TWI905696B - Bread making improver and bread improvement method - Google Patents

Abstract

An objective of the present invention is to provide a bread-making improving agent that exhibits an effect equivalent to the effect of adding gluten to bread. The bread improving agent that consists of soy protein, phospholipase, and a polypeptide elastinase consisting of a specific amino acid sequence is a substitute for active gluten.

Description

Bread improvers and bread improvement methods

This invention relates to providing a bread improver that can replace gluten and a method for improving bread using the improver.

Due to droughts caused by climate change and geopolitical factors, wheat crop failures and reductions in cultivated area have become significant, leading to a global gluten shortage. Since gluten is an essential component for producing the shape and texture of bread or noodles made primarily from wheat flour, the decrease in the gluten content of wheat flour and the insufficient supply of gluten in the market are related to reduced bread and noodle manufacturing efficiency and product quality.

The role of gluten in bread can be primarily described in four aspects: increased water absorption, improved physical properties, increased volume, and improved texture. Previous bread improvers could also improve several of these four functions individually or in combination. However, compared to the effects of adding gluten, these improvements are more heterogeneous, and it is unknown whether any bread improver can improve all four functions in the same way and uniformly as adding gluten, using a single agent. [Previous Art Documents] [Patent Documents]

[Patent Document 1]

[The problem the invention aims to solve]

The present invention relates to a bread improver that can replace gluten and a method for improving bread products using the improver. [Means for solving the problem]

The inventors of this case, through active and repeated research to solve the aforementioned problems, discovered a bread improver containing soybean protein, phospholipase, and elastin-degrading enzyme. Even when the amount of active gluten added is reduced and replaced with this improver, it can still show the same and similar effects as active gluten, thus completing this invention.

That is, the present invention provides: (1) a bread improver, comprising: a raw material including soy protein, phospholipase and elastin-degrading enzyme; (2) the bread improver as described in (1), wherein the elastin-degrading enzyme described in (1) is a polypeptide having the characteristics of (a) to (c) below: (a) a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence represented by sequence number 1 and having elastin-degrading activity; (b) a polypeptide derived from the genus *Streptococcus*; (c) a polypeptide with an optimal reaction pH of 7.0–11.0 and an optimal reaction temperature of 50–80°C. (3) The bread improver as described in (1) or (2) above, wherein the phospholipase activity per 100 parts by weight of the bread improver is 10,000 U to 20,000 U, and the elastin-degrading enzyme activity is 1 U to 1,000 U. (4) A method for improving bread, wherein 0.01 to 10 parts by weight of the bread improver described in (1) to (3) above replace 1 part by weight of gluten. [Invention Effect]

If the bread improver of this invention is added to wheat flour with reduced gluten content or quantity as a bread improver, as an active gluten, or as a substitute for a bread improver with active gluten as the main ingredient, then the functions of gluten—increasing water absorption, improving physical properties, increasing volume, and improving taste—can be improved to the same or similar state as the aforementioned gluten with a single agent.

The bread improver of this invention contains raw materials including soy protein, phospholipase, and elastin-degrading enzymes, and replaces the function of gluten. A small amount of this bread improver can achieve the same effect as gluten. The effect obtained by adding 1 part by weight of active gluten to 100 parts by weight of wheat flour can be replaced by 0.01 to 10 parts by weight of this bread improver, increasing water absorption in the bread-making process, creating dough properties with good handling efficiency, increasing the volume of baked bread, achieving a soft texture and high resilience, and maintaining the quality of bread containing sufficient gluten.

The term gluten refers to the protein obtained by applying physical forces such as kneading or stirring after the gluten and gluten in wheat flour absorb water. The gluten in this invention includes both intrinsic and extrinsic gluten in wheat flour, both of which can be replaced by the bread improver of this invention. Here, the intrinsic gluten in wheat flour refers to the gluten contained in wheat flour used as a raw material for bread (the gluten formed after kneading wheat flour), and its content tends to decrease when the quality of the wheat flour is poor. The term "added gluten" refers to gluten added as a byproduct to supplement the function when the intrinsic gluten is insufficient. Examples of gluten added as a byproduct include wet gluten, which is made by soaking and washing wheat flour in water to remove the cereal components other than gluten; active gluten, which is made by drying wet gluten into powder; and gluten preparations with active gluten as the main ingredient. The main functions of gluten in bread making, as described in this manual, are to increase water absorption during the bread-making process, create dough with good handling properties, increase the volume of baked bread, and produce bread with a soft texture and high resilience.

