阅读说明：本技术 使用γ谷氨酰基转肽酶松弛面团 (Relaxing dough using gamma glutamyl transpeptidase ) 是由 L·卡卢 S·M·兰德维克 I·V·马特维 S·T·约根森 K·延森 于 2018-06-20 设计创作，主要内容包括：一种用于改善面团拉伸性的方法,该方法包括a)将γ谷氨酰基转肽酶添加至面粉或至包含面粉的面团中；以及b)制作该面团。这些γ谷氨酰基转肽酶可获得自地衣芽孢杆菌和堀越氏芽孢杆菌。(A method for improving the stretchability of a dough, the method comprising a) adding gamma glutamyl transpeptidase to the flour or to the dough comprising the flour; and b) preparing the dough. These gamma glutamyl transpeptidases can be obtained from bacillus licheniformis and bacillus horikoshii.)

1. A method for improving the stretchability of a dough, the method comprising

a) Adding gamma glutamyl transpeptidase (e.c.2.3.2.2) to flour or to a dough comprising flour; and

b) the dough is prepared.

2. The method of claim 1 wherein a flat dough is produced from the dough.

3. The method according to any of the preceding claims, wherein the dough is made into an edible product selected from the group consisting of: bread, flat bread, crackers, pasta, noodles, laminated baked products, biscuits, french baguette bread, hamburgers, and pizza.

4. The method according to any one of the preceding claims, wherein 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.

5. The process according to any one of the preceding claims, wherein the gamma glutamyl transpeptidase is a bacterial gamma glutamyl transpeptidase.

6. The method according to any one of the preceding claims, wherein the gamma glutamyl transpeptidase has at least 60% identity with SEQ ID No. 1.

7. The process according to any of the preceding claims, wherein the gamma glutamyl transpeptidase is added in an amount in the range of 0.01-100mg of enzyme protein per kg of flour.

8. The method of any one of the preceding claims, wherein glutathione is additionally added.

9. The method according to any of the preceding claims, wherein the dough has a stretchability that is better than the stretchability of a dough prepared under the same conditions but not treated with gamma glutamyl transpeptidase.

10. The method according to any of the preceding claims, wherein the dough further comprises one or more enzymes selected from the group consisting of: amylases, maltogenic amylases, beta-amylases, aminopeptidases, carboxypeptidases, catalases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, glucan 1, 4-alpha-maltotetraohydrolases, glucanases, galactanases, alpha-galactosidases, beta-galactosidases, glucoamylases, glucose oxidases, alpha-glucosidases, beta-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidoglutaminases, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminase, and xylanases.

11. The method according to any one of the preceding claims, wherein the flat bread is selected from the group consisting of: tortillas, pita bread, arabic bread, indian bread, wheat bread, and gluten-free bread.

12. A pre-mix comprising gamma glutamyl transpeptidase (e.c.2.3.2.2) and flour.

13. The premix according to claim 12, wherein the premix further comprises one or more enzymes selected from the group consisting of: amylases, maltogenic amylases, beta-amylases, aminopeptidases, carboxypeptidases, catalases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, glucan 1, 4-alpha-maltotetraohydrolases, glucanases, galactanases, alpha-galactosidases, beta-galactosidases, glucoamylases, glucose oxidases, alpha-glucosidases, beta-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidoglutaminases, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminase, and xylanases.

14. Use of gamma glutamyl transpeptidase (e.c.2.3.2.2) to increase the stretchability of dough.

15. A composition comprising a gamma glutamyl transpeptidase (e.c.2.3.2.2), wherein said gamma glutamyl transpeptidase has at least 60% identity to SEQ ID NO: 1.

Technical Field

The present invention relates to a method of improving the stretchability of a dough (e.g., flat dough) when producing, for example, bread, flat bread, crackers, pizza, pasta, noodles, laminated baked products, biscuits, french baguette and hamburgers.

Background

Currently, in industrial dough making processes, it is known to add dough conditioning additives to the dough to improve parameters such as texture, volume, extensibility and mechanical ability of the dough.

Reducing agents such as glutathione (gluthathione), cysteine, malt, proteases, sorbic acid and non-leavening yeast are known dough improving additives for improving the extensibility of the dough.

