Enzymatic method for reducing usage amount of fat and oil in bakery product

Abstract

Disclosed is a method for reducing the usage amount of edible fat and oil in a bakery product, the method comprising mixing at least one maltose alpha-amylase and edible fat and oil into dough, and baking same to prepare a bakery product. The usage amount of the edible fat and oil in the dough can be reduced by at least 10 wt % compared with not using the enzyme treatment; and the enzyme may also include cellulase and/or phospholipase. The method does not reduce or substantially reduce the quality of the bakery product, and allows same to have a shelf life of at least 4 days. Also involved is baked fat and oil prepared from the above-mentioned enzyme and edible fat and oil.

Claims

1. A method for reducing the usage amount of edible fat and oil in a baked product prepared from a dough, comprising the steps of: (a) incorporating at least one maltose alpha-amylase into the dough; (b) incorporating edible fat and oil added in the dough; and (c) preparing the baked product from the dough by baking, wherein the amount of edible fat and oil in the dough can be reduced by at least 10% by weight relative to the amount of edible fat and oil in the dough under the same conditions, except the maltose alpha-amylase is not added to the dough, and wherein a sensory evaluation of the baked product determines the baked product does not worsen or substantially worsen compared with a baked product prepared under the same conditions except the maltose alpha-amylase-is not added into the dough and the amount of edible fat and oil is not reduced.

2. The method according to claim 1, wherein the dough additionally comprises a cellulase.

3. The method according to claim 1, wherein the content of the edible fat and oil in the baked product is at least 1% (w/w) by weight relative to the baked product.

4. The method according to claim 1, wherein the amount of edible fat and oil in the dough can be reduced by at least 15% relative to that under the same conditions except for not adding the maltose alpha-amylas.

5. The method according to claim 1, wherein the dough further comprises cellulase and/or phospholipase.

6. The method according to claim 1, wherein the baked product is bread, cake, Chinese pastry, soft bread, puff bread, toast, French roll, bun, sponge cake, or chiffon cake.

7. The method according to claim 1, wherein the baked product has a shelf life of at least 4 days or the baked product at day 4 has a lower hardness value and/or higher elasticity value compared with a baked product prepared under the same conditions except the maltose alpha-amylase is not added to the dough and the amount of edible fat and oil is not reduced.

8. The method according to claim 1, wherein in the dough the amount of the maltose amylase is 10-1000 MANU relative to each kilogram of flour.

9. The method according to claim 1, wherein the edible fat and oil is butter, artificial butter, vegetable oil, margarine and/or shortening.

10. The method according to claim 1, wherein the dough further comprises flour, edible salt, edible sugar, edible essence, yeast and/or vitamin C.

11. The method according to claim 1, wherein the baked product is a bread and the sensory evaluation is a comprehensive evaluation of touch softness, bread crumb structure, taste softness, taste moisture, olfactory fragrance, and gustatory aroma.

12. The method according to claim 1, wherein the baked product is a cake, and the sensory evaluation is a comprehensive evaluation of taste softness, taste moisture, melt-in-the-mouth effect, and viscidity of the cake.

13. The method according to claim 1, wherein the amount of edible fat and oil in the dough can be reduced by at least 25% relative to that under the same conditions except for not adding the maltose alpha-amylase.

14. The method according to claim 1, wherein the amount of edible fat and oil in the dough can be reduced by at least 35% relative to that under the same conditions except for not adding the maltose alpha-amylase.

15. The method according to claim 1, wherein the baked product at day 14 has a lower hardness value and/or higher elasticity value compared with a baked product prepared under the same conditions except the maltose alpha-amylase is not added to the dough and the amount of edible fat and oil is not reduced.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

(1) Baked Product

(2) The process of the present invention can be used for any baked product from a dough, whether of soft or crisp properties, and whether of a white, light or dark type. In the baked product prepared by the process of the present invention, the content of the edible fat and oil is at least 1% (w/w) or at least 2% (w/w), preferably at least 3% (w/w) relative to the baked product, or the content of the edible fat and oil is at least 4% (w/w) relative to the baked product, or the content of the edible fat and oil is at least 5% (w/w) relative to the baked product, or the content of the edible fat and oil is at least 6% (w/w) relative to the baked product. Although there is still a certain content of edible fat and oil in the baked product, using the process of the present invention has significantly reduced the content of the edible fat and oil relative to that without using the process of the present invention, in the case that the sensory evaluation of the prepared baked product does not worsen or substantially worsen.

(3) In a preferred embodiment of the present invention, the typical examples of the baked products are breads, for example soft breads, preferably bun, toast, French roll and milk bar breads. In a preferred embodiment of the present invention, the examples of the baked products also can be puff bread, such as croissant and pull-apart bread. In a preferred embodiment of the present invention, the typical examples of the baked products are cakes, for example sponge cakes or chiffon cakes. In a preferred embodiment of the present invention, the examples of the baked products also can be Chinese snacks with a relatively high oil content, such as moon cake.

(4) In a preferred embodiment of the present invention, due to the use of the process of the present invention, the usage amount of edible fat and oil in the dough can be reduced by at least 15%, for example at least 20%, preferably at least 25%, at least 30%, at least 35%, or more preferably at least 40% by weight relative to that under the same condition except for the absence of maltose -amylase or the enzyme composition of maltose -amylase and cellulase.

(5) In a preferred embodiment of the present invention, when preparing toast, since the usage amount of edible fat and oil generally accounts for about 10% by weight of that of flour during the preparation of currently general commercially available toast, using the process of the present invention, the usage amount of edible fat and oil can be reduced to about 6% by weight of that of flour, that is to say, the usage amount of edible fat and oil in the dough can be reduced by 40%. However, if the usage amount of fat and oil only is reduced by about 25%, it is also entirely feasible.

