSOMAX® Oil is due to the fact that LYSOMAX® Oil is active on the sn2 position of NAPE whilst POWERBAKE® is active on the sn1 position in polar lipids (including MGDG and DGDG and phospholipases—including NAPE).
  • Example 1 it was shown that the optimal dosage of LYSOMAX® Oil was 100 ppm in combination with POWERBAKE® 4080. In order to further study the dosage response, LYSOMAX® Oil was tested in dosage from 25 ppm to 200 ppm in combination, with POWERBAKE® 4080. Results are shown in Table 3.
  • the baking results confirm a synergistic effect of 30 ppm POWERBAKE® 4080 combined with LYSOMAX® Oil.
  • a minimum of 75 ppm LYSOMAX® Oil is needed to see the synergistic effect and the optimum dosage in 100 ppm.
  • POWERBAKE® 4080 was tested in combination with MAXAPAL® or LYSOMAX® Oil in Hard Crust Roll recipe.
  • MAXAPAL® is a phospholipase with high PLA2 specificity.
  • the total dough and bread score is calculated as the sum of the individual score apart from stickiness score, which is added as (10-stickiness)
  • MAXAPAL® is very active on NAPE during formation of NALPE.
  • the enzyme is very specific for NAPE and no significant formation of NAGPE is observed. This might explain why Maxapal is tolerant to different dosage.
  • MAXAPAL® has no activity on DGDG, but small activity on MGDG illustrated as MGMG formation was observed.
  • the specificity of MAXAPAL® with regard to hydrolysis of NAPE to NALPE explains why this enzyme has positive synergistic effect in combination with POWERBAKE® 4080 and this also explains why MAXAPAL® cannot easily be overdosed. It is however seen that combination of POWERBAKE® 4080 and MAXAPAL® produces small amount of NAGPE.
  • MAXAPAL® produces sn1-NALPE which is a more preferred substrate for POWERBAKE® 4080 than sn2-NALPE, because POWERBAKE® 4080 is active on the fatty acid at the sn1 position.
  • POWERBAKE® 4080 When POWERBAKE® 4080 is combined with MAXAPAL® it is possible to increase the amount of C16:0_NALPE, and as shown in Table 7 this enzyme combination is also active on galactolipids like DGDG and MGDG in the dough during production of DGMG and MGMG.
  • the positive synergistic effect of POWERBAKE® 4080 and MAXAPAL® on baking performance was explained by the combined effect on galactolipids and NAPE during formation of DGMG, MGMG and 16:0_NALPE.
  • MAXAPAL® is also active on other phospholipids like PC and PE in the dough, and it is known that these components also have more saturated fatty acid at the sn1 position. It is therefore expected that LPC and LPE produced in the dough also has a higher amount of saturated (c16:0) fatty acid.
  • the enzymes were tested according to the procedure for Hard Crust Rolls (Example 1) and specific bread volume and dough and bread properties were evaluated.
  • PLA2 enzymes showed synergistic effect in combination with a sn1 specific enzyme POWERBAKE® 4080. This enzyme also has sn1 specific phospholipase activity. In this test other sn1 specific phospholipases were tested in combination with MAXAPAL®, PLA2 as shown in Table 10.
  • Baking experiments have shown that a combination of a sn1 specific enzyme POWERBAKE® 4080 and sn2 specific enzyme MAXAPAL® has a positive synergistic effect on bread volume when used in baking. It is however known that the amount of phospholipids in flour is rather limited. The aim of this test was to investigate the effect of these enzymes when the dough was enriched with soya lecithin.
  • the baking experiment was conducted according to the procedure for Hard Crust Rolls (Example 1) with enzymes and lecithin as shown in Table 11.
  • Baking test with lecithin combined with enzymes Enzyme dosage based on flour. Baking POWERBAKE ® MAXAPAL ® Bread test 4080 #4313 SOLEC TM B-10, volume no. ppm ppm lecithin % (ccm/g) 1 5.90 2 0.2 6.15 3 500 0.2 6.26 4 30 0.2 5.91 5 30 500 0.2 7.08 6 0.5 6.43 7 500 0.5 6.57 8 30 0.5 6.58 9 30 500 0.5 7.13
  • the baking results from table 12 confirm the synergistic effect by combination of MAXAPAL® PLA2 and a glycolipase POWERBAKE® when these enzymes were tested in an American flour.
