Physical property-improving composition for cereal flour dough and use method of same

Abstract

[Problem] To provide: a composition for promoting improvement in the extensibility of cereal flour dough and formation of SS bonds; and a method for improving cereal flour dough by using the composition. [Solution] It has been found that, by degrading y-type and x-type subunits 5 among HMW-GSs that form glutenin by using a polypeptide having elastin degradation activity, the extensibility of cereal flour dough improves, and at the same time, new SS bonds are formed as a result of increased mobility of cysteine residues in the glutenin.

Description

Composition for improving physical properties of cereal flour dough and method of using the same

[0001] The present invention relates to a composition comprising a polypeptide with elastinolytic activity that improves the extensibility of cereal flour dough and promotes the formation of SS bonds.

[0002] The processing suitability of foods made primarily from flour varies significantly depending on the flour proteins used. Flour proteins are classified into albumin, globulin, prolamin, and glutelin based on their solubility; the former two are metabolic proteins, and the latter two are storage proteins. Wheat flour storage proteins, such as gliadin (equivalent to prolamin) and glutenin (equivalent to glutelin), constitute gluten. Wheat flour glutenin forms a network-like polymer via disulfide bonds, significantly affecting the volume and dough extensibility of foods such as bread. When glutenin is separated by molecular weight using polyacrylamide gel electrophoresis (SDS-PAGE), it is divided into two groups: high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS). HMW-GS can be further divided into x-type, which has a short migration distance, and y-type, which has a long migration distance.

[0003] The migration pattern of glutenin in SDS-PAGE varies depending on the wheat variety, and is therefore utilized for screening and breeding wheat varieties with excellent taste and manufacturing suitability. According to conventional knowledge, for example, in the case of bread, wheat with good bread-making properties lacks LMW-GS and retains the combination of subunits 5 and 10 of HMW-GS (hereinafter abbreviated as 5+10), with subunit 5 corresponding to the x-type and subunit 10 corresponding to the y-type.

[0004] The x-type and y-type HMW-GS differ in the number of cysteine ​​residues they contain. The x-type has been reported to have two or four cysteine ​​residues, while the y-type has three or seven. In either case, the x-type has fewer cysteine ​​residues than the y-type. Exceptionally, subunit 5 of the 5+10 structure is an x-type but contains three or five cysteine ​​residues. Not all of these cysteine ​​residues participate in disulfide bonds, and research is still ongoing to determine which cysteine ​​residues are involved in disulfide bonds. However, all reports indicate that the y-type has more cysteine ​​residues than the x-type, and that even in the x-type, the cysteine ​​residue contained only in subunit 5 is disulfide bonded. The y-type and subunit 5 not only increase the number of bonding sites but also facilitate the construction of a polymer structure from a linear to a network structure, imparting strong elasticity to bread dough. Therefore, varieties that have subunit 5 in the x-type as well as the y-type tend to be wheat varieties with excellent taste and manufacturing suitability.

[0005] However, when 5+10 HMW-GS and some LMW-GS with strong gluten content coexist, the resilience of the flour dough becomes too strong, making it difficult to stretch and resulting in a decrease in the specific volume of the bread. Wheat with this combination of subunits has also been bred, and a representative variety is the domestic wheat variety "Yumechikara." When using "Yumechikara" as an ingredient in bread, it is necessary to blend in an appropriate amount of medium-strength flour to loosen the dough, or the baker or bread maker must use their high level of skill to correct the dough's characteristics when making bread.

[0006] Not only bread, but all wheat flour foods produced on a factory scale must be mechanically and freezing resistant due to the diversification of manufacturing processes and sales formats, and even if the SS bonds in wheat flour dough are strengthened, good dough extensibility is required. The effect of supporting SS bonds and the effect of decomposing gluten and starch to improve dough extensibility can be adjusted by combining appropriate amounts of redox agents and enzymes. Various quality improvers have been developed that are formulated with this antagonistic balance in mind.

[0007] On the other hand, elastin is a protein found specifically in animal proteins. Elastin is a protein found in the tendons and fibers of meat, making meat foods difficult to eat. However, efforts have been made to improve the texture by combining elastase with various other enzymes.

[0008] Yasuo Matsumura; The significance of the Grain Science Consortium and factors determining the physical properties of wheat flour products, Chemistry and Biology, 52(6), pp387-394; (2014) Masaharu Seguchi, Seiichi Yoshino; Gluten - Precipitation factors - 1, New Food Indust. , 63(6), pp1-18; (2021) Goro Ishikawa, Mika Saito, Hiroyuki Ito, Masato Taira, Hidekazu Maejima, Yoshinori Taniguchi, Toshiki Nakamura; Development of PCR DNA markers for discriminating wheat high-molecular-weight glutenin subunit "5+10" and its application to bread varieties for the Tohoku region, Tohoku National Agriculture and Food Research Institute Research Bulletin, 103, pp27-37; (2005) Yi Li, Jiahui Fu, Qun Shen, Dong Yang; High-Molecular-Weight Glutenin Subunits: Genetics, Structures, and Relation to End Use Qualities, Int. J. Mol. Sci., 22(184), (2021)

[0009] The problem to be solved by the present invention is to provide a composition that improves the extensibility of grain flour dough and promotes the formation of SS bonds, and a method for improving grain flour dough using said composition.

[0010] As a result of extensive research into solving the above-mentioned problems, the inventors have discovered that the above-mentioned problems can be solved by degrading the y-type HMW-GS and x-type subunit 5 that make up glutenin with a polypeptide having elastinolytic activity, thereby improving the extensibility of grain flour dough and at the same time increasing the mobility of cysteine ​​residues in the glutenin to form new SS bonds.

