Improved baking compositions

Abstract

The present invention relates to an improved baking composition. It has been found that a combination of enzymes, in particular a combination of a thermophilic serine protease and a lipase, is capable of improving the softness of a baked product. Compositions comprising these enzymes, the use of such a combination of enzymes, and methods of preparing a baked product using a combination of a thermophilic serine protease and a lipase are provided herein.

Description

[0001] This application is a divisional application of the invention patent application filed on July 10, 2017, with application number 201780042881.5 and invention title "Improved Baking Composition". Technical Field

[0002] This invention relates to the improvement of the short bite of baked goods. Background Technology

[0003] When purchasing baked goods, consumers consider a range of parameters such as appearance, softness, moisture, and aroma. The edibility of the baked goods is also important; they should, for example, be easy to bite into without requiring extensive chewing, as this aspect is considered a characteristic of freshness. Consumers also prefer bread with the lowest possible (labeled) additives.

[0004] During aging, the characteristics of baked goods change. In particular, the aroma changes, the product becomes harder, drier, and more difficult to chew, thus baked goods are considered "not so fresh".

[0005] The softness of a baked product can be defined as the ease with which a piece of baked product can be bitten into or torn apart. This is reflected by the force required to break the sample and the number of chews required to break the sample down to a consistency ready to be swallowed. To some extent, softness is the opposite of chewiness. Furthermore, softness is very different from tenderness. In fact, the tenderness of bread is reflected by the force required to compress the sample to a certain deformation. Tenderness is also the opposite of hardness. Therefore, bread can be tender without being soft, and vice versa.

[0006] Several methods have been proposed to improve the softness of baked goods. EP0776604 describes the use of unsaturated glycerol monoesters to produce microwaveable crisp bread rolls with a soft texture. WO2009138447 describes the use of intermediate heat-stable or heat-stable serine or metalloproteinases to improve the softness of baked goods.

[0007] However, these methods have been observed to have their limitations. In Europe, the addition of monoglycerides or other emulsifiers requires a specific E-number marking for baked goods, some of which has prevented some consumers from purchasing the product.

[0008] The use of intermediate or thermally stable serine or metalloproteinases has improved the softness of baked goods to some extent, but this improvement is limited in a certain way because high levels of enzymes tend to be detrimental to other properties of baked goods, such as crumbliness and resilience.

[0009] Therefore, new methods and products are needed to further improve softness. Summary of the Invention

[0010] The inventors have discovered that in baking applications, particularly in bread making, the combination of thermophilic serine proteases and lipases, which are optimally active at temperatures above 70°C, has a synergistic effect on softness.

[0011] Therefore, in a first aspect, the present invention relates to a composition comprising: - At least one first enzyme, wherein the first enzyme is a thermophilic serine protease that exhibits optimal activity at temperatures above 70°C, preferably above 80°C; and - At least one second enzyme, wherein the second enzyme is a lipase.

[0012] In a particular embodiment, the compositions disclosed herein provide that the first enzyme is a thermophilic serine protease, wherein the ratio between the protease activity at the optimal temperature and the protease activity at 25°C is greater than 10, preferably greater than 15.

[0013] In certain embodiments, the compositions disclosed herein provide that the first enzyme is a neutral or basic thermophilic serine protease.

[0014] In certain embodiments, the composition as disclosed herein provides that the first enzyme is preferably derived from... Aquatic habitat bacteria ( Thermus aquaticus Taq protease isolated from [the source] is preferably aqualysin I or aqualysin II, more preferably aqualysin I, and even more preferably [from] [the source]. Aquatic thermophilic bacteria Aquaporinase I isolated from LMG8924.

[0015] In a particular embodiment, the composition disclosed herein provides the first enzyme present in an amount of 100 to 1200 units / 100 kg flour, preferably 200 to 900 units / 100 kg flour, more preferably 350 to 700 units / 100 kg flour.

[0016] In certain embodiments, the compositions disclosed herein provide that the second enzyme is a triacylglycerol lipase or triacylglycerol acyl hydrolase as defined in enzyme entry EC 3.1.1.3, preferably selected from... cotton Thermophilic filamentosa ( Thermomyces lanuginosus ), Rhizopus oryzae ( Rhizopus oryzae )and Rhizopus oryzae ( Rhizomucor miehei The lipase obtained.

[0017] In a particular embodiment, the compositions disclosed herein provide the second enzyme present in amounts of 5 to 100 LU from cottony thermophilic filamentous ... Lipase / 100 kg flour; 0.023 to 0.360 LU from Rhizopus oryzae Lipase / 100 kg flour; and 50 to 200 LU from Rhizopus oryzae Lipase per 100 kg of flour.

[0018] Therefore, in another aspect, the present invention relates to the use of the compositions disclosed herein in baking applications.

[0019] In certain embodiments, the compositions disclosed herein are used in bread improvers.

[0020] In certain embodiments, the compositions disclosed herein are used in soft-baked products and hard-crust baked products, preferably bread, soft rolls, donuts, rolls, microwaveable rolls, Danish pastries, croissants, hamburger rolls, pizzas and pita breads, and cakes.

[0021] Therefore, in another aspect, the present invention relates to a bread improver comprising the composition disclosed herein.

[0022] Therefore, in another aspect, the present invention relates to a method for preparing baked goods, comprising adding the following steps to dough or batter prior to baking: - At least one first enzyme, wherein the first enzyme is a thermophilic serine protease that exhibits optimal activity at temperatures above 70°C, preferably above 80°C; and - At least one second enzyme, wherein the second enzyme is a lipase.

