Relaxing dough using gamma-glutamyl transpeptidase

Abstract

A method for improving the extensibility of dough, the method comprising a) adding a gamma glutamyl transpeptidase to flour or to a dough comprising flour; and b) making the dough. The gamma glutamyl transpeptidases can be obtained from Bacillus licheniformis and Bacillus horikoshii.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a method for improving the stretchability of dough, such as flat dough, when producing, for example, bread, flatbreads, crackers, pizza, pasta, noodles, laminated bakery products, biscuits, baguettes and hamburgers. Background Art

[0004] Currently, in industrial dough making processes, it is known to add dough improving additives to the dough in order to improve parameters such as texture, volume, extensibility and mechanical capabilities of the dough.

[0005] Reducing agents such as gluthathione, cysteine, malt, protease, sorbic acid and non-fermenting yeast are known dough improving additives for improving the extensibility of dough.

[0006] When making products such as bread, flatbreads, crackers, pizza, pasta, noodles, laminated bakery products, cookies, baguettes and hamburgers, there is still a need to find solutions to improve the extensibility in dough production with no or only little effect on other dough parameters. Summary of the invention

[0007] Surprisingly, the inventors have found that gamma glutamyl transpeptidase (EC 2.3.2.2) increases dough extensibility with no or only little effect on other dough parameters, and therefore we claim:

[0008] A method for improving dough extensibility, the method comprising

[0009] a) adding gamma glutamyl transpeptidase to flour or a dough comprising flour; and

[0010] b) making the dough.

[0011] In one embodiment, a flat dough is produced from the dough.

[0012] In one embodiment, the dough is formed into an edible product selected from the group consisting of bread, flatbreads, crackers, pizza, pasta, noodles, laminated bakery products, cookies, baguettes, and hamburgers.

[0013] In one embodiment, the flour is selected from the group consisting of wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum flour, and combinations thereof.

[0014] In one embodiment the gamma glutamyl transpeptidase is a bacterial gamma glutamyl transpeptidase, particularly a Bacillus gamma glutamyl transpeptidase.

[0015] In one embodiment, the gamma glutamyl transpeptidase is at least 60% identical to SEQ ID NO:1.

[0016] In one embodiment, the gamma glutamyl transpeptidase is added in an amount of 0.01-100 mg enzyme protein / kg flour.

[0017] In one embodiment, additional glutathione is added to the dough.

[0018] In one embodiment, the dough has extensibility that is better than the extensibility of a dough prepared under the same conditions but not treated with gamma glutamyl transpeptidase.

[0019] In one embodiment, the dough further comprises one or more enzymes selected from the group consisting of amylase, maltogenic amylase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzyme, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-α-maltotetraosyl hydrolase, glucanase, galactanase, α-galactosidase, β-galactosidase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase and xylanase.

[0020] In one embodiment, the flatbread is selected from the group consisting of tortillas, pita bread, Arabic bread, Indian flatbreads (including wheat-based flatbreads, and gluten-free flatbreads).

[0021] In one embodiment, a premix is ​​claimed, the premix comprising gamma glutamyl transpeptidase (EC 2.3.2.2) and flour.

[0022] In one embodiment, the premix further comprises one or more enzymes selected from the group consisting of amylases, maltogenic amylases, β-amylases, aminopeptidases, carboxypeptidases, catalases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, glucanases, galactanases, α-galactosidases, β-galactosidases, glucoamylases, glucose oxidases, α-glucosidases, β-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidylglutaminases, peroxidases, phospholipases, phytases, polyphenol oxidases, proteolytic enzymes, ribonucleases, transglutaminases, and xylanases.

[0023] In one embodiment, use of a gamma glutamyl transpeptidase (EC 2.3.2.2) for increasing the extensibility of dough is claimed.

[0024] In one embodiment, we claim a composition comprising a gamma glutamyl transpeptidase (EC 2.3.2.2), wherein the gamma glutamyl transpeptidase has at least 60% identity to SEQ ID NO:1. DETAILED DESCRIPTION

[0025] definition

[0026] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0027] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48: 443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite (The European Molecular Biology Open Software Suite), Rice et al., 2000, TrendsGenet. [Genetics Trend] 16: 276-277) (preferred 5.0.0 version or updated version). The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output (obtained using non-simplified (-nobrief) option) of the "longest identity" marked by Needle is used as the identity percentage and is calculated as follows:

[0028] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0029] Variant: The term "variant" means a polypeptide having gamma-glutamyl transpeptidase activity comprising an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding one or more amino acids adjacent to and immediately following the amino acid occupying a position.

[0030] Improved properties: When a gamma glutamyl transpeptidase according to the present invention is incorporated into flour and / or dough in an effective amount, one or more properties are improved compared to flour and / or dough without added enzyme.

[0031] The improved properties can be determined by comparing dough and / or baked products prepared according to the methods described below, with and without the addition of an enzyme of the invention.

[0032] Sensory quality may be assessed using procedures that are well established in the baking industry, and may include, for example, the use of a panel of trained taste testers.

[0033] Improved stretchability: The term "improved stretchability of the dough" is defined herein as the property of the dough that it can withstand increased stretching without breaking.

[0034] Increased stretchability is a very important parameter since it means that it is possible, for example, to obtain very thin doughs.

[0035] Increased strength: The term "increased strength of the dough" is defined herein as a property of the dough that generally has a greater elastic property and / or requires more work input to be molded and shaped.