[0011] That is, the present invention provides: (1) a composition for increasing disulfide bonds in glutenin polymer proteins by degrading high molecular weight glutenin subunits contained in cereal flour with a polypeptide having elastinolytic activity; (2) the composition of (1) above, characterized in that the high molecular weight glutenin subunits contained in cereal flour are y-type and / or x-type subunits5; (3) the composition of (1) or (2), in which the polypeptide having elastinolytic activity includes a monomer with a molecular weight of 18 kDa; and (4) a method for increasing disulfide bonds in glutenin polymer proteins by degrading high molecular weight glutenin subunits contained in cereal flour with a polypeptide having elastinolytic activity in an amount of 0.3 U (units) or more but less than 3.0 U per 100 g of wheat flour.

[0012] According to the present invention, by adding a polypeptide having elastinolytic activity to cereal flour dough and subjecting it to an enzymatic degradation reaction, high-molecular-weight glutenin polymer proteins are degraded, the mobility of free cysteine ​​residues in the glutenin increases, and the number of disulfide bonds in the glutenin polymer proteins increases. As a result, the cereal flour dough has improved extensibility, but because it contains many disulfide bonds, excessive resilience is suppressed, improving bread-making properties. Furthermore, wheat flour containing 5+10 HMW-GS and some LMW-GS with strong gluten content has such high dough resilience that breadmaking was previously impossible without the addition of all-purpose flour. However, by applying this improved method, bread can be made without the use of all-purpose flour.

[0013] SDS-polyacrylamide electrophoresis patterns of polypeptide powders; the left lane is a molecular weight marker. Comparison of wheat gluten degradation rates (test enzyme powder 0.1 mg / mL); the vertical axis of the graph indicates gluten residual rate (%), the horizontal axis indicates elapsed time (hours). The solid line (A) in the graph indicates test group 1, the dotted line (B) indicates comparison group 1, and the dotted line (C) indicates the control group. Comparison of wheat gluten degradation levels (test enzyme powder 0.5 mg / mL); the vertical and horizontal axes and (A) to (C) in the graph are the same as in Figure 1. Comparison of wheat gluten degradation levels (test enzyme powder 1.0 mg / mL); the vertical and horizontal axes and (A) to (C) in the graph are the same as in Figure 1. Amount of disulfide bonds in gluten aggregates (test enzyme powder 0.5 mg / mL); (A) indicates the control group, (B) indicates test group 1, and (C) indicates comparison group 1. Appearance of gluten aggregates when enzyme activity inhibitors are added. Elastase activity when enzyme activity inhibitors are added; the vertical axis of the graph represents elastase activity (U / L). Changes in protein concentration in hydrophobic chromatography; the horizontal axis of the graph represents fraction number, the left axis represents protein concentration (mg / ml), and the right axis represents ammonium sulfate concentration (%). The solid line in the graph represents protein concentration, and the dotted line represents ammonium sulfate concentration. Presence or absence of gluten aggregates in the collected fractions. SDS-PAGE gels for each fraction; (E) represents elastase, (M) represents molecular weight marker. SDS-PAGE gels of gluten hydrolyzate; (Cont.) represents the control group, (A) represents experimental group 1.

[0014] The present invention will be described in detail below. The composition of the present invention contains at least a polypeptide having the activity of specifically hydrolyzing elastin (hereinafter referred to as a polypeptide having elastin-degrading activity), and thus has elastin-degrading activity.

[0015] The polypeptide having elastinolytic activity used in the present invention may be prepared in accordance with standard enzyme purification methods from animal tissues such as those of humans, pigs, mice, and rats, bacteria such as those of the genus Pseudomonas and Streptomyces, actinomycetes, fungi, etc. The polypeptide having elastinolytic activity used in the present invention may also be a commercially available enzyme or enzyme preparation having elastinolytic activity.

[0016] Commercially available elastase or elastase preparations include, for example, elastase or elastase preparations derived from human pancreas, human leukocytes, human purulent sputum leukocytes, porcine pancreas, mouse pancreas, mouse macrophage, rat pancreas, Pseudomonas, etc.

[0017] The polypeptide having elastinolytic activity used in the present invention may be obtained by biosynthesis in Escherichia coli or the like in accordance with known genetic engineering techniques, or may be obtained by chemical synthesis techniques such as the Boc method or the Fmoc method. When using chemical synthesis techniques, it is convenient to use a peptide synthesizer.

[0018] In addition to the above-mentioned methods, the peptides having elastinolytic activity used in the present invention can be isolated from microorganisms of the genus Streptomyces. Examples include Streptomyces erythraeus, Streptomyces griseus, Streptomyces omiyaensis, Streptomyces fradiae, and Streptomyces roseoflavus. The culture medium for culturing these microorganisms may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, and inorganic salts that can be assimilated by the cells of the microorganism.

[0019] Examples of carbon sources that can be used include glucose, acetic acid, ethanol, glycerol, molasses, and sulfite pulp waste liquor. Examples of nitrogen sources that can be used include urea, ammonia, ammonium sulfate, ammonium chloride, and nitrates. Phosphate, potassium, and magnesium sources can be ordinary industrial raw materials such as superphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, and magnesium chloride, with inorganic salts such as zinc, copper, manganese, and iron ions being added as needed. Furthermore, vitamins, amino acids, nucleic acid-related substances, and the like may be added as needed. Organic substances such as casein, yeast extract, meat extract, and peptone may also be added.

[0020] Culture conditions may be appropriately selected depending on the type of medium, culture method, etc., and are not particularly limited as long as they allow the cells of the above-mentioned microorganism to grow and produce the polypeptide having elastinolytic activity used in the present invention. For example, the pH is adjusted to 3.0 to 9.0, preferably 5.0 to 8.5, and more preferably 6.0 to 8.0, and the microorganism is cultured in a liquid medium under aerobic conditions such as shaking culture or aeration and agitation culture at 25 to 35°C, preferably 27 to 30°C, for 6 to 96 hours. The pH may be adjusted to a constant value during culture. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like.