(6) In a preferred embodiment of the present invention, when preparing pull-apart bread, since the usage amount of edible fat and oil generally accounts for about 25% by weight of that of flour during the preparation of currently general commercially available toast, using the process of the present invention, the usage amount of edible fat and oil in the dough can be reduced by at least 15%, at least 20%, preferably at least 25%, at least 30%, at least 35%, or more preferably at least 40% by weight during the preparation process relative to the usage amount of edible fat and oil under the same condition except for the absence of the treatment by the enzyme.

(7) In a preferred embodiment of the present invention, the prepared baked product (for example at day 4, day 7 or day 14) has a lower hardness value and/or higher elasticity value compared with the prepared baked product under the same condition except for not adding maltose amylase or the enzyme composition of maltose -amylase and cellulase into the dough and not reducing the usage amount of edible fat and oil.

(8) In a preferred embodiment of the present invention, the sensory evaluation of the prepared baked product does not worsen or substantially worsen compared with the prepared baked product under the same condition except for not adding maltose amylase or the enzyme composition of maltose -amylase and cellulase into the dough and not reducing the usage amount of edible fat and oil. Preferably, for bread, the sensory evaluation is the comprehensive evaluation of touch softness, bread crumb structure, taste softness and taste moisture. More preferably, the sensory evaluation also comprises the evaluation of olfactory fragrance and gustatory aroma.

(9) In a preferred embodiment of the present invention, although it significantly reduces the usage amount of edible fat and oil, the prepared baked product has a shelf life of at least 4 days, or at least 7 days or at least 14 days.

(10) In a preferred embodiment of the present invention, the baked product prepared in the present invention has an improved shelf life, the shelf life thereof is at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.

(11) The shelf life can be measured as follows: comparing the baked product prepared by the process of the present invention and the control baked product (i.e. compared with that under the same condition except for not adding the enzymes into the dough and not reducing the usage amount of edible fat and oil); measuring the hardness of the baked product by a texture analyzer, and comparing it with that of the control baked product stored under the same condition. The improved shelf life is defined as the baked product which is not as hard as (i.e. softer than) the control when being measured by a texture analyzer.

(12) Maltose -Amylase

(13) Maltose -amylase is an enzyme classified in EC3.2.1.133. The enzymatic activity does not need the non-reducing terminus of the substrate. The main enzymatic activity results in that amylopectin and amylose are degraded to maltose or a relatively long maltodextrin. The enzyme can hydrolyze amylopectin and amylose to maltose in alpha-configuration, and can also hydrolyze maltotriose and cyclodextrin.

(14) In a preferred embodiment of the present invention, maltose -amylase is selected from the group consisting of: (a) a polypeptide, which comprises or consists of the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3; (b) a polypeptide, which is derived from (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence of (a); (c) a polypeptide, having at least 80% sequence identity to the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

(15) In a preferred embodiment of the present invention, the maltose -amylase provided by the present invention is the polypeptide comprising the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and a modified polypeptide or homologous polypeptide thereof.

(16) In a preferred embodiment of the present invention, for example, the polypeptide comprising the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 comprises the polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, the polypeptide constituted by adding a signal peptide sequence into the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the polypeptide obtained by adding an appropriate marker sequence at the N-terminus and/or C-terminus of the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

(17) The modified polypeptide in the present invention refers to the protein which comprises the amino acid sequence obtained by deleting, substituting, inserting or adding one or more amino acids in the amino acid sequence show by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and has the maltose -amylase activity.

(18) In a preferred embodiment of the present invention, the modification of the amino acid in said modified polypeptide or homologous polypeptide thereof is a conservative modification. For example, conservative substitution refers to substituting one or more amino acid residues with other chemically similar amino acids without substantially changing the protein activity. Examples are a case in which a hydrophobic residue is substituted by other hydrophobic residues and a case in which a polar residue is substituted by other polar residues having the same charge.

(19) A similarly functional amino acid which can be subjected to such a conservative substitution is well known in the corresponding art of each amino acid. Specifically, as a non-polar (hydrophobic) amino acid, examples are alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like. As a polar (neutral) amino acid, examples are glycine, serine, threonine, tyrosine, glutamic acid, aspartic acid, cysteine and the like. As a positively charged (basic) amino acid, examples are arginine, histidine, lysine and the like. In addition, as a negatively charged (acidic) amino acid, examples are aspartic acid, glutamic acid and the like.

(20) In one aspect, the maltose -amylase according to the present invention differs by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids from the amino acids of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

(21) The homologous polypeptide of the present invention refers to comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least preferably 92%, at least preferably 93%, at least preferably 94%, at least preferably 95%, at least preferably 96%, at least preferably 97%, at least preferably 98%, at least preferably 99%, more preferably 100% homology (sequence identity) to the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

(22) For purposes of the present invention, the sequence identity between two amino acid sequences is determined by using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) which is implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled longest identity (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(23) (Identical Residues100)/(Length of AlignmentTotal Number of Gaps in Alignment)

(24) The polypeptide of the present invention may be natural, synthetic, semi-synthetic, or recombinantly produced. The polypeptide of the present invention can be produced by genetic engineering, by known peptide synthesis or by digesting the polypeptides of the present invention with appropriate peptidases. Preferably, the polypeptide of the present invention may a polypeptide product produced and encoded by the recombinant DNA sequence in the host cell according to a bioengineering method, also may be synthesized according to solid phase or liquid phase chemical technique, for example, it may be synthesized according to the method described by Steward and Young (Steward, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, Ill., (1984)) using an Applied Biosystem synthesizer or a Pioneer peptide synthesizer according to solid phase chemical technique.