[0023] In a particular embodiment, the method disclosed herein provides that the baked product exhibits improved softness, preferably wherein, compared with a reference baked product prepared without the first or second enzyme, the maximum force required to destroy the baked product prepared using at least one first enzyme and at least one second enzyme is reduced by at least 15%, wherein the at least one first enzyme is a thermophilic serine protease having optimal activity at temperatures above 70°C, preferably above 80°C, and the at least one second enzyme is a lipase.

[0024] Therefore, in a particular embodiment, the compositions disclosed herein are used to prepare baked goods with improved fluffiness, wherein preferably, the maximum force required to destroy the prepared baked goods is reduced by at least 15% compared to a reference baked goods prepared without the first or second enzyme.

[0025] In certain implementations, the methods disclosed herein provide no observed adverse effects on dough rheology, breadcrumb structure, and the volume of the resulting baked product.

[0026] Therefore, in another aspect, the present invention relates to a baking product prepared from dough or batter comprising the compositions disclosed herein. Attached Figure Description

[0027] Figures 1A to 1C This refers to different aspects of assessing softness using a texture analyzer. Figure 1A This indicates that it is equipped with a pizza stretching device (a texture analyzer with two probes and pins). Figure 1B This indicates the actual measurement setting, in which a small round bread is attached to a pizza stretching device, and in which the upper probe moves upward at a constant speed until the small round bread breaks. Figure 1C A typical graph of the measurement is shown, where the required force, expressed in grams (g), is measured as a function of time (s). Detailed Implementation

[0028] Before describing the present products, compositions, uses, and methods of the invention, it should be understood that the invention is not limited to the specific products, compositions, uses, and methods or combinations described, as these products, compositions, uses, and methods, and combinations can certainly vary. It should also be understood that the terminology used herein is not intended to be limiting, as the scope of the invention will be defined only by the appended claims.

[0029] As used in this article, the singular forms “a,” “an,” and “the” include both singular and plural objects, unless the context explicitly indicates otherwise.

[0030] As used herein, the terms “comprising,” “containing,” and “consisting of” are synonymous with “including,” “containing,” or “having,” and are inclusive or open-ended, and do not exclude additional unlisted components, elements, or method steps. It should be understood that the terms “comprising,” “containing,” and “consisting of” as used herein include the terms “consisting of,” “consists,” and “consists of.”

[0031] The numerical range represented by the endpoints includes all numbers and fractions that fall into the corresponding range, as well as the endpoints themselves.

[0032] When referring to measurable values ​​such as parameters, quantities, durations, etc., the terms "about" or "approximately" as used herein refer to variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and even more preferably + / -0.1% or less, of the specified value, which, where appropriate, are suitable for performing the disclosed invention. It should be understood that the values ​​referenced by the modifier "about" or "approximately" are themselves specifically and preferably disclosed.

[0033] While the terms “one or more” or “at least one”, such as one or more or at least one member of a group of members, are self-evident, by further example, the term specifically includes references to any one of the members, or to any two or more of the members, such as any ≥3, ≥4, ≥5, ≥6 or ≥7 of the members, and up to all of the members.

[0034] All references cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings of all references specifically mentioned herein are incorporated herein by reference.

[0035] Unless otherwise defined, all terms used in this disclosed invention (including technical and scientific terms) have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance, including terminology definitions, is provided to better understand the teachings of this invention.

[0036] The various aspects of the invention are defined in more detail in the following paragraphs. Each aspect thus defined may be combined with any other one or more aspects unless explicitly indicated to the contrary. In particular, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

[0037] Throughout this specification, references to "one embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout the specification are not necessarily, but can all refer to the same embodiment. Furthermore, as will readily be apparent to those skilled in the art from this disclosure, in one or more embodiments, particular features, structures, or characteristics can be combined in any suitable manner. Moreover, while some embodiments described herein include certain, but not others, features included in other embodiments, combinations of features from different embodiments are also within the scope of the invention and form different embodiments, as will be understood by those skilled in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0038] The inventors were surprised to find that by using a new combination of enzymes, particularly a combination of thermophilic serine protease and lipase, they could achieve a more significant improvement in tenderness than the expected improvement in tenderness when the enzymes were used alone.

[0039] Softness, sometimes referred to as the opposite of chewiness and / or toughness, is used to specify the force required to break down a sample of baked goods and / or the number of chews required to chew a sample of baked goods to a consistency ready to be swallowed. Softness can be readily measured by trained taste-testing teams and can be reasonably quantified using any softness scale. Such techniques are well-known in the food industry and are often referred to as sensory testing. In this approach, an initial training session is organized to familiarize team members with the range of products to be tested. During this session, reference standards are laid out to train team members to identify differences between the attributes of the products being measured. In a second step, team members receive numbers or samples of the products on a scale from 0 to 10 to rate their softness.

[0040] Softness can also be assessed using a texture analyzer. In this method, small round rolls are attached to a pizza stretching device (with two probes and pins) (see...). Figure 1A The upper probe will move upward at a constant speed and break the small round loaf in this way (see...). Figure 1B The required force, expressed in grams (g), is measured using a texture analyzer. Throughout the process, the required force increases until the small loaf of bread cracks, at which point the force decreases. A typical graph of this measurement is shown in [image / graph]. Figure 1C The maximum force (F) measured is shown in the figure. max () is a common parameter used to assess softness.

[0041] The inventors were surprised to find that using sufficient amounts of thermophilic serine protease and sufficient amounts of lipase simultaneously in the dough before baking showed an unexpected synergistic effect on improving the softness of baked products.