[0036] Increased elasticity: The term "increased elasticity of the dough" is defined herein as the property of a dough that has a higher tendency to recover its original shape after being subjected to a certain physical stress.

[0037] Increased stability of the dough: The term "increased stability of the dough" is defined herein as the property of a dough that is less susceptible to mechanical abuse, therefore better retaining its shape and volume, and is assessed by the height:width ratio of a cross-section of the bread after normal and / or extended proofing.

[0038] Reduced stickiness of dough: The term "reduced stickiness of dough" is defined herein as a property of dough that has less tendency to stick to surfaces, such as in a dough production machine, and is assessed empirically by a skilled test baker or measured using a texture analyzer as known in the art (e.g., TAXT2).

[0039] Improved mechanical capabilities: The term "improved mechanical capabilities of the dough" is defined herein as properties of a dough that is generally less sticky and / or harder and / or more elastic.

[0040] Increased volume of dough / baked product: The term "increased volume of dough / baked product" is a measurement of the volume of dough or a given loaf of bread. The volume can be determined by the rapeseed displacement method, or by a skilled baker, or by using a food volume meter (Volscan profiler) 600 as described in Example 2.

[0041] Improved crumb structure of baked products: The term "improved crumb structure of baked products" is defined herein as the following properties about baked products: crumb uniformity, cell wall thickness, and size of individual cell cavities on a bread slice.

[0042] The crumb structure of baked products is typically assessed visually by the baker or by digital image analysis as known in the art (e.g., C-cell, Calibre Control International Ltd, Appleton, Warrington, UK).

[0043] Improved softness of baked products: The term "improved softness of baked products" is the opposite of "hardness" and is defined herein as the property of a baked product that is more easily compressed and is assessed empirically by a skilled test baker or measured using a texture analyzer as known in the art (e.g., TAXT2 or TA-XT Plus from Stable Micro Systems Ltd, Surrey, UK).

[0044] Dough composition for making dough

[0045] As used herein, "dough" means any dough used to prepare baked or culinary products.

[0046] According to the present invention, the dough used to prepare the baked or culinary product may be made from any suitable dough ingredients comprising flour.

[0047] As used herein, "flatbread" means a dough that typically has a thickness of one millimeter to several centimeters.

[0048] According to the present invention, the flat dough can be used to make, for example, flat breads, crackers, pizza, pasta, noodles, laminated dough and biscuits.

[0049] Flatbreads can be made from a simple mixture of flour, water and salt, and then well rolled into a flat dough. Flatbreads have a very fast baking time (usually <2 minutes).

[0050] Flatbreads can be unleavened (ie, made without yeast) or leavened (ie, made with yeast).

[0051] The flatbread may further include optional ingredients such as olive oil, sesame oil, shortening, and spices.

[0052] Examples of flatbreads include tortillas, pita bread, Arabic bread, and Indian flatbreads, including wheat-based flatbreads and gluten-free flatbreads.

[0053] Additional non-limiting examples of flatbreads include lavash, baladi, barbari, Sangak, tanoor, taftoon, shami, halabi, mafrood, burr, bairuti, pocket bread, naan, phulka, hapati, paratha, Arabic pita, Lebanese, mafrood, chapati, Sangak, roti, taboon, shrak, mashrouh, nasir, tannoor, lavash, and taftan.

[0054] The dough used to prepare the flatbread product can be made from any suitable flour source, such as flour derived from cereals, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, potato flour, soy flour, flour from legumes, and combinations thereof.

[0055] Any flatbread method may be used to prepare the flatbreads. Methods for preparing flatbreads generally involve the following sequential steps: making a dough (with an optional proofing step), sheeting or dividing the dough, shaping and / or rolling, and proofing, which steps are well known in the art.

[0056] The flat dough according to the invention can also be used to make pizza. Pizza is a typical yeast flat bread, covered with, for example, tomato sauce and cheese, and baked in an oven.

[0057] The flat dough according to the present invention can also be used to make crackers. Crackers are baked food products made from flat dough. As known in the art, condiments or seasonings (e.g., salt, herbs, seeds, and / or cheese) can be added to or sprinkled on the dough before baking. Crackers come in many shapes and sizes - round, square, triangular, etc. Crackers are an ancient flat bread.

[0058] The flat dough according to the invention can also be used to make noodles and pasta.

[0059] Noodles are made from unleavened dough that is stretched, squeezed or flattened and then cut into one of a variety of shapes. Noodles are usually cooked in boiling water, sometimes with the addition of cooking oil and / or salt. They can be pan-fried or deep-fried.

[0060] Pasta is generally noodles made from an unleavened dough of durum wheat flour mixed with water and / or eggs and formed into sheets or various shapes and then cooked by boiling. Pasta can also be made from flours from other cereals or grains.

[0061] The flat dough according to the invention can also be used to prepare laminated bakery products.

[0062] Laminated dough is a culinary preparation consisting of many thin layers of butter-separated dough produced by repeated folding and rolling. Such doughs can contain multiple layers, i.e., more than 10 layers. During baking, the water in the butter evaporates and expands, causing the dough to expand and separate, while the lipids in the butter essentially fry the dough, resulting in a light, flaky product. Examples of laminated doughs include breakfast croissants and other pastries, such as Danish pastry, flaky pastry, and puff pastry.

[0063] The flat dough according to the invention can also be used to make biscuits.

[0064] The dough according to the invention can also be used to produce any baked or cooked product, in particular bread, flatbreads, crackers, pizza, pasta, noodles, laminated baked products, biscuits, baguettes and hamburgers.