In the production of products such as bread, loaves, crackers, pizza, panini, noodles, laminated bakery products, biscuits, baguette and hamburgers, there is still a need to find solutions that improve the stretchability in dough production but have no or only little effect on other dough parameters.

Disclosure of Invention

Surprisingly, the inventors have found that gamma glutamyl transpeptidase (e.c.2.3.2.2) increases the extensibility of dough with no or only little effect on other dough parameters, and we claim therefore:

a method for improving the stretchability of a dough, the method comprising

a) Adding gamma glutamyl transpeptidase to flour or flour containing dough; and

b) the dough is prepared.

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

In one embodiment, the dough is made into an edible product selected from the group consisting of: bread, flat bread, crackers, pizza, pasta, noodles, laminated baked products, biscuits, french baguette bread, and hamburgers.

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.

In one embodiment, the gamma glutamyl transpeptidase is a bacterial gamma glutamyl transpeptidase, in particular a bacillus gamma glutamyl transpeptidase.

In one embodiment, the gamma glutamyl transpeptidase has at least 60% identity with SEQ ID NO: 1.

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

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

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

In one embodiment, the dough further comprises one or more enzymes selected from the group consisting of: amylases, maltogenic amylases, beta-amylases, aminopeptidases, carboxypeptidases, catalases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, glucan 1, 4-alpha-maltotetraohydrolases, glucanases, galactanases, alpha-galactosidases, beta-galactosidases, glucoamylases, glucose oxidases, alpha-glucosidases, beta-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidoglutaminases (peptidoglutaminases), peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminase, and xylanases.

In one embodiment, the flat bread is selected from the group consisting of: tortillas, pita, arabic bread, indian bread (including wheat bread, and gluten-free bread).

In one embodiment, a premix is claimed comprising gamma glutamyl transpeptidase (e.c.2.3.2.2) and flour.

In one embodiment, the premix further comprises one or more enzymes selected from the group consisting of: amylases, maltogenic amylases, beta-amylases, aminopeptidases, carboxypeptidases, catalases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, glucanases, galactanases, alpha-galactosidases, beta-galactosidases, glucoamylases, glucose oxidases, alpha-glucosidases, beta-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidoglutamidases, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminase, and xylanases.

In one embodiment, the use of gamma glutamyl transpeptidase (e.c.2.3.2.2) to increase the stretchability of dough is claimed.

In one embodiment, we claim a composition comprising a gamma glutamyl transpeptidase (e.c.2.3.2.2), wherein the gamma glutamyl transpeptidase has at least 60% identity with SEQ ID NO: 1.

Detailed Description

Definition of

Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For The purposes of The present invention, sequence identity between two amino acid sequences is determined using The Needman-Wunsch algorithm (Needleman-Wunsch) (Needleman and Wunsch,1970, J.Mol.biol. [ J.Mol.Biol ]48:443-453), as implemented in The Nidel (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, trends GeneGenet. [ genetic trends ]16:276-277) (preferably version 5.0.0 or more). The parameters used are the gap opening penalty of 10, the gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The output of the "longest identity" of the nidel label (obtained using the non-reduced (-nobrief) option) was used as a percentage of identity and was calculated as follows:

(same residue x 100)/(alignment Length-total number of vacancies in alignment)

Variants: the term "variant" means a polypeptide having gamma glutamyl transpeptidase activity comprising alterations (i.e., substitutions, insertions, and/or deletions) at one or more (e.g., several) positions. Substitution means the substitution of an amino acid occupying a position with a different amino acid; deletion means the removal of an amino acid occupying a position; and an insertion means the addition of one or more amino acids adjacent to and immediately following the amino acid occupying a position.

Improved properties: when the gamma glutamyl transpeptidase according to the invention is incorporated in an effective amount into flour and/or dough, one or more properties are improved compared to flour and/or dough without the addition of enzyme.

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 the enzyme of the invention.

Sensory quality can be assessed using procedures well established in the baking industry and may include, for example, the use of a trained set of taste testers.

Improved stretchability: the term "improved extensibility of the dough" is defined herein as the property of the dough that can withstand increased extension without breaking.

Increased stretchability is a very important parameter as it means that it is possible, for example, to obtain very thin doughs.

Increased strength: the term "increased strength of the dough" is defined herein as the property of the dough that generally has greater elastic properties and/or requires more work input to mold and shape.

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.