[0042] In fact, the combination of two types of enzymes gives a greater degree of tenderness than the sum of the effects of using the enzymes alone. A synergistic effect exists when the combined effect of the amount of thermoserious protease x and the amount of lipase y is greater than the sum of the effects of the amount of thermoserious protease x and the amount of lipase y.

[0043] Therefore, in a first aspect, the present invention relates to a composition comprising: - At least one first enzyme, wherein the first enzyme is a thermophilic serine protease; and - At least one second enzyme, wherein the second enzyme is a lipase.

[0044] In a particular embodiment, the first enzyme is a thermophilic serine protease that exhibits optimal activity at temperatures above 70°C, preferably above 75°C, and more preferably above 80°C.

[0045] It has been further discovered that the synergistic effect mentioned herein is particularly present when the thermophilic serine protease mentioned herein exhibits optimal activity at temperatures above 70°C, preferably above 75°C, and more preferably above 80°C.

[0046] As used herein, the term "protease" generally refers to an enzyme that hydrolyzes peptide bonds (also called peptidases or proteases) that link amino acids together in a polypeptide chain, preferably as defined in enzyme entry EC 3.4. They are classified into several categories based on their catalytic residues. Among these categories, serine proteases (or serine endopeptidases) are proteases that cleave peptide bonds in proteins, where serine serves as a nucleophilic amino acid at the active site. Serine proteases are defined by enzyme entry EC 3.4.21. Serine proteases can be further subdivided according to their substrate specificity into trypsin-like, chymotrypsin-like, thrombin-like, elastase-like, or subtilisin-like substances.

[0047] In the context of this invention, casein cross-linked with azuril (AZCL-casein) is used as a substrate to measure protease activity. Hydrolysis of the protease produces water-soluble staining fragments, and the release rate of these fragments (e.g., an increase in absorbance at 590 nm) is directly correlated with enzyme activity (Protazyme AK tablets, Megazyme, Ireland). Further details regarding protease activity measurements are given in the examples. Protease activity can also be measured using other assays known to those skilled in the art for protease activity. One of these is a calorimetric method using casein as a substrate followed by detection of released amino acids using Folin & Ciocalteu's phenol reagent.

[0048] As used herein, the term "thermophilic," and in particular "thermophilic protease," as used herein, refers to a protease that becomes active at elevated temperatures. In particular, thermophilic proteases exhibit optimal activity at temperatures above 70°C, preferably above 75°C, and more preferably above 80°C.

[0049] As used herein, the term "lipase" generally refers to a triacylglycerol lipase or triacylglycerol acylhydrolase as defined by enzyme entry EC 3.1.1.3. Lipases are defined herein as enzymes that catalyze the hydrolysis of triacylglycerols to produce free fatty acids, diacylglycerols, monoacylglycerols, and glycerol. Lipases used in compositions defined herein may include enzymatic activities, such as phospholipase activity.

[0050] In the context of this invention, p-nitrobenzene palmitate (pNPP) is used as a substrate and lipase activity is measured according to the methods described herein. Enzyme activity can also be measured using other assays for lipase activity known to those skilled in the art (see, for example, Stoytcheva M. & al, 2012, Current Analytical Chemistry, Vol. 8, p. 400 for a review).

[0051] Specifically, p-nitrophenyl palmitate (pNPP) was used as a substrate to measure lipase activity. The release of yellow p-nitrophenol caused by lipase hydrolysis of p-nitrophenyl palmitate was measured spectrophotometrically at 414 nm. One milli-unit of lipase (LmU) is defined as the amount of enzyme required to release 1 nanomolar (nmol) of p-nitrophenol per minute from p-nitrophenyl palmitate at 45°C and pH 7.5. Further details regarding the measurement of lipase activity are given in the examples. Enzyme activity can also be measured using other assays known to those skilled in the art for lipase activity (see, for example, Stoytcheva M. & al, 2012, Current Analytical Chemistry, Vol. 8, p. 400 for a review).

[0052] In a particular embodiment, the compositions disclosed herein provide that the first enzyme is a thermophilic serine protease, wherein the ratio between the protease activity at the optimal temperature and the protease activity at 25°C is greater than 10, preferably greater than 15. By providing said ratio greater than 10, the thermophilic serine protease used herein provides an improved effect on tenderness.

[0053] In certain embodiments, the compositions disclosed herein provide that the first enzyme is obtained by synthesis or by genetic engineering, through extraction from naturally occurring eukaryotes or prokaryotes. In certain embodiments, the compositions disclosed herein provide that the first enzyme is a neutral or basic thermophilic serine protease. While fungal proteases are sensitive to high temperatures, bacterial neutral and basic proteases are more tolerant of higher heat treatments.

[0054] In certain embodiments, the composition as disclosed herein provides that the first enzyme is preferably derived from... Aquatic habitat bacteria The Taq protease isolated from [the source] is preferably lysozyme I or lysozyme II, more preferably lysozyme I, and even more preferably [from [the source]]. Aquatic thermophilic bacteria Aquaporinase I isolated from LMG8924.

[0055] In a particular embodiment, the composition disclosed herein provides the first enzyme, preferably hydrolysin I, in an amount of 100 to 1200 units / 100 kg flour, preferably 200 to 900 units / 100 kg flour, more preferably 350 to 700 units / 100 kg flour. Advantageously, hydrolysin I is added to dough / paste at an amount of 100 to 1200 units / 100 kg flour, preferably 200 to 900 units / 100 kg flour, more preferably 350 to 700 units / 100 kg flour, and enzyme activity is obtained using the method described herein.

