Frozen bread dough modifier

Abstract

To provide a technique for improving the quality of bread produced using frozen bread dough.SOLUTION: The present technique provides frozen bread dough modifier containing carbohydrate oxidase. The frozen bread dough modifier according to the present technique has a volume increasing effect, a food texture improving action, and an appearance improving action. The present technique also provides: a method for producing frozen bread dough comprising a dough preparing step for adding carbohydrate oxidase to a bread dough material and preparing bread dough, and a freezing step for freezing the prepared dough; and a method for producing bread comprising a thawing step for thawing frozen bread dough containing carbohydrate oxidase, a fermentation step for fermenting the thawed bread dough, and a heating step for heating the fermented bread dough.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a modifier for frozen dough. More specifically, the present invention relates to a modifier for frozen dough using a carbohydrate oxidase, a method for producing frozen dough, and a method for producing bread. [Background technology]

[0002] In recent years, techniques for improving bread quality using various enzymes have been developed. For example, Patent Document 1 discloses a technique for improving dough hardness, adhesion, stability, and toughness by using carbohydrate oxidase. Patent Document 2 discloses a technique for improving the rheological properties of flour lump by using an oxidoreductase capable of oxidizing maltose. Patent Document 3 discloses a technique for stabilizing the gluten network of dough by using enzymes such as hexose oxidase and glucose oxidase.

[0003] Furthermore, Patent Document 4 discloses a technique in which carbohydrate oxidase is used to tighten bread dough, reduce stickiness during the production process, and make the dough easier to handle. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2001-526058 [Patent Document 2] Special Publication No. 11-506338 [Patent Document 3] Special Publication No. 2017-510282 [Patent Document 4] International Publication No. 2014 / 042237 Brochure Summary of the Invention [Problem to be solved by the invention]

[0005] With the development of freezing technology, the distribution of frozen dough has become widespread, but bread made with frozen dough has a problem of being inferior in volume compared to bread made without the freezing process. This is thought to be due to the destruction of the gluten structure in the dough during freezing storage and the death of yeast due to the formation of ice crystals in the dough.

[0006] Furthermore, in countries where freezing technology is still developing, the quality of the freezing equipment is not high, so there is a demand to improve the quality of the frozen dough itself.

[0007] Therefore, a main object of the present technology is to provide a technology for improving the quality of bread produced using frozen dough. [Means for solving the problem]

[0008] It has been reported that the bread production technology proposed in the above-mentioned Reference 4, which does not involve a freezing process, uses carbohydrate oxidase to tighten the bread dough, resulting in a decrease in the volume of the produced bread and an increase in hardness. However, the inventors of the present application unexpectedly discovered that bread produced by freezing and storing bread dough using carbohydrate oxidase and then thawing and fermenting the dough has an increased volume, which led to the completion of the present technology.

[0009] That is, the present technology first provides a frozen dough modifier containing carbohydrate oxidase, which has the effects of increasing volume, improving texture, and improving appearance. In the present technology, the carbohydrate oxidase can be one that has the property of acting on one or more carbohydrates selected from glucose, maltotriose, maltose, galactose, maltotetraose, lactose, cellobiose, and maltodextrin. Furthermore, in the present technology, a carbohydrate oxidase consisting of a polypeptide shown in any one of (1) to (3) below can be used as the carbohydrate oxidase. (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1; (2) A polypeptide in which one or more amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and which exhibits substrate specificity equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; and (3) A polypeptide having an amino acid sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 and exhibiting substrate specificity equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0010] In the present technology, next, a dough preparation step of adding carbohydrate oxidase to the dough material to prepare the dough; A freezing step of freezing the prepared dough; The present invention provides a method for producing frozen bread dough, which comprises the steps of: In addition, the present technology includes a thawing step of thawing frozen bread dough containing carbohydrate oxidase; A fermentation process for fermenting the thawed dough; A heating step of heating the fermented dough; Also provided is a method for producing bread in which the above steps are carried out. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] A preferred embodiment for carrying out the present invention will be described below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention is not to be interpreted narrowly by this embodiment.