The so-called soy protein-containing raw materials used in the bread improver of this invention can include soybeans, defatted soy flour, concentrated soy flour, concentrated soy protein, isolated soy protein, soy milk, etc., as well as the proteins contained therein, or extracts obtained under specific conditions, soy powder, etc. The properties and form of the extracted soy protein can be granular, fragmented, pelleted, or powdered, etc., although not particularly limited to these, but from the viewpoint of easy mixing and dispersion with bread-making grains, pelleted or powdered form is preferred, and powdered form is even more preferred. Soy protein can be obtained by using commercially available products or by using methods known to be effective. As commercially available products, examples include Fujipro AL, Fujipro SEH, Proleena 700, Proleena 800, and Proleena 900 manufactured by Fuji Oil Co., Ltd.; Solpea 4000H and Solpea 5000H manufactured by Nissin Oriyo Co., Ltd.; Propham 649, Propham 974, Propham 781, and Propham 825 manufactured by ADM Co., Ltd.; and SUPRO XT219D and SUPRO PM manufactured by Solae Co., Ltd. In cases where preparation is carried out using well-known methods, it can also be prepared and used using the methods described in Japanese Patent Application Publication No. 8-173052 or Japanese Patent Application Publication No. 9-121780. For example, in the case of raw materials containing soy protein, an inorganic salt solution with a concentration of 1.0% to 7.5% by weight is added to extract the soy protein, and the extract is desalted by electrodialysis or ultrafiltration, and then phytic acid and its salts are removed by isoelectric point precipitation. The admixture ratio of the raw material containing soy protein in the bread improver of the present invention is preferably 30 parts by weight or more and 95 parts by weight, more preferably 50 parts by weight or more and 90 parts by weight, and even more preferably 60 parts by weight or more and 85 parts by weight. If the amount is less than 30 parts by weight, the dough will lack elasticity and have reduced water absorption. If it is more than 95 parts by weight, it will not be able to produce elasticity and extensibility, and will become a hard, clay-like bread dough, which cannot meet the function of being a substitute for gluten.

The phospholipases used in the bread improver of this invention are enzymes that hydrolyze phospholipids such as glycerophospholipids and neurolipids. Depending on the location of the catalytic reaction, they are classified as phospholipases A1 and A2, B, C, and D. However, there are no particular restrictions as long as they do not impair the effects of other active ingredients contained in the bread improver of this invention. Furthermore, one type of phospholipase or a combination of two or more types can be used. Phospholipase A1 (enzyme number: EC 3.1.1.32) is a general term for enzymes that hydrolyze the acetyl group at position 1 of phospholipids, and phospholipase A2 (enzyme number: EC 3.1.1.4) is a general term for enzymes that hydrolyze the acetyl group at position 2, both of which produce lysophospholipids and fatty acids. Phospholipase B (EC 3.1.1.5, etc.) is an enzyme that hydrolyzes both the acetyl group at position 1 and position 2 of phospholipids; it is also known as lysophospholipase. Unless otherwise stated, phospholipase A1 and phospholipase A2 may include phospholipase B. Phospholipase C (EC 3.1.4.3, etc.) is an enzyme that breaks down phospholipids on the hydrophobic side of the phosphate group. Phospholipase D (EC 3.1.4.4, etc.) is an enzyme that breaks down phospholipids on the hydrophilic side of the phosphate group. The source of the phospholipase used in this invention is not particularly limited. As long as it possesses the enzymatic activity described above, it can be derived from microorganisms such as animals, plants, fungi, or bacteria; enzymes produced using gene recombination technology; DNA-modifying enzymes; well-known phospholipases; or artificially modified homologues of these. The phospholipase used in the bread improver of this invention can be a commercially available product or one obtained through appropriate manufacturing. Examples of commercially available phospholipases include Lysonase (manufactured by Sanyo Fine Co., Ltd.), DENABAKE RICH, PLA2 Nagase 10P/R, and PLA2 Nagase L/R (all manufactured by Nagase ChemteX Co., Ltd.). The phospholipase may contain other components or may not contain other components. The phospholipase contained in the bread improver of this invention is preferably 5000U to 50000U, more preferably 10000U to 20000U, per 100 parts by weight of bread improver. The bread improver of this invention is designed to replace 1 part by weight of gluten with 0.01 to 10 parts by weight. Its purpose is to make the effect exhibited by gluten closer to the target quality. Although the activity value of phospholipase can be appropriately adjusted, the aforementioned range is more suitable due to factors such as ease of use. If too much phospholipase is added, the bread crumb tends to become overly soft; if too little is added, the dough tends to lack extensibility and softness, resulting in poor texture.