[0065] The dough according to the invention may also contain other conventional dough relaxing ingredients, such as glutathione, protease, malt, sorbic acid, L-cysteine ​​and / or yeast extract.

[0066] There may be a synergistic effect between gamma-glutamyl transpeptidase and glutathione.

[0067] There may be a synergistic effect between gamma-glutamyl transpeptidase and protease.

[0068] There may be a synergistic effect between γ-glutamyl transpeptidase and malt.

[0069] There may be a synergistic effect between gamma-glutamyl transpeptidase and sorbic acid.

[0070] There may be a synergistic effect between gamma-glutamyl transpeptidase and L-cysteine.

[0071] There may be a synergistic effect between gamma-glutamyl transpeptidase and yeast extract.

[0072] Dough according to the present invention can also comprise one or more emulsifiers.Emulsifiers can be used to improve the extensibility of dough.The example of suitable emulsifier is monoglyceride or diglyceride, polyoxyethylene stearate, diacetyl tartaric acid ester of monoglyceride, sugar ester of fatty acid, propylene glycol ester of fatty acid, polyglycerol ester of fatty acid, lactic acid ester of monoglyceride, acetate ester of monoglyceride, lecithin or phospholipid or ethoxylated monoglyceride.Specific emulsifier comprises monoglyceride, diacetyl tartaric acid ester (DATEM) of monoglyceride and sodium stearoyl lactylate (SSL).

[0073] Other conventional ingredients that can be added to the dough include proteins, such as milk powder, gluten, and soy; eggs (whole eggs, egg yolks or egg whites); oxidants, such as ascorbic acid, potassium bromide, potassium iodide, azodicarbonamide (ADA), ammonium persulfate or potassium persulfate; sugars, such as sucrose, dextrose, etc.; salts, such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate, diluents (such as silicon dioxide), starch from different sources. Other conventional ingredients also include hydrocolloids, such as CMC, guar gum, xanthan gum, locust bean gum, etc. Modified starches can also be used.

[0074] The dough according to the invention may be a fibrous dough, for example the dough may contain cereals (e.g. whole wheat) and / or be enriched with additional fibre in the form of e.g. cereal brans (e.g. wheat bran). Wheat bran is produced as a by-product of grinding wheat into white flour.

[0075] Typically, fibers are classified into fine fibers, medium fibers, and coarse fibers as is known in the art. Fine fibers are particularly useful in the present invention.

[0076] In addition to the preparation of fresh flat dough or fresh flat dough products, the invention also relates to a method for preparing frozen flat dough or frozen flat dough products.

[0077] The invention is particularly useful for preparing flat doughs and products obtained therefrom in an industrial process, wherein these products are prepared mechanically using automated or semi-automated equipment.

[0078] Enzymes

[0079] γ-glutamyl transpeptidase

[0080] Gamma glutamyl transpeptidase (EC 2.3.2.2) plays an important role in glutathione metabolism, where the enzyme catalyzes the transfer of gamma glutamyl groups from gamma glutamyl compounds to amino acids, peptide acceptors or water (Tate and Meister, 1981, Mol. Cell. Biochem. [Molecular and Cellular Biochemistry] 39: 357-368). For example, gamma glutamyl transpeptidase catalyzes the hydrolysis of glutathione to produce glutamic acid and transfers the gamma glutamyl group of glutathione to amino acids.

[0081] For example, gamma-glutamyl transpeptidases have been reported from Bacillus subtilis (JP 4281787), Bacillus natto (JP 2065777), and Bacillus agaradhaerens (WO 02 / 077009).

[0082] According to the present invention, a preferred gamma glutamyl transpeptidase is a bacterial gamma glutamyl transpeptidase; in particular, a Bacillus gamma glutamyl transpeptidase; in particular, a Bacillus licheniformis or Bacillus horikoshii gamma glutamyl transpeptidase.

[0083] Preferably, the gamma glutamyl transpeptidase is an enzyme having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1.

[0084] In one embodiment, the gamma glutamyl transpeptidase is an enzyme having the amino acid sequence shown in SEQ ID NO: 1 herein:

[0085]

[0086] In another embodiment, the gamma glutamyl transpeptidase is an enzyme having the amino acid sequence shown in SEQ ID NO: 2 herein:

[0087]

[0088] These amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (e.g., a polyhistidine stretch, an antigenic epitope, or a binding domain).

[0089] Examples of conservative substitutions are within the group consisting of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0090] Gamma glutamyl transpeptidase may typically be added in an effective amount such as in the range from 0.01-100 mg enzyme protein / kg flour, such as 0.1-50 mg enzyme protein / kg flour, such as 0.5-50 mg enzyme protein / kg flour, such as 1-50 mg enzyme protein / kg flour.

[0091] Other enzymes

[0092] Optionally, one or more additional enzymes (such as amylases, maltogenic amylases, β-amylases, aminopeptidases, carboxypeptidases, catalases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, glucan 1,4-α-maltotetraosyl hydrolases, glucanases, galactanases, α-galactosidases, β-galactosidases, glucoamylases, glucose oxidases, α-glucosidases, β-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidylglutaminases, peroxidases, phospholipases, phytases, polyphenol oxidases, proteolytic enzymes, ribonucleases, transglutaminases and xylanases) can be used together with the enzyme composition according to the invention.

[0093] The one or more additional enzymes may be of any origin, including mammalian origin, plant origin, and microbial (bacterial, yeast or fungal) origin.