[0056] In certain embodiments, the compositions disclosed herein provide that the second enzyme is a triacylglycerol lipase or triacylglycerol acyl hydrolase as defined in enzyme entry EC3.1.1.3, preferably selected from... cottony thermophilic filaments bacteria , Rhizopus oryzae and Rhizopus oryzae The obtained lipase.

[0057] In certain embodiments, the compositions disclosed herein provide the second enzyme present in amounts of 5 to 100 LU, preferably 15 to 75 LU, more preferably 20 to 60 LU. cottony thermophilic filamentous ... Lipase / 100 kg flour; 0.023 to 0.360 LU, preferably 0.09 to 0.18 LU from Rhizopus oryzae Lipase per 100 kg of flour; and 50 to 200 LU, preferably 90 to 160 LU, more preferably about 138 LU from Rhizopus oryzae Lipase / 100 kg flour, enzyme activity was obtained using the method described herein.

[0058] In certain embodiments, the compositions disclosed herein provide a ratio of the amount of the first enzyme to the amount of the second enzyme between 5 and 35 (for Taq protease and from...). cottony thermophilic filamentous ... The lipases (for Taq proteins and those derived from...) ranged between 1950 and 7800 (for Taq proteins and those derived from...). Rhizopus oryzae lipase), and / or between 2 and 8 (for Taq protease and from Rhizopus oryzae (lipase).

[0059] In certain embodiments, the compositions disclosed herein are bread improvers, French pastry mixtures, or French pastry premixes, preferably bread improvers.

[0060] During baking, bread improvers (also known as dough conditioners, dough improvers, or flour improvers) are typically added to the dough to improve the texture, volume, flavor, and freshness of baked goods, as well as the processability and stability of the dough. Typically, bread improvers include or consist of the following: one or more enzymes (e.g., amylase, xylanase, phospholipase, oxidase, lipoxygenase, dehydrogenase, and laccase), one or more oxidizing or reducing agents (e.g., ascorbic acid, glutathione, cysteine), one or more emulsifiers (e.g., monoglycerides (or mixtures of monoglycerides and diglycerides), monoglyceride derivatives (e.g., succinylated lactylated or acetylated monoglycerides, diacetyl tartrate of monoglycerides (DATEM), glyceryl monostearate (GMS), propylene glycol monoester, etc.), sorbitol emulsifiers (sorbitol monostearate), polysorbates, sodium stearoyl lactylate (SSL), polyglycerides, sucrose esters, and lecithin), one or more lipid materials (e.g., margarine, butter, oil, shortening), one or more vitamins (e.g., pantothenic acid and vitamin E), one or more gums, and / or one or more fiber sources (e.g., oat fiber). French pastry mixes typically contain all the ingredients of a French pastry product recipe except for water, fats (oil, butter, margarine), and eggs. French pastry premixes are typically French pastry mixtures in which all or part of the flour and sugar have been removed.

[0061] In a particular embodiment, the composition disclosed herein is a dough or paste comprising flour and at least one first enzyme and at least one second enzyme, wherein the first enzyme is a thermophilic serine protease that preferably has optimal activity at a temperature above 70°C, more preferably above 75°C, and even more preferably above 80°C; wherein the second enzyme is a lipase.

[0062] Specifically, the second enzyme is present in amounts of 5 to 100 LU, preferably 15 to 75 LU, more preferably 20 to 60 LU. cottony thermophilic filamentous ... Lipase / 100 kg flour; 0.023 to 0.360 LU, preferably 0.09 to 0.18 LU from Rhizopus oryzae Lipase per 100 kg of flour; and 50 to 200 LU, preferably 90 to 160 LU, more preferably about 138 LU from rice Mucor Lipase / 100 kg flour, enzyme activity was obtained using the method described herein.

[0063] In another embodiment, the compositions disclosed herein may further comprise appropriate amounts of the following substances: one or more enzymes (e.g., amylase, xylanase, phospholipase, oxidase, lipoxygenase, dehydrogenase, and laccase), one or more oxidizing or reducing agents (e.g., ascorbic acid, glutathione, cysteine), one or more emulsifiers (e.g., monoglycerides (or mixtures of monoglycerides and diglycerides), monoglyceride derivatives (e.g., succinylated lactylated or acetylated monoglycerides, diacetyl tartrate of monoglycerides (DATEM), glyceryl monostearate (GMS), propylene glycol monoester, etc.), sorbitol emulsifiers (sorbitol monostearate), polysorbates, sodium stearoyl lactylate (SSL), polyglycerides, sucrose esters, and lecithin), one or more lipid materials (e.g., margarine, butter, oil, shortening), one or more vitamins (e.g., pantothenic acid and vitamin E), one or more gums, and / or one or more fiber sources (e.g., oat fiber).

[0064] In yet another embodiment, the composition disclosed herein comprises: - At least one first enzyme, wherein the first enzyme is a thermophilic serine protease that has optimal activity at a temperature above 70°C, preferably above 75°C, and more preferably above 80°C. - One or more monoglycerides; and - At least one second enzyme, wherein the second enzyme is a lipase.

[0065] As used in this article, the term "monoglyceride" generally refers to a class of glycerides composed of glycerol molecules linked to fatty acids via ester bonds.

[0066] Monoglycerides are one of many types of emulsifiers used in baking applications: these include monoglycerides (or mixtures of monoglycerides and diglycerides; designated E471 by the International System for Food Additives (INS) or 184.1505 by the U.S. Food and Drug Administration), monoglyceride derivatives (such as succinylated lactylated or acetylated monoglycerides, diacetyl tartrate of monoglycerides (DATEM), glyceryl monostearate (GMS), propylene glycol monoester, etc.), sorbitol emulsifiers (sorbitol monostearate), polysorbates, sodium stearoyl lactylate (SSL), polyglycerides, sucrose esters, and lecithin.