The activity units of phospholipases A1, A2, and B used in this invention can be determined by the following steps. The activity of phospholipases A1, A2, or B can be determined by co-culturing the enzyme with lecithin in the presence of oxygen and by measuring the enzyme-dependent degradation of lecithin at the SN-1 or SN-2 positions. Specifically, the enzyme reaction can be performed using, for example, a 1% L-α-lecithin solution (0.1M Tris-HCl buffer (pH 8.0), 5mM CaCl2). The degradation of lecithin at the SN-1 or SN-2 positions can be measured, for example, by the formation of free fatty acids. The formation of free fatty acids can be measured, for example, using the NEFA C-Test Wako (manufactured by Wako Junya Pharmaceutical Co., Ltd.). The amount of enzyme that breaks down 1 μmol of lecithin at the SN-1 or SN-2 position in 1 minute at 37°C and pH 8.0 is defined as 1 U (unit). Furthermore, the activities of phospholipases A1, A2, and B can be individually determined by quantifying the free fatty acids generated at the SN-1 and SN-2 positions. The activity unit of phospholipase C was determined and defined as follows: Phospholipase C activity was measured by adding a solution containing 12.5 μM WH-15 and sufficient phospholipase C enzyme to a reaction buffer solution containing 50 mM HEPES (pH 7.2), 70 mM KCl, 1 mM CaCl, and 2.2 mM DTT. The reaction was incubated at room temperature for 90 minutes, and the reaction was stopped with EGTA (final concentration 0.05 mM). Use a suitable spectrophotometer and record the excitation and emission spectra (λex/em = 344/530nm). This assay can also be performed using commercially available assay kits, such as KXTbio (manufactured by KXT Corporation). The active unit of phospholipase D is determined and defined as follows: Mix 0.1 mL of enzyme solution with 0.9 mL of lecithin-containing matrix solution and react at 37°C for 30 minutes. After the reaction is stopped, add 50 μL of reaction solution to 1 mL of cholinesterase-containing colorimetric solution and react for 5 minutes. After the reaction is stopped, measure the amount of pigment generated by choline. Using lecithin as the matrix, the amount of enzyme that releases 1 μmol of choline per minute at 37°C is defined as 1 U (unit).

The elastin-degrading enzyme of this invention is a polypeptide (hereinafter also referred to as the polypeptide of this invention) containing the amino acid sequence of sequence number 1, and is an amino acid sequence having at least 80%, at least 90%, and at least 95% sequence identity with respect to that amino acid sequence. Sequence identity is, for example, a value calculated by properly aligning the query sequence with the sequence of sequence number 1. The sequence identity in this invention is a value calculated using the Clustal algorithm.

The polypeptide of this invention may also contain one or more (e.g., several) modifications. Modifications may be substitutions, insertions, or deletions of amino acids, and may contain one or more modifications simultaneously. Substitution means that an amino acid present at any position in the amino acid sequence is replaced by a different amino acid; deletion means that an amino acid present at any position in the amino acid sequence is lost; insertion means that a different amino acid is added to any position in the amino acid sequence, thereby altering the amino acid sequence following that position.