Increased stability of the dough: the term "increased stability of the dough" is defined herein as the property of the dough that is not susceptible to mechanical abuse and therefore retains its shape and volume better and is evaluated by the ratio to the height to width of the cross-section of the bread after normal and/or extended proofing.

Reduced stickiness of the dough: the term "reduced stickiness of a dough" is defined herein as the characteristic of a dough that has, for example, a tendency to have less sticking surfaces in a dough production machine and is evaluated empirically by a skilled test baker or measured using a texture analyzer (e.g., TAXT2) as is known in the art.

Improved mechanical capability: the term "improved mechanical ability of a dough" is defined herein as the property of a dough that is generally less sticky and/or harder and/or more elastic.

Increased volume of dough/baked product the term "increased volume of dough/baked product" is as measured on the volume of dough or the volume of a given piece of bread. The volume can be determined by the rapeseed displacement method, or by a skilled baker, or by using a food volume measuring instrument (Volscan profile) 600 as described in example 2.

Improved crumb structure of the baked product: the term "improved crumb structure of a baked product" is defined herein as follows with respect to the characteristics of the baked product: pulp uniformity, pore wall thickness, and size of each cell on the bread slice.

The crumb structure of the baked product 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, alpyton, wolington, uk).

Improved softness of the baked product: the term "improved softness of the baked product" is in contrast to "firmness" and is defined herein as the characteristic of a baked product that is more easily compressed and is evaluated 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 microsystems Ltd, sally, uk).

Dough composition for making dough

As used herein, "dough" means any dough used to prepare a baked or cooked product.

The dough used to prepare the baked or cooked product according to the present invention may be made from any suitable dough ingredients comprising flour.

As used herein, "flat bread" means dough that typically has a thickness of one millimeter to several centimeters.

The flat dough may be used in accordance with the present invention to make, for example, flat bread, crackers, pizza, pasta, noodles, laminated dough, and cookies.

Flat bread can be made from a simple mixture of flour, water and salt, and then rolled well into a flat dough. Flat bread has a very fast baking time (typically <2 minutes).

Flat bread can be unfermented (i.e., made without yeast) or fermented (e.g., made with yeast).

The flat bread may further include optional ingredients such as olive oil, sesame oil, shortening, and spices.

Examples of flat bread include tortillas, pita bread, arabic bread, and indian bread, including wheat bread and gluten-free bread.

Non-limiting examples of additional flat bread include submenu bread (lavash), baladi bread (baladi), barbarrari bread (barbai), sangka bread (Sangak), tanoor bread (tanoor), tafton bread (taftoon), sago bread (shami), halabi bread (halabi), mufu de bread (mafrood), bur bread (burr), bairi bread (bairuti), pocket bread (pocket break), indian pancake (naan), ebony (phka), hapati, indian throw (paratha), Arabic pita cake (arabpita), libanese (Lebanese), mahofuran bread (maftood), qia pancake (papachachachai), sangka (sanggak), thai cake (royai), tenuasu cake (eastern), tenuan cake (tak), tenuatan (taan), and sanora (sweet), tenuara bread (taan).

The dough used to prepare the flat bread product may be made from any suitable flour source, for example, flours derived from grains, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, potato flour, soybean flour, flours from beans, and combinations thereof.

Any flat bread method can be used to prepare flat bread. The method of preparing flat bread generally involves the following sequential steps: preparing a dough (with an optional proofing step), sheeting or dividing, shaping and/or rolling the dough (rolling), and proofing, which steps are well known in the art.

The flat dough according to the invention can also be used for making pizza. Pizza is typically a yeast flat bread covered with, for example, ketchup and cheese, and baked in an oven.

The flat dough according to the present invention may also be used to make crackers. Crackers are baked food products made from flat dough. Seasonings or flavorings (e.g., salt, herbs, seeds, and/or cheese) can be added to or sprayed on the dough prior to baking, as is known in the art. The soda biscuits have various shapes and sizes-round, square, triangular and the like. The biscuit is an old flat bread.

The flat dough according to the invention can also be used for making noodles and spaghetti.

The noodles are made from an unfermented dough that is stretched, extruded or flattened and then cut into one of a variety of shapes. The noodles are usually cooked in boiling water, sometimes with the addition of edible oils and/or salt. They may be fried or deep-fried.