[0094] Suitable commercial alpha-amylase compositions include, for example, BAKEZYME P 300 (available from DSM) and FUNGAMYL 2500BG, FUNGAMYL 4000BG, FUNGAMYL 800L, FUNGAMYL ULTRA BG and FUNGAMYL ULTRA SG (available from Novozymes A / S).

[0095] The maltogenic alpha-amylase (EC 3.2.1.133) may be derived from Bacillus. A maltogenic alpha-amylase from Bacillus stearothermophilus strain NCIB 11837 is available from Novozymes under the trade name Commercially available.

[0096] The maltogenic alpha-amylase may also be a variant of a maltogenic alpha-amylase from Bacillus stearothermophilus, such as disclosed in WO 1999 / 043794, WO 2006 / 032281, or WO 2008 / 148845, e.g. 3D.

[0097] The anti-staling amylase used in the present invention may also be an amylase (glucan 1,4-α-maltotetraose hydrolase (EC 3.2.1.60)) from Pseudomonas saccharophilia or a variant thereof, such as any amylase disclosed in WO 1999 / 050399, WO 2004 / 111217 or WO 2005 / 003339.

[0098] Glucoamylases useful in the present invention include Aspergillus niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. [Journal of the European Molecular Biology Association] 3 (5), pp. 1097-1102), Aspergillus awamori glucoamylase disclosed in WO 84 / 02921, or Aspergillus oryzae glucoamylase (Agric. Biol. Chem. [Agriculture, Biology and Chemistry] (1991), 55 (4), pp. 941-949). Suitable commercial glucoamylases are available from Novozymes.

[0099] The glucose oxidase may be a fungal glucose oxidase, in particular an Aspergillus niger glucose oxidase (eg Available from Novozymes AB).

[0100] The xylanase may be of microbial origin, e.g. derived from bacteria or fungi, e.g. a strain of Aspergillus (particularly Aspergillus aculeatus, Aspergillus niger, Aspergillus awamori or Aspergillus tubingensis), or from a strain of Trichoderma (e.g. Trichoderma reesei), or from a strain of Humicola (e.g. Humicola insolens).

[0101] Suitable commercially available xylanase preparations for use in the present invention include PANZEA BG, PENTOPAN MONO BG, and PENTOPAN 500BG (available from Novozymes), GRINDAMYL POWERBAKE (available from Danisco), and BAKEZYME BXP 5000 and BAKEZYME BXP 5001 (available from DSM).

[0102] The protease may be from Bacillus, such as Bacillus amyloliquefaciens. Suitable proteases may be obtained from

[0103] The phospholipase may have phospholipase A1, A2, B, C, D or lysophospholipase activity; it may or may not have lipase activity. It may have animal origin, for example from pancreas, snake venom or bee venom, or it may have microbial origin, for example from filamentous fungi, yeast or bacteria, for example Aspergillus or Fusarium, for example Aspergillus niger, Aspergillus oryzae or Fusarium oxysporum. Preferred lipases / phospholipases from Fusarium oxysporum are disclosed in WO 98 / 26057. Moreover, the variants described in WO 00 / 32758 may be used.

[0104] Suitable phospholipase compositions are LIPOPAN F and LIPOPAN XTRA (available from Novozymes) or PANAMORE GOLDEN and PANAMORE SPRING (available from DSM).

[0105] Enzyme treatment

[0106] The gamma glutamyl transpeptidase according to the invention is added to the dough ingredients, for example indirectly to the dough by adding it to the flour used to prepare the dough, or directly to the dough itself.

[0107] The gamma glutamyl transpeptidase can be added to the flour or dough in any suitable form, such as for example in liquid form (particularly a stabilized liquid), or it can be added to the flour or dough as a substantially dry powder or granules, therefore, we also claim a granule comprising the gamma glutamyl transpeptidase according to the invention, and a stabilized liquid comprising the gamma glutamyl transpeptidase according to the invention.

[0108] For example, granules may be produced as disclosed in U.S. Pat. No. 4,106,991 and U.S. Pat. No. 4,661,452. Liquid enzyme preparations may be stabilized, for example, by adding sugars or sugar alcohols or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art.

[0109] Premix

[0110] It is often advantageous to provide a mixture of one or more enzymes used in the process of the invention with other ingredients for improving the properties of the dough product. These are often referred to in the art as "premixes" which typically contain flour.

[0111] Thus, in a further aspect, the present invention relates to a premix for improving the quality of dough for the preparation of a flatbread product or a plurality of flatbread products, the premix comprising a gamma-glutamyl transpeptidase and one or more dough ingredients, in particular flour, e.g. flour from cereals, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour and any combination thereof.

[0112] The premix further comprises one or more enzymes selected from the group consisting of amylase, maltogenic amylase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzyme, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-α-maltotetraose hydrolase, glucanase, galactanase, α-galactosidase, β-galactosidase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase and xylanase.

[0113] In another embodiment, the present invention relates to a premix comprising a gamma-glutamyl transpeptidase of the present invention and flour, e.g., flour from a cereal, e.g., wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum flour, and any combination thereof, and one or more additional enzymes as previously described.

[0114] The premix composition may be in liquid form or in dry or substantially dry form.

[0115] The invention described and claimed herein is not limited to the scope of the specific embodiments disclosed herein, as these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments, as well as combinations of one or more of these embodiments, are intended to be included within the scope of the invention.