[0021] After the culture, the culture broth is subjected to a common solid-liquid separation method such as centrifugation or filter press to obtain a culture supernatant. The culture supernatant can be used as is for preparing the composition of the present invention, but it may also be purified by ultrafiltration, hydrophobic chromatography, ion exchange chromatography, gel filtration chromatography, etc., and the resulting purified product may be used as is or as a dried product by a drying method that does not impair the enzyme activity for preparing the composition of the present invention.

[0022] The polypeptide having elastinolytic activity used in the present invention is preferably a polypeptide that exhibits the following properties in terms of elastinolytic activity: Optimum reaction pH: pH 7.0 to 11.0 Optimum reaction temperature: 50 to 80°C pH stability: stable at pH 7.0 or lower Temperature stability: stable at 5 to 60°C

[0023] Here, the optimal reaction pH refers to the pH at which 80% or more of the activity is maintained, with the activity at pH 8.0 being 100%. The optimal reaction temperature refers to the temperature at which 80% or more of the activity is maintained, with the activity at 70°C being 100%. The pH stability refers to the pH at which 90% or more of the pre-reaction activity is maintained even after standing at 25°C for 24 hours at a predetermined pH. The temperature stability refers to the temperature at which 70% or more of the pre-reaction activity is maintained even after standing at pH 7.5 for 10 minutes at a predetermined temperature.

[0024] The polypeptide having elastin degrading activity used in the present invention may be a polypeptide that exhibits a molecular weight of approximately 18 kDa in SDS-polyacrylamide gel electrophoresis. To exhibit this degrading activity, it is sufficient for the polypeptide to contain at least a monomer having a molecular weight of approximately 18 kDa, and it may exist in the form of a dimer or trimer.

[0025] Specific examples of polypeptides having elastin decomposition activity that can be used in the present invention include polypeptides having any of the following amino acid sequences (a) to (e) and having elastin decomposition activity:

[0026] The polypeptides having the amino acid sequence set forth in SEQ ID NO: 1 shown below include polypeptides showing a molecular weight of approximately 18 kDa in SDS-polyacrylamide gel electrophoresis: (a) the amino acid sequence set forth in SEQ ID NO: 1; (b) an amino acid sequence in which 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids have been added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 1; and (c) an amino acid sequence having at least 80%, preferably 90%, more preferably 95% sequence identity or sequence homology with the amino acid sequence set forth in SEQ ID NO: 1.

[0027] The deletion, substitution, or addition of amino acids in (b) above may occur simultaneously, and the substituted or added amino acids may be natural or non-natural. Examples of natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0028] Furthermore, the amino acid sequence homology or identity in (c) above can be calculated by appropriately aligning a query sequence (sequence to be evaluated) with the sequence of SEQ ID NO: 1 using analysis software such as BLAST, FASTA, or CLUSTAL. In the present application, the value calculated using the CLUSTAL algorithm is used.

[0029] The content of each polypeptide in the composition of the present invention depends on the specific activity of the elastin-degrading activity of the polypeptide (enzyme activity per unit polypeptide), but typically, the content is preferably an amount that results in an elastin-degrading activity of 0.1 to 1,000 units (U), more preferably 1 to 100 units (U), per gram of the composition of the present invention.

[0030] Elastin degrading activity can be calculated using Elastin Congo Red (Nacalai Tesque), which is finely divided elastin with the dye Congo Red attached, as a substrate, with the activity that causes a change in absorbance at 495 nm of 1 per hour being defined as 1 unit (U: enzyme activity unit).

[0031] The composition of the present invention may be combined with other ingredients in appropriate amounts as needed, as long as the effect of the present invention, i.e., the elastin-degrading activity, is not impaired and glutelin-degrading activity is not unnecessarily imparted. For example, organic acid salts include sodium citrate, potassium citrate, calcium citrate, sodium acetate, sodium ascorbate, sodium lactate, calcium lactate, sodium sulfate, potassium sulfate, calcium sulfate, potassium carbonate, sodium carbonate, calcium carbonate, and calcium glycerophosphate. Furthermore, polysaccharides include alginic acid, sodium alginate, pectin, carboxymethylcellulose, carrageenan, guar gum, curdlan, starch, gum arabic, welan gum, cassia gum, xanthan gum, chitosan, psyllium seed gum, gellan gum, tamarind seed gum, dextran, furcellaran, pullulan, and hyaluronic acid. In addition, the composition may contain various edible fats and oils, dairy products, fruit juices, grain flours, etc.; emulsifiers such as monoglycerides, succinic acid monoglyceride, diacetyltartaric acid monoglyceride, sucrose fatty acid esters, lecithin, enzymatically hydrolyzed lecithin, sodium stearoyl lactylate and calcium stearoyl lactylate; enzymes such as α-amylase, β-amylase, glucoamylase, hemicellulase (pentosanase), cellulase, and proteases other than those of the present invention; amino acids such as cysteine, cystine, methionine, alanine, aspartic acid, and glycine; inorganic salts such as sodium chloride, potassium chloride, ammonium chloride, and calcium dihydrogen phosphate; nucleic acids such as sodium inosinate and sodium guanylate; vitamins such as vitamin B1, vitamin B2, vitamin C (L-ascorbic acid), and vitamin E; alcohols such as ethanol and glycerol; sugars such as sucrose, glucose, maltose, and lactose; and excipients such as dextrin and various starches. These additive components may be added singly or in combination.

[0032] The term "cereal flour" as used herein refers to flour prepared by grinding cereal grains, cereal endosperm, or the endosperm with the germ and / or epidermis attached. Examples of cereal grains described herein include wheat, rye, and barley, and examples of cereal flour include wheat flour (e.g., strong flour, semi-strong flour, medium-strength flour, weak flour, whole wheat flour, etc.), barley flour, and rye flour (whole rye flour or peeled rye that is finely ground, medium-ground, coarsely ground, or stone-ground).