  • Sponge and Dough bread making procedure has traditionally been used and is still widely used in the US baking industry.
  • the Sponge and Dough procedure is characterized by two step dough mixing.
  • the sponge is made by mixing flour (70% of total flour), water and yeast, which is fermented for quite a long time (3 hr).
  • the sponge is then mixed with the remaining flour, water, sugar, salt and other ingredients.
  • Normally enzymes are also added to the dough, but in the case of adding two enzymes which will compete for the same substrate, it is possible to add one enzyme at the sponge side, and then add the other enzymes at the dough side.
  • POWERBAKE® 4090 is an enzyme that acts on a polar lipid at the sn1 position. In particular, it is a fungal lipolytic enzyme having PLA1 activity on polar lipids and having SEQ ID NO: 1 disclosed herein. POWERBAKE® 4090 with an enzyme activity of 15,500 TIPU was used.
  • Test Description 1 Control 2 MAXAPAL ®. 250 ppm 3 POWERBAKE ® 4090. 3.23 ppm 4 MAXAPAL ®/POWERBAKE ® 4090. 250 ppm/3.23 ppm
  • MAXAPAL® and POWERBAKE® 4090 separately showed need for higher force compared to control (no MAXAPAL® or POWERBAKE® 4090 added) indicating harder bread.
  • Combining MAXAPAL® and POWERBAKE® 4090 provided the best softness. Same combination also showed the highest synergy in respect to volume (data not presented here).
  • the bread softness results, as shown in FIG. 2 confirm that a positive synergistic effect is obtained by adding a combination of MAXAPAL® and POWERBAKE® 4090. This synergistic effect was observed in the production of white pan bread.
  • Test Description 1 Control 2 MAXAPAL ®. 250 ppm 3 POWERBAKE ® 4090. 3.23 ppm 4 MAXAPAL ®/POWERBAKE ® 4090. 250 ppm/3.23 ppm
  • PLA2 alone showed increased softness compared to the control (no MAXAPAL® or POWERBAKE® 4090 added) at both day 1 and day 3.
  • POWERBAKE®4090 showed softness on level (or lower) than control (no Maxapal® or POWERBAKE® 4090 added).
  • TMS trimethyl silyl derivatives
  • Oven program 1 2 3 4 Oven temperature, ° C. 80 200 240 360 Isothermal, time, min. 2 0 0 10 Temperature rate, ° C./min. 20 10 12
  • Evaporated sample is dissolved in 1.5 ml Heptane:Pyridin, 2:1. 500 ⁇ l sample solution is transferred to a crimp vial, 100 ⁇ l MSTFA (N-Methyl-N-trimethylsilyl-trifluoraceamid) is added and reacted for 15 minutes at 60° C.
  • MSTFA N-Methyl-N-trimethylsilyl-trifluoraceamid
  • CRC08335 A synthetic gene (CRC08335) encoding a fungal phospholipase A2 type-2 was ordered from Generay (http://www.generay.com.cn/english/) as a codon-optimized gene for expression in Trichoderma reesei .
  • the protein sequence of CRC08335 (SEQ ID NO. 4) ( FIG. 5 ) was identified from an internal Myceliophthora thermophile strain and shares 95% identity with its closest homolog in the NCBI database (a secretory phospholipase A2 from Thermothelomyces thermophila ATCC 42464 with the NCBI accession number XP_003666499.1).
  • CRC08335 has an N-terminal signal peptide sequence per prediction by SignalP 4.0 (SignalP 4.0: discriminating signal peptides from transmembrane regions. Thomas Nordahl Petersen, Soren Brunak, Gunnar von Heijne & Henrik Nielsen. Nature Methods, 8:785-786, 2011), suggesting that it is an extracellular enzyme.
  • the synthetic gene of CRC08335 (SEQ ID NO. 5) ( FIG. 6 ), which retains its N-terminal native signal peptide, was cloned into pGXT (the same as the pTTTpyr2 vector described in published PCT Application WO2015/017256, incorporated by reference herein).
  • pGXT the same as the pTTTpyr2 vector described in published PCT Application WO2015/017256, incorporated by reference herein.