[0012] <1. Modifier for frozen bread dough> The frozen dough improver according to the present technology contains carbohydrate oxidase as an active ingredient, and has the effects of increasing volume, improving texture, and improving appearance.

[0013] The volume increasing effect is an effect of increasing the volume of bread produced using the frozen bread dough modifier according to the present technology.

[0014] The texture improving effect refers to the effect of improving the texture of bread produced using the frozen dough modifier of the present technology, and in the present technology, it mainly has the effect of improving the crispness of the bread.

[0015] The appearance improving effect is an effect of improving the appearance of bread produced using the frozen dough modifier according to the present technology. For example, in French breads such as baguettes and batards, the cuts on the surface called coupes are important in terms of appearance, and the present technology has the effect of improving the spread of these cuts on the surface such as coupes.

[0016] The carbohydrate oxidase that can be used in the present technology will be described in detail below.

[0017] (1) Carbohydrate oxidase The carbohydrate oxidase that can be used in the present technology is not particularly limited as long as it is an enzyme that can oxidize carbohydrates, but is preferably an enzyme that oxidizes disaccharides or more. Specific examples include proteins having the physicochemical properties described below.

[0018] A.Action A carbohydrate oxidase that can be used in the present technology oxidizes a sugar, which will be described later, in the presence of oxygen to generate a sugar acid. More specifically, when a carbohydrate oxidase that can be used in the present technology is allowed to act on a sugar, which will be described later, in the presence of oxygen, a sugar acid and hydrogen peroxide are generated.

[0019] B. Substrate specificity The carbohydrate oxidase that can be used in the present technology is a protein that exhibits activity against one or more carbohydrates selected from glucose, maltotriose, maltose, galactose, maltotetraose, lactose, cellobiose, and dextrin. The relative activities against each substrate, when the activity against glucose is taken as 100%, are as follows: maltotriose: about 92%, maltose: about 86%, galactose: about 79%, maltotetraose: about 60%, lactose: about 58%, cellobiose: about 53%, and maltodextrin: about 24%. In the present invention, a substrate was determined to be "a substrate on which the enzyme acts well" if its relative activity was 50% or more, taking the activity when glucose was used as the substrate (100%) as the standard.

[0020] In this way, by using a carbohydrate oxidase that is active not only on monosaccharides such as glucose but also on a wide range of carbohydrates of disaccharides or greater, it is possible to make the carbohydrate oxidation function in a wide range of fields that cannot be addressed by existing glucose oxidase or oligosaccharide oxidase.

[0021] C.Km value In the present technology, the specific calculation method of the Km value (Michaelis constant) of a protein is not particularly limited, and a known method can be freely selected for calculation. Examples of methods for calculating the Km value of a protein include Lineweaver-Burk plot, Eadie-Hofstee plot, Hanes-Woolf plot, etc., and preferably Hanes-Woolf plot. The Km value of the carbohydrate oxidase that can be used in the present technology is not particularly limited, but is preferably [Km value of glucose] / [Km value of maltose]≦1, and more preferably 0.4≦[Km value of glucose] / [Km value of maltose]≦1.

[0022] D. Molecular mass The carbohydrate oxidase that can be used in this technique is one having a molecular mass of approximately 63 kDa as determined by SDS-PAGE.

[0023] E. Optimum pH As the carbohydrate oxidase that can be used in the present technology, a carbohydrate oxidase that has the highest carbohydrate oxidase activity at about pH 5.0 to 9.0 under reaction conditions of 37° C. for 5 minutes can be used.

[0024] F. Stable pH range As the carbohydrate oxidase that can be used in the present technology, a carbohydrate oxidase that is stable at about pH 5.0 to 10.5 under treatment conditions of 37° C. for 15 minutes can be used.