[0067] Furthermore, in another aspect, the present invention relates to the use of the compositions disclosed herein in baking applications. In the context of this invention, baking applications refer to applications related to bread and French pastry products. In particular, the baked products are soft baked products and / or hard-crust baked products, preferably bread, soft rolls, donuts, rolls, microwaveable rolls, Danish pastries, croissants, hamburger rolls, pizzas and pita breads, and cakes.

[0068] In certain embodiments, the use of the compositions disclosed herein in bread improvers, French pastry mixtures, or French pastry premixes is provided.

[0069] Therefore, the object of the present invention is to provide the use of the compositions according to the invention for improving the softness of baked products, which includes adding a sufficient amount of one or more thermophilic serine proteases and a sufficient amount of one or more lipases disclosed herein to the dough prior to baking.

[0070] In certain embodiments, the compositions disclosed herein are used to improve the softness of baked goods.

[0071] In another aspect, this article discloses a method for preparing baked goods, comprising adding the following steps to dough or batter prior to baking: - At least one first enzyme, wherein the first enzyme is a thermophilic serine protease; and - At least one second enzyme, wherein the second enzyme is a lipase.

[0072] In particular, the first enzyme is a thermophilic serine protease that exhibits optimal activity at temperatures above 70°C, preferably above 75°C, and even more preferably above 80°C.

[0073] In the context of this invention, casein cross-linked with azuril (AZCL-casein) is used as a substrate to measure protease activity. Hydrolysis of the protease produces water-soluble staining fragments, and the release rate of these fragments (e.g., an increase in absorbance at 590 nm) is directly correlated with enzyme activity (Protazyme AK tablets, Megazyme, Ireland). Further details regarding protease activity measurements are given in the examples. Protease activity can also be measured using other assays known to those skilled in the art for protease activity. One of these is a calorimetric method using casein as a substrate followed by detection of released amino acids using Folin & Ciocalteu's phenol reagent.

[0074] In the context of this invention, p-nitrobenzene palmitate (pNPP) is used as a substrate and lipase activity is measured according to the methods described herein. Enzyme activity can also be measured using other assays for lipase activity known to those skilled in the art (see, for example, Stoytcheva M. & al, 2012, Current Analytical Chemistry, Vol. 8, p. 400 for a review).

[0075] In a particular embodiment, the method disclosed herein provides that the first enzyme is a thermophilic serine protease, wherein the ratio between the protease activity at the optimal temperature and the protease activity at 25°C is greater than 10, preferably greater than 15. By providing said ratio greater than 10, the thermophilic serine protease used herein provides an improved effect on tenderness.

[0076] In certain embodiments, the methods disclosed herein provide that the first enzyme is obtained by synthesis or by extraction from naturally occurring eukaryotes or prokaryotes through genetic engineering. In certain embodiments, the compositions disclosed herein provide that the first enzyme is a neutral or basic thermophilic serine protease.

[0077] In certain embodiments, the method disclosed herein provides that the first enzyme is preferably derived from... Aquatic thermophilic bacteria The Taq protease isolated from [the source] is preferably lysozyme I or lysozyme II, more preferably lysozyme I, and even more preferably [from [the source]]. Aquatic thermophilic bacteria Aquaporinase I isolated from LMG8924.

[0078] In certain embodiments, the method disclosed herein provides the addition of the first enzyme, preferably hydrolysin I, to dough or batter at an amount of 100 to 1200 units / 100 kg flour, preferably 200 to 900 units / 100 kg flour, and more preferably 350 to 700 units / 100 kg flour. Advantageously, hydrolysin I is added to dough / batter at an amount of 100 to 1200 units / 100 kg flour, preferably 200 to 900 units / 100 kg flour, and more preferably 350 to 700 units / 100 kg flour, and enzyme activity is obtained using the method described herein.

[0079] In certain embodiments, the methods disclosed herein provide that the second enzyme is a triacylglycerol lipase or triacylglycerol acyl hydrolase as defined in enzyme entry EC 3.1.1.3, preferably selected from... cottony thermophilic filamentous ... , Migen mold and Rhizopus oryzae The obtained lipase.

[0080] In certain embodiments, the method disclosed herein provides that the second enzyme is added to dough or batter in the following amounts: 5 to 100 U, preferably 15 to 75 U, more preferably 20 to 60 U. cottony thermophilic filamentous ... Lipase / 100 kg flour; 0.023 to 0.360 U, preferably 0.09 to 0.18 U from Rhizopus oryzae Lipase per 100 kg of flour; and 50 to 200 U, preferably 90 to 160 U, more preferably about 138 U from Rhizopus oryzae Lipase / 100 kg flour, enzyme activity was obtained using the method described herein.

[0081] In certain embodiments of the methods disclosed herein, a suitable amount of enzyme may be added to the dough or batter during its preparation or directly before the ingredients are mixed. In other embodiments, the enzyme may be added as part of a (bread) improver, a French pastry mixture, or a premix, preferably as part of a bread improver. In particular, the enzyme or bread improver is added before proofing.

[0082] In a particular embodiment, this document discloses a method for preparing baked goods, comprising adding the following steps to dough or batter prior to baking: - At least one first enzyme, wherein the first enzyme is a thermophilic serine protease that has optimal activity at a temperature above 70°C, preferably above 75°C, and more preferably above 80°C. - At least one second enzyme, wherein the second enzyme is a lipase; and - One or more monoglycerides.