Spaghetti is typically noodle noodles made from unfermented dough of hard wheat flour mixed with water and/or eggs and formed into a sheet or various shapes and then cooked by boiling. Spaghetti can also be made from flours from other cereals (cereal) or grains (grain).

The flat dough according to the present invention may also be used for preparing laminated baked products.

Laminated dough is a culinary preparation consisting of a number of thin layers of butter separated dough, produced by repeated folding and rolling (culinary preparation). Such doughs may contain multiple layers, i.e., more than 10 layers. During baking, the moisture in the butter evaporates and expands, causing the dough to expand and separate, while the lipids in the butter essentially fry the dough apart, resulting in a light chip product. Examples of laminated doughs include breakfast croissants and other pastries such as Danish pastries (Danish dough), Flaky dough (Flaky dough) and Puff dough (Puff dough).

The flat dough according to the present invention may also be used to make biscuits.

The dough according to the present invention may also be used for the production of any baked or cooked product, in particular bread, flat bread, crackers, pizza, pasta, noodles, laminated baked products, biscuits, french baguette and hamburgers.

The dough according to the present invention may also comprise other conventional dough relaxing ingredients such as glutathione, proteases, malt, sorbic acid, L-cysteine and/or yeast extract.

There may be a synergy between gamma glutamyl transpeptidase and glutathione.

There may be a synergy between gamma glutamyl transpeptidase and protease.

There may be a synergy between gamma glutamyl transpeptidase and malt.

There may be a synergy between gamma glutamyl transpeptidase and sorbic acid.

There may be a synergy between gamma glutamyl transpeptidase and L-cysteine.

There may be a synergy between gamma glutamyl transpeptidase and yeast extract.

The dough according to the present invention may also comprise one or more emulsifiers. Emulsifiers may be used to improve the stretchability of the dough. Examples of suitable emulsifiers are mono-or diglycerides, polyoxyethylene stearate, diacetyl tartaric acid esters of monoglycerides, sugar esters of fatty acids, propylene glycol esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, lecithin or phospholipids, or ethoxylated monoglycerides. Specific emulsifiers include monoglycerides, diacetyl tartaric acid ester of monoglycerides (DATEM) and Sodium Stearoyl Lactylate (SSL).

Other conventional ingredients that may be added to the dough include proteins such as milk powder, gluten, and soy; eggs (whole egg, yolk or white); oxidizing agents, such as ascorbic acid, potassium bromide, potassium iodide, azodicarbonamide (ADA), ammonium persulfate, or potassium persulfate; sugars such as sucrose, dextrose, and the like; salts, for example sodium chloride, calcium acetate, sodium or calcium sulphate, diluents (such as silica), starches of different origin. Other conventional ingredients also include hydrocolloids such as CMC, guar gum, xanthan gum, locust bean gum, and the like. Modified starches may also be used.

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

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

In addition to preparing fresh flat dough or fresh flat dough products, the present invention also relates to methods for preparing frozen flat dough or frozen flat dough products.

The invention is particularly useful for preparing flat dough and products obtained from flat dough in an industrial process, wherein the products are prepared mechanically using automated or semi-automated equipment.

Enzyme

Gamma glutamyl transpeptidase

Gamma glutamyl transpeptidase (e.c.2.3.2.2) plays an important role in glutathione metabolism, where it catalyzes the transfer of gamma glutamyl groups from gamma glutamyl compounds to amino acids, peptide receptors or water (Tate and Meister,1981, mol. cell. biochem. [ molecular and cellular biochemistry ]39: 357-. For example, gamma glutamyl transpeptidase catalyzes the hydrolysis of glutathione to produce glutamic acid, and transfers the gamma glutamyl group of glutathione to an amino acid.

Gamma glutamyl transpeptidases have been reported, for example, from Bacillus subtilis (JP 4281787), Bacillus natto (JP 2065777), and Bacillus agaricus (Bacillus agaradhaerens) (WO 02/077009).

Preferred gamma glutamyl transpeptidases according to the invention are bacterial gamma glutamyl transpeptidases; in particular, bacillus gamma glutamyl transpeptidase; in particular, Bacillus licheniformis (Bacillus licheniformis) or Bacillus horikoshii (Bacillus horikoshii) gamma glutamyl transpeptidase.

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.