  • the pTTT-pyr2 expression vector contained the Trichoderma reesei cbhI-derived promoter (cbhI) and cbhI terminator regions allowing for a strong inducible expression of the gene of interest.
  • nidulans amdS and pyr2 selective markers confer growth of transformants on acetamide as a sole nitrogen source, and the Trichoderma reesei telomere regions allow for non-chromosomal plasmid maintenance in a fungal cell.
  • the resultant plasmid was labelled pZKY512-1.
  • a plasmid of pZKY512-1 is provided in FIG. 7 .
  • the protein sequence of CRC08335 identified from an internal Myceliophthora thermophile strain is set forth as SEQ ID NO. 4.
  • the polypeptide sequence of the predicted signal peptide is MKFLSTALCLASSVLA (SEQ ID NO: 6).
  • the plasmid pZKY512-1 was transformed into a suitable Trichoderma reesei strain (method described in published PCT application WO 05/001036) using protoplast transformation (Te'o et al. (2002) J. Microbiol. Methods 51:393-99). Transformants were selected on a solid medium containing acetamide as the sole source of nitrogen. After 5 days of growth on acetamide plates, transformants were collected and subjected to fermentation in 250 mL shake flasks in defined media containing a mixture of glucose and sophorose.
  • Enzyme specificity for CRC08335 was determined according to ‘Assay for the Determination of phospholipase activity and sn1 and sn2 position specificity on PC (phosphatidylcholine)’.
  • the assay use PC substrate with a tailored FFA (free fatty acid) composition analysing the liberated FFA by GLC analysis. Results are outlined in table 16.
  • NAPE activity of CRC08335 was evaluated by lipid profile analysis of dough from baking trials conducted with and without enzyme addition. Baking application was conducted according to the procedure for Hard Crust Rolls (Example 1).
  • NAPE activity was verified by HPLC analysis of dough lipid.
  • Dough lipids were extracted from fully proofed, freeze dried doughs according to procedure for extraction of lipids form dough. The isolated lipids were analysed by HPLC using a HILIC DIOL column 1.7 ⁇ m, 50*2.1 mm (Fortis Technologies Ltd, UK).
  • the solvents used were solvent A: 96% Acetone, 4% Methanol, 1 mM Ammonium formate and solvent B: 60% Acetone, 34% Methanol, 6% MiliQ water, 1 mM Ammonium formate with the following gradient: 0-20 minutes 100% solvent A to 100% solvent B. 20-30 minutes 100% solvent B, 30-40 minutes 100% solvent A.
  • NAPE and NALPE were quantified using a charged aerosol detector and 1-palmitoyl-sn-glycero-3-phosphoethanolamine-N-linoeoyl (Avanti Polar Lipids, Alabama, USA) as internal standard.
  • the results from the HPLC analysis are shown in table 17.
  • POWERBAKE® 4080 and POWERBAKE® 4090 is a glycolipase with sn1 activity on both galactolipids and phospholipids in dough.
  • NAPE has different fatty acid composition at the sn1 and the sn2 position with typically more saturated fatty acids (C16:0 and C18:0) at the sn1 position.
  • HPLC/MS analysis it was shown that Maxapal contributed to a strong increase in C16:0_NALPE in dough.
  • C16:0_NALPE has a stronger improvement on dough stability than C18:2 NALPE, because NALPE in aquatics system forms different mesomorphic phases depending on the fatty acid composition.
  • MAXAPAL® on its own did however not contribute with much effect on bread volume, but when it was combined with either POWERBAKE® 4080 or POWERBAKE® 4090 a strong synergistic effect is formed. This can be explained by the reaction products C16:0_NALPE, MGMG and DGMG produced by a combination of the two enzymes.
  • MAXAPAL® and POWERBAKE® 4080 when used in combination may compete for the NAPE substrate. This can be mitigated in certain bread making procedures where the dough is mixed in two steps, e.g. in the so called Sponge and Dough procedure.
  • MAXAPAL® or other PLA2 with activity on NAPE
  • POWERBAKE® 4080 or POWERBAKE® 4090 at the dough side for production of DGMG and MGMG.
  • a further advantage of adding MAXAPAL® at the sponge side was that the reaction products (e.g. NALPE) were readily available during dough mixing which contributes to improved dough properties.

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