[0033] In the present invention, high molecular weight glutenin subunits (HMW-GS) are proteins that constitute glutenin contained in wheat flour. They have a molecular weight of 70,000-90,000 and are composed of approximately 600-800 amino acid residues. The amino acid sequence is divided into three structural domains: a non-repetitive N-terminal domain of approximately 100 residues, a repetitive central domain of approximately 500-700 residues, and a non-repetitive domain of approximately 40 residues. Furthermore, HMW-GS can be classified into x-type and y-type based on their SDS-PAGE migration patterns. HMW-GS contained in cereal flours other than wheat flour are called high molecular weight secalin in rye flour and D-hordein in barley. While high molecular weight secalin in rye flour can be divided into x-type and y-type units, similar to wheat flour, barley D-hordeins cannot be separated. The polypeptides having elastinolytic activity of the present invention exert their effects regardless of the differences in high molecular weight glutenin subunits, high molecular weight secalin, and D-hordein, and change the structure of each of the glutenin, secalin, and hordein polymers (called glutenin polymer proteins only in the case of wheat flour).

[0034] HMW-GS contained in wheat flour is divided into x-type, which migrates relatively short distances on SDS-PAGE, and y-type, which migrates longer distances, and each detected band is assigned a number. For example, HMW-GS numbered 5 is designated as subunit 5. Wheat flour is a hexaploid with the genomic composition "AABBDD." HMW-GS is located at Glu-1 (locus) on the long arms of chromosomes 1A, 1B, and 1D, and is designated Glu-A1, Glu-B1, and Glu-D1 for each genome. Some subunits encoded by Glu-B1 or Glu-D1 are often inherited as a set; x-type subunit 5 and y-type subunit 10 are located on Glu-D1 and are known to be inherited together.

[0035] In wheat, wheat flour containing both 5+10 HMW-GS and some LMW-GS with strong gluten refers to wheat flour containing both the Glu-D1d gene encoding the 5+10 HMW-GS and the Glu-B3b or Glu-B3g gene encoding the LMW-GS with strong gluten, and representative varieties include Yumechikara, Ginga no Chikara, and Hanamanten. In the present invention, wheat flour other than those listed above can be treated equally as long as it contains a set of genes Glu-D1d and Glu-B3b or Glu-D1d and Glu-B3g and the expression of these genes is not inhibited, and the effects of the polypeptides with elastinolytic activity can be obtained.

[0036] By contacting a composition containing a polypeptide having elastinolytic activity of the present invention with cereals, it is possible to improve the extensibility of dough made from the cereal flour while simultaneously increasing SS bonds. Furthermore, wheat flour containing 5+10 HMW-GS and LMW-GS, which has a strong gluten content, produces dough with such high resilience that breadmaking is not possible without the addition of all-purpose flour. However, by adding the improver, bread can be made without the use of all-purpose flour.

[0037] There are no particular limitations on the method for treating grains with a composition containing a polypeptide having elastinolytic activity of the present invention, as long as it is under general conditions established by those skilled in the art. For example, any of the following methods may be used: a method in which the composition of the present invention is premixed with other flours and then dough is made, or a method in which the composition of the present invention is dispersed in water or liquid oil and then kneaded as part of the raw materials during the mixing process.

[0038] When contacting a composition containing a polypeptide having elastin-degrading activity of the present invention with grain flour, the amount, temperature, and time of each component can be adjusted appropriately depending on the amount of glutenin polymer protein in the grain flour, and the specific activity and amount of each polypeptide contained in the composition of the present invention.

[0039] For example, when a composition containing a polypeptide having elastinolytic activity of the present invention is brought into contact with 100 g of gluten (glutenin polymer), the amount added may be an amount such that the polypeptide having elastinolytic activity contains 0.3-30 units (U), preferably 1.0-25 units (U), and more preferably 3.0-21 units (U) of elastinolytic activity. When the composition is brought into contact with 100 g of wheat flour, the amount added is 0.1-10 units (U), preferably 0.2-7 units (U), and more preferably 0.3-3 units (U) per 100 g of wheat flour, assuming that the wheat flour contains 13% gluten.

[0040] When a composition containing a polypeptide having elastinolytic activity of the present invention is brought into contact with cereal flour, glutenin molecules are degraded, but the formation of disulfide bonds between glutenins or between glutenin and gliadin is promoted. Among glutenin molecules, the y-type and x-type subunits 5 of HMW-GS are particularly susceptible to degradation. These glutenin molecules are divided into three regions: the N-terminal domain, the central repeat domain, and the C-terminal domain. The central repeat domain contains more hydrophobic amino acids, such as alanine, than other molecules. Because the polypeptide having elastinolytic activity used in the present invention is believed to cleave alanine residues, the y-type and x-type subunits 5 are presumably degraded at the alanine residues. While the mechanism by which disulfide bonds are increased is unclear, it is thought that hydrolysis of the y-type and x-type subunits 5 in the central repeat domain by the polypeptide having elastinolytic activity of the present invention increases the mobility of cysteine ​​residues within HMW-GS, thereby increasing the freedom to form disulfide bonds with other molecules.

[0041] The present invention will be specifically described below using examples and comparative examples, but the present invention is not limited to these.

[0042] Example (1) Production of Polypeptide Having Elastolytic Activity A spore suspension (10 ７ One loopful of the culture medium (30 g / L of soluble corn starch, 30 g / L of corn steep liquor, 1 g / L of ammonium sulfate, 0.5 g / L of magnesium sulfate, and 3 g / L of calcium carbonate, adjusted to pH 7.0) was inoculated with one loopful of the culture medium, and the medium was cultured in a 200 mL Erlenmeyer flask at 28°C and 200 rpm for 12 hours to obtain a completed seed culture solution.

