Method for preparing fat-reduced bakery product

Abstract

The present invention relates to methods, uses and products for preparing bakery products comprising reduced fat. The method comprises the following steps: a) preparing a dough with one or more lipases or / and one or more proteases, b) moulding and / or tabletting the dough, and c) baking the dough. The baked product is selected from the group consisting of biscuits, cookies, 5, and crackers. The fat of the bakery product is reduced by at least 10 wt.% to 25 wt.% compared to bakery products prepared without the use of the one or more lipases or / and the one or more proteases.

Description

[0001] References to sequence listings

[0002] This application contains a sequence listing in computer readable form. This computer readable form is incorporated herein by reference. Technical Field

[0003] The present invention relates to a method for preparing a baked product. In particular, the present invention relates to a method for preparing a baked product comprising reduced fat. Background Art

[0004] The baking industry is one of the largest sectors in the food processing industry and the biscuit industry is the largest segment in the baking industry due to its nutritional profile, ready-to-eat nature of the product, rich in energy, an excellent source of dietary supplement, easy digestibility and high hygienic properties. Generally, the basic ingredients of biscuits and cookies are flour, oil and sugar. The other ingredients may vary depending on the type of biscuit or cookie and the recipe followed. Most biscuits and cookies are high in fat and calories. Currently, there is a great deal of attention on the contribution of commercial baked biscuits and cookies to the growing obesity epidemic, especially among children. The industry is focusing on providing commercial baked biscuits and cookies with lower calorie content by simply reducing the fat content and / or sugar content in the biscuit or cookie formulation. When the fat content in the formulation is reduced, the baked biscuits and cookies become drier, more crumbly, have other unacceptable characteristics and may have a reduced shelf life.

[0005] Any fat constituent in the dough can play a role in lubricating the dough, extending the shelf life of commercially baked biscuits and cookies, and improving the texture of biscuits and cookies. The shelf life of commercially baked biscuits and cookies is extended primarily by allowing fat to lubricate and spread in the dough to coat the sugar constituent of the dough, thereby preventing air from entering the sugar constituent. However, as the fat content is reduced, the ability of the remaining fat constituent to spread and coat the sugar is also reduced.

[0006] In addition, the cost of different types of oils used in baking (e.g., soybean oil, palm oil, sunflower oil, rapeseed oil, safflower oil, corn oil, or cottonseed oil) is rising for various reasons. By reducing the amount of oil used when baking biscuits and cookies, the overall manufacturing price of biscuits and cookies can be reduced.

[0007] The use of organically derived phospholipids, emulsifiers or modified starches in baking biscuits and cookies is known in the prior art, but these substitutes are used in high amounts, about 3% or more, which is expensive and also affects the texture, mouthfeel and taste of the baked biscuits and cookies.

[0008] Therefore, there is a need to cost-effectively reduce the fat and / or sugar content of baked biscuits and cookies without sacrificing flavor, texture and desired taste. Summary of the invention

[0009] The present invention relates to a method for preparing a baked product.

[0010] In one aspect, the present invention relates to a method for preparing a baked product comprising reduced fat. The method comprises the steps of: a) preparing a dough with one or more lipases and / or one or more proteases, b) molding and / or sheeting the dough, and c) baking the dough. The baked product is selected from the group consisting of biscuits, cookies, laminated biscuit variants and crackers.

[0011] In another aspect, the present invention relates to the use of one or more lipases and / or one or more proteases in the production of bakery products to reduce the fat and / or sugar used in the recipe for making the bakery product.

[0012] In yet another aspect, the present invention relates to a reduced fat baked product obtained by preparing the dough with one or more lipases or / and one or more proteases.

[0013] Sequence Listing Overview

[0014] SEQ ID NO: 1 is the mature amino acid sequence of the 1,3 lipase from Thermomyces lanuginosus.

[0015] SEQ ID NO: 2 is the mature amino acid sequence of a phospholipase from Fusarium oxysporum.

[0016] SEQ ID NO: 3 is the mature amino acid sequence of a phospholipase from Thermomyces lanuginosus.

[0017] SEQ ID NO: 4 is the mature amino acid sequence of a phospholipase from Thermomyces lanuginosus.

[0018] SEQ ID NO: 5 is the mature amino acid sequence of α-amylase from Aspergillus oryzae

[0019] SEQ ID NO: 6 is the mature amino acid sequence of a protease derived from Bacillus amyloliquefaciens

[0020] SEQ ID NO: 7 is the mature amino acid sequence of a xylanase from Thermomyces lanuginosus

[0021] SEQ ID NO: 8 is the mature amino acid sequence of a protease derived from Nocardia violacea

[0022] SEQ ID NO: 9 is the mature amino acid sequence of a protease derived from Bacillus licheniformis

[0023] SEQ ID NO: 10 is the mature amino acid sequence of a protease derived from Fusarium oxysporum

[0024] SEQ ID NO: 11 is the mature amino acid sequence of a protease derived from Aspergillus oryzae

[0025] SEQ ID NO: 12 is the mature amino acid sequence of a protease derived from Aspergillus oryzae BRIEF DESCRIPTION OF THE DRAWINGS

[0026] A more complete understanding of the invention may be obtained by referring to the detailed description and claims when considered in conjunction with the accompanying drawings.

[0027] Figure 1 is a graph showing the effect of low amounts of lipase on the spreading coefficient and height coefficient of cream cookies compared to the effect of higher amounts of phospholipase on the cookies.

[0028] Figure 2 The texture of the cream cookies with 10% less fat in the negative control sample and the lipase-containing test sample is shown.

[0029] Figure 3 The texture of the cream cookies with 20% less fat in the negative control sample and the lipase-containing test sample is shown.

[0030] Figure 4 It is shown that the stacking height of the cream cookies with 25% fat reduction increased slightly for both the positive control sample and the test samples containing lipase and protease.

[0031] Figure 5 Shrinkage and hardness of a laminated biscuit variant with 10% fat reduction in a negative control sample versus a positive control sample and a test sample containing lipase and protease are shown.

[0032] Figure 6 Shown are the stack heights of laminated biscuit variants with 10% reduction in fat for the negative control sample and the test samples containing lipase and protease.

[0033] definition

[0034] The disclosed embodiments relate to methods, uses, and products for preparing baked products comprising reduced fat.

[0035] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the specification and the relevant art, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein. For the sake of brevity and / or clarity, well-known functions or structures may not be described in detail.

[0036] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0037] Throughout this disclosure, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or groups of steps or elements.

[0038] The term "consisting of is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present. The term "consisting essentially of is meant to include any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the listed elements.

[0039] As used herein, the term lipase is a 1,3 specific lipase that acts only on triglycerides.