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

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

these amino acid changes may 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; typically a small deletion of 1-30 amino acids; small amino-terminal or carboxy-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by altering the net charge or another function (e.g., a polyhistidine segment, an epitope, or a binding domain).

Examples of conservative substitutions are within the following groups: 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 which do not generally alter specific activity are known in The art and are described, for example, by H.Neurath and R.L.Hill,1979, in The Proteins, Academic Press, N.Y.. 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.

Gamma glutamyl transpeptidase may typically be added in an effective amount, e.g.in the range of from 0.01-100mg enzyme protein per kg flour, e.g.0.1-50 mg enzyme protein per kg flour, e.g.0.5-50 mg enzyme protein per kg flour, e.g.1-50 mg enzyme protein per kg flour.

Additional enzymes

Optionally, one or more additional enzymes (such as amylases, maltogenic amylases, beta amylases, aminopeptidases, carboxypeptidases, catalases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, glucan 1, 4-alpha-maltotetraohydrolases, glucanases, galactanases, alpha-galactosidases, beta-galactosidases, glucoamylases, glucose oxidases, alpha-glucosidases, beta-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidoglutaminases, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminase, and xylanases) may be used with the enzyme compositions according to the invention.

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

Suitable commercial alpha-amylase compositions include, for example, BAKEZYME P300 (available from DSM), FUNGAMYL 2500BG, FUNGAMYL 4000BG, FUNGAMYL 800L, FUNGAMYL ULTRA BG, and FUNGAMYLLULTRA SG (available from Novozymes A/S)).

The maltose alpha-amylase (EC 3.2.1.133) may be derived from Bacillus. The maltose alpha-amylase from Bacillus stearothermophilus strain NCIB 11837 is available from Novoxin under the trade nameThe following are commercially available.

The maltose alpha-amylase may also be a variant of a maltose alpha-amylase from Bacillus stearothermophilus, e.g. as disclosed in WO 1999/043794, WO 2006/032281, or WO 2008/148845, e.g.

3D。

The anti-aging amylase for use in the present invention may also be an amylase (glucan 1, 4-alpha-maltotetraohydrolase (ec3.2.1.60)), from Pseudomonas saccharophila (Pseudomonas saccharophila) or variants thereof, such as any of the amylases disclosed in WO 1999/050399, WO 2004/111217 or WO 2005/003339.

Glucoamylases useful in the present invention include Aspergillus niger G1 or G2 glucoamylase (Boel et al (1984), EMBOJ. [ journal of the European society of molecular biology ]]3(5), pages 1097-1102), Aspergillus awamori glucoamylase as disclosed in WO 84/02921, or Aspergillus oryzae glucoamylase (agric. biol. chem. [ agricultural, biological and chemical)](1991) 55(4), pages 941-949). Suitable commercial glucoamylases are available from Novitin Inc

The glucose oxidase may be a fungal glucose oxidase, specifically an Aspergillus niger glucose oxidase (e.g. Aspergillus niger glucose oxidase)

Can be obtained fromWiki corporation).

The xylanase may be of microbial origin, e.g. derived from a bacterium or fungus, such as a strain of aspergillus, in particular aspergillus aculeatus, aspergillus niger, aspergillus awamori or aspergillus tubingensis, or derived from a strain of trichoderma, e.g. trichoderma reesei, or derived from a strain of humicola, e.g. humicola insolens.

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

The protease may be from the genus Bacillus, e.g., Bacillus amyloliquefaciens. Suitable proteases may be obtained from

The phospholipase may have phospholipase a1, a2, B, C, D or lysophospholipase activity; it may or may not have lipase activity. It may be of animal origin, for example from pancreas, snake venom or bee venom, or it may be of microbial origin, for example from filamentous fungi, yeasts or bacteria, for example of the genus aspergillus or fusarium, for example aspergillus niger, aspergillus oryzae or fusarium oxysporum. Preferred lipases/phospholipases from Fusarium oxysporum are disclosed in WO 98/26057. Furthermore, the variants described in WO00/32758 may be used.

Suitable phospholipase compositions are LIPOPAN F and LIPOPAN XTRA (available from Novoxin), or PANAMORE GOLDEN and PANAMORE SPRING (available from DSM).

Enzyme treatment

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.