[0043] 1 mL of the resulting seed culture solution was transferred to 2000 mL of a main culture medium (a medium containing soluble corn starch 300 g / L, corn steep liquor 150 g / L, defatted soybean flour 250 g / L, ammonium sulfate 10 g / L, magnesium sulfate 5 g / L, and calcium carbonate 30 g / L, and adjusted to pH 7.0), and cultured in a 10 L flask at 28°C and 200 rpm for 40 hours to obtain a main culture solution.

[0044] After completion of the main culture, the liquid was filtered to remove solid matter such as bacteria, and then treated with an ultrafiltration membrane with a molecular weight cutoff of 6,000 to recover a concentrated liquid fraction, which was then spray-dried to obtain a polypeptide powder.

[0045] Example (2) Elastolytic activity of polypeptide The polypeptide powder was subjected to SDS-polyacrylamide electrophoresis and stained by Coomassie brilliant blue staining (Sigma). As a result, as shown in Figure 1, bands were confirmed at approximately 18 kDa, approximately 14 kDa, and approximately 11 kDa.

[0046] The amino acid sequences of the proteins contained in the bands of about 18 kDa, about 14 kDa, and about 11 kDa were analyzed by a standard method, and it was found that all of the proteins were the same protein that had been shortened by processing from the N-terminus, and the amino acid sequence of the protein was set forth in SEQ ID NO: 1. Furthermore, the full length of the gene encoding the polypeptide was determined by a standard method, and it was estimated to be a gene consisting of a 726 bp base sequence.

[0047] The gene consisting of the 726 bp nucleotide sequence was cloned into the multicloning site of pNCMO2 (Takara Bio Inc.), and expression analysis was performed using a Brevibacillus expression system (Takara Bio Inc.). As a result, it was confirmed that the proteins of approximately 18 kDa, approximately 14 kDa, and approximately 11 kDa also exhibited elastinolytic activity.

[0048] The elastin degradation activity was determined by using Elastin Congo Red (manufactured by Nacalai Tesque) as a substrate, and the enzyme activity that causes a change in absorbance at 495 nm of 1 per hour was defined as 1 U.

[0049] Example (3) Comparison of Wheat Gluten Decomposition Rates The decomposition rates of wheat gluten were compared for the polypeptides with elastinolytic activity obtained in Example (1). The test group to which the polypeptides were added was designated Experimental Group 1. Distilled water was used as the control group, and Orientase 90N (manufactured by HI Corporation) was used as Comparative Group 1. Orientase 90N is a powdered endo-type neutral protease. For convenience, the peptides with elastinolytic activity obtained in Example (1) and Orientase 90N are hereinafter collectively referred to as the test enzyme powder.

[0050] - Method - The evaluation method is described below. 1) Wheat gluten (Regular Gluten A: manufactured by Mitsubishi Corporation Life Sciences) was dispensed into 1.5 mL microtubes to a final concentration of 20 mg / mL. 2) Test enzyme powders were added to the microtubes in 1) to final concentrations of 0.1, 0.5, and 1.0 mg / mL. Distilled water was added to the control group. 3) The enzyme reaction was carried out in a thermomixer (30°C, 1100 rpm), and the residual gluten rate was determined 3 and 6 hours after the start of the reaction. 4) The residual gluten rate was determined by leaving each test sample to stand for a certain period of time, removing the supernatant by decantation, drying the precipitate, and measuring its weight to calculate the decomposition rate.

[0051] Results: Figures 2 to 4 show the residual wheat gluten over time for each dose of test enzyme powder. Figure 2 shows the evaluation when the test enzyme powder was added at a final concentration of 0.1 mg / mL, Figure 3 shows the results for 0.5 mg / mL, and Figure 4 shows the results for 1.0 mg / mL. In Figures 2 to 4, the control group, indicated by the dotted line (angle), maintained a 100% residual rate even 6 hours after the start of the enzyme reaction. This indicates that wheat gluten is not degraded under conditions of 30°C and 1100 rpm. In Experimental Group 1, indicated by the solid line, the residual rate decreased slightly depending on the elapsed time and the added amount, but never fell below 80%. In contrast, in Comparison Group 1, indicated by the dashed line, the residual rate decreased significantly depending on the elapsed time and the added amount. These results indicate that polypeptides with elastinolytic activity have the ability to degrade wheat gluten, but the degradation rate is slow, while Orientase 90N rapidly degrades the same substrate.

[0052] Example (4) Quantitative Evaluation of SS Bond Amount in Gluten Aggregates After adding the test enzyme powder to wheat gluten and allowing the enzyme reaction to occur, the amount of SS bonds contained in the product obtained as gluten aggregates was quantified. The test group to which the polypeptide was added was designated as Experimental Group 1. Distilled water was used as the control group, and Orientase 90N (manufactured by HI Corporation) was used as Comparative Group 1.

[0053] -Method- 1) Wheat gluten (regular gluten A) was dispensed into 1.5 mL microtubes to a final concentration of 20 mg / mL. 2) Test enzyme powders were added to the microtubes in 1) to a final concentration of 0.5 mg / mL. Distilled water was added to the control group. 3) The enzyme reaction was carried out in a thermomixer (30°C, 1110 cp, 3 hours). 4) Each test group was centrifuged, and the centrifugal precipitate was washed several times with distilled water and then lyophilized. 5) The lyophilized material was solubilized with 8 M urea, and free cysteine ​​residues were measured using Ellman's reagent. The total amount of cysteine ​​residues was determined in the same manner after adding 2-mercaptoethanol.

[0054] Results: The amount of disulfide bonds contained in gluten aggregates is shown in Figure 5. Figure 5 shows the results of adding the test enzyme powder at a final concentration of 0.5 mg / mL. The amount of disulfide bonds in the control group was approximately 0.6 μmol / g, while in experimental group 1 it was 2.9 μmol / g. By enzymatically reacting with a polypeptide with elastinolytic activity, approximately five times as many disulfide bonds were generated in the gluten aggregates. On the other hand, in comparison group 1, the amount decreased to 0.3 μmol / g, indicating that the addition of Orientase 90N reduced the amount of disulfide bonds. These results demonstrate that when polypeptides with elastinolytic activity are enzymatically reacted with wheat gluten, they produce gluten aggregates and increase the number of disulfide bonds in the product.