[0040] As used herein, the term fat reduction refers to the use of a lower amount of fat when preparing the dough for a baked product (eg, biscuits, cookies, laminated biscuit variations, and / or crackers).

[0041] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of "longest identity" using Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453), which is implemented in the Needle program of EMBOSS software package (EMBOSS:TheEuropean Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16:276-277) (preferred 6.6.0 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. In order to make Needle program report the longest identity, non-simplified options must be specified in the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0042] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0043] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of "longest identity" using Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same), which is implemented by the Needle program of EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, the same) (preferred 6.6.0 version or updated version). The parameters used are gap open penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order to make Needle program report the longest identity, non-simplified options must be specified in the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0044] (number of identical deoxyribonucleotides × 100) / (length of alignment – ​​total number of gaps in alignment)

[0045] Although certain aspects of the present disclosure will be described hereinafter with reference to embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the claims. DETAILED DESCRIPTION

[0046] The present invention relates to methods, uses and products for preparing bakery products comprising reduced fat.

[0047] In order to provide reduced fat in baked products (such as but not limited to biscuits, cookies, laminated biscuit variants or crackers), the inventors have found that adding a small amount (parts per million) of one or more lipases when preparing the dough for the baked product and reducing the fat in the dough by 10%-25% by replacing the reduced fat with flour will provide the baked product with the same or improved quality or characteristics of the baked product (e.g., texture, dough properties, size, mouthfeel, balanced flavor / aroma, etc.). The one or more lipases act as emulsifiers, which are added to the dough in small amounts compared to other commercially known emulsifiers that need to be added in higher amounts (3-5% by weight). In addition, the inventors have found that adding a small amount of one or more lipases when preparing the dough for the baked product and reducing the fat in the dough by 10%-25% and 1wt.%-5wt.% of sugar will not impair the quality or characteristics of the baked product. In addition, the inventors have found that adding a small amount (parts per million) of one or more proteases when preparing dough for baked products and reducing the fat in the dough by 10%-25% by replacing the reduced fat with flour provides a baked product that is the same as or improved with some qualities or characteristics of the baked product, such as texture, dough properties, size, mouthfeel, balanced flavor / aroma, etc. In one aspect, the present invention relates to a method for preparing a baked product, the method comprising the steps of: a) preparing dough with one or more lipases or / and one or more proteases; b) molding and / or sheeting the dough; and c) baking the dough. Compared with a baked product prepared without the use of the one or more lipases or / and one or more proteases, the fat of the baked product is reduced by at least 10 weight % (wt.%). In another embodiment, compared with a baked product prepared without the use of the one or more lipases or / and one or more proteases, the fat of the baked product is reduced by at least 20 wt.%. In another embodiment, compared with a baked product prepared without the use of the one or more lipases or / and one or more proteases, the fat of the baked product is reduced by at least 25 wt.%. The baked product is selected from the group consisting of biscuits, cookies, laminated biscuit variants and crackers.

[0048] In an embodiment of the present invention, the step of preparing dough comprises adding one or more sugars, one or more flours, one or more lipases or / and one or more proteases and one or more fats. The dough comprises one or more added sugars of 10wt.% to 30wt.%, preferably one or more added sugars of 15wt.% to 25wt.%, more preferably 20wt.%. In another embodiment, compared with a baked product prepared without using the one or more lipases or / and one or more proteases, the sugar content of the baked product is further reduced. Compared with a baked product prepared without using the one or more lipases or / and one or more proteases, the sugar of the baked product is reduced by 1wt.% to 5wt.%.

[0049] In an embodiment of the present invention, the dough for cookies comprises 40 wt.% to 70 wt.% of one or more added flours, preferably 55 wt.% to 65 wt.% of one or more added flours. The one or more flours are selected from the group consisting of wheat flour, all-purpose flour, whole wheat flour, rice flour, corn flour, millet flour, soy flour, oat flour, pastry flour, bread flour, gluten-free flour, cassava flour and self-raising flour.

[0050] In an embodiment of the present invention, the dough for cookies comprises 0.00004 wt.% to 0.0002 wt.% of one or more added lipases, preferably 0.00006 wt.% to 0.0001 wt.% of one or more added lipases.

[0051] In an embodiment of the invention, the dough comprises 0.4 to 2 milligrams (mg) enzyme protein / kilogram (kg) of flour of one or more added lipases, preferably 0.6 to 1 mg enzyme protein / kg of flour.

[0052] In an embodiment of the present invention, the dough for cookies contains 20 to 100 parts per million (ppm) of one or more added lipases, preferably 25 to 55 ppm of one or more added lipases. The lipase is a wild-type lipase of Thermomyces lanuginosus (formerly known as Humicola lanuginosus). In a preferred embodiment, the lipase is a 1,3 specific lipase that acts only on triglycerides.

[0053] In an embodiment of the present invention, the dough for the laminated biscuit variant comprises 0.00001 wt.% to 0.0006 wt.% of one or more added lipases, preferably 0.000014 wt.% to 0.000016 wt.%.

[0054] In an embodiment of the invention, the dough for the laminated biscuit variant comprises 0.1 to 0.6 milligram (mg) enzyme protein / kilogram (kg) of flour of one or more added lipases, preferably 0.14 to 0.16 mg enzyme protein / kg of flour.

[0055] In an embodiment of the present invention, the dough for the laminated biscuit variant contains 5 to 30 parts per million (ppm) of one or more added lipases.

[0056] In an embodiment of the invention, the one or more lipases are polypeptides selected from the group consisting of:

[0057] (a) a polypeptide having at least 80% sequence identity to SEQ ID NO: 1,

[0058] (b) a polypeptide derived from SEQ ID NO: 1 having 1 to 30 alterations such as substitutions, deletions and / or insertions, for example 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions,

[0059] (c) a polypeptide derived from the polypeptide of (a), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids, and

[0060] (d) a fragment of the polypeptide of (a).

[0061] In another embodiment, the polypeptide has 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%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:1.

[0062] In a preferred embodiment of the present invention, the lipase is a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:1.

[0063] In embodiments, the polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, such as 1-5 amino acids.

[0064] In another aspect, the polypeptide is derived from SEQ ID NO: 1 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 1 by substitution, deletion or addition of one or several amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO: 1 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 1 is up to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The 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 small deletions of 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 changing the net charge or another function (such as a polyhistidine segment, an antigenic epitope or a binding module).

[0065] In an embodiment of the invention, the dough comprises 12 wt.% to 35 wt.% of one or more fats. The one or more fats are selected from the group consisting of palm oil; palm olein, butter, clarified butter, anhydrous milk fat, corn oil, margarine, olive oil, hydrogenated vegetable fat, sunflower oil, rapeseed oil, rice bran oil, lard, soybean oil and eggs. In a preferred embodiment, the fat is palm oil or rapeseed oil.