The polypeptide of this invention exhibits molecular weights of approximately 18 kDa, approximately 14 kDa, and approximately 11 kDa in SDS-polyacrylamide gel electrophoresis. The polypeptide can be composed of monomers or a combination of two or more monomers. Examples include compositions of approximately 18 kDa and approximately 14 kDa and approximately 11 kDa, approximately 18 kDa and approximately 14 kDa, approximately 18 kDa and approximately 11 kDa, and approximately 14 kDa and approximately 11 kDa.

The polypeptide of this invention can be manufactured using genetic engineering. The gene encoding the polypeptide can be selected and utilized from the base sequence encoding sequence number 1, or from the base sequence encoding an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with sequence number 1. The sequence identity of the base sequence is a value calculated using the Clustal algorithm.

The polypeptide of this invention can be manufactured using a performance vector, for example, by using actinomycetes of the genus *Streptomyces * as a host. Any vector used for actinomycetes can be employed, such as the vector described in Japanese Patent Application Publication No. 2014-207898. The actinomycetes used as the host can be any commonly available actinomycetes; however, it is particularly preferred to use actinomycetes belonging to the genus *Streptomyces *. Specifically, examples include *Streptomyces erythraeus *, * Streptomyces griseus *, *Streptomyces omiyaensis* , * Streptomyces fradiae* , *Streptomyces roseoflavus * , * Streptomyces septatus* , *Streptomyces lividans * , * Streptomyces lavendulae *, * Streptomyces virginia * , and * Streptomyces coelicolor *. In the production of the recombinant polypeptide of this invention, the culture medium composition, culture medium pH, culture temperature, culture time, etc., can be efficiently produced by appropriately determining the most suitable conditions.

In addition to genetic production via recombination, the polypeptides of this invention can also be isolated from microorganisms of the genus Streptomyces . Examples include Streptomyces erythraeus , Streptomyces griseus , Streptomyces omiyaensis , Streptomyces fradiae , and Streptomyces roseoflavus . General proteins can be isolated from these microorganisms, and polypeptides containing the amino acid sequence of sequence number 1, or polypeptides containing an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of sequence number 1, exhibiting a molecular weight of approximately 18 kDa, approximately 14 kDa, or approximately 11 kDa.

In order to obtain the polypeptide of this invention by microbial culture, the culture medium used in the culture step is preferably a culture medium containing carbon source, nitrogen source, and inorganic salts that can be utilized by cells in this invention. Any of natural culture medium or synthetic culture medium can be used.

In order to obtain the polypeptide of this invention through microbial culture, the culture conditions can be appropriately selected according to the type of culture medium and the culture method. If the cells will proliferate and produce the enzyme of this invention, there are no special restrictions.

To separate the polypeptides of this invention from microorganisms, separation can be performed by separating the cells from the culture medium components after culture and obtaining the culture supernatant. Separation methods can include conventional methods such as centrifugation and filter press. The culture supernatant can be used directly; however, it can also be processed and purified using methods such as UF concentration, hydrophobic chromatography, ion exchange chromatography, and gel filtration chromatography to obtain a solution containing the polypeptides.

The above-mentioned solutions containing peptides can be used directly as bread improvers, or they can be dried. There are no restrictions on the drying method; any suitable method can be chosen as long as it does not damage the enzyme activity.

The peptides obtained by the previous method can be used to determine the activity value of elastin-degrading enzymes. The determination method is based on the elastin Congo red method. Elastin Congo red is used as a matrix, and the activity value of a change of 1 in absorbance at 495 nm over 1 hour is set as 1 U.

The elastin-degrading enzyme used in the bread improver of this invention is preferably between 1 U and 1000 U per 100 parts by weight of the bread improver, more preferably between 10 U and 500 U, and even more preferably between 10 U and 100 U. If the amount added is more than 1000 U, the dough tends to be sticky; if it is less than 1 U, the dough has low extensibility and low mechanical toughness.