[0083] In a particular embodiment, the method disclosed herein provides that the baked product exhibits improved fluffiness, preferably wherein, compared to a reference baked product prepared without the first or second enzyme, the maximum force required to destroy the baked product prepared using at least one first enzyme and at least one second enzyme is reduced by at least 15%, wherein the at least one first enzyme is a thermophilic serine protease having optimal activity at temperatures above 70°C, preferably above 75°C, and most preferably above 80°C; and the at least one second enzyme is a lipase. In a particular embodiment, compared to a reference baked product prepared without the first or second enzyme, the maximum force required to destroy the baked product prepared using the first and second enzymes is reduced by at least 20%. In a particular embodiment, fluffiness is measured on the day following baking.

[0084] In a particular embodiment, the method disclosed herein provides that the baked product exhibits improved fluffiness, preferably wherein, compared to a reference baked product prepared without the first enzyme or the second enzyme, the maximum force required to destroy the baked product prepared using at least one first enzyme and at least one second enzyme is reduced by at least 15%, wherein the at least one first enzyme is a thermophilic serine protease having optimal activity at temperatures above 70°C, preferably above 75°C, and most preferably above 80°C; and the at least one second enzyme is a lipase. In a particular embodiment, compared to a reference baked product prepared using the first enzyme or the second enzyme, the maximum force required to destroy the baked product prepared using the first enzyme and the second enzyme is reduced by at least 20%. This indicates that the combination of the first enzyme and the second enzyme provides a synergistic effect, which provides a better effect on fluffiness than expected. In a particular embodiment, fluffiness is measured on the same day after baking.

[0085] In certain embodiments, the methods disclosed herein provide that no adverse effects have been observed on dough rheology, breadcrumb structure, and the volume of the resulting baked products.

[0086] Furthermore, in another aspect, the present invention relates to baked products prepared from dough or batter comprising the compositions disclosed herein.

[0087] In the context of this invention, baked products are baked or French pastry products known in the art, such as those selected from the group consisting of: bread, soft bread rolls, bagels, donuts, Danish pastries, hamburger rolls, pizzas, pita bread, Italian bread, sponge cakes, ice cream cakes, pound cakes, muffins, cupcakes, steamed cakes, waffles, chocolate cakes, cake donuts, yeast-leavened donuts, baguettes, bread rolls, thin biscuits, rolls, cookies, pie crusts, sweet bread crisps, and other baked products. More preferably, the invention relates to bread, baguettes, and bread rolls. In particular, the baked products are soft baked products and hard-crust baked products, preferably bread, soft bread rolls, donuts, rolls, microwaveable rolls, Danish pastries, croissants, hamburger rolls, pizzas, pita bread, and cakes.

[0088] Example

[0089] Example 1: Determination of enzyme activity

[0090] Protease activity was measured on azurophilic cross-linked casein (AZCL-casein). It was prepared by staining and cross-linking casein to produce a material that is hydrated in water but insoluble in water. Hydrolysis of the protease yields water-soluble stained fragments, and the release rate of these fragments (e.g., an increase in absorbance at 590 nm) is directly correlated with enzyme activity (Protazyme AK tablets, Megazyme, Ireland). Protazyme AK tablets (pH 7.0) were incubated at 60°C in 100 mM Na₂HPO₄·2H₂O for 5 minutes. An aliquot of enzyme (1.0 ml) was added, and the reaction was allowed to continue for a precise 10 minutes. The reaction was terminated by adding trisodium phosphate (10 ml, 2% w / v, pH 12.3). The tube was allowed to stand at room temperature for approximately 2 minutes, and the contents were filtered. The absorbance of the filtrate was measured at 590 nm against a substrate blank.

[0091] Activity is expressed as: mU (milliunit) / ml = (34.2) (Abs 590 Enzyme-Abs 590 (Blank) + 0.6) / dilution. One unit corresponds to 1000 mU.

[0092] Lipase activity was measured using p-nitrophenyl palmitate (pNPP) as a substrate. The release of yellow p-nitrophenol caused by lipase hydrolysis of p-nitrophenyl palmitate was measured spectrophotometrically at 414 nm. One milliunit (LmU) of lipase was defined as the amount of enzyme required to release 1 nanomolar (nmol) of p-nitrophenol per minute from p-nitrophenyl palmitate at 45°C and pH 7.5. For the test, 120 µl of 1 mM pNPP solution (0.05 M sodium phosphate buffer containing 0.69 M acetone and 0.0049 M Triton X-100 at pH 7.5) was mixed with 60 µl of enzyme sample and incubated at 45°C for 30 min.

[0093] The absorbance was measured at 414 nm in a 96-well microtiter plate with a substrate blank as a reference.

[0094] Activity is expressed as: LmU / ml = (((Abs enzyme - Abs blank) / 30) x 0.18)) / (13380 x 0.06)) x sample dilution x 1000000 [30 = reaction time in minutes; 0.18 = reaction volume in ml; 13380 = molar extinction coefficient at 414 nm (M -1 cm -1 ); 0.06 = enzyme sample volume in ml; 1,000,000 is used to convert to LmU / ml] One lipase unit (LU) corresponds to 1000 lipase milliunits (LmU).

[0095] Example 2: Soft small round bread

[0096] Soft, round bread rolls were prepared using the dough compositions in Table 1. The enzymes used were as follows: - TlLip: In Aspergillus oryzae Expressed in (Nopase-Novozymes, Denmark) cotton Thermophilic filamentosa Lipase.