[0066] In an embodiment, the step of preparing the dough comprises adding:

[0067] i. 10 wt.% to 30 wt.% of one or more sugars,

[0068] ii. 40wt.% to 70wt.% of one or more flours,

[0069] iii. 0.2 wt.% to 2 wt.% of one or more lipases; and

[0070] iv. 12 to 35 wt.% of one or more fats.

[0071] In an embodiment, the step of preparing the dough comprises adding:

[0072] i. 10 wt.% to 30 wt.% of one or more sugars,

[0073] ii. 40wt.% to 70wt.% of one or more flours,

[0074] iii. 20 ppm to 100 ppm of one or more lipases; and

[0075] iv. 12 to 35 wt.% of one or more fats.

[0076] In an embodiment, the step of preparing the dough comprises adding one or more proteases.

[0077] In an embodiment of the present invention, the dough for cookies comprises 0.0.00015 wt.% to 0.003 wt.% of one or more added proteases, preferably 0.00003 wt.% to 0.0006 wt.%.

[0078] In an embodiment of the invention, the dough for cookies comprises 1.5 to 30 milligrams (mg) enzyme protein / kilogram (kg) of flour of one or more added proteases, preferably 0.3 to 6 mg enzyme protein / kg of flour.

[0079] In an embodiment of the present invention, the dough for cookies contains 5 to 100 parts per million (ppm) of one or more added proteases.

[0080] In an embodiment of the present invention, the dough for the laminated biscuit variant comprises 0.00015 wt.% to 0.009 wt.% of one or more added proteases, preferably 0.00003 wt.% to 0.00018 wt.%.

[0081] In an embodiment of the invention, the dough for the laminated biscuit variant comprises 1.5 to 90 milligrams (mg) enzyme protein / kilogram (kg) of flour of one or more added proteases, preferably 0.3 to 18 mg enzyme protein / kg of flour.

[0082] In an embodiment of the present invention, the dough for the laminated biscuit variant contains 5 to 300 parts per million (ppm) of one or more added proteases.

[0083] In an embodiment of the invention, the one or more proteases are derived from Bacillus amyloliquefaciens.

[0084] In an embodiment of the invention, the one or more proteases are polypeptides selected from the group consisting of:

[0085] (a) a polypeptide having at least 80% sequence identity to SEQ ID NO: 6,

[0086] (b) a polypeptide derived from SEQ ID NO: 6 having 1 to 30 alterations such as substitutions, deletions and / or insertions, for example 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions,

[0087] (c) a polypeptide derived from the polypeptide of (a), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids, and

[0088] (d) a fragment of the polypeptide of (a).

[0089] In another embodiment, the polypeptide has 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%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:6.

[0090] In a preferred embodiment of the present invention, the protease is a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:6.

[0091] In embodiments, the polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, such as 1-5 amino acids.

[0092] In another aspect, the polypeptide is derived from SEQ ID NO:6 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:6 by substitution, deletion or addition of one or several amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:6 comprising substitution, deletion and / or insertion at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:6 is up to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The 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 small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by changing the net charge or another function (such as a polyhistidine segment, antigenic epitope or binding module).

[0093] In yet another embodiment, the step of preparing the dough comprises adding one or more lipases and one or more proteases in combination.

[0094] In another embodiment, the step of preparing the dough comprises adding one or more leavening agents. The dough comprises 0.3 wt.% to 3 wt.% of one or more added leavening agents. The leavening agent is selected from the group consisting of sodium bicarbonate, ammonium bicarbonate, sodium acid pyrophosphate (SAPP), sodium phosphate, monocalcium phosphate, sodium aluminum sulfate, potassium hydrogen tartrate, sodium aluminum phosphate, glucono-delta-lactone and adipic acid and combinations thereof.

[0095] In yet another embodiment, the step of preparing the dough further comprises adding other ingredients, such as invert syrup, salted or unsalted butter, lecithin, milk powder, distilled mono- and diglycerides (DMG), salt, liquid glucose, cheese, fresh milk, colorants, flavorings, sodium metabisulfite, sweetened condensed milk, spices and condiments, glyceryl monostearate (GMS), diacetyltartaric acid esters of mono- and diglycerides (DATEM), and / or water.

[0096] In an embodiment, the steps of preparing the dough include creaming, mixing and keeping the dough at an appropriate temperature. While preparing the dough, the one or more flours and the one or more lipases or / and one or more proteases are added during the mixing step. If the one or more lipases or / and one or more proteases are added during mixing, a rest period is not required. The present invention improves dough properties, such as dough pH, ​​dough texture, dough temperature and dough sheeting.

[0097] In an embodiment, the dough is molded in one or more dough molds. In another embodiment, the dough is sheeted in one or more sheeting machines.

[0098] In an embodiment, the dough is cut in one or more cutters after sheeting / lamination.

[0099] In another embodiment, the sheeted dough is laminated and folded one or more times to form a laminated biscuit variation.

[0100] In an embodiment, the dough is baked in an oven. The oven is selected from the group consisting of a tunnel oven, a rotary oven and a layer oven. The present invention improves the size of the baked product (e.g., cookies). The size can be weight, stack height, average length, average width, volume, density, spread coefficient, height coefficient, etc.

[0101] On the other hand, the present invention relates to the purposes of one or more lipases or / and one or more proteases in producing baked products, in order to reduce the use of fat and / or sugar when preparing baked products.When preparing the dough for baked products, one or more characteristics of baked products are kept after reducing fat and / or sugar.These one or more characteristics are selected from the group consisting of mouthfeel, texture, taste, chewiness, spreadability, color-height-length, volume, moisture, intensity, firmness, crispness and shelf life.Compared with the baked products prepared without using these one or more lipases or / and one or more proteases, the fat of the dough of these baked products has reduced by at least 10wt.%.

[0102] In another aspect, the present invention relates to a baked product obtainable by the process defined in the above examples, or using one or more lipases and / or one or more proteases.

[0103] The following numbered paragraphs describe particular embodiments of the present disclosure:

[0104] 1. A method for preparing a baked product comprising reduced fat, the method comprising the following steps:

[0105] a. Prepare the dough by adding at least the following:

[0106] i. one or more sugars,

[0107] ii. one or more flours,

[0108] iii. one or more lipases and / or one or more proteases; and

[0109] iv. one or more fats;

[0110] b. molding the dough in one or more dough molds and / or sheeting the dough in one or more dough sheeting machines;

[0111] c. baking the dough;

[0112] Wherein the baked product has at least 10 wt.% less fat compared to a baked product prepared without the one or more lipases and / or one or more proteases.