(25) In a preferred embodiment of the present invention, the maltose -amylase is derived from the genus Bacillus, especially those derived from Bacillus lichenformis, Bacillus stearothermophilus or Bacillus amyloliquefaciens. The term derived from refers to it can be generated or expressed in the original wild type strain and another strain, i.e. the term encompasses the expression of wild type and naturally generated proteins, and the expression of any recombinant, genetically engineered or synthases in the host.

(26) The maltose -amylase can be the maltose -amylase as disclosed in e.g., WO 1999/043794, WO 2006/032281, or WO 2008/148845 or the variants of the enzyme.

(27) Suitable commercial maltose -amylases comprise Novamyl, OPTICAKE 50 BG, Novamyl Boost and Novamyl 3D (obtainable from Novozymes A/S).

(28) In a preferred embodiment of the present invention, the maltose -amylase added into a dough comprises the maltose -amylase shown by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a modified polypeptide or homologous polypeptide thereof or a mixture thereof.

(29) In a preferred embodiment of the present invention, the maltose -amylase added into a dough is a mixture comprising the maltose -amylase shown by SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3, such as a mixture comprising the maltose -amylase shown by SEQ ID NO: 2 and/or SEQ ID NO: 3.

(30) In a preferred embodiment of the present invention, the maltose -amylase added into a dough comprises 10%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 90%-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3, preferably comprises 20%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 80%-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3, preferably comprises 30%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 70%-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3, preferably comprises 40%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 60%-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3, preferably comprises 50%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 50%-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3, preferably comprises 60%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 40-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3, preferably comprises 70%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 30%-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3, preferably comprises 80%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 20%-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3, and preferably comprises 90%-95% (w/w) of the maltose -amylase shown by SEQ ID NO: 2 and 10%-5% (w/w) of the maltose -amylase shown by SEQ ID NO: 3.

(31) The amount of the maltose -amylase generally in the range of 1-1500 ppm, preferably 10-100 ppm per kilogram of flour. Calculated by the enzymatic activity, the additive amount of the maltose -amylase preferably is 10-1000 MANU/kg flour, such as 50-500 MANU/kg. In a preferred embodiment of the present invention, one ppm maltose amylase corresponds to about 2.7 MANU of the enzymatic activity. One MANU (maltose amylase Novo unit) can be defined as the amount of enzyme required to release 1 mol maltose per minute in 30 minutes at the substrate concentration of 10 mg maltotriose (Sigma M 8378) in 0.1 M citrate buffer per milliliter at 37 C. and pH 5.0.

(32) Cellulases

(33) As used herein, it should be understood that the term cellulases comprises the enzyme composition or enzyme mixture of cellulases, especially endoglucanases (EC 3.2.1.4).

(34) In one embodiment, the used cellulases according to the present invention is the enzyme composition comprising endoglucanases (EC 3.2.1.4).

(35) The cellulases may comprise a carbohydrate-binding module (CBM) which enhances the binding of the enzyme to a cellulose-containing fiber and increases the efficacy of the catalytic active part of the enzyme. A CBM is defined as contiguous amino acid sequence within a carbohydrate-active enzyme with an independent fold having carbohydrate-binding activity. For further information on CBMs, see, e.g., the CAZy internet server or Tomme et al., 1995, in Enzymatic Degradation of Insoluble Polysaccharides (Saddler & Penner, eds.), Cellulose-binding domains: classification and properties. pp. 142-163, American 25 Chemical Society, Washington.

(36) Endoglucanases (E.C. 3.2.1.4) catalyze endo hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin, -1,4 bonds in mixed -1,3 glucans such as cereal -D-glucans or xyloglucans and other plant material containing cellulosic parts.

(37) The endoglucanase activity can be determined according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, such as by the hydrolysis of carboxymethylcellulose (CMC).

(38) In addition to endoglucanase, the cellulase mixture also can comprises cellobiohydrolase (E.C.3.2.1.91) and/or -glucosidase (E.C.3.2.1.21), especially cellobiohydrolase.

(39) Cellobiohydrolase catalyzes the hydrolysis of 1,4--D-glucosidic linkages in cellulose, cellooligosaccharides, or any -1,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain.

(40) The examples of cellobiohydrolase comprise CBH I and CBH II from Trichoderma reesei.

(41) In some embodiments, the cellulase or cellulase mixture can be derived from a strain of the genus Trichoderma, such as a strain of Trichoderma reesei, or a strain of the genus Humicola, such as a strain of Humicola insolens, or a strain of the genus Aureosporium, preferably a strain of Chrysosporium lucknowense.

(42) In some embodiments, the enzyme composition for use in the method and/or use of the present invention can be the product (such as fermentation product) of expressing one or more enzymes in a suitable host cell.

(43) Preferably, cellulase composition can be obtained from (for example, obtained from) genus Trichoderma, preferably obtained from Trichoderma reesei.

(44) The example of commercial cellulase product produced by Trichoderma reesei is Celluclast BG and is obtainable from Novozymes A/S.

(45) The cellulase can be applied at an additive amount of an effective amount, for example, based on per kilogram of flour, at least 0.5 ppm, at an amount in a range of 0.5-5000 ppm, preferably, the usage amount of the cellulase can be in a range of 1-10 ppm, but it can be added as required.

(46) Phospholipase

(47) Phospholipase can have the activity of phospholipases A1, A2, B, C, D or lysophospholipase, and it may have or may not have lipase activity. It may be of animal origin, e.g., from pancreas, snake venom or bee venom, or it may be of microbial origin, e.g., from filamentous fungi, yeast or bacteria, such as Aspergillus or Fusarium, e.g., A. niger, A. oryzae or F. oxysporum. A preferred lipase/phospholipase from Fusarium oxysporum is disclosed in WO 98/26057. Also, the variants described in WO 00/32758 may be used.