The gamma glutamyl transpeptidase can be added to the flour or dough in any suitable form, such as for example in liquid form (in particular a stabilized liquid), or it can be added to the flour or dough as a substantially dry powder or granules, thus 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.

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

Premix compound

It is generally advantageous to provide a mixture of one or more enzymes used in the process of the present invention with other ingredients for improving the properties of the dough product. These are commonly referred to in the art as "premixes," which typically comprise flour.

Thus, in a further aspect, the present invention relates to a premix for improving the quality of a dough (for preparing a flat bread product or flat bread products), the premix comprising gamma glutamyl transpeptidase and one or more dough ingredients, especially flours, e.g. flours from cereals, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour and any combination thereof.

The premix further comprises one or more enzymes selected from the group consisting of: amylases, maltogenic amylases, beta-amylases, aminopeptidases, carboxypeptidases, catalases, cellulolytic enzymes, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, glucan 1, 4-alpha-maltotetraohydrolases, glucanases, galactanases, alpha-galactosidases, beta-galactosidases, glucoamylases, glucose oxidases, alpha-glucosidases, beta-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidoglutaminases, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminase, and xylanases.

In another embodiment, the invention relates to a premix comprising the gamma glutamyl transpeptidase of the invention and flour, e.g., flour from cereals, such as 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.

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

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since 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 present invention.

Various references are cited herein, the disclosure of which is incorporated by reference in its entirety. The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

Examples of the invention

Example 1

Gamma Glutamyl Transpeptidase (GGT) expression (SEQ ID NO:1)

The Gamma Glutamyl Transpeptidase (GGT) gene was identified in Bacillus licheniformis ((ATCC PTA-7992) encoding a GGT protein (SEQ ID NO: 1).

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 a Ribosome Binding Site (RBS) from the B.clausii (Bacillus clausii) alkaline protease gene before insertion of the GGT signal peptide.

The forward primer (SEQ ID NO:3) incorporates EcoRI and SacI sites before the alkaline protease RBS before GGT initiation.

The downstream primer (SEQ ID NO:4) incorporates MluI and BamHI after the GGT stop codon.

SEQ ID NO:3：

5'-GACTGAATTCGAGCTCTATAAAAATGAGGAGGGAACCGAATGAGACGGTTAGCTTTCTTAG-3'

SEQ ID NO:4：

5'-GACTGGATCCACGCGTTACTATTTAGCCGATGTCTTAATGT-3'

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 where the annealing temperature was gradually lowered from 62 ℃ to 52 ℃ (1 ℃ reduction per step) and then held constant at 57 ℃ for 20 cycles.

A PCR fragment of approximately 1.8 kb was obtained, digested with SacI + MluI, and cloned into a 3.3kb SacI-MluI vector fragment from pSJ6814 (described in EP 1766002B 1).

The ligation mixture was transformed into E.coli laboratory strains (selection for ampicillin resistance) by electroporation, and transformants with the correct DNA sequence of the PCR amplified segment were retained.

The 2.35 kb EcoRI-MluI fragment containing the cryIIIA _ sta-ggt construct was excised from the transformants and ligated to the 4.75kb MluI-EcoRI fragment of pSJ6869 (described in US 20140106457).

The ligation mixture was transformed into a laboratory strain of Bacillus subtilis, erythromycin (2. mu.g/ml) resistance was selected at 30 ℃ and the correct transformants were retained.

The correct transformants were transformed into the Bacillus subtilis strain conjugated donor PP289-5(US 6066473) to produce a strain which served as the donor conjugated to the Bacillus licheniformis host strain PP1897-3(US 8431382).

The tetracycline sensitive trans-adapter was isolated and after plasmid integration at 50 ℃ colonies were selected by ErmR, isolating colonies with a very weak or missing amylase phenotype. These integrants were propagated at 30 ℃ to allow plasmid replication and loss and to obtain amylase-negative, erythromycin-sensitive strains.

Such Bacillus licheniformis strains as known in the art of growth, and as known in the art of recovery of GGT (SEQ ID NO: 1).

Example 2

Gamma glutamyl transpeptidase in baking (SEQ I)D NO:1)

Bread was prepared using the direct dough leavening (straight dough) procedure according to the following recipe and process conditions. All chemicals applied were food grade. Fungamyl 2500BG (2500FAU/g) was obtained from Novoxil.