[0025] G.Optimal temperature As the carbohydrate oxidase that can be used in the present technology, a carbohydrate oxidase that has the highest carbohydrate oxidase activity at about 20° C. to 55° C. under reaction conditions of pH 7.0 for 5 minutes can be used.

[0026] H. Temperature stability The carbohydrate oxidase that can be used in this technology is one that can maintain 80% or more of its activity even when treated at temperatures up to 45°C under treatment conditions of pH 7.0 for 15 minutes.

[0027] I. Origin The origin of the carbohydrate oxidase that can be used in the present technology described above is not particularly limited, but examples thereof include those derived from microorganisms belonging to the genus Acremonium, such as Acremonium chrysogenum.

[0028] Here, "carbohydrate oxidase derived from Acremonium chrysogenum" means a carbohydrate oxidase produced by a microorganism classified as Acremonium chrysogenum (whether it is a wild-type strain or a mutant strain), or a carbohydrate oxidase obtained by a genetic engineering technique using a carbohydrate oxidase gene. Therefore, a recombinant produced by a host microorganism into which a carbohydrate oxidase gene obtained from Acremonium chrysogenum (or a gene obtained by modifying said gene) has been introduced also falls under "carbohydrate oxidase derived from Acremonium chrysogenum."

[0029] Examples of Acremonium chrysogenum from which carbohydrate oxidases that can be used in the present technology are derived include Acremonium chrysogenum NBRC30055 (NITE, Japan), ATCC15006 (ATCC, USA), and DSM880 (DSMZ, Germany).

[0030] J. Amino acid sequence The amino acid structure of the carbohydrate oxidase that can be used in the present technology is not limited, but as an example, it can be specified by the following amino acid sequence.

[0031] Specifically, the carbohydrate oxidase that can be used in the present technology can be identified by the amino acid sequence shown in SEQ ID NO:1.

[0032] Generally, when a portion of the amino acid sequence of a protein is modified, the modified protein may have the same function as the unmodified protein. In other words, the modification of the amino acid sequence may not have a substantial effect on the function of the protein, and the function of the protein may be maintained before and after the modification. Thus, in another aspect, the present invention provides a protein having carbohydrate oxidase activity, which comprises an amino acid sequence represented by SEQ ID NO: 1 in which one to several amino acids are deleted, substituted and / or added. "Deletion, substitution and / or addition of one to several amino acids constituting the amino acid sequence" typically refers to a partial difference in the amino acid sequence.

[0033] The difference in the amino acid sequence here is permissible as long as the carbohydrate oxidase activity can be maintained (some variation in activity is acceptable). As long as this condition is met, the position at which the amino acid sequence differs is not particularly limited, and differences may occur at multiple positions. Here, multiple means, for example, a number corresponding to less than about 30% of the entire amino acid sequence, and preferably less than about 20%. More preferably, the number corresponds to less than about 10%, even more preferably, the number corresponds to less than about 5%, and most preferably, the number corresponds to less than about 1%. That is, it refers to having, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, even more preferably about 95% or more, and most preferably about 99% or more identity with the amino acid sequence of SEQ ID NO:1.

[0034] Also, preferably, a method for obtaining a protein is used in which a conservative amino acid substitution is made in an amino acid residue that is not essential for the carbohydrate oxidase activity. Here, "conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families according to their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues in the same family.

[0035] Incidentally, the identity (%) of two amino acid sequences or two nucleic acids (hereinafter, the term "two sequences" is used to include both) can be determined, for example, by the following procedure. First, the two sequences are aligned to enable optimal comparison. In this case, for example, gaps may be introduced into the first sequence to optimize the alignment with the second sequence. When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as a molecule at the corresponding position in the second sequence, it can be said that the molecules at that position are identical. The identity of two sequences is a function of the number of identical positions common to the two sequences (i.e., identity (%) = number of identical positions / total number of positions x 100), and preferably, the number and size of gaps required for optimizing the alignment are also taken into consideration.