[0116] Various references are cited herein, the disclosures of which are incorporated herein by reference in their entireties.The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0117] Examples

[0118] Example 1

[0119] Gamma glutamyl transpeptidase (GGT) expression (SEQ ID NO: 1)

[0120] The gamma glutamyl transpeptidase (GGT) gene was identified in Bacillus licheniformis (ATCC PTA-7992) encoding the GGT protein (SEQ ID NO: 1).

[0121] Two oligonucleotide primers (SEQ ID NO: 3 and SEQ ID NO: 4) were designed that allowed PCR amplification of the entire GGT open reading frame (ORF) with the ribosome binding site (RBS) from the alkaline protease gene from Bacillus clausii inserted in front of the GGT signal peptide.

[0122] The upstream primer (SEQ ID NO: 3) incorporated EcoRI and Sad sites before the alkaline protease RBS before the GGT initiation.

[0123] The downstream primer (SEQ ID NO: 4) incorporated MluI and BamHI after the GGT stop codon.

[0124] SEQ ID NO:3:

[0125] 5'-GACTGAATTCGAGCTCTATAAAAATGAGGAGGGAACCGAATGAGACGGTTAGCTTTCTTAG-3'

[0126] SEQ ID NO:4:

[0127] 5'-GACTGGATCCACGCGTTACTATTTAGCCGATGTCTTAATGT-3'

[0128] Chromosomal DNA from Bacillus licheniformis PL1980 (US 8431382) was used as template in a PCR reaction with primers SEQ ID NO:3 and SEQ ID NO:4, wherein the annealing temperature was stepwise decreased from 62°C to 52°C (1°C per step) and then kept constant at 57°C for 20 cycles.

[0129] A PCR fragment of approximately 1.8 kb was obtained, digested with Sad+MluI and cloned into a 3.3 kb Sad-MluI vector fragment from pSJ6814 (described in EP 1766002 B1).

[0130] The ligation mixture was transformed into an E. coli laboratory strain (selecting for ampicillin resistance) by electroporation, and transformants with the correct DNA sequence of the PCR amplified segment were retained.

[0131] The 2.35 kb EcoRI-MluI fragment containing the cryIIIA_stab-ggt construct was excised from the transformant and ligated to the 4.75 kb MluI-EcoRI fragment of pSJ6869 (described in US 20140106457).

[0132] The ligation mixture was transformed into a laboratory strain of Bacillus subtilis, selecting for resistance to erythromycin (2 micrograms / ml) at 30°C, and correct transformants were retained.

[0133] Correct transformants were transformed into Bacillus subtilis conjugation donor strain PP289-5 (US 6066473), thereby generating a strain that was used as a donor for conjugation with Bacillus licheniformis host strain PP1897-3 (US 8431382).

[0134] Tetracycline-sensitive transconjugants were isolated and, after plasmid integration at 50°C, colonies with a very weak or absent amylase phenotype were isolated using ErmR selection. These integrants were propagated at 30°C to allow for plasmid replication and loss, and amylase-negative, erythromycin-sensitive strains were obtained.

[0135] This Bacillus licheniformis strain was grown as known in the art, and GGT (SEQ ID NO: 1) was recovered as known in the art.

[0136] Example 2

[0137] Gamma glutamyl transpeptidase in baking (SEQ ID NO: 1)

[0138] Bread was prepared using a straight dough procedure according to the following recipe and process conditions. All chemicals used were food grade. Fungamyl 2500BG (2500 FAU / g) was available from Novozymes.

[0139] Gamma glutamyl transpeptidase (GGT - SEQ ID NO: 1) was prepared as described in Example 1.

[0140] Table 1: Dough recipe

[0141]

[0142] program:

[0143] Weigh out all ingredients. Add salt, sucrose, yeast, ascorbic acid, calcium propionate and enzymes to a mixing bowl. Weigh out tap water and adjust the temperature with ice (to approximately 9°C-10°C in order to achieve a dough temperature of 27°C after mixing) and add to the mixing bowl. Add 2500g flour (2000g Kolibri and 500g Victory) to the mixing bowl and use a spiral mixer (Diesna Dierks & GmbH, Germany) all ingredients were mixed at 63 rpm for 3 min and at 90 rpm for 7 min. The mixed dough was removed from the mixing bowl and the temperature was controlled and the dough parameters were determined manually (as described in the section - Manual dough evaluation).

[0144] The dough is divided into 450g each, made into a round shape by hand, and then covered with plastic after standing at room temperature for 15min. The dough pieces after standing are formed into bread in a sheeting machine (MO671 MPB-001, Glimek, Sweden) and transferred to a greased 1400ml dish (top 230x 115x 68mm). The bread is proofed at 32°C, 86% humidity for 60min. The proofed bread is baked at 225°C with steam for 35min in a cabinet oven (Piccolo, Wachtel, Germany). The bread is taken out from the dish and allowed to cool to room temperature. The volume of the bread is determined as described in the volume determination.

[0145] Table 2: Manual dough evaluation

[0146] The dough properties were evaluated approximately 5 min after mixing. A scale between 0-10 was used and the dough properties were evaluated relative to a control without added GGT. The control was given a value of 5. Detailed information on the definitions, evaluations and scales is found in the table below.