Gamma glutamyl transpeptidase (GGT-SEQ ID NO:1) was prepared as described in example 1.

Table 1: dough formulation

The procedure is as follows:

all ingredients were weighed out. Salt, sucrose, yeast, ascorbic acid, calcium propionate, and enzymes are added to the mixing bowl. Tap water was weighed out and temperature adjusted with ice (to about 9-10 ℃ to reach a dough temperature of 27 ℃ after mixing) and added to the mixing bowl. 2500g of flour (2000g of Kolibri and 500g of Victory) was added to the mixing bowl and a Spiral mixer (Spiral mixer) (DIOSNA Dierks) was used&

GmbH, germany) all ingredients were mixed at 63rpm for 3min and 90rpm 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 chapter-manual dough evaluation).

The dough was divided into 450g portions, rounded by hand, and then left to stand at room temperature for 15min and covered with plastic. The resting dough pieces were formed into bread in a tablet press (MO671 MPB-001, Glimek company, sweden) and transferred into greased 1400ml pans (top 230x 115x 68 mm). The bread was proofed at 32 deg.C and 86% humidity for 60 min. The proofed bread was baked with steam at 225 ℃ for 35min in a cabinet oven (Piccolo, wahetel, germany). The bread was removed from the pan and allowed to cool to room temperature. The volume of the bread is determined as described in the volume determination.

Table 2: manual dough evaluation

Dough properties were evaluated approximately 5min after mixing. Dough properties were evaluated using a scale between 0 and 10 and relative to a control without added GGT. A control value of 5 is given. Detailed information about the definition, evaluation and scaling is found in the table below.

And (3) volume determination:

the specific volume was measured using a food volumeter 600 (Stable microsystems, uk) running on food volumeter software. Each bread was embedded in the machine. The weight of each bread was automatically determined by the built-in balance of the Volscan instrument. The volume of each bread was calculated from the 3D image created by the instrument, while each bread was rotated at 1.5 revolutions per second, scanning with the laser beam at 3mm vertical steps per revolution. The specific volume of each bread was calculated according to the following formula:

specific volume (ml/g) ═ volume (ml)/weight (g)

The reported values are the average of 2 breads from the same dough.

Table 3: results

Conclusion

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

Example 3

Gamma glutamyl transpeptidase (SEQ ID NO:2)

The Gamma Glutamyl Transpeptidase (GGT) gene was identified in Bacillus horikoshii strain.

Horikoshi's bacillus strain was found in New Zealand, and the registration date was 5/15 in 1982.

Horikoshi's bacillus gamma glutamyl transpeptidase (SEQ ID NO:2) has the following mature protein sequence:

as known in the art, SEQ ID NO 2 is expressed as an extracellular protein in Bacillus subtilis.

SEQ ID NO 2 shows 68% sequence identity with SEQ ID NO 1.

Example 4

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

The dough was prepared using the direct dough leavening (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.

Table 4: the dough formula comprises:

the procedure is as follows:

all ingredients were weighed out. Stock solutions containing salt, sucrose and ascorbic acid were prepared in tap water and stored for use in ice until use. A yeast stock solution was further prepared in tap water. Weighing tap water; the temperature was adjusted (to achieve a dough temperature of 26 ℃ after mixing) and then added to the mixing bowl.

100g flour (80g Kolibri and 20g Victory), salt/sugar/ascorbic acid stock solution, GGT and yeast solution were added to the mixing bowl and mixed for 5min using a 100g mixer ((National manufacturing Co., National MFG Co., Nebras, model 100) 200A.) the mixed dough was removed from the mixing bowl, rounded to form a ball and the temperature was recorded.

Manual dough evaluation:

dough properties were evaluated approximately 2min after mixing.

Dough properties were evaluated using a scale between 0 and 10 and relative to a control without added GGT. Controls were run in triplicate and values given as 5.

Softness and elasticity were first assessed and the dough ball was then cut in half using a sharp knife. The tack was measured on fresh incisions. The stretchability of each dough ball was evaluated twice (2 and a half pieces). Further details regarding definitions, evaluations and scales are found in table 5 below.

Table 5: dough evaluation parameters

Table 6: results

Conclusion

Both SEQ ID No.1 and SEQ ID No.2 show a significant increase in dough stretchability, while no or little effect on other dough properties is observed.

Sequence listing

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