[0036] Comparison of two sequences and determination of identity can also be achieved using a mathematical algorithm. A specific example of a mathematical algorithm that can be used for sequence comparison includes, but is not limited to, the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search can be performed using the NBLAST program with score=100 and wordlength=12.

[0037] To obtain an amino acid sequence equivalent to the polypeptide molecule of the present invention, for example, a BLAST polypeptide search may be performed with the XBLAST program with score=50 and wordlength=3. To obtain a gapped alignment for comparison, Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25(17):3389-3402 may be used. When using BLAST and Gapped BLAST, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) may be used. For details, see http: / / www.ncbi.nlm.nih.gov.

[0038] The carbohydrate oxidase that can be used in the present technology may be a part of a larger protein (e.g., a fusion protein). Examples of sequences added to the fusion protein include sequences that are useful for purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

[0039] The protein having the above amino acid sequence can be easily prepared by genetic engineering techniques. For example, it can be prepared by transforming an appropriate host cell (e.g., Escherichia coli, yeast, filamentous fungus) with DNA encoding the present protein and recovering the protein expressed in the transformant. The recovered protein is appropriately prepared according to the purpose. If the present protein is obtained as a recombinant protein in this way, various modifications are possible. For example, if the DNA encoding the present protein and other appropriate DNA are inserted into the same vector and the recombinant protein is produced using the vector, the present protein can be obtained as a recombinant protein to which any peptide or protein is linked. In addition, sugar chains and / or lipids may be added, or modifications may be made to cause N-terminal or C-terminal processing. The above modifications make it possible to simplify the extraction and purification of the recombinant protein, or to add biological functions, etc.

[0040] K. Carbohydrate Oxidase Content in Frozen Bread Dough Modifiers The content of carbohydrate oxidase in the frozen dough modifier according to the present technology can be freely set as long as it does not impair the effect of the present technology. When the frozen dough modifier is in liquid form, the content can be, for example, 0.001U / mL to 10000U / mL, preferably 0.01U / mL to 5000U / mL, more preferably 0.1U / mL to 1000U / mL, and even more preferably 1U / mL to 500U / mL. When the frozen dough modifier is in powder form, the content can be, for example, 0.001U / g to 10000U / g, preferably 0.01U / g to 5000U / g, more preferably 0.1U / g to 1000U / g, and even more preferably 1U / g to 500U / g.

[0041] (2) Other ingredients The frozen dough modifier according to the present technology may be composed of only the carbohydrate oxidase described above, or may contain one or more other components selected freely as long as the effect of the present technology is not impaired. Examples of other components that can be used include excipients, pH adjusters, colorants, flavorings, disintegrants, lubricants, stabilizers, and other components that are normally used in formulations. Furthermore, components having known or future functions can be used in combination as appropriate depending on the purpose.

[0042] <2. Method of manufacturing frozen dough and method of manufacturing bread> The method for producing frozen dough according to the present technology is a method that at least includes a dough preparation step and a freezing step. The method for producing bread according to the present technology is a method that at least includes a thawing step, a fermentation step, and a heating step. In addition, depending on the type of bread, it is possible to perform general bread production steps before, after, or simultaneously with each step, as long as the effect of the present technology is not impaired. Each step will be described in detail below in chronological order.

[0043] (1)Dough preparation process The dough preparation step is a step of adding carbohydrate oxidase to dough ingredients to prepare bread dough. Specifically, it is a step of kneading the dough ingredients to which carbohydrate oxidase has been added to prepare dough. The specific kneading method in the dough preparation step is not particularly limited as long as it does not impair the effects of the present invention, and any kneading method commonly used in bread manufacturing methods can be freely selected and used. For example, the dough ingredients can be mixed using a cooking utensil such as a rubber spatula, and then kneaded by hand, or kneaded using a machine capable of kneading.