[0055] Example (5) Identification of the Enzyme That Forms Gluten Aggregates 1 From Examples (3) and (4), it was inferred that when a polypeptide having elastinolytic activity is enzymatically reacted with wheat gluten, it slowly decomposes gluten and generates SS bonds in gluten aggregates. We investigated whether this change in wheat gluten is due to the elastinolytic enzyme (elastase) contained in the polypeptide or other contaminating enzymes.

[0056] Method: 1) Wheat gluten (regular gluten A) was dispensed into 1.5 mL microtubes to a final concentration of 20 mg / mL. 2) A polypeptide with elastinolytic activity was added to the microtube in step 1 to a final concentration of 0.5 mg / mL. Five test plots were set up, some of which also contained enzyme inhibitors. Test plot 1 (T1): Wheat gluten + polypeptide with elastinolytic activity Test plot 2 (T2): Wheat gluten + polypeptide with elastinolytic activity + EDTA Test plot 3 (T3): Wheat gluten + polypeptide with elastinolytic activity + PMSF Test plot 4 (T4): Wheat gluten + polypeptide with elastinolytic activity + TPCK Test plot 5 (T5): Wheat gluten + polypeptide with elastinolytic activity + AAPV-CMK. The enzyme inhibitors used in Test plots 2 to 5 have the following characteristics: EDTA (ethylenediaminetetraacetic acid): Inhibits the enzymatic activity of metalloproteases and nucleases. PMSF (phenylmethylsulfonyl fluoride): Inhibits the enzymatic activity of serine proteases and cysteine ​​proteases. TPCK (N-tosyl-L-phenylalanine chloromethyl ketone): Inhibits the enzymatic activity of serine proteases. AAPV-CMK (Ala-Ala-Pro-Val-chloromethyl ketone): Inhibits the enzymatic activity of elastase. 3) The enzyme reaction was carried out in a thermomixer (30°C, 1110 cp, 3 hours). 4) The presence or absence of gluten aggregates was observed, and the enzymatic activity of elastase was further examined using Elastin Congo Red (Nacalai Tesque) as a substrate. 1 U of enzyme activity was defined as the change in absorbance at 495 nm of 1 per hour.

[0057] Results: Figure 6 shows the appearance of gluten aggregates, and Figure 7 shows elastase activity. Test group 1 (wheat gluten + polypeptide with elastinolytic activity) confirmed gluten aggregates in the reaction solution, similar to test group 1 in Example (4). Furthermore, this test group exhibited high elastase activity, approximately 20 U / L, indicating that it maintained high activity. Test group 2 (wheat gluten + polypeptide with elastinolytic activity + EDTA) confirmed gluten aggregates in the reaction solution. Furthermore, this test group exhibited high elastase activity, approximately 20 U / L, similar to test group 1. Test group 3 (wheat gluten + polypeptide with elastinolytic activity + PMSF) confirmed no gluten aggregates in the reaction solution. Furthermore, no elastase activity value was obtained for this test group, indicating that the enzymatic activity of PMSF was inhibited. Test group 4 (wheat gluten + polypeptide with elastinolytic activity + TPCK) contained gluten aggregates in the reaction solution. Furthermore, the elastase activity in this test group was equivalent to that in test groups 1 and 2 (approximately 20 U / L), demonstrating that it maintained a high level of activity. Test group 5 (wheat gluten + polypeptide with elastinolytic activity + AAPV-CMK) contained no gluten aggregates in the reaction solution. Furthermore, no elastase activity value was obtained for this test group, indicating that the enzymatic activity of AAPV-CMK had been inhibited. Based on the above, test groups 1, 2, and 4, in which gluten aggregates were observed in the reaction solution, maintained high levels of elastase activity, whereas test groups 3 and 5, in which no gluten aggregates were observed, showed inhibited elastase activity. Since gluten aggregates could not be observed when elastase activity was inhibited, it was inferred that the component that produced gluten aggregates and increased the amount of SS bonds among polypeptides with elastin-degrading activity was elastase.

[0058] Example (6) Identification of the Enzyme That Forms Gluten Aggregates 2 In Examples (3) to (5), it was speculated that the elastase activity of the polypeptide having elastinolytic activity is a factor that degrades gluten and generates SS bonds in gluten aggregates. Therefore, the polypeptide having elastinolytic activity was further fractionated by hydrophobic chromatography to examine whether elastase was contained in the fractions having enzymatic activity.

[0059] -Method- 1) Polypeptides with elastin degrading activity were subjected to hydrophobic chromatography. Separation conditions were as follows: Column: Phenyl-Sepharose; Eluent: Eluent A) 20 mM phosphate buffer, pH 6.0, 20% ammonium sulfate; Eluent B) 20 mM phosphate buffer, pH 6.0; Gradient: 0% Eluent B (50 min) → 0%-100% Eluent B (175 min) → 100% Eluent B (75 min); Flow rate: 0.5 mL / min; Temperature: 25°C. 2) The eluate was collected every 2 minutes, and the elapsed time was recorded as a fraction number. 3) Wheat gluten (regular gluten A) was dispensed into 1.5 mL microtubes to a final concentration of 20 mg / mL. 4) 0.1 mL of the fraction obtained in 2) was added to the microtube in 3) and the presence or absence of gluten aggregates was observed. 5) The fraction obtained in 2), molecular weight markers, and separately purified elastase were subjected to SDS-PAGE, and the molecular weights of the proteins contained in each fraction were compared.