[0113] 2. The method for preparing the baked product as described in paragraph 1, wherein the baked product is selected from the group consisting of biscuits, cookies, laminated biscuit variants and crackers.

[0114] 3. The method for preparing the baked product as described in paragraph 1 or 2, wherein the dough comprises 10 wt.% to 30 wt.% of one or more added sugars.

[0115] 4. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the flour is selected from the group consisting of wheat flour, all-purpose flour, whole wheat flour, rice flour, corn flour, millet flour, soy flour, oat flour, pastry flour, bread flour, gluten-free flour, tapioca flour and self-raising flour.

[0116] 5. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough comprises 40 wt.% to 70 wt.% of one or more additional flours.

[0117] 6. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the lipase is a Thermomyces lanuginosus (formerly known as Humicola lanuginosus) wild-type lipase.

[0118] 7. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the one or more lipases are polypeptides selected from the group consisting of:

[0119] (a) a polypeptide having 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 1;

[0120] (b) a polypeptide derived from SEQ ID NO: 1 having 1 to 30 alterations such as substitutions, deletions and / or insertions, for example 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0121] (c) a polypeptide derived from the polypeptide of (a), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids; and

[0122] (d) a fragment of the polypeptide of (a).

[0123] 8. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the one or more lipases is a polypeptide having the sequence of SEQ ID NO: 1.

[0124] 9. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the cookies comprises 0.00001 wt.% to 0.0002 wt.% of one or more added lipases, preferably 0.00006 wt.% to 0.0001 wt.% of one or more added lipases.

[0125] 10. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the laminated biscuit variant comprises 0.00001 wt.% to 0.0006 wt.% of one or more added lipases, preferably 0.000014 wt.% to 0.000016 wt.% of one or more added lipases.

[0126] 11. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the cookies comprises 0.4 to 2 mg enzyme protein / kg flour of one or more added lipases, preferably 0.6 to 1 mg enzyme protein / kg flour.

[0127] 12. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the laminated biscuit variant comprises 0.1 to 0.6 mg enzyme protein / kg flour of one or more added lipases, preferably 0.14 to 0.16 mg enzyme protein / kg flour.

[0128] 13. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough comprises 20 to 100 parts per million (ppm) of one or more added lipases.

[0129] 14. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the laminated biscuit variant comprises 5 to 30 parts per million (ppm) of one or more added lipases.

[0130] 15. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the step of preparing the dough comprises adding one or more proteases.

[0131] 16. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the protease is derived from Bacillus amyloliquefaciens.

[0132] 17. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the one or more proteases are polypeptides selected from the group consisting of:

[0133] (a) a polypeptide having 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%, at least 99%, or 100% sequence identity to SEQ ID NO:6;

[0134] (b) a polypeptide derived from SEQ ID NO: 6 having 1-30 alterations such as substitutions, deletions and / or insertions, for example 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0135] (c) a polypeptide derived from the polypeptide of (a), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids; and

[0136] (d) a fragment of the polypeptide of (a).

[0137] 18. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the one or more proteases are a polypeptide having the sequence of SEQ ID NO: 6.

[0138] 19. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the cookies comprises 0.00015 wt.% to 0.003 wt.% of one or more proteases, preferably 0.00003 wt.% to 0.0006 wt.%.

[0139] 20. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the laminated biscuit variant comprises 0.00015 wt.% to 0.009 wt.% of one or more proteases, preferably 0.00003 wt.% to 0.00018 wt.%.

[0140] 21. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the cookies comprises 1.5 to 30 mg enzyme protein / kg flour of one or more added proteases, preferably 0.3 to 0.6 mg enzyme protein / kg flour.

[0141] 22. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the laminated biscuit variant comprises 1.5 to 90 mg enzyme protein / kg flour of one or more added proteases, preferably 0.3 to 18 mg enzyme protein / kg flour.

[0142] 23. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the cookies comprises 5 to 100 parts per million (ppm) of one or more added proteases.

[0143] 24. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough for the laminated biscuit variant comprises 5 to 300 parts per million (ppm) of one or more added proteases.

[0144] 25. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the step of preparing the dough comprises adding one or more lipases in combination with one or more proteases.

[0145] 26. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the fat is selected from the group consisting of: palm oil; palm olein, butter, clarified butter, anhydrous milk fat, corn oil, margarine, olive oil, hydrogenated vegetable fats, sunflower oil, rapeseed oil, rice bran oil, lard, soybean oil and eggs.

[0146] 27. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the fat is palm oil.

[0147] 28. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the dough comprises 12 wt.% to 35 wt.% of one or more fats.

[0148] 29. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the step of preparing the dough comprises adding:

[0149] i. 10 wt.% to 30 wt.% of one or more sugars,

[0150] ii. 40wt.% to 70wt.% of one or more flours,

[0151] iii. 0.00001 wt.% to 0.0002 wt.% of one or more lipases and / or one or more proteases; and

[0152] iv. 12 to 35 wt.% of one or more fats.

[0153] 30. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the step of preparing the dough comprises adding:

[0154] i. 10 wt.% to 30 wt.% of one or more sugars,

[0155] ii. 40wt.% to 70wt.% of one or more flours,

[0156] iii. 0.1 to 2 mg enzyme protein / kg flour of one or more added lipases or / and one or more proteases; and

[0157] iv. 12 to 35 wt.% of one or more fats.

[0158] 31. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the step of preparing the dough comprises adding:

[0159] i. 10 wt.% to 30 wt.% of one or more sugars,

[0160] ii. 40wt.% to 70wt.% of one or more flours,

[0161] iii. 5 ppm to 300 ppm of one or more lipases and / or one or more proteases; and

[0162] iv. 12 to 35 wt.% of one or more fats.

[0163] 32. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the method further reduces sugars in the baked product compared to the baked product prepared without using the one or more lipases and / or one or more proteases.

[0164] 33. The method for preparing the baked product of paragraph 24, wherein the sugar of the baked product is reduced by 1 wt.% to 5 wt.% compared to the baked product prepared without using the one or more lipases and / or one or more proteases.

[0165] 34. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the fat of the baked product is reduced by at least 20 wt.% compared to a baked product prepared without using the one or more lipases and / or one or more proteases.

[0166] 35. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the fat of the baked product is reduced by at least 25 wt.% compared to the baked product prepared without using the one or more lipases and / or one or more proteases.

[0167] 36. The method for preparing the baked product as described in any of the preceding paragraphs, wherein the step of preparing the dough further comprises adding one or more leavening agents.