[0147]

[0148] Volume determination:

[0149] The specific volume was measured using a Food Volume Analyzer 600 (Stable microsystems, UK) running on the Food Volume Analyzer software. Each loaf of bread was inserted into the machine. The weight of each loaf of bread was automatically determined by the built-in balance of the Volscan instrument. The volume of each loaf of bread was calculated from the 3D image created by the instrument while it was rotated at a speed of 1.5 revolutions per second while being scanned with a laser beam at 3 mm vertical steps per revolution. The specific volume of each loaf of bread was calculated according to the following formula:

[0150] Specific volume (ml / g) = volume (ml) / weight (g)

[0151] The reported values ​​are the average of 2 loaves from the same dough.

[0152] Table 3: Results

[0153]

[0154]

[0155] in conclusion

[0156] Surprisingly, the addition of GGT resulted in a significantly more extensible dough, while no effect on other dough properties was observed. No effect on bread volume was observed.

[0157] Example 3

[0158] Gamma glutamyl transpeptidase (SEQ ID NO: 2)

[0159] The gamma-glutamyl transpeptidase (GGT) gene was identified in the Bacillus Horikoshi strain.

[0160] The Bacillus Horikoshi strain was discovered in New Zealand and was registered on May 15, 1982.

[0161] Bacillus Horikoshi gamma-glutamyl transpeptidase (SEQ ID NO: 2) has the following mature protein sequence:

[0162]

[0163] As is known in the art, SEQ ID NO: 2 is expressed as an extracellular protein in Bacillus subtilis.

[0164] SEQ ID NO:2 shows 68% sequence identity with SEQ ID NO:1.

[0165] Example 4

[0166] Gamma glutamyl transpeptidase in baking (SEQ ID NO: 1 and SEQ ID NO: 2)

[0167] The dough was prepared using the straight dough procedure according to the following recipe and process conditions. All chemicals applied were food grade. Gamma glutamyl transpeptidase (GGT) was added at the concentrations described in Table 4.

[0168] Table 4: Dough recipe:

[0169]

[0170]

[0171] program:

[0172] Weigh out all ingredients. A stock solution containing salt, sucrose and ascorbic acid was prepared in tap water and stored in ice until use. A yeast stock solution was further prepared in tap water. Weigh out tap water; adjust the temperature (to achieve a dough temperature of 26°C after mixing) and then add to the mixing bowl.

[0173] 100 g flour (80 g Kolibri and 20 g Victory), salt / sugar / ascorbic acid stock solution, GGT and yeast solution were added to a mixing bowl and mixed for 5 min using a 100 g mixer ((National MFG Co, Nebraska, Model 100-200A). The mixed dough was taken out of the mixing bowl, rounded to form a ball, and the temperature was recorded. The dough parameters were manually determined by hand.

[0174] Manual dough assessment:

[0175] Dough properties were evaluated approximately 2 min after mixing.

[0176] A scale between 0-10 was used and dough properties were assessed relative to a control without GGT addition. The control was performed in triplicate and given a value of 5.

[0177] Softness and elasticity were assessed first, then the dough balls were cut in half using a sharp knife. Stickiness was measured on the fresh cuts. The stretchability of each dough ball was assessed twice (2 halves). Further details on the definitions, assessments and scales are found in Table 5 below.

[0178] Table 5: Dough evaluation parameters

[0179]

[0180] Table 6: Results

[0181]