(48) Suitable phospholipase compositions are LIPOPAN F and LIPOPAN XTRA (obtainable from Novozymes A/S) or PANAMORE GOLDEN and PANAMORE SPRING (obtainable from DSM).

(49) For example, suitable commercial lipase preparation is LIPOPAN, for example, LIPOPAN 50 BG which is obtainable from Novozymes A/S.

(50) Additional Enzymes

(51) Optionally, additional Enzymes, such as amylase, alpha-amylase, 0-amylase, fungal alpha-amylase, carboxypeptidase, catalase, lipolytic enzyme, galactanase, alpha-galactosidase, 0-galactosidase, glucanase, glucoamylase, glucose oxidase, lipase, oxidase, peroxidase, phytase, polyphenoloxidase, protease and/or xylanase can be used with the enzymes related to the process according to the present invention.

(52) Dough

(53) The present invention also relates to methods for preparing a dough or baked product, these methods comprises incorporating an effective amount of an enzyme to control or reduce the usage amount of edible fat and oil in the preparation process of a baked product.

(54) Phrase incorporating is defined herein as adding the related materials according to the present invention into a dough, into any ingredients of a dough to be made, and/or into any mixture of dough ingredients of a dough to be made.

(55) In other words, the baking composition of the present invention can be added in any steps of the dough preparation, and can be added in one, two, or more steps. The composition can be added into the dough ingredients for kneading and baking by using a method well known in the art.

(56) Term effective amount is defined herein as according to the preparation requirement of a baked product, which is sufficient to provide a measurable effect on at least one property of interest of a dough and/or baked product.

(57) Term dough is defined herein as a mixture of flour and other ingredients, and the dough is hard enough for kneading or rolling.

(58) The dough of the present invention can comprise flour derived from any grains, including wheat, barley, rye, oat, corn, sorghum, rice, millet, and any mixture thereof.

(59) In a preferred embodiment of the present invention, the dough also comprises the group consisting of: flour, yeast, edible salt, edible sugar, vitamin C and/or edible essence. Preferably, in consideration of the significant reduction in the usage amount of edible fat and oil, although the prepared baked product has no changes or substantial changes in terms of touch softness, bread crumb structure, taste softness and taste moisture, an appropriate amount of edible essence can be added to make up for the partial change of the olfactory fragrance and/or gustatory aroma caused by the massive reduction in the usage amount of edible fat and oil. Especially, since margarine can contain edible essence, this part of edible essence and the reduction in the usage amount of edible fat and oil may have some effects on the odor, especially the odor after the storage, in preferred embodiment of the present invention, this can be made up for by adding an appropriate amount of edible essence.

(60) The dough also can contain other dough ingredients, such as protein, such as milk powder, gluten and soybean; egg (whole egg, yolk or egg white); oxidants, such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; amino acids, such as L-cysteine; starch; and/or salts, such as sodium chloride, calcium acetate, sodium sulfate, calcium propionate or calcium sulfate.

(61) Starch can be wheat starch, corn starch, maize starch, tapioca starch, cassava starch, potato starch; and/or sugar, such as sucrose, cane sugar, lactose or high fructose corn syrup.

(62) In the present invention, the edible fats and oils are natural fats and oils or reprocessed fats and oils, wherein, for example, the natural fats and oils are vegetable fats and oils and animal fats and oils, for example vegetable oils or butters, wherein the vegetable oils for example, are rapeseed oils or soybean oils, and for example, the reprocessed fats and oils are margarine, shortening, non-dairy cream etc.

(63) In some embodiments of the present invention, for some applications, it does not need emulsifiers; but for some applications, it may need emulsifiers. The emulsifiers suitable for the present invention preferably are emulsifiers selected from the group consisting of: diacetyl tartaric acid esters of monoglycerides (DATEM), sodium stearyl lactate (SSL), calcium stearyl lactate (CSL), ethoxylated mono and diglycerides (EMG), distilled monoglycerides (DMG), polysorbate (PS), and succinylated monoglycerides (SMG).

(64) The dough of the present invention can be fresh, frozen or partially baked (pre-baked).

(65) The dough of the present invention is generally a fermented dough or a dough to be subjected to fermentation. The dough can be fermented in a variety of ways, such as fermenting the dough by adding a chemical starter (e.g., sodium bicarbonate) or by adding a starter (fermented dough), but preferably by adding a suitable yeast culture such as Saccharomyces cerevisiae (bread yeast) culture (such as a commercially available Saccharomyces cerevisiae strain).

(66) The present invention also relates to a baking fat and oil for baking, comprising edible fat and oil, cellulase and at least one maltose -amylase, and preferably, the fat and oil also contains an emulsifier and/or antioxidant. When preparing a baked product from a dough by using the baking fat and oil, due to the addition of cellulase and at least one maltose -amylase, the usage amount of edible fat and oil in the preparation process can be reduced by at least 10% by weight relative to that under the same condition except for not adding the enzymes into the baking fat and oil, and preferably, in the prepared baked product, the content of the edible fat and oil is at least 1% (w/w), for example at least 2% (w/w), preferably at least 3% (w/w) relative to the baked product.

(67) In some embodiments of the present invention, the edible fats and oils contained in the fats and oils for baking are vegetable oils, for example rapeseed oil, soybean oil, beef tallow, cream, coconut oil or palm kernel oil. Preferably, in the fats and oils for baking, the emulsifier is at least one of fatty acid glycerides, span 60, polyglyceryl esters, sucrose esters and sodium stearyl lactates.