[0168] 37. The method for preparing the baked product as described in paragraph 36, wherein the leavening agent is selected from the group consisting of sodium bicarbonate, ammonium bicarbonate, sodium acid pyrophosphate (SAPP), sodium phosphate, monocalcium phosphate, sodium aluminum sulfate, potassium hydrogen tartrate, sodium aluminum phosphate, glucono-delta-lactone and adipic acid and combinations thereof.

[0169] 38. The method for preparing the baked product of paragraphs 36-37, wherein the dough comprises 0.3 wt.% to 3 wt.% of one or more additional leavening agents.

[0170] 39. A method for preparing the baked product as described in any of the preceding paragraphs, wherein the step of preparing the dough further comprises adding invert syrup, salted or unsalted butter, lecithin, milk powder, distilled mono- and diglycerides (DMG), salt, liquid glucose, cheese, fresh milk, colorants, flavorings, sodium metabisulfite, sweetened condensed milk, spices and condiments, glyceryl monostearate (GMS), diacetyltartaric acid esters of mono- and diglycerides (DATEM) and / or water.

[0171] 40. Use of one or more lipases in the production of bakery products to reduce the fat and / or sugar used in the preparation of the bakery products.

[0172] 41. The use of paragraph 40, wherein one or more properties of the baked product are maintained.

[0173] 42. The use of paragraph 40 or 41, wherein the one or more characteristics are selected from the group consisting of mouthfeel, texture, taste, chewiness, spreadability, color-height-length, volume, moisture, strength, hardness, crispness and shelf life.

[0174] 43. The use as described in any of paragraphs 40-42, wherein the baked product has at least 10 wt.% less fat, preferably at least 20 wt.% less fat, even more preferably at least 25 wt.% less fat compared to a baked product prepared without using the one or more lipases and / or one or more proteases.

[0175] 44. A baked product obtainable by the method of any of paragraphs 1 to 39, or by the use of any of paragraphs 40 to 43.

[0176] Examples

[0177] The following examples are not intended to be an exhaustive list of all the different ways in which the present disclosure may be implemented or an exhaustive list of all the features that may be added to the present disclosure. It will be appreciated by those skilled in the art that various changes and additions may be made to the various embodiments without departing from the present disclosure. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0178] Unless otherwise indicated, the percentages listed in the following examples are weight percentages based on the total weight of the composition.

[0179] Materials and methods

[0180] The experiments were conducted in Denmark using standard Danish cookie making ingredients. To prepare the cookies, standard ingredients and a standard procedure were used, which are listed below in Example 1.

[0181] Enzymes used in the assay:

[0182]

[0183] Example 1 - Process flow for preparing cream cookies

[0184] 1. Emulsification :

[0185] Weigh canola oil, invert syrup, salted butter, lecithin, distilled mono- and diglyceride (DMG) paste, and flavors into a mixing bowl and mix at medium speed for 2 minutes. Add sugar and skim milk powder (SMP) to the mixing bowl and mix at medium speed for 3 minutes. Dissolve salt, sodium bicarbonate, and ammonium bicarbonate in water and add to the mixing bowl. Mix the ingredients in the mixing bowl at medium speed for 5 minutes.

[0186] 2. mix:

[0187] Add wheat flour and mix these ingredients for 5-6 minutes to get the right consistency of dough. Add water as required to adjust the consistency of dough.

[0188] 3. temperature:

[0189] Keep the temperature of the dough at 23-25 ​​degrees Celsius (℃)

[0190] 4. Molding / Tableting:

[0191] The dough was molded in a cookie molder and pressed in a dough sheeter to a thickness of 3.5 mm.

[0192] 5. Baking:

[0193] The molded and sheeted dough was baked at a temperature of 235°C for 6-7 minutes.

[0194] 6. Cooling and Packaging:

[0195] Chill cookies for 1.5 times the baking time and wrap in aluminum foil.

[0196] Example 2 - Lipase Screening:

[0197] A trial was conducted using 2 lipases to achieve a 20% fat reduction.

[0198] 1. Lipase – 1,3-specific lipase that acts only on triglycerides (SEQ ID NO: 1)

[0199] 2. Phospholipases: Phospholipase 1 (SEQ ID NO: 2), phospholipase 2 (SEQ ID NO: 3) and phospholipase 3 (SEQ ID NO: 4) that act on triglycerides and polar lipids.

[0200] During the mixing step of the process in Example 1, lipase was added with the wheat flour.

[0201] Table 1: Height coefficients of cookies using different lipases:

[0202]

[0203]

[0204] Table 2: Order of Cookie Stacking Height Based on Enzyme Dosage

[0205] Sort by Stacking height order Enzyme dosage in parts per million (ppm) First Lipase 10ppm second Phospholipase 1 100ppm third Phospholipase 2 50ppm

[0206] Tables 1 and 2 show that 10 ppm of lipase (SEQ ID NO: 1) showed better performance in terms of stacking height of cookies compared to phospholipases (SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4).

[0207] Cookies baked with lipase scored higher on the sensory attributes compared to cookies baked with phospholipase. Figure 1 It is shown that lipase provides similar or better results at very low dosages as low as 10ppm compared to phospholipases. The spreading coefficient and height coefficient of cookies with 20% fat reduction and 10ppm lipase content are similar or higher than those of control cookies and cookies baked using 50ppm or more phospholipases. Therefore, the use of lipase is a cost-effective method for baking cookies.

[0208] Example 3 - Process for preparing cream cookies with 10%-20% reduced fat

[0209] The cream cookies were prepared using the process steps defined in Example 1. The quantities of the ingredients used in the process of preparing the cream cookies are mentioned in Table 3 (10% fat reduction) and Table 4 (20% fat reduction).

[0210] Table 3: Composition of cream cookies with 10% reduced fat

[0211]

[0212]

[0213] Table 4: Composition of cream cookies with 20% reduced fat

[0214]

[0215]

[0216] 1. Emulsification :

[0217] Three samples were prepared: a positive control, a negative control, and a test containing lipase (SEQ ID NO: 1).

[0218] 2. mix:

[0219] Wheat flour was added to each of the three samples. Lipase (SEQ ID NO: 1) was added only in the sample-containing lipase test. No resting time was required for the dough.

[0220] 3. temperature:

[0221] Keep the temperature of the dough at 23-25 ​​degrees Celsius (℃)

[0222] 4. Molding / Tableting:

[0223] The dough was molded in a cookie molder and pressed in a dough sheeter to a thickness of 3.5 mm.