[0097] - TaProt: Aquatic thermophilic bacteria Taq1 protease (water cell lysin I) is described in WO2009138447A1. This enzyme has optimal activity at 80°C.

[0098] Table 1 It contains flour, mono- and diacetyl tartrates (E472e) of glycerol mono- and diacetyl esters of fatty acids, dextrose, ascorbic acid, and enzymes (α-amylase, xylanase). The ingredients were mixed in a Diosna (SP24) spiral mixer at low speed for 2 minutes and then at high speed for 6 minutes. The final dough temperature was 27°C. After the dough had rested for 5 minutes, 1500 g of dough was rounded and rested for 10 minutes at baking temperature (25°C) and humidity (50-55%). 50 g dough pieces were prepared using an Eberhardt molder. These dough pieces were then rested for 85 minutes in a Koma proofbox at 35°C and 95% relative humidity. The loaves were then baked at 250°C for 9 minutes in a MiweCondo countertop oven without steam. It will be apparent to those skilled in the art that the same final results can be obtained by using equipment from other suppliers.

[0099] On the same day after baking, the pizza was stretched using the TA-XT2, which is equipped with a pizza stretching device that operates at a speed of 20 mm / s. TMA texture analyzer was used to assess the softness of loaves of bread. This allows for the measurement of force (the maximum force required to break a loaf, expressed in grams (g)). Because factors such as flour batch, ambient temperature and humidity, and the time between baking and testing can affect this parameter, the measurements were compared to a reference using the same ingredients, baked and tested in parallel. For each test, 10 loaves were evaluated. The standard deviation of the measurements was 33. Confidence intervals were calculated by multiplying the standard deviation by the coefficient of Stieth's Law, which was obtained using a 5% alpha risk value and 5 degrees of freedom. The confidence interval was 85.

[0100] If the combined effect of two components is greater than the sum of the effects of using the components individually, then the two components have a synergistic effect, considering a 95% confidence interval. In other words, a synergistic effect exists when the effect of (xg enzyme A + yg enzyme B) is greater than the sum of the effects of xg enzyme A and yg enzyme B.

[0101] Using TA-XT2 TM The results of the softness measurement performed by the texture analyzer are listed in Table 2.

[0102] Table 2

[0103] The results showed that when the actual reduction in force obtained when the enzymes were combined was below the lower limit of the confidence interval of the theoretical value (additional effect), it indicated a synergistic effect on softness.

[0104] The softness of the small round loaves was also assessed on the same day after baking by a trained panel of baking experts. They were asked to label the products using a linear scale of 0 to 10 (where 0 is low (chewy) and 10 is high (soft)). The standard deviation of the sensory softness measurement was 0.3. Confidence intervals were calculated using a 5% alpha risk value and a number of degrees of freedom equal to 5 by multiplying the standard deviation by the coefficient of Stieth's Law. The confidence interval was 0.5.

[0105] The results are listed in Table 3.

[0106] Table 3

[0107] The results showed that when the enzymes were combined, the actual increase in softness obtained was higher than the upper limit of the confidence interval of the theoretical value (additional effect), indicating a synergistic effect on softness.

[0108] Example 3a: Other lipases (1)

[0109] Soft mini round breads were prepared according to the formula and the method of Example 2 using the enzyme combinations listed in Table 4. The enzymes used are as follows: - RmLip: In Aspergillus oryzae The expression in Rhizopus oryzae Lipase (Palatase 2000L - Novozymes, Denmark).

[0110] - TaProt: As in Example 1

[0111] Table 4

[0112] The softness of the small round breads was evaluated using the same method as in Example 1. The standard deviation and confidence interval for texture analysis were 38 and 97, respectively. The standard deviation and confidence interval for sensory analysis were 0.2 and 0.5, respectively.

[0113] The results are shown in Table 5 (texture analysis) and Table 6 (sensory analysis).

[0114] Table 5

[0115] Table 6

[0116] Synergistic effects of enzyme combinations were observed.

[0117] Example 3b: Other lipases (2)

[0118] Soft round bread rolls were prepared according to the formula and the method of Example 1 using the enzyme combinations listed in Table 7. The enzymes used are as follows: - RoLip: Rhizopus oryzae Lipase (Bakezyme L 80000B–DSM) - TaProt, as in Example 1 Table 7

[0119] The softness of the small round bread was evaluated using the same method as in Example 1.

[0120] The results are shown in Table 8.

[0121] Table 8

[0122] A synergistic effect was observed using a third lipase.

[0123] Example 4: Other proteases

[0124] Soft round bread rolls were prepared according to the formula and the method of Example 1 using the enzyme combinations listed in Tables 9 and 10. The enzymes used are as follows: - TlLip: In Aspergillus oryzae The expression in (Nopase-Novozymes) cottony thermophilic filamentous ... Lipase.

[0125] - SuProt: From Bacillus licheniformis The serine protease (subtilisin-P5380-1G, Sigma Aldrich) has an optimal temperature activity of 60°C.

[0126] - ThProt: From Geobacillus stearothermophilus Metalloproteinases (thermophilic protease-P1512-1G, Sigma Aldrich)

[0127] Table 9

[0128] Table 10

[0129] The softness of the small round bread was evaluated using the texture analysis method described in Example 1.

[0130] The results are shown in Tables 11 and 12.

[0131] Table 11

[0132] Table 12

[0133] No synergistic effect was observed with proteases having optimal activity at 65°C and with metalloproteinases.