[0060] Results: Figure 8 shows the progression of protein concentration during hydrophobic chromatography, Figure 9 shows the presence or absence of gluten aggregates in the collected fractions, and Figure 10 shows SDS-PAGE gels for each fraction. As shown in Figure 8, polypeptides with elastinolytic activity were detected after 23 minutes of elution and continued to be detected until 40 minutes. When fractions from this period were enzymatically reacted with wheat gluten, gluten aggregates were detected in fractions 31-37, as shown in Figure 9. Furthermore, when molecular weights were separated by SDS-PAGE, bands with an estimated molecular weight of approximately 18 kDa were detected in fractions 31 and 35. The estimated molecular weights matched those of the elastase bands separated by cation exchange resin, confirming that the proteins detected in fractions 31 and 35 were elastase. These results confirm that the proteins detected near the estimated molecular weight of 18 kDa in the polypeptides with elastinolytic activity are factors that cause gluten aggregate formation and are elastase.

[0061] Example (7) Changes in gluten proteins degraded with peptides or elastase having elastinolytic activity Example (3) confirmed that when wheat gluten is degraded with peptides or elastase having elastinolytic activity, the gluten is gently degraded. In order to investigate which components of gluten are degraded and which are involved in the increase in the amount of SS bonds, a qualitative evaluation of the proteins contained in the gluten aggregates in the control group and experimental group 1 was carried out.

[0062] -Method- The gluten hydrolysates obtained in the control group and experimental group 1 of Example 3 were subjected to SDS-PAGE to determine the molecular weight of the protein contained in each test group and estimate the protein.

[0063] Results: Figure 11 shows an SDS-PAGE gel of the gluten hydrolysates obtained in the control and experimental groups. In the control group, clear bands were observed near 130 kDa, 100 kDa, 90 kDa, and 80 kDa. In contrast, in experimental group 1, bands were observed near 100 kDa and 80 kDa, but were somewhat unclear, and the band near 90 kDa was barely discernible. The protein with an estimated molecular weight of approximately 100 kDa is known to represent the x-type subunit 5 of HMW-GS. In addition, proteins near 80 kDa and 90 kDa are known to be y-type HMW-GS, suggesting that these proteins are degraded by peptides with elastinolytic activity or elastase. From the above, it was found that the decomposition of wheat gluten confirmed in Example (3) was the x-type subunit 5 of HMW-GS and some of the y-type HMW-GS among the glutenin molecules contained in gluten.

[0064] Summary: The results of Examples (3) to (7) demonstrated that the polypeptide or elastase having elastase activity of the present invention degrades the x-type subunit 5 of HMW-GS contained in glutenin in wheat gluten, as well as some y-type HMW-GS. Degradation of specific sites is unlikely to occur with other proteases, and has not been observed with, for example, endo-type neutral proteases. Furthermore, because degradation occurs at specific sites, the degradation rate may be slow. On the other hand, it was confirmed that the amount of disulfide bonds increased when the polypeptide or elastase having elastase activity of the present invention was enzymatically reacted with wheat gluten. This behavior is thought to be due to the fact that degradation of the HMW-GS increases the mobility of cysteine ​​residues present in the molecule, making it easier to form disulfide bonds.

[0065] Example (8) Preparation of Yumechikara Roll Bread with Added Polypeptide Having Elastase Activity Examples (3)-(7) demonstrated that polypeptides or elastases with elastase activity can (i) degrade x-type subunit 5 of HMW-GS contained in wheat gluten and some y-type HMW-GS, and (ii) increase the mobility of cysteine ​​residues present in the molecule through degradation of HMW-GS, thereby increasing the amount of disulfide bonds. (i) may improve dough extensibility, and (ii) may improve dough elasticity. Meanwhile, domestic Yumechikara wheat was engineered to incorporate a combination of HMW-GS subunits 5 and 10 to create a wheat variety with excellent flavor and manufacturing suitability. However, because it also retains LMW-GS, the dough is known to have excessively strong physical properties, resulting in poor breadmaking. Therefore, we investigated whether we could improve breadmaking properties by enzymatically reacting a polypeptide with elastase activity with Yumechikara.

[0066] - Bread making - The composition of the roll bread is shown in Table 1, and the manufacturing process is shown in Table 2.

[0067]

[0068]

[0069] Method: Roll bread (straight method) can be prepared according to the following procedure. For formulations containing a polypeptide with elastinolytic activity, the polypeptide was prepared in advance as a 1% aqueous solution and added according to the recipe. Specifically, strong flour (Yumechikara), medium-strength flour (Kitahonami), sugar, salt, skim milk powder, yeast (Dia Yeast, Mitsubishi Corporation Life Sciences), yeast food (Pandaia C-500, Mitsubishi Corporation Life Sciences), water, and the polypeptide with elastinolytic activity were weighed and kneaded in a vertical mixer. The agitation speed of the mixer was adjusted appropriately, and after confirming that gluten had formed in the dough, shortening was added and the dough was kneaded again. Mixing was stopped once the dough and shortening were sufficiently mixed and gluten had re-formed in the dough. The dough temperature at this point was recorded as the kneading temperature. The dough was cooled appropriately during mixing to maintain the kneading temperature within 28±0.5°C. The kneaded dough was allowed to undergo primary fermentation in a constant temperature oven (floor). After primary fermentation was complete, the dough was divided, rolled into balls, and allowed to rest during bench time. After bench time, the rolled dough was shaped using a molder. The shaped dough was allowed to undergo secondary fermentation in a proofer, and after fermentation was complete, it was baked in a reel oven or fixed oven. After baking, the bread was cooled at room temperature for 90 minutes, packed in a plastic bag, and stored in a constant temperature oven maintained at 25°C until use in the evaluation tests.

[0070] - Results - During the series of processes, differences were noticed in the texture of the bread dough when it was rolled after being divided. Comparison area 3, which was made entirely from Yumechikara wheat flour, felt a resilience when the dough was rolled, but areas 2, 3, and 4, which had added polypeptides with elastinolytic activity, had a reduced resilience and were easier to roll. Comparing the appearance of the bread after baking, comparison area 3 had a tendency for the dough to become unruly and the roll came off, but this was not observed in areas 2 and 3, and the shape was good.