[0044] In the dough preparation process, it is possible to knead all of the ingredients used in the bread dough at the same time, but depending on the types of ingredients to be included in the bread dough, the ingredients can be kneaded in any way that suits the ingredients used and the purpose, such as mixing some of the ingredients and then mixing the remaining ingredients and kneading them together, or mixing several types of ingredients separately and then kneading the mixed ingredients together.

[0045] In the dough preparation process, it is preferable that fermentation does not proceed excessively. If fermentation proceeds excessively before freezing, freezing damage may occur to the yeast, and the fermentation power after thawing may decrease. Therefore, in the dough preparation process, it is preferable to prepare the bread dough under conditions where the temperature of the dough ingredients is 22°C or lower. As a specific method for lowering the temperature of the dough ingredients, one or more general dough cooling methods can be freely selected and used, such as using ice, using refrigerated ingredients, or cooling the cooking utensils used for kneading, as long as the effect of the present invention is not impaired.

[0046] This technology also includes the production of bread using the leaven method. A typical production method using the leaven method is to mix part of the dough ingredients with yeast and water, then ferment to prepare a leaven, mix the remaining dough ingredients with the leaven, ferment the main dough, and then divide, shape, and perform final fermentation before baking. In the dough preparation step of this technology, it is also possible to prepare the main dough by mixing the leaven, the remaining dough ingredients, and carbohydrate oxidase. In this case, it is preferable that the final fermentation does not proceed excessively.

[0047] The amount of carbohydrate oxidase added in the dough preparation process can be freely set as long as it does not impair the effect of the present technology. The lower limit of the amount added can be, for example, 0.01 mU or more, preferably 0.1 mU or more, more preferably 0.5 mU or more, and even more preferably 1 mU or more per 1 g of dough ingredients. The upper limit of the amount added can be, for example, 1 U or less, preferably 100 mU or less, and more preferably 10 mU or less per 1 g of dough ingredients.

[0048] The dough ingredients used in the dough preparation process can be any combination of ingredients used in general bread dough, as long as they do not impair the effects of the present technology. For example, wheat flour such as weak flour, medium flour, semi-strong flour, and strong flour, wheat flour derived from durum wheat, rice flour, rye flour, barley flour, oat flour, buckwheat flour, barnyard millet flour, foxtail millet flour, corn flour, and starches from various grains; yeast such as dry yeast and live yeast; additives for activating yeast such as yeast food; various seasonings such as salt, mayonnaise, sauce, soy sauce, and amino acids; various ingredients whose main ingredient is milk, such as skim milk powder, whey powder, and butter powder; oils and fats; eggs (including whole eggs, egg yolks, egg whites, and powdered eggs); and various additives such as flavorings, emulsifiers, bread-making improvers, thickeners, stabilizers, bacteriostatic agents, and enzymes other than carbohydrate oxidase.

[0049] (2) Freezing process The freezing step is a step of freezing the dough prepared in the dough preparation step. The freezing method in the freezing step is not limited as long as it does not impair the effects of the present technology, and any general freezing method can be freely selected and used.

[0050] The freezing conditions in the freezing step are not limited as long as they do not impair the effects of the present technology. For example, the temperature conditions for freezing are preferably -25°C or lower, more preferably -30°C or lower, and quick freezing is preferred. The frozen storage conditions of the frozen dough are also not limited as long as they do not impair the effects of the present technology. The temperature conditions can be appropriately adjusted depending on the type and size of the dough, and for example, the temperature conditions during frozen storage are preferably -8 to -22°C, more preferably -12 to -20°C.

[0051] (3) Thawing process The thawing step is a step of thawing the frozen bread dough containing carbohydrate oxidase. The thawing method in the thawing step is not limited as long as it does not impair the effect of the present technology, and a general thawing method can be freely selected and used. For example, room temperature thawing (1 to 30°C), room temperature thawing (15 to 25°C), refrigerated thawing (1 to 10°C), microwave thawing, etc. can be mentioned.