[0182] in conclusion

[0183] Both SEQ ID No. 1 and SEQ ID No. 2 showed a significant increase in dough extensibility, while no or little effect was observed on other dough properties. Sequence Listing <110> Novozymes A / S <120> Relaxing dough using gamma-glutamyl transpeptidase <130> 14491-WO-PCT <160> 4 <170> PatentIn Version 3.5 <210> 1 <211> 585 <212> PRT <213> Bacillus licheniformis <400> 1 Met Arg Arg Leu Ala Phe Leu Val Val Ala Phe Cys Leu Ala Val Gly 1 5 10 15 Cys Phe Phe Ser Pro Val Ser Lys Ala Glu Gly Val Met Ser Gly Gly 20 25 30 Gly Gly Asp Lys Val Ala Val Gly Lys Asp Gly Met Val Ala Thr Ala 35 40 45 His Pro Leu Ala Ser Lys Ile Gly Ala Glu Val Leu Lys Lys Gly Gly 50 55 60 Asn Ala Ile Asp Ala Ala Ile Ala Ile Gln Tyr Ala Leu Asn Val Thr 65 70 75 80 Glu Pro Met Met Ser Gly Ile Gly Gly Gly Gly Phe Met Met Val Tyr 85 90 95 Asp Gly Glu Thr Lys Glu Thr Ser Ile Ile Asn Ser Arg Glu Arg Ala 100 105 110 Pro Glu Gly Ala Lys Pro Asp Met Phe Leu Asp Gly Asp Gly Lys Val 115 120 125 Ile Pro Phe Ser Glu Arg Ser Arg His Gly Asn Ala Val Gly Val Pro 130 135 140 Gly Thr Leu Lys Gly Leu Glu Ala Ala His Lys Lys Trp Gly Thr Lys 145 150 155 160 Lys Met Glu Asp Leu Ile Ser Pro Ser Ile Lys Leu Ala Glu Glu Gly 165 170 175 Phe Pro Ile Asp Ser Val Leu Ala Asp Ala Ile Lys Asp His Gln Asp 180 185 190 Lys Leu Ser Lys Thr Ala Ala Lys Asp Ile Phe Leu Pro Asp Gly Glu 195 200 205 Pro Leu Lys Glu Gly Asp Ile Leu Val Gln Lys Asp Leu Ala Lys Thr 210 215 220 Phe Lys Leu Ile Arg Lys Glu Gly Ser Lys Ala Phe Tyr Asp Gly Glu 225 230 235 240 Ile Gly Arg Ala Ile Ala Asp Val Val Gln Asp Phe Gly Gly Ser Met 245 250 255 Thr Pro Asp Asp Leu Ser Arg Tyr Glu Val Thr Thr Asp Lys Pro Ile 260 265 270 Trp Gly Glu Tyr His Gly Tyr Asp Ile Ala Ser Met Pro Pro Pro Ser 275 280 285 Ser Gly Gly Val Phe Met Leu Gln Met Leu Lys Leu Ile Asp Asp Phe 290 295 300 His Leu Ser Gln Tyr Asp Pro Lys Ser Phe Glu Lys Tyr His Leu Leu 305 310 315 320 Ala Glu Thr Met His Leu Ser Tyr Ala Asp Arg Ala Ala Tyr Ala Gly 325 330 335 Asp Pro Glu Phe Val Asp Val Pro Leu Arg Gly Leu Leu Asp Pro Asp 340 345 350 Tyr Ile Lys Glu Arg Gln Lys Leu Ile Ser Leu Asp Ser Met Asn Arg 355 360 365 Asp Val Lys Glu Gly Asp Pro Trp Lys Tyr Glu Glu Gly Glu Pro Asn 370 375 380 Tyr Glu Ile Val Pro Gln Pro Glu Asp Lys Thr Ile Gly Glu Thr Thr 385 390 395 400 His Phe Thr Val Thr Asp Gln Trp Gly Asn Val Val Ser Tyr Thr Thr 405 410 415 Thr Ile Glu Gln Leu Phe Gly Thr Gly Ile Leu Val Pro Gly Tyr Gly 420 425 430 Leu Phe Leu Asn Asn Glu Leu Thr Asp Phe Asp Ala Val Pro Gly Gly 435 440 445 Ala Asn Glu Val Gln Pro Asn Lys Arg Pro Leu Ser Ser Met Thr Pro 450 455 460 Thr Ile Val Phe Lys Asp Glu Lys Pro Val Leu Thr Val Gly Ser Pro 465 470 475 480 Gly Gly Thr Thr Ile Ile Ala Ser Val Phe Gln Thr Ile Leu Asn Tyr 485 490 495 Phe Glu Tyr Gly Met Ser Leu Gln Asp Ala Ile Glu Glu Pro Arg Ile 500 505 510 Tyr Thr Asn Ser Leu Thr Ser Tyr Arg Tyr Glu Ser Gly Met Pro Glu 515 520 525 Asp Val Arg Arg Lys Leu Asn Asp Phe Gly His Lys Phe Gly Ala Asn 530 535 540 Pro Val Asp Ile Gly Asn Val Gln Ser Ile Phe Ile Asp Arg Glu Asn 545 550 555 560 Lys Thr Phe Met Gly Val Ala Asp Ser Ser Arg Asn Gly Thr Ala Val 565 570 575 Gly Val Asn Ile Lys Thr Ser Ala Lys 580 585 <210> 2 <211> 558 <212> PRT <213> Bacillus horikoshii <400> 2 Gln Lys Pro Val Lys Gly Ser Asn Glu Val Ala Val Gly Lys Asp Gly 1 5 10 15 Met Val Ser Thr Ser His Pro Leu Ala Ser Glu Ile Gly Ala Asp Ile 20 25 30 Leu Arg Lys Gly Gly Asn Ala Met Asp Ala Ala Ile Ala Val Gln Phe 35 40 45 Ala Leu Asn Val Val Glu Pro Met Met Ser Gly Ile Gly Gly Gly Gly 50 55 60 Phe Met Met Val Tyr Asp Ala Glu Thr Asp Glu Thr Thr Ile Val Asn 65 70 75 80 Ser Arg Glu Arg Ala Pro Ala Gly Ala Thr Pro Asp Met Phe Leu Asn 85 90 95 Pro Asp Gly Ser Leu Ile Pro Phe Gln Glu Arg Val Arg His Gly Asn 100 105 110 Ser Val Gly Val Pro Gly Thr Leu Lys Gly Leu Glu Ala Ala His Glu 115 120 125 Lys Trp Gly Thr Arg Pro Phe Gln Gln Leu Ile Thr Pro Ala Phe Gln 130 135 140 Leu Ala Gln Asn Gly Phe Ser Val Asp Arg Gln Leu Ala Leu Gln Ile 145 150 155 160 Glu Asn Asn Lys Glu Lys Leu Ala Gly Thr Ala Ala Lys Glu Val Phe 165 170 175 Leu Pro Lys Gly Glu Pro Ile Lys Glu Gly Asp Trp Leu Val Gln Lys 180 185 190 Asp Leu Ala Lys Thr Phe Lys Leu Ile Arg Ser His Gly Ser Glu Val 195 200 205 Phe Tyr Asp Gly Glu Ile Gly Glu Ala Leu Ala Ala Thr Val Gln Asp 210 215 220 Phe Gly Gly Ser Met Thr Ile Glu Asp Leu Gln Asn Tyr Gly Val Thr 225 230 235 240 Glu Asp Glu Pro Val Trp Gly Glu Tyr Lys Gly Tyr Asp Ile Ala Ser 245 250 255 Met Pro Pro Pro Ser Ser Gly Gly Leu Phe Leu Leu Gln Met Leu Lys 260 265 270 Thr Leu Asp Ser Phe Asp Ile Ser Gln Tyr Asp Arg Arg Ser Lys Glu 275 280 285 Val Tyr His Leu Leu Ala Glu Ala Met His Leu Ser Tyr Ala Asp Arg 290 295 300 Gly Ala Tyr Ala Gly Asp Pro Glu Phe Val Glu Val Pro Met Ile Gly 305 310 315 320 Leu Leu His Pro Asp Tyr Ile Ala Glu Arg Ser Ala Leu Ile Asp Ile 325 330 335 Asn Ser Val Asn Thr Asn Pro Gln Pro Gly Asp Pro Trp Gln Tyr Glu 340 345 350 Asp Val Asp Pro Asn Tyr Asn Val Ile Lys Gln Asn Asp Glu Lys Asp 355 360 365 Ile Gly Glu Thr Thr His Phe Thr Val Ala Asp Arg Trp Gly Asn Leu 370 375 380 Val Ser Tyr Thr Thr Thr Ile Glu Gln Val Phe Gly Ser Gly Ile Met 385 390 395 400 Val Pro Gly Tyr Gly Phe Met Leu Asn Asn Glu Leu Thr Asp Phe Asp 405 410 415 Ala Arg Pro Gly Gly Ala Asn Glu Val Gln Pro Asn Lys Arg Pro Leu 420 425 430 Ser Ser Met Thr Pro Thr Ile Val Phe Glu Asp Gly Lys Pro Ile Met 435 440 445 Ser Val Gly Ser Pro Gly Gly Pro Thr Ile Ile Thr Ser Val Leu Gln 450 455 460 Val Val Leu Asn Val Met Asp Tyr Glu Met Gly Leu Glu Glu Ala Ile 465 470 475 480 Ala Glu Pro Arg Ile Tyr Thr Asn Thr Ile Asn Ser Tyr Arg Tyr Glu 485 490 495 Asp Gly Ile Ser Ala Glu Val Leu Ser Glu Leu Asn Ala Met Gly His 500 505 510 Arg Phe Pro Ser Asn Ser Glu Leu Ile Gly Asn Val Gln Ser Ile Leu 515 520 525 Ile Asp Tyr Glu Lys Asp Glu Tyr Val Gly Val Ala Asp Ala Arg Arg 530 535 540 Asp Gly Ala Ser Val Gly Tyr Thr Arg Pro Gly Lys Arg Lys 545 550 555 <210> 3 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gactgaattc gagctctata aaaatgagga gggaaccgaa tgagacggtt agctttctta 60 g 61 <210> 4 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 4 gactggatcc acgcgttat atttagccga tgtcttaatg t 41