Claims

1. A dough or batter comprising a composition and flour, said composition comprising: - At least one first enzyme, wherein, The first enzyme is Aquatic thermophilic bacteria ( Thermus aquaticus Taq1 protease, water cell lysin I; and - At least one second enzyme, wherein the second enzyme is a triacylglycerol lipase or triacylglycerol acyl hydrolase as defined in enzyme entry EC 3.1.1.3, wherein the second enzyme is selected from... cottony thermophilic filamentous ... ( Thermomyces lanuginosus ), Rhizopus oryzae ( Rhizopus oryzae )and Rhizopus oryzae ( Rhizomucor miehei The lipase obtained.

2. The dough or batter according to claim 1, wherein, The first enzyme is from Aquatic thermophilic bacteria Separated from LMG8924 Aquatic thermophilic bacteria Taq1 protease is a water cell lysin I.

3. The dough or batter according to any one of claims 1 to 2, wherein, The first enzyme is present in an amount of 100 to 1200 units / 100 kg flour, preferably 200 to 900 units / 100 kg flour, and more preferably 350 to 700 units / 100 kg flour.

4. The dough or batter according to any one of claims 1 to 3, wherein, The second enzyme is added to the dough or batter in the following amounts: - When measurements are performed according to the description in Example 1, when the second enzyme is from cottony thermophilic filamentous ... When the lipase content is high, it is 5 to 100 LU / 100 kg flour; - When measurements are performed according to the description in Example 1, when the second enzyme is from Rhizopus oryzae When the lipase content is 0.023 to 0.360 LU / 100 kg flour; or - When measurements are performed according to the description in Example 1, when the second enzyme is from Rhizopus oryzae When the lipase content is 50 to 200 LU / 100 kg flour.

5. The dough or batter according to any one of claims 1 to 4, wherein, When the second enzyme is from cottony Thermophilic filamentous fungi When the lipase is present, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 5 to 35; when the second enzyme is derived from... Rhizopus oryzae When the lipase is present, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 1950 to 7800; or when the second enzyme is derived from... Rhizopus oryzae When lipase is involved, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 2 to 8.

6. Use of the dough or batter according to any one of claims 1 to 5 in baking applications.

7. The use according to claim 6, for soft-baked products and hard-crust baked products, preferably bread, soft rolls, donuts, rolls, microwaveable rolls, Danish pastries, croissants, hamburger rolls, pizzas and pita breads, and cakes.

8. A bread improver comprising the composition defined in any one of claims 1 to 5.

9. A method for preparing baked goods, comprising adding the following steps to dough or batter prior to baking: - At least one first enzyme, wherein, The first enzyme is Aquatic thermophilic bacteria Taq1 protease hydrolysate I; and - At least one second enzyme, wherein the second enzyme is a triacylglycerol lipase or triacylglycerol acyl hydrolase as defined in enzyme entry EC 3.1.1.3, wherein the second enzyme is selected from... cottony thermophilic filamentous ... , Rhizopus oryzae and Mihegen Mucor The obtained lipase.

10. The method according to claim 9, wherein, The first enzyme is present in an amount of 100 to 1200 units / 100 kg flour, preferably 200 to 900 units / 100 kg flour, and more preferably 350 to 700 units / 100 kg flour.

11. The method according to claim 9 or 10, wherein, The second enzyme is present in the following amounts: - When measurements are performed according to the description in Example 1, when the second enzyme is from cottony thermophilic filamentous ... When the lipase content is high, it is 5 to 100 LU / 100 kg flour; - When measurements are performed according to the description in Example 1, when the second enzyme is from Rhizopus oryzae When the lipase content is 0.023 to 0.360 LU / 100 kg flour; or - When measurements are performed according to the description in Example 1, when the second enzyme is from Rhizopus oryzae When the lipase content is 50 to 200 LU / 100 kg flour.

12. The method according to any one of claims 9 to 11, wherein, When the second enzyme is from cottony thermophilic Hylocereus When the lipase is present, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 5 to 35; when the second enzyme is derived from... Rhizopus oryzae When the lipase is present, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 1950 to 7800; or when the second enzyme is derived from... Rhizopus oryzae When lipase is involved, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 2 to 8.

13. Use of the dough or batter according to any one of claims 1 to 5 in the preparation of baked goods having improved fluffiness, wherein, Preferably, the maximum force required to destroy the prepared baked product is reduced by at least 15% compared to a reference baked product prepared without using the first enzyme or the second enzyme.

14. A baked product prepared from dough or batter according to any one of claims 1 to 5.

15. The baked product according to claim 14, wherein, The first enzyme is present in an amount of 100 to 1200 units / 100 kg flour, preferably 200 to 900 units / 100 kg flour, and more preferably 350 to 700 units / 100 kg flour.

16. The baked product according to claim 14 or 15, wherein, The second enzyme is present in the following amounts: - When measurements are performed according to the description in Example 1, when the second enzyme is from cottony thermophilic filamentous ... When the lipase content is high, it is 5 to 100 LU / 100 kg flour; - When measurements are performed according to the description in Example 1, when the second enzyme is from Rhizopus oryzae When the lipase content is 0.023 to 0.360 LU / 100 kg flour; or - When measurements are performed according to the description in Example 1, when the second enzyme is from Rhizopus oryzae When the lipase content is 50 to 200 LU / 100 kg flour.

17. The baked product according to any one of claims 14 to 16, wherein, When the second enzyme is from cottony Thermophilic filamentosa When the lipase is present, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 5 to 35; when the second enzyme is derived from... Rhizopus oryzae When the lipase is present, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 1950 to 7800; or when the second enzyme is derived from... Rhizopus oryzae When lipase is involved, the ratio of the units of the first enzyme to the units of the second enzyme is in the range of 2 to 8.

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