[0052] (4) Fermentation process The fermentation process is a process for fermenting the bread dough thawed in the thawing process. The fermentation method in the fermentation process is not limited as long as it does not impair the effects of the present technology, and any general fermentation method can be freely selected and used. The fermentation process here corresponds to the fermentation of the dough (first fermentation (floor time), bench time, and second fermentation (final fermentation, proofing)). In other words, it is not the fermentation when preparing a leaven such as in the liquid seed method.

[0053] The fermentation temperature in the fermentation step can be freely set according to the type of yeast used and the state of the dough, as long as it does not impair the effects of the present invention. For example, the fermentation temperature in the fermentation step is preferably set to 23 to 40°C, more preferably 26 to 35°C.

[0054] The fermentation time in the fermentation step can also be freely set according to the type and amount of yeast used and the state of the dough, as long as it does not impair the effects of the present invention. For example, the fermentation time in the fermentation step is preferably set to 30 minutes to 24 hours, more preferably 1 to 16 hours.

[0055] The fermentation process can be divided into several stages, for example, a method of carrying out each stage in the following order: primary fermentation (floor time) → division process (described later) → bench time → shaping process (described later) → secondary fermentation (final fermentation, proofing).

[0056] In the fermentation step, an enzymatic reaction by a carbohydrate oxidase can be allowed to proceed under the same conditions as the fermentation conditions, but an enzymatic treatment step can also be provided separately from the fermentation step. In that case, the conditions for the enzymatic treatment can be freely set as long as they do not impair the effects of the present technology. For example, the pH, temperature, and reaction time can be set according to the physicochemical properties of the carbohydrate oxidase used, such as the optimum pH, stable pH range, optimum temperature, and temperature stability. The pH can be set, for example, to pH 5.0 to 10.5, preferably pH 5.0 to 7.5. The temperature can be set, for example, to 20° C. to 55° C., preferably 30° C. to 50° C., and more preferably 35° C. to 45° C. The reaction time can be set, for example, to 1 to 12 hours, preferably 4 to 10 hours. It is preferable to simultaneously carry out the fermentation step and the enzymatic treatment step.

[0057] (5)Dividing process The dividing step is a step of dividing the prepared bread dough. In the present technology, the dividing step is not essential and can be appropriately performed depending on the type of bread to be produced, etc.

[0058] The dividing step can be carried out at any time after the dough preparation step. For example, after the dough preparation step, the dough can be appropriately rested until it settles, divided into pieces of desired size, and the divided pieces can be frozen. In addition, the frozen dough can be thawed, divided into pieces of desired size, appropriately shaped, fermented, and then subjected to the heating step described below.

[0059] (6) Molding process The shaping step is a step of shaping the prepared bread dough. In the present technology, the shaping step is not essential and can be appropriately performed depending on the type of bread to be produced, etc.

[0060] The shaping step can be carried out at any time after the dough preparation step. For example, after the dough preparation step, the dough can be appropriately rested until it settles, and then shaped into a desired shape and frozen in the shaped state. In addition, the frozen dough can be thawed, shaped into a desired shape, fermented appropriately, and then heated, as described below.

[0061] (7)Heating process The heating step is a step of heating the fermented bread dough. The heating method in the heating step is not limited as long as it does not impair the effect of the present technology, and any common heating method can be freely selected and used. For example, baking, deep frying, steaming, microwave heating, etc. can be mentioned.

[0062] The heating temperature in the heating step can be freely set depending on the type and shape of the bread to be produced, the heating method, etc., as long as the effects of the present invention are not impaired. For example, the heating temperature in the heating step is preferably set to 140 to 280°C, more preferably 160 to 240°C.

[0063] The heating time in the heating step can also be freely set according to the type and shape of the bread to be produced, the heating method, etc., as long as it does not impair the effects of the present invention. For example, the heating time in the heating step is preferably set to 10 minutes to 2 hours, and more preferably set to 15 minutes to 1 hour. EXAMPLES

[0064] The present invention will be described in more detail below with reference to examples. Note that the examples described below are representative examples of the present invention, and the scope of the present invention should not be construed as being narrowed by these examples.