(68) Industrial Process

(69) The present invention is particularly useful for preparing a dough and baked product in an industrialized process, wherein the dough for preparing the baked product is prepared in a mechanistic way by using automated or semi-automated equipment.

(70) The process for preparing bread generally relates to following sequential steps: making dough (and optional proofing step), sheeting or dividing, shaping or rolling, and proofing the dough, these steps are well known in the art.

(71) If using the optional proofing step, then preferably adding more flour and can add a base to neutralize the acid generated in this step or to be generated in the second proofing step. In the production process of industrial baking according to the present invention, one or more of these steps are carried out by using automated or semi-automated equipment.

(72) The invention described and claimed herein is not limited to the scope of the specific embodiments disclosed herein, and these embodiments are only illustrative of several aspects of the invention.

Embodiment 1: Use of Enzyme for Reducing Oil in Toast

(73) Direct dough fermentation process was used for preparing toast, wherein all used raw materials were at food grade, fungal amylase was Fungamyl 2500 SG, xylanase was Pentopan Mono BG, glucose oxidase was Gluzyme Mono 10000BG, cellulase was Celluclast BG, all were commercial products of Novozymes A/S, and maltose amylase (the compounding of SEQ ID NO: 2 and SEQ ID NO: 3) was derived from Bacillus stearothermophilus.

(74) According to the ingredients list of table 1 and the protocol of table 2, flour, edible salt, sucrose, yeast, calcium propionate, SSL, CSL, and DMG were weighed and added into a flour cylinder (a vertical stirring and kneading machine, DIOSNA brand), enzymes, water and milk flavor were added and stirred, and the water temperature was adjusted such that the temperature of the dough was maintained at 23 C. after being mixed. Artificial butter was added, the mixture was stirred continuously until the gluten was extended. The made dough was standing for 10 min, then the dough was divided at 450 g/dough. The divided doughs were stood for 10 min after being preliminarily shaped, then were further shaped twice by a pressing and shaping machine. The dough was placed into a mould, then was placed into a proofing box and proofed for 120 min at the proofing temperature of 35 C. and the proofing humidity of 85%. The proofed dough was placed into a oven for baking, the oven temperature (broiling 185 C., baking 185 C.). The baked toast was cooled and prepared into a product.

(75) TABLE-US-00001 TABLE 1 Recipe of raw materials Baking percentage Recipe % (based on flour) Pengtai wheat flour 100 (Bimbo toast slice flour) Water 68 Yeast 1.2 (Angel high activity dry yeast) Sucrose 15 Artificial butter 6 or 10 (Namchow vege bake margarine) Edible salt 1 Vitamin C 0.01 Calcium propionate 0.2 (Madale) Sodium stearyl lactate 0.125 (Henan Zhengtong, SSL6425) Calcium stearyl lactate 0.125 (Henan Zhengtong, CSL6024) Molecular distilled monoglyceride 0.2 (DuPont, PH200) Maxim milk flavor 0.098 (W1310456) Basal enzymes Xylanase (30 ppm), fungal amylase (12 ppm), glucose oxidase (30 ppm) Oil reducing enzymes 60 ppm

(76) TABLE-US-00002 TABLE 2 Protocol Usage amount Maltose Batch of fat and oil amylase Cellulases A 10% 0 ppm 0 ppm B 6% 0 ppm 0 ppm C 6% 60 ppm 0 ppm D 6% 55 ppm 5 ppm

(77) Method for determining hardness: The toast was divided by using a toast slicer (the thickness of each bread slice was 1.2 cm), two sliced bread slices were in one group (the thickness was 2.4 cm), and were determined by a TA.XT Plus texture analyzer. Gram was used as the unit, the higher the hardness value, represented that the bread softness of the prepared bread was lower, and the quality was poorer.

(78) Method for determining elasticity: The toast was divided by using a toast slicer (the thickness of each bread slice was 1.2 cm). Two sliced bread slices were in one group (the thickness was 2.4 cm), and were determined by a TA.XT Plus texture analyzer. % was used as the unit. The higher the elasticity value, represented that the quality of the prepared bread was better.

(79) Sensory evaluation: Touch softness, bread crumb structure, olfactory fragrance, taste softness, taste moisture, and gustatory aroma of above-mentioned each batch of prepared toasts were scored by 5 skilled test baker (the bread prepared in batch A was scored 5.0 points and used as the baseline, the score which was not less than 4.0 represented that it did not substantially worsen). The mean was taken for a comprehensive evaluation, wherein the higher the score of the mean, represented that the quality of the prepared bread was better.

(80) The total sensory evaluation of each batch of prepared breads at 24 hours after being prepared (i.e. day 1) was: the mean of batch A was 5.0 points, the mean of batch B was 4.8 points, the mean of batch C was 5.2 points, and the mean of batch D was 5.3 points; and the sensory evaluation of each batch of prepared breads at 336 hours after being prepared (i.e. day 14) was: the mean of batch A was 5.0 points, the mean of batch B was 4.2 points, the mean of batch C was 5.4 points, and the mean of batch D was 5.6 points.

(81) It can be seen that in the preparation process of toast, although the usage amount of edible fat and oil was greatly reduced (the usage amount was reduced 40%), the preparation cost of bread was greatly saved and the fat and oil content was greatly reduced. However, due to the addition of the maltose amylase, the sensory evaluation of the prepared bread did not worsen relative to the reference solution, even was improved to some extent, wherein when maltose amylase and cellulase were added into the dough, the sensory evaluation of the prepared bread was better, so there is a kind of synergistic effect between maltose amylase and cellulase.