Claims

1. A method for improving dough extensibility, the method comprising: include: a) adding gamma glutamyl transpeptidase (EC 2.3.2.2) to the flour or to a dough comprising the flour; and b) preparing the dough, wherein the gamma-glutamyl transpeptidase has at least 98% identity to SEQ ID NO: 1, The dough has better extensibility than dough prepared under the same conditions but not treated with gamma-glutamyl transpeptidase. Wherein, the gamma-glutamyl transpeptidase is Bacillus licheniformis gamma-glutamyl transpeptidase.

2. The method according to claim 1, wherein a flat dough is produced from the dough.

3. The method according to claim 1, wherein the dough is formed into an edible product selected from the group consisting of: composition: Bread, noodles, laminated bakery products, biscuits, hamburgers and pizza.

4. The method of claim 3, wherein the bread is a flatbread or a baguette.

5. The method according to claim 1, wherein the flour is selected from the group consisting of composition: Wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum flour and combinations thereof.

6. The method according to claim 1, wherein the gamma-glutamyl transpeptidase is added in an amount ranging from 0.01 to 100 mg of enzyme protein / kg of flour.

7. The method according to claim 1, wherein glutathione is additionally added.

8. The method according to claim 1, wherein the dough further comprises one or more enzymes selected from the group consisting of: composition: Amylases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, esterases, glucan 1,4-α-maltotetraose hydrolases, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, hemicellulolytic enzymes, invertases, oxidases, pectinolytic enzymes, peptidylglutaminases, peroxidases, phytases, proteolytic enzymes and transglutaminases.

9. The method of claim 8, wherein the amylase is a maltogenic amylase, a β-amylase, or a glucoamylase.

10. The method of claim 8, wherein the esterase is a deoxyribonuclease, a ribonuclease, a lipase or a phospholipase.

11. The method according to claim 8, wherein the proteolytic enzyme is an aminopeptidase or a carboxypeptidase.

12. The method according to claim 8, wherein the oxidase is glucose oxidase or polyphenol oxidase.

13. The method of claim 12, wherein the polyphenol oxidase is laccase.

14. The method of claim 8, wherein the hemicellulolytic enzyme is a glucanase, a xylanase, a mannosidase or a galactanase.

15. The method of claim 8, wherein the peroxidase is catalase or haloperoxidase.

16. The method according to claim 4, wherein the flat bread is selected from the group consisting of composition: Tortillas, pita bread, Arabic bread, Indian flatbread, wheat flatbread, and gluten-free flatbread.

17. The method of claim 3, wherein the edible product is a soda cracker.

18. The method of claim 3, wherein the edible product is pasta.

19. Use of gamma-glutamyl transpeptidase (EC 2.3.2.2) for increasing the extensibility of dough, in, The gamma glutamyl transpeptidase has at least 98% identity to SEQ ID NO: 1, wherein the gamma glutamyl transpeptidase is a Bacillus licheniformis gamma glutamyl transpeptidase.

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