[0065] In the present technique, the following method was used to measure the activity of carbohydrate oxidase. [Glucose oxidase activity measurement method] An appropriate amount of enzyme was weighed, and dissolved or uniformly dispersed in cooled pH 7.0 potassium phosphate-sodium hydroxide buffer (0.1 mol / L) to make 50 mL, which was used as the sample solution. 2.50 g of D(+)-glucose was weighed, dissolved in water to make 25 mL, which was used as the substrate solution. 0.5 mL of the substrate solution, 2 mL of potassium phosphate-sodium hydroxide buffer (0.1 mol / L, pH 7.0, containing phenol), 0.5 mL of peroxidase test solution (25 units / mL), and 0.1 mL of 4-aminoantipyrine solution (1→250) were placed in a quartz cell and heated at 37°C for 10 minutes. 0.1 mL of the sample solution was added to this solution, mixed well, and heated at 37°C to make the test solution. Separately, potassium phosphate-sodium hydroxide buffer (0.1 mol / L) at pH 7.0 or water was used instead of the sample solution, and the procedure was repeated as in the preparation of the test solution to make a control solution. The absorbance at 500 nm was measured for the test solution and the control solution 2 minutes and 5 minutes after the addition of the sample solution. The amount of glucose oxidized was quantified from the molar extinction coefficient of the quinone imine dye produced. The amount of enzyme required to oxidize 1 μmol of glucose per minute under the conditions was defined as 1 unit.

[0066] In this example, an enzyme purified by the method described in WO2014 / 042237 was used as an example of a carbohydrate oxidase.

[0067] <Experimental Example 1> In experimental example 1, the effects on the volume, appearance (extent of cuts on the surface), and texture (crispness) of the bread produced using no enzymes, the use of glucose oxidase, and the use of carbohydrate oxidase in a bread producing method that does not involve a freezing process were investigated.

[0068] (1) Bread production The ingredients other than yeast shown in Table 1 below were placed in a mixer and mixed at low speed for about 5 minutes until the ice melted. After mixing at high speed for about 2 minutes, yeast was added and mixing at high speed was continued until the dough came together to prepare bread dough. 2.1 kg of the prepared bread dough was weighed out, formed into balls, and rested for 5 minutes. The rested bread dough was divided into 70 g portions, rolled into balls, and shaped into cylindrical shapes with a length of 8 to 10 cm, then placed in a mold and arranged vertically so that the length was about 6 cm. After fermentation at 28 ° C for 15 hours, each piece was cut vertically and baked in a 170 ° C oven for 17 to 20 minutes with steam added for 8 to 10 seconds to produce bread.

[0069] (2) Evaluation The volume (specific volume (mL / g)) of the produced bread was measured. In addition, the appearance (extent of the cuts on the surface) and texture (crispness) were evaluated according to the following evaluation criteria.

[0070] <Appearance (extension of surface cuts)> +: The coupe is not wide and is poor quality. ++: The coupe is wide and has a generally good appearance. +++: The coupe has a good spread and has an excellent appearance

[0071] <Texture (crispy)> +: Not crispy, defective ++: Crispy and generally good texture +++: Has a good crispiness and excellent texture

[0072] (3) Results The results are shown in Table 1 below.

[0073] [Table 1]

[0074] (4) Discussion As shown in Table 1, in bread manufacturing methods that do not involve a freezing process, the evaluation results were similar regardless of whether enzymes were added or the type of enzyme used.

[0075] <Experimental Example 2> In Experimental Example 2, when bread was produced through a process of freezing bread dough, the effects on the volume, appearance (extent of cuts on the surface), and texture (crispness) of the produced bread were investigated when no enzymes, glucose oxidase, or carbohydrate oxidase were used.