(82) In addition, it can be known from table 3 bellow, compared with the product of batch A as the control, when the usage amount of edible fat and oil in the preparation process was reduced from 10% to 6% based on the flour weight, the elasticity value of the prepared toast was relatively good, but the hardness value was significantly higher. However, under the same low level of usage amount of fat and oil, the bread prepared by adding maltose amylase into the dough exhibited very excellent softness and elasticity. Especially, when maltose amylase and cellulase were added into the dough, the hardness value of the prepared bread was the lowest. All toast prepared by a method related to an enzymatic method exhibited more obvious advantages in terms of softness and elasticity as the storage time of the breads was increasing. For example, the quality at day 14 of the bread prepared in batch C was better than the level at day 7 of the bread prepared in batch A, and the texture at day 14 of the bread prepared in batch D was obviously better than the level at day 7 of the bread prepared in batch A, especially raising the softness while maintaining a very good elasticity.

(83) TABLE-US-00003 TABLE 3 Hardness and elasticity of products of each batch Day 1 Day 4 Day 7 Day 14 Batch (Hardness/elasticity) (Hardness/elasticity) (Hardness/elasticity) (Hardness/elasticity) A 220.23 g/60.21% 352.50 g/54.26% 454.36 g/55.10% 619.74 g/51.59% B 288.12 g/61.01% 481.42 g/56.91% 550.97 g/55.48% 748.71 g/52.94% C 222.63 g/63.01% 311.68 g/59.44% 332.54 g/58.63% 437.75 g/58.86% D 209.36 g/62.93% 303.97 g/60.96% 308.20 g/60.25% 404.84 g/59.66%

(84) It suggests that in the preparation process of toast with a relatively high oil content, by adding maltose amylase into a dough, preferably complex cellulase, it can ensure that the overall evaluation of key indicators of the prepared toast did not worsen or substantially worsen, while the usage amount of edible fat and oil in the preparation was greatly reduced, which not only reduced preparation cost of the baked product greatly, but also can significantly reduce the usage amount of edible fat and oil in the preparation process, therefore the prepared baked product also was more aligned with the concept of healthy food.

Embodiment 2: Use of Enzyme for Reducing Oil in Toast

(85) Direct dough fermentation process was used for preparing toast, wherein all used raw materials were at food grade, fungal amylase was Fungamyl 2500 SG, xylanase was Pentopan Mono BG, glucose oxidase was Gluzyme Mono 10000BG, cellulase was Celluclast BG, all were commercial products of Novozymes A/S, and maltose amylase (SEQ ID NO: 1 or SEQ ID NO: 2), were all derived from Bacillus stearothermophilus.

(86) According to the ingredients list of table 4 and the protocol of table 5, flour, edible salt, sucrose, yeast, calcium propionate, SSL, CSL, and DMG were weighed and added into a flour cylinder (a vertical stirring and kneading machine, DIOSNA brand), enzymes, water and milk flavor were added and stirred, and the water temperature was adjusted such that the temperature of the dough was maintained at 23 C. after being mixed. Artificial butter was added, the mixture was stirred continuously until the gluten was extended. The made dough was standing for 10 min, then the dough was divided at 450 g/dough. The divided doughs were stood for 10 min after being preliminarily shaped, then were further shaped twice by a pressing and shaping machine. The dough was placed into a mould, then was placed into a proofing box and proofed for 120 min at the proofing temperature of 35 C. and the proofing humidity of 85%. The proofed dough was placed into a oven for baking, the oven temperature (broiling 185 C., baking 185 C.). The baked toast was cooled and prepared into a product.

(87) TABLE-US-00004 TABLE 4 Recipe of raw materials Baking percentage Recipe % (based on flour) Pengtai wheat flour 100 (Bimbo toast slice flour) Water 62 Yeast 1.2 (Angel high activity dry yeast) Sucrose 15 Artificial butter 6 or 10 (Namchow vege bake margarine) Edible salt 1 Vitamin C 0.01 Calcium propionate 0.2 (Madale) Sodium stearyl lactate 0.125 (Henan Zhengtong, SSL6425) Calcium stearyl lactate 0.125 (Henan Zhengtong, CSL6024) Molecular distilled monoglyceride 0.2 (DuPont, PH200) Maxim milk flavor 0.098 (W1310456) Basal enzymes Xylanase (30 ppm), fungal amylase (12 ppm), glucose oxidase (30 ppm) Oil reducing enzymes 60 ppm

(88) TABLE-US-00005 TABLE 5 Protocol: Usage Maltose amylase Maltose amylase Cellulase amount of (SEQ ID NO: 2)/ (SEQ ID NO: 1)/ Celluclast Batch fat and oil % ppm ppm BG/ppm A 10 0 0 0 B 6 60 0 0 C 6 55 0 5 D 6 0 60 0

(89) According to the parameter characterization method in embodiment 1, sensory evaluation was performed on each batch of prepared breads at 24 hours (i.e. day 1), 96 hours (i.e. day 4), 168 hours (i.e. day 7), and 336 hours (i.e. day 14) after being prepared, as shown in table 6:

(90) TABLE-US-00006 TABLE 6 Sensory evaluation of each batch of breads Batch Day 1 Day 4 Day 7 Day 14 A 5.0 5.0 5.0 5.0 B 5.1 5.4 5.5 5.6 C 5.3 5.4 5.5 5.6 D 5.2 5.2 5.4 5.3

(91) It can be seen that in the preparation process of toast, although the usage amount of edible fat and oil was greatly reduced (the usage amount was reduced 40%), the preparation cost of bread and the fat and oil content were greatly saved. However, due to the addition of the maltose amylase (SEQ ID NO: 1 or SEQ ID NO: 2), the sensory evaluation of the prepared bread did not worsen relative to the reference solution, even was improved to some extent.