[0224] Table 5: Dough properties of cream cookies with 10% fat reduction

[0225] Dough properties Positive Control Negative control Enzyme test Dough pH 8.2 8.2 8.3 Dough texture 5 4 (dry and not too smooth) 5.5 Much better texture Dough temperature 24 25 24.5 Dough sheeting 5 4 6

[0226] Table 6: Dough properties of cream cookies with 20% fat reduction

[0227]

[0228] The dough properties of the test samples containing lipase were similar or improved compared to the positive control sample. At 10% and 20% fat reduction, the test dough containing lipase was better than the control dough in terms of machinability, while the fat-reduced dough without lipase (negative control) was very dry and rough during the sheeting / molding process. Thus, lipase exhibited emulsifying activity in wheat flour to achieve fat reduction.

[0229] 5. Baking:

[0230] The sheeted and molded dough was baked at a temperature of 235°C for 6-7 minutes.

[0231] 6. Cooling and Packaging:

[0232] Chill cookies for 1.5 times the baking time and wrap in aluminum foil.

[0233] Table 7: Dimensions of cream cookies with 10% fat reduction

[0234]

[0235] Table 8: Dimensions of cream cookies with 20% fat reduction

[0236]

[0237] The size of the cream cookies for the sample-containing lipase test were similar or improved compared to the cream cookies for the positive control sample.

[0238] Figure 2 and Figure 3 The positive control sample and the lipase-containing test sample showed similar texture. Figure 2 and Figure 3As shown, reducing fat by 10% and 20% in the cream cookie baking process using lipase does not impair the texture of the cream cookie.

[0239] The test cookies containing lipase showed a significant increase in the stacking height of the cookies at both 10% and 20% fat reduction compared to the positive control and the negative control (reduced fat but without lipase). The density of the test cookies containing lipase was also lower than the control.

[0240] As the lipase dosage becomes higher, the sugar in the cookie can also be reduced, as the emulsifying properties of the lipase increase the sugar perception in the cookie. Thus, at a lipase dosage of 50 ppm, 2% of the sugar was reduced and replaced with wheat flour, while the fat was reduced by 20%.

[0241] Example 4 - Triangle Test-1

[0242] A total of 15 panelists served as tasters for the triangle test. Each panelist was presented with three samples (two identical samples and one different sample) at the same time, and then asked to identify the sample that was different from the other two samples. The following are six ways to present the samples to the panelists, and each sample is coded with a 3-digit number.

[0243] Code describe 1 630，815 Control - Standard formula 2 731，324 Fat reduction 10% + enzymes Code describe 1 254，375 Fat reduction 20% + enzymes 2 603，514 Control - Standard formula

[0244] Panelists are asked to identify codes on a scorecard that represent different samples. This method requires panelists to choose between samples. Interpretation is based on the minimum number of correct responses required to achieve significance at a predetermined significance level, given the total number of responses received. The goal is to identify different samples in a triangle test.

[0245] Table 9: Triangle test results for 10% fat reduction

[0246]

[0247] Table 10: Triangle test results for 20% fat reduction

[0248]

[0249]

[0250] None of the panelists found or observed any hardness in the cookie texture caused by the reduction in fat and the corresponding increase in the percentage of wheat flour in the samples-containing lipase test. Therefore, lipase maintained the texture of the cookies while baking the cookies after reducing the fat by 10% and 20%.

[0251] The panelists perceived that the flavor / aroma of the control sample was unbalanced, while the flavor / aroma of the trial was well-harmonized / balanced.

[0252] For the 10% fat reduction, several panelists observed that the test samples were slightly sweeter than the control, which was due to the emulsifying properties of the lipase, and this was a positive attribute as the increase in sweetness also improved the mouthfeel and overall acceptability of the cookie.

[0253] For the 20% fat reduction, 2% sugar reduction and replacement with wheat flour, several panelists (2 / 15) observed that the control sample was slightly sweeter than the test. Thus, lipase also aids in sugar reduction as well as fat reduction at high doses.

[0254] Example 5 - Screening of other enzymes with lipase to reduce fat by 25% and sugar by 2% in cream cookies

[0255] The following experiments in Table 11 were performed to evaluate the synergistic effect of other enzymes with lipase

[0256] Table 11: Tests to evaluate the synergistic effect of other enzymes with lipase

[0257]

[0258]

[0259]

[0260] The cookie sample containing the combination of lipase and protease 1 had a similar texture to the positive control sample with 25% less fat, so this combination was tested further.

[0261] Example 6 - Process for preparing cream cookies with 25% reduced fat

[0262] The cream cookies were prepared using the process steps defined in Example 1. The quantities of the ingredients used in the process of preparing the cream cookies are mentioned in Table 12 (Fat Reduction 25%).

[0263] Table 12: Composition of cream cookies with 25% reduced fat and 2% reduced sugar

[0264]

[0265]

[0266] 1. Emulsification :

[0267] Three samples were prepared: a positive control, a test containing lipase, and a test containing lipase and protease 1.

[0268] 2. mix:

[0269] Wheat flour was added to each of the three samples. Lipase was added only in the sample-containing lipase test. Lipase and protease 1 were added only in the sample-containing lipase and protease 1 test. No resting time was required for the dough.

[0270] 3. temperature:

[0271] Keep the temperature of the dough at 23-25 ​​degrees Celsius (℃)

[0272] 4. Molding / Tableting:

[0273] The dough was molded in a cookie molder and pressed in a dough sheeter to a thickness of 3.5 mm.

[0274] Table 13: Dough properties of cream cookies with 25% fat reduction

[0275] Dough properties Positive Control Tests containing lipase and protease 1 Dough pH 8.2 8.3 Dough texture 5 6(soft) Dough temperature 24 24 Dough sheeting 5 6(soft)

[0276] The dough properties of the test samples containing lipase and protease 1 were similar or improved compared to the positive control sample. Therefore, lipase and protease 1 exhibited emulsifying activity in wheat flour, thereby achieving fat reduction.

[0277] 5. Baking:

[0278] The sheeted and molded dough was baked at a temperature of 235°C for 6-7 minutes.

[0279] 6. Cooling and Packaging:

[0280] Chill cookies for 1.5 times the baking time and wrap in aluminum foil.

[0281] Table 14-A: Dimensions of Cream Cookies with 25% Reduced Fat

[0282]

[0283] At a lipase dosage of 25% fat reduction (60 ppm), the bulk height dropped dramatically.The test sample containing lipase (60 ppm) had a harder texture and floury mouthfeel than the positive control sample.

[0284] Cookie shrinkage and harder cookie texture clearly indicated the need for dough relaxation when reducing fat at higher percentages. To overcome the cookie shrinkage and harder texture, an additional enzyme (Protease 1) was used to achieve a texture similar to the positive control.

[0285] Table 14-B: Dimensions of Cream Cookies with 25% Reduced Fat

[0286]

[0287] The size of the cream cookies for the samples - the trials containing lipase and protease 1 were similar or improved compared to the cream cookies for the positive control sample.