[0076] (1) Making bread dough The ingredients other than yeast shown in Table 2 below were placed in a mixer and mixed at low speed for about 5 minutes until the ice melted. After mixing at high speed for about 2 minutes, yeast was added and mixing at high speed was continued until the dough came together to prepare bread dough. Note that the temperature was regulated by using water in ice form, using refrigerated ingredients, or using a jacketed mixer as appropriate so that the temperature of the dough did not exceed 22°C.

[0077] 2.1 kg of the prepared dough was weighed out, formed into balls, and rested for 5 minutes. The rested dough was divided into 70 g portions, rolled into balls, and shaped into cylindrical shapes with a length of 8 to 10 cm. The dough was then placed in a mold and arranged vertically so that the length was about 6 cm. In this state, the dough was frozen at -35°C for about 30 minutes until the core temperature of the dough reached -10°C, after which it was placed in a plastic bag, sealed, and stored in a storage freezer at -18°C for 1 to 6 weeks.

[0078] (2) Bread production The frozen dough produced above was removed from the plastic bag and arranged on a flat tray with holes at intervals of about 10 cm so that they would not touch each other. In this state, it was thawed at room temperature for about 1 hour, and fermented at 28°C for 14 hours. After fermentation, each piece was cut vertically and baked in a 170°C oven for 17 to 20 minutes while adding steam for 8 to 10 seconds to produce bread.

[0079] (3) Evaluation The volume (specific volume (mL / g)) of the produced bread was measured. In addition, the appearance (extent of the cuts on the surface) and texture (crispness) were evaluated according to the same evaluation criteria as in Experimental Example 1.

[0080] (4) Results The results are shown in Table 2 below.

[0081] [Table 2]

[0082] (5) Discussion As shown in Table 2, when bread is produced through a process of freezing bread dough, the bread of Comparative Example 2, which used glucose oxidase, lost its volume, whereas the breads of Examples 4 to 6, which used carbohydrate oxidase, had a volume equal to or greater than that of the control after a one-week freezing period. Furthermore, after a six-week freezing period, the volume was reduced compared to the control, but the loss in volume was suppressed in Examples 4 to 6 compared to Comparative Example 2.

[0083] Regarding appearance (spread of cut surface), the bread of Comparative Example 2, which used glucose oxidase, had the same result as the control, whereas the breads of Examples 4 to 6, which used carbohydrate oxidase, were clearly evaluated as being good.

[0084] Regarding texture (crispy texture), the evaluation of the control bread in which no enzyme was used and the bread in Comparative Example 2 in which glucose oxidase was used decreased as the freezing period increased. On the other hand, as shown by the results for the 5-week and 6-week freezing periods, the breads in Examples 4 to 6 in which carbohydrate oxidase was used had a good texture (crispy texture) even after a long freezing period.

Claims

1. A frozen bread dough improver containing carbohydrate oxidase.

2. The frozen dough modifier according to claim 1, which has a volume increasing effect.

3. The frozen bread dough modifier according to claim 1 or 2, which has a texture improving effect.

4. A frozen bread dough modifier according to any one of claims 1 to 3, which has an appearance improving effect.

5. The frozen bread dough improver according to any one of claims 1 to 4, wherein the carbohydrate oxidase has the property of acting on one or more carbohydrates selected from glucose, maltotriose, maltose, galactose, maltotetraose, lactose, cellobiose, and maltodextrin.

6. A frozen bread dough improver according to any one of claims 1 to 5, wherein the carbohydrate oxidase comprises a polypeptide shown in any one of (1) to (3) below. (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1, (2) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and which exhibits substrate specificity equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; and (3) A polypeptide having an amino acid sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 and exhibiting substrate specificity equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

1.

7. A dough preparation step of adding carbohydrate oxidase to dough ingredients to prepare dough; A freezing step of freezing the prepared dough; A method for producing frozen bread dough comprising the steps of:

8. A thawing step of thawing frozen bread dough containing carbohydrate oxidase; A fermentation process for fermenting the thawed dough; A heating step of heating the fermented dough; A method for producing bread.

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