(92) In addition, it can be known from table 7 bellow, compared with the product of batch A as the control, under the low level of usage amount of fat and oil, the bread prepared by adding maltose amylase into the dough exhibited very excellent softness and elasticity at day 1 and day 14, wherein when maltose amylase and cellulase were added into the dough, the sensory evaluation of the prepared bread was better, so there is a kind of synergistic effect between maltose amylase and cellulase. The evaluation spanning 14 days is sufficient for short-shelf life breads such as toast to suggest that by adding maltose amylase into a dough, it can ensure that the overall evaluation of key indicators of the prepared toast did not worsen or substantially worsen, or even can be improved, while the usage amount of edible fat and oil in the preparation was greatly reduced.

(93) TABLE-US-00007 TABLE 7 Hardness and elasticity of products of each batch Day 1 Day 14 Batch (Hardness/elasticity) (Hardness/elasticity) A 169.15 g/61.85% 484.00 g/50.71% B 162.98 g/65.98% 265.94 g/64.11% C 160.64 g/65.67% 238.01 g/63.81% D 150.61 g/65.61% 382.80 g/58.95%

Embodiment 3: Use of Enzyme for Reducing Oil in French Roll

(94) Direct dough fermentation process was used for preparing toast, wherein all used raw materials were at food grade, fungal amylase was Fungamyl 2500 SG, xylanase was Pentopan Mono BG, glucose oxidase was Gluzyme Mono 10000BG, cellulase was Celluclast BG, all were commercial products of Novozymes A/S, and maltose amylase (the compounding of SEQ ID NO: 2 and SEQ ID NO: 3) was derived from Bacillus stearothermophilus.

(95) According to the following ingredients list of table 8, flour, edible salt, sucrose, yeast, CSL and sodium dehydroacetate materials were weighed and added into a flour cylinder (a vertical stirring and kneading machine, DIOSNA brand), mixed uniformly, eggs, syrup, water and enzymes were added and stirred, shortening was added, the mixture was stirred until the gluten was extended, and the temperature of the dough was maintained at 23 C. The dough was divided at 200 g/dough, then pressed 30 times until the gluten is complete. The dough was rolled, divided at 25 g/dough, placed into a mould, then was placed into a proofing box and proofed for 90 min at the proofing temperature of 35 C. and the proofing humidity of 85%. The proofed dough was placed into a oven for baking, the oven temperature (broiling 200 C., baking 155 C.), and the baking temperature is 10 min. The baked French roll was cooled and packaged.

(96) TABLE-US-00008 TABLE 8 Recipe of raw materials Baking percentage Recipe % (based on flour) Pengtai wheat flour 100 Water 35 Eggs 10 Yeast 1.5 (Angel high activity dry yeast) Sucrose 20 Shortening 12.5 (Nanqiao Kinsco sheet margarine) Fructose-glucose syrup F42 3 (Henan Feitian) Edible salt 0.8 Full cream formulated milk powder 5 (Nestl) Vitamin C 100 ppm Sucrose esters (Henan Wanbang) 0.08 Calcium stearyl lactate 0.08 (Henan Zhengtong, CSL6024) Sodium dehydroacetate 0.1 (Madale) Basal enzymes Xylanase (30 ppm), fungal amylase (12 ppm), glucose oxidase (30 ppm) Compounded oil reducing enzymes Maltose amylase and cellulase

(97) TABLE-US-00009 TABLE 9 Protocol Maltose Batch Shortening % amylase/ppm Cellulase/ppm A 12.5 0 0 B 8 0 0 C 8 94 6

(98) Sensory evaluation: touch softness, tactile elasticity, bread crumb structure, olfactory fragrance, taste softness, taste moisture, and gustatory aroma of above-mentioned each batch of prepared French rolls were scored by 5 skilled test baker (the roll prepared in batch A was scored 5.0 points and used as the baseline). The mean was taken for a comprehensive evaluation, wherein the higher the score of the mean, represented that the quality of the prepared roll was better.

(99) Sensory evaluation was performed on each batch of prepared French rolls at 24 hours (i.e. day 1), 72 hours (i.e. day 3), 336 hours (i.e. day 14), 504 hours (i.e. day 21) and 672 hours (i.e. day 28) after being prepared:

(100) TABLE-US-00010 TABLE 10 Sensory evaluation of each batch of breads Batch A Batch B Batch C Day 1 5.0 4.7 4.9 Day 3 5.0 4.7 5.0 Day 14 5.0 4.8 5.1 Day 21 5.0 4.9 5.1 Day 28 5.0 4.7 5.3

(101) As shown in table 10 above, in the preparation process of French roll, although the usage amount of the shortening was greatly reduced (the usage amount was reduced 36%), the preparation cost of bread was saved. However, due to the addition of the compounded oil reducing enzyme of maltose amylase and cellulase, the sensory evaluation of the prepared bread did not worsen, even was improved to some extent. The evaluation spanning 28 days is sufficient for middle-shelf life breads such as French roll to suggest that in the preparation process of French roll with a relatively high oil content, by adding maltose amylase into a dough, preferably complex cellulase, it can ensure that the overall evaluation of key indicators of the prepared toast did not worsen or substantially worsen, while the usage amount of edible fat and oil in the preparation was greatly reduced, which not only reduced preparation cost of the baked product greatly, but also can significantly reduce the usage amount of edible fat and oil in the preparation process, therefore the prepared baked product also was more aligned with the concept of healthy food.

Embodiment 4: Use of Enzyme for Reducing Oil in Sponge Cake

(102) Whole egg stirring process was used for preparing sponge cake, wherein all used raw materials were at food grade.