[0288] Figure 4 It is shown that the positive control sample and the test sample containing lipase and protease 1 have similar texture. Figure 4 As shown, a 25% fat reduction in the cream cookie baking process using lipase and protease 1 did not compromise the texture of the cream cookie. The stacking height of the test sample was also slightly increased compared to the positive control.

[0289] The test cookies containing lipase and protease 1 showed a significant increase in the stacking height of the cookies at a 25% reduction in fat compared to the positive control. The density of the test cookies containing lipase and protease 1 was also lower than the positive control.

[0290] As the dosage of lipase and protease 1 becomes higher, the sugar in the cookie can also be reduced because the emulsifying properties of lipase and protease 1 increase the sugar perception in the cookie. Thus, at a lipase dosage of 50 ppm and a protease 1 dosage of 8 ppm, 2% of the sugar was reduced and replaced with wheat flour, while the fat was reduced by 25%.

[0291] Example 7 - Triangle Test 2

[0292] A total of 20 panelists served as tasters for the triangle test. The triangle test was conducted in the same manner as in Example 4 using the cream cookie sample of Example 6.

[0293] Table 15: Triangle test results for 25% fat reduction

[0294]

[0295] None of the panelists found or observed any hardness in the texture of the cookies caused by the reduction in fat and the corresponding increase in the percentage of wheat flour in the test sample - containing lipase and protease 1. Most of the panelists preferred the test sample to the control sample due to the improved texture.

[0296] Example 8 - Process for Preparing Laminated / Hard Dough Biscuit Variations

[0297] 1. Emulsification :

[0298] Weigh fat, invert syrup, glyceryl monostearate (GMS), DATEM, spices, add to mixing bowl and mix at medium speed for 2 minutes. Add sugar and skim milk powder (SMP) to mixing bowl and mix at medium speed for 3 minutes. Dissolve salt, sodium bicarbonate and ammonium bicarbonate in water and add to mixing bowl. Mix the ingredients in mixing bowl at medium speed for 5 minutes.

[0299] 2. mix:

[0300] Add wheat flour in a mixing bowl. Add SMBS in dissolved form along with water in the mixing bowl. Mix these ingredients for 5-6 minutes to get the right consistency of dough. Add water as required to adjust the consistency of dough.

[0301] 3. temperature:

[0302] Keep the temperature of the dough at 40-42 degrees Celsius (℃)

[0303] 4. Standing time

[0304] Allow the dough to rest for 20 minutes.

[0305] 5. Tableting:

[0306] After a rest period, the dough is laminated in a sheeting machine and the dough sheets are folded. The lamination and folding process is repeated 6 times. The thickness of the final sheet after lamination is 2 mm.

[0307] 6. Cutting:

[0308] The laminated dough sheets were cut using a Teflon cutter with dhoker pin holes.

[0309] 7. Baking:

[0310] After cutting, the dough pieces were baked at a temperature of 270° C. for 5-6 minutes.

[0311] 8. Cooling and Packaging:

[0312] Chill cookies for 1.5 times the baking time and wrap in aluminum foil.

[0313] Example 9 - Lipase for 10% Fat Reduction in Laminated / Hard Dough Biscuit Variations

[0314] The laminated / hard dough cookie variant was prepared using the process steps defined in Example 8. The laminated / hard dough cookie variant was tested for fat reduction using lipase alone. The initial trial was designed to reduce the fat of the laminated / hard dough cookie variant by 10% using only 30 ppm of lipase.

[0315] Table 16: Dough properties and biscuit dimensions using lipase alone in laminated / hard dough biscuit variants with 10% fat reduction

[0316]

[0317]

[0318] Results: A decrease in volume and an increase in density were observed in the test samples.

[0319] Sensory Observations: The test samples were drier than the positive control sample. The reduced fat samples were observed to have a firmer texture than the positive control sample.

[0320] in conclusion:

[0321] The shrinkage and hardening of the texture of the biscuits clearly indicated that dough relaxation was required when reducing fat at higher percentages in making laminated / hard dough biscuit variants. Therefore, the use of additional enzymes (protease) was required to achieve a texture similar to that of the positive control.

[0322] Example 10 - Protease Screening for 10% Fat Reduction in Laminated / Hard Dough Biscuit Variations

[0323] The laminated / hard dough biscuit variant was prepared using the process steps defined in Example 8. The following 8 proteases were tested to reduce fat by 10% in the laminated / hard dough biscuit variant.

[0324] 1. Protease 1

[0325] 2. Blend 1

[0326] 3.Protease 2

[0327] 4. Blend 2

[0328] 5.Protease 3

[0329] 6.Protease 4

[0330] 7. Blend 3

[0331] 8. Blend 4

[0332] The details of protease screening are as follows:

[0333] 1. Positive control based on finished product (FG) containing 10.64% full fat

[0334] 2. Negative control based on FG to reduce fat by 10% and fat percentage of 9.35% without enzyme

[0335] 3. The test was based on FG with a fat reduction of 10% and a fat percentage of 9.35%, and containing different proteases.

[0336] Table 17: Dough properties and biscuit dimensions using proteases 1-4

[0337]

[0338]

[0339]

[0340] Table 18: Dough properties and biscuit dimensions using proteases 5-8

[0341]

[0342]

[0343]

[0344] All proteases showed equal or better dough properties, biscuit size, sensory properties compared to the positive control. Biscuits containing protease 1 showed better dough properties, biscuit size, and sensory properties compared to other types of proteases. Protease 1 worked at very low doses, so protease 1 was shortlisted for further testing with lipase.

[0345] Example 11 - Process for Preparing Laminated / Hard Dough Biscuit Variations with 10% Reduced Fat

[0346] The laminated / hard dough biscuit variant was prepared using the process steps defined in Example 8. Trials were carried out using lipase and protease 1 with a 10% fat reduction.

[0347] Table 19: Dough properties and biscuit dimensions with 10% fat reduction using lipase and protease 1

[0348]

[0349]

[0350]

[0351] Results: It was observed that the assay containing proteinase 1 and lipase had better spreading and stacking height coefficients similar to the positive control.

[0352] Using protease 1 alone, similar volume and density as the positive control was obtained, but the mouthfeel was dry in terms of sensory properties. Therefore, lipase was added to match the mouthfeel similar to the positive control. The mouthfeel matched the combination of protease 1 and lipase, as the lipase enhanced the emulsifying properties.

[0353] Example 12 - Process for Preparing Laminated / Hard Dough Biscuit Variations with 10% Reduced Fat

[0354] Laminated / hard dough biscuits variant was prepared using the process steps defined in Example 8. The quantities of various ingredients used in the process of preparing such biscuits are mentioned in Table 20 (10% reduction in fat in laminated variant).