[0071] Example (9) Measurement of dough length during shaping In order to evaluate the extensibility of bread dough, which can be perceived intuitively during bread making, the dough length during shaping was examined.

[0072] -Method- Bread dough was molded in a molder according to the conditions listed in Table 2, and the length of the dough unwound from the molder was measured (n=6).

[0073] Results: The results of the dough length for each test group are shown in Table 3. Experimental group 2, experimental group 3, and comparison group 4, which contained peptides with elastinolytic activity, had longer dough lengths than comparison group 3, which used only Yumechikara flour, suggesting improved extensibility.

[0074]

[0075] Example (10) Evaluation of the Physical Properties of Bread Dough The extension resistance and degree of extension of bread dough to which a polypeptide having elastinolytic activity had been added were measured using an Extensograph (manufactured by Brabender).

[0076] -Method- 1) After the flooring process in Table 2, the bread dough was divided into 100g portions and rolled using the rounder attached to the Extensograph. 2) Bench time was allowed (room temperature, 20 minutes). 3) Roll-molded using a molder. 4) Measured using the Extensograph.

[0077] - Results - When comparing comparison area 3, implementation area 2, implementation area 3, and comparison area 4, which used only Yumechikara in the wheat flour, an increase in extensibility was observed in implementation area 2, implementation area 3, and comparison area 4, which added a polypeptide with elastinolytic activity. In addition, when comparing the extension resistance of the same test areas, the measured values ​​increased in implementation area 2, which added 50 ppm of polypeptide with elastinolytic activity, and implementation area 3, which added 100 ppm, but the extension resistance did not increase in comparison area 4, which added 300 ppm. When comparing comparison area 2, implementation area 2, implementation area 3, and comparison area 4, which used Yumechikara and Kita Honami in combination with the wheat flour, the extensibility of implementation area 2, implementation area 3, and comparison area 4, which added a polypeptide with elastinolytic activity, did not achieve values ​​equivalent to comparison area 2, but was close in implementation area 3. On the other hand, when comparing the elongation resistance of the same test sections, the measured values ​​increased in all test sections to which polypeptides with elastinolytic activity were added (Test Section 2, Test Section 3, Comparison Section 4). However, despite the fact that Comparison Section 4 contained the highest amount of polypeptides with elastinolytic activity, the elongation resistance decreased. From the above, it was found that adding polypeptides with elastinolytic activity to Yumechikara can improve dough extensibility while producing a strong, elastic dough. However, it was found that adding 300 ppm or more of the polypeptide relative to the flour significantly increased the elasticity of the bread dough, and reduced the degree of extensibility and elongation resistance.

[0078]

[0079] Example (11) Evaluation of Specific Volume of Bread Roll The specific volume of bread containing the polypeptide having elastinolytic activity was measured according to the rapeseed substitution method, and the bread was measured one day after baking.

[0080] -Method- 1) Prepare a container slightly larger than the bread to be measured, fill it with rapeseed, and level the top. 2) Remove the rapeseed from the container, place the bread in the container, return the rapeseed to the container, and then level the top. 3) Measure the volume of the rapeseed that overflowed with a measuring cylinder, and use this volume of rapeseed as the volume of the bread being measured.

[0081] - Results - The results of the specific volume for each test group are shown in Table 5. There was no difference in specific volume except for comparison group 4, where the specific volume clearly decreased. From the above, it was found that when a polypeptide with elastinolytic activity is added, even if only Yumechikara is used in the wheat flour, it can produce the same volume as when Yumechikara and Kitahonami are used in combination.

[0082]

[0083] Summary: Using polypeptides with elastinolytic activity as bread ingredients has been shown to improve dough extensibility and produce strong, elastic dough. Since polypeptides with elastinolytic activity specifically degrade y-type HMW-GS and x-type subunit 5 of glutenin contained in wheat gluten, increasing the amount of disulfide bonds, we investigated the effect of adding them to bread made with Yumechikara, a type of wheat flour that contains these HMW-GS but has poor breadmaking properties. Typically, when using Yumechikara wheat flour, bread is made by mixing approximately 30% all-purpose flour (e.g., Kitahonami) to improve breadmaking properties. However, adding an appropriate amount of this polypeptide enabled the production of bread dough with good extensibility and high elasticity, even without all-purpose flour. Furthermore, bread with good appearance (volume and peeling) after baking was also produced. However, it was also suggested that if the polypeptide is added in excess, the amount of SS bonds in the bread dough may become too high, and the improvement in dough extensibility and increase in specific volume may reach a plateau.

[0084] As explained above, according to the present invention, a polypeptide having elastinolytic activity specifically degrades y-type HMW-GS and x-type subunit 5 of the glutenins contained in wheat gluten, thereby increasing the amount of disulfide bonds. This effect of addition is particularly effective for domestic wheat, which has an unbalanced glutenin composition due to wheat breeding, and simply adding this polypeptide to the wheat enables the production of high-quality bread with excellent bread-making properties.

Claims

1. A composition for decomposing high molecular weight glutenin subunits contained in cereal flour with a polypeptide having elastolytic activity to increase disulfide bonds in gluten polymer proteins.

2. The composition according to claim 1, wherein the high molecular weight glutenin subunit contained in the cereal flour is a y-type and / or x-type subunit 5.

3. The composition according to claim 1 or 2, wherein the polypeptide having elastolytic activity contains a monomer with a molecular weight of 18 kDa.

4. A method for decomposing high molecular weight glutenin subunits contained in cereal flour with a polypeptide having an elastolytic activity of 0.3 U (unit) or more and less than 3.0 U per 100 g of wheat flour to increase disulfide bonds in gluten polymer proteins.

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