(103) According to the ingredients list of table 11 and the protocol of table 12, calcium propionate, baking soda, whole milk powder, flour and enzyme were mixed and sieved for later use. Eggs, edible sugar, edible salt and enzymes were added to a stirring tank (a vertical stirring tank, DIOSNA brand) and stirred for 2 minutes; all liquid and powder ingredients except oil were added, stirred for 1 minute, the tank was scraped, and the mixture was stirred continuously for 2 minutes; vegetable oil was added, stirred for 1 minute, the tank was scraped, then the batter was poured into a mould at 300 g/portion, placed into a oven for baking, the oven temperature (broiling 180 C., baking 170 C.). The baked cake was cooled and prepared into a product. The used oil reducing enzyme is the compounding of cellulase and maltose amylase, wherein the cellulase is Celluclast BG (commercial products of Novozymes A/S), and maltose amylase (the compounding of SEQ ID NO: 2 and SEQ ID NO: 3) was derived from Bacillus stearothermophilus.

(104) TABLE-US-00011 TABLE 11 Recipe of raw materials Baking percentage Recipe % (based on batter) Flour 25 (Queen low gluten flour) Eggs 28 (purchased from Deqingyuan) Powdered sugar 27 Vegetable oil (purchased 6 or 11 from Golden dragon fish) Water 5 Whole milk powder 1 (purchased from Nestl) Edible salt 0.3 Baking soda 0.3 (purchased from Puratos) Calcium propionate 0.4 (Madale) Oil reducing enzymes 200 ppm

(105) TABLE-US-00012 TABLE 12 Protocol Usage amount Oil reducing Batch of fat and oil enzymes A 11% 0 ppm B 6% 0 ppm C 6% 200 ppm
Parameter Characterization

(106) Method for determining hardness: The cake was smoothly divided (the height of each cake was 45 cm), and determined by a TA.XT Plus texture analyzer. Gram was used as the unit, the higher the hardness value, represented that the bread softness of the prepared cake was lower, and the quality was poorer.

(107) The ratio of the energy (positive peak area integration under the curve) of the two pressing processes in the texture test was used as the texture of samples to test viscidity. The viscidity reflects the taste quality of the cake. The higher the viscidity, the better the viscidity of the prepared cake and the better the quality.

(108) Sensory evaluation: Taste softness, taste viscidity, taste moisture, and melt-in-the-mouth effect of above-mentioned each batch of prepared cakes were scored by 5 skilled test baker (the cake prepared in batch A was scored 5.0 points and used as the baseline, a gap of 0.5 points represented that there is a substantial quality difference). The mean was taken for a comprehensive evaluation, wherein the higher the score of the mean, represented that the quality of the prepared cake was better.

(109) The total sensory evaluation of each batch of prepared cakes at 72 hours after being prepared (i.e. day 3) was: the mean of batch A was 5 points, the mean of batch B was 3 points, and the mean of batch C was 5 points; each batch of prepared cakes at 168 hours after being prepared (i.e. day 7) was: the mean of batch A was 5 points, the mean of batch B was 4 points, and the mean of batch C was 5.8 points; and each batch of prepared cakes at 360 hours after being prepared (i.e. day 15) was: the mean of batch A was 5 points, the mean of batch B was 4 points, and the mean of batch C was 5.3 points.

(110) It can be seen that in the preparation process of cake, although the usage amount of edible fat and oil was greatly reduced (the usage amount was reduced about 45%), the preparation cost of cake was greatly saved and the fat and oil content was reduced. However, due to the addition of the oil reducing enzyme, the sensory evaluation of the prepared cake did not worsen relative to the reference solution, even was improved to some extent, and the sensory evaluation of the prepared cake was better.

(111) TABLE-US-00013 TABLE 13 Sensory evaluation of each batch of products Day 3 Day 7 Day 15 (softness/ (softness/ (softness/ moisture/melt-in- moisture/melt-in- moisture/melt-in- Batch the-mouth effect) the-mouth effect) the-mouth effect) A 5/5/5 5/5/5 5/5/5 B 3/3/3 4/4/4 4/4/4 C 5/5/5 6/6/5.5 5.5/5.5/5

(112) In addition, it can be known from table 14 bellow, compared with the product of batch A as the control, when the usage amount of edible fat and oil in the preparation process was reduced from 11% to 6%, the viscidity value of the cake prepared by batch B was relatively good, but the hardness value was significantly higher. However, under the same low level of usage amount of fat and oil, the cake prepared by adding oil reducing enzyme into the formula of cakes exhibited very excellent softness and viscidity. The cake prepared by a method related to an enzymatic method exhibited more obvious advantages in terms of softness and viscidity as the storage time of the cakes was increasing. For example, the texture at day 15 of the cake prepared in batch C was obviously better than the level at day 7 of the cake prepared in batch A, especially raising the softness while raising the viscidity.

(113) TABLE-US-00014 TABLE 14 Hardness and viscidity of products of each batch Day 3 Day 7 Day 15 Batch (hardness/viscidity) (hardness/viscidity) (hardness/viscidity) A 432 g/0.63 510 g/0.56 597 g/0.52 B 439 g/0.66 579 g/0.59 623 g/0.52 C 319 g/0.75 371 g/0.66 492 g/0.59

(114) It suggests that in the preparation process of cake with a relatively high oil content, by adding oil reducing enzyme into a formula, it can ensure that the overall evaluation of key indicators of the prepared cake did not worsen or substantially worsen, while the usage amount of edible fat and oil in the preparation was greatly reduced, which not only reduced preparation cost of the baked product greatly, but also can significantly reduce the usage amount of edible fat and oil in the preparation process, therefore the prepared baked product also was more aligned with the concept of healthy food.