[0355] Table 20: Ingredients of 10% Reduced Fat Cookies

[0356]

[0357] 1. Emulsification :

[0358] Three samples were prepared: a positive control, a negative control, and a test containing lipase and protease 1.

[0359] 2. mix:

[0360] Wheat flour was added to each of the three samples. Lipase and Protease 1 were added only in the sample - the test containing Lipase and Protease 1.

[0361] 3. temperature:

[0362] Keep the temperature of the dough at 40-42 degrees Celsius (°C). Let the dough rest for 20 minutes.

[0363] 4. Tableting:

[0364] The dough was laminated in a sheeting machine and the dough sheets were folded. The lamination and folding process was repeated 6 times. The thickness of the final sheet after lamination was 2 mm.

[0365] Table 21: Dough properties of laminated / hard dough biscuit variants with 12% fat reduction

[0366]

[0367] At 10% fat reduction, the experimental dough containing lipase and protease 1 outperformed the control dough in terms of machinability, whereas the fat-reduced dough (negative control) without lipase and protease 1 dried and shrank during the sheeting process. This indicates that lipase and protease 1 have emulsifying and dough relaxing activities in wheat flour, leading to fat reduction.

[0368] 5. Cutting:

[0369] The laminated dough sheets were cut using a Teflon cutter with dhoker pin holes.

[0370] 6. Baking:

[0371] The laminated dough sheets were baked at a temperature of 270°C for 5-6 minutes.

[0372] 7. Cooling and Packaging:

[0373] Chill the crackers for 1.5 times the baking time and wrap in aluminum foil.

[0374] Table 22: Dimensions of laminated biscuit variants with 10% reduced fat

[0375]

[0376]

[0377] The size of the crackers for the samples - the trial containing lipase and protease 1 were similar or improved compared to the crackers for the positive control sample.

[0378] Figure 5 and Figure 6 It is shown that the positive control and the test sample containing lipase and protease 1 have similar texture. Figure 5 and Figure 6 As shown, a 10% fat reduction in the baking process of the laminated biscuit variant using lipase and protease 1 did not compromise the texture of the crackers.

[0379] The experimental crackers containing lipase and protease 1 showed a slight increase in the stacking height of the crackers at a 10% reduction in fat compared to the positive control and the negative control (reduced fat but without lipase and protease 1). The density of the experimental crackers containing lipase and protease 1 was also lower than that of the control.

[0380] Example 13 - Triangle Test 3

[0381] A total of 20 panelists served as tasters for the triangle test. The triangle test was conducted in the same manner as in Example 4 using the cracker samples of Example 8.

[0382] Table 23: Triangle test results for 12% fat reduction in laminated biscuit variants

[0383]

[0384]

[0385] None of the panelists found or observed any hardness in the cracker texture caused by the 10% reduction in fat and the corresponding increase in the percentage of wheat flour in the sample - containing lipase and protease 1.

Claims

1. A method for preparing a baked product comprising reduced fat, the method comprising the following steps: a. Prepare the dough by adding at least the following: i. one or more sugars, ii. one or more flours, iii. one or more lipases and / or one or more proteases; and iv. one or more fats; b. molding the dough in one or more dough molds and / or sheeting the dough in one or more dough sheeting machines; c. baking the dough; The baked product has at least 10 weight percent (wt.%) to 25 wt.% fat reduction compared to a baked product prepared without the one or more lipases and / or one or more proteases.

2. The method for preparing the baked product as claimed in claim 1, wherein the baked product is selected from the group consisting of biscuits, cookies, laminated biscuit variants and crackers.

3. The method for preparing the baked product as claimed in claim 1 or 2, wherein the dough comprises 10 wt.% to 30 wt.% of one or more added sugars.

4. The method for preparing the baked product as claimed in any one of the preceding claims, wherein the flour is selected from the group consisting of wheat flour, all-purpose flour, whole wheat flour, rice flour, corn flour, millet flour, soya flour, oat flour, pastry flour, bread flour, gluten-free flour, tapioca flour and self-raising flour.

5. The method for preparing the baked product as claimed in any one of the preceding claims, wherein the dough comprises 40 wt.% to 70 wt.% of one or more additional flours.

6. The method for preparing the baked product as claimed in any one of the preceding claims, wherein the lipase is a Thermomyces lanuginosus (formerly known as Humicola lanuginosus) wild-type lipase.

7. The method for preparing the baked product as claimed in any one of the preceding claims, wherein the one or more lipases are polypeptides selected from the group consisting of: (a) a polypeptide having 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 1; (b) a polypeptide derived from SEQ ID NO: 1 having 1 to 30 alterations such as substitutions, deletions and / or insertions, for example 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (c) a polypeptide derived from the polypeptide of (a), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids; and (d) a fragment of the polypeptide of (a).

8. The method for preparing the baked product as claimed in any one of the preceding claims, wherein the one or more lipases is a polypeptide having the sequence of SEQ ID NO:

1.

9. The method for preparing the baked product as claimed in any one of the preceding claims, wherein the one or more proteases are polypeptides selected from the group consisting of: (a) a polypeptide having 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%, at least 99%, or 100% sequence identity to SEQ ID NO:6; (b) a polypeptide derived from SEQ ID NO: 6 having 1-30 alterations such as substitutions, deletions and / or insertions, for example 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (c) a polypeptide derived from the polypeptide of (a), wherein the N- and / or C-terminus has been extended by the addition of one or more amino acids; and (d) a fragment of the polypeptide of (a).

10. The method for preparing the baked product as claimed in any one of the preceding claims, wherein the one or more proteases is a polypeptide having the sequence of SEQ ID NO:

6.

11. The method for preparing the baked product as claimed in any one of the preceding claims, wherein the dough comprises 12 to 35 wt.% of one or more fats.

12. The method for preparing the baked product according to any one of the preceding claims, wherein the baked product has 1 to 5 wt.% reduction in sugars compared to a baked product prepared without the one or more lipases or / and one or more proteases.

13. Use of one or more lipases and / or one or more proteases in the production of baked products for reducing the fat used in the preparation of the baked products by 10 wt.% to 25 wt.% and / or reducing the sugar by 1 wt.% to 5 wt.%.

14. Use according to claim 13, wherein one or more properties of the baked product are maintained, wherein at least one of these properties is selected from the group consisting of mouthfeel, texture, taste, chewiness, spreadability, color-height-length, volume, moisture, strength, firmness, crispness and shelf life.

15. A baked product obtainable by the method according to any one of claims 1 to 12, or by the use according to any one of claims 13 to 14.

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