The bread improver of this invention can also be further formulated by mixing other food raw materials commonly used in food manufacturing, or additives, flavorings, colorings, etc., into soy protein, phospholipase, and the polypeptide of this invention. For example, as organic acids, examples include sodium citrate, potassium citrate, calcium citrate, sodium acetate, sodium lactate, calcium lactate, sodium sulfate, potassium sulfate, calcium sulfate, potassium carbonate, sodium carbonate, calcium carbonate, and calcium glycerophosphate. Furthermore, examples of polysaccharides include alginic acid, sodium alginate, pectin, carboxymethyl cellulose, carrageenan, guar gum, catalana polysaccharide, starch, gum arabic, welan gum, cassia gum, xanthan gum, chitosan, psyllium husk gum, gelatan, tamarind seed gum, dextrins, fucoidan alginate, polytranquilized glucose, and hyaluronic acid. Additionally, examples of proteins include egg whites and wheat gluten. In addition, it may also contain various edible oils, dairy products, fruit juices, grain flours, etc., or emulsifiers such as monoglycerides, succinic monoglycerides, diacetyl tartrate monoglycerides, sucrose fatty acid esters, lecithin, enzymes that break down lecithin, sodium stearyl lactate and calcium stearyl lactate, α-amylase, β-amylase, glucoamylase, pentosanase, cellulase, glucose oxidase, and proteases other than the elastin-degrading enzyme of this invention. Amino acids such as cysteine, cystine, methionine, alanine, aspartic acid, and glycine; collagen and peptides other than the peptides of this invention; inorganic salts such as sodium chloride, potassium chloride, ammonium chloride, calcium sulfate, calcium carbonate, and calcium dihydrogen phosphate; nucleic acids such as sodium inosinate and sodium guanylate; vitamins such as vitamin B1, vitamin B2, vitamin C, and vitamin E; alcohols such as ethanol and glycerol; sugars such as sucrose, glucose, maltose, and lactose; excipients such as grain flour, dextrin, and various starches. When using grain flour as an excipient, the grain flour may be gluten-containing or gluten-free, and these may be mixed in any proportion. Only one of these additives may be selected, or multiple additives may be combined.

The form of the bread improver of this invention is not particularly limited. Various forms such as solid, jelly, paste, and liquid can be selected. It is preferable to determine its form according to the target material and the manner in which the bread improver will be used.

There are no particular restrictions on the manufacturing method of the bread improver of this invention. However, each component can be a refined product with few impurities, or raw materials containing each component can be used. In this case, it is preferable to use raw materials with stable specifications. The components can be added by weighing them first, or by weighing the pre-formulated components first. When the bread improver of this invention is formulated, it can be mixed with raw materials used in general bread products, without affecting its ability to replace gluten.

The bread improver of this invention, in order to partially or completely replace the intrinsic or added gluten in cereal flour, preferably adds 0.01 to 10 parts by weight of gluten, more preferably 0.05 to 0.5 parts by weight, and even more preferably 0.1 to 0.3 parts by weight, relative to 1 part by weight of gluten. For example, if the wheat flour used in the raw materials has a gluten content of 10%, and the goal is to increase the gluten content to 12%, conventional techniques would add 2 parts by weight of active gluten. However, if the bread improver of this invention is used, 1 part by weight of the 2 parts by weight of active gluten can be replaced by the bread improver of this invention. In this case, the bread improver of the present invention, at a ratio of 0.1 to 1 part by weight, replaces 1 part by weight of the active gluten. Furthermore, the bread improver of the present invention can not only replace part of the active gluten, but also replace it entirely. In the above cases, active gluten can also be omitted, and the bread improver of the present invention, at a ratio of 0.2 to 2 parts by weight, can be used instead. If the added amount is less than 0.01 parts by weight relative to 1 part by weight of gluten, the functions produced by the gluten cannot be obtained; if it is greater than 10 parts by weight, it will affect the dough's compactness, stickiness, and rubbery texture, as well as its bread-making properties.

In this invention, whether it is a bread improver that can replace gluten is determined by evaluating whether the increase in water absorption, improvement in physical properties, increase in volume, and improvement in taste are equivalent and homogeneous compared to the test area with added active gluten. The increase in water absorption means that, compared with the active gluten addition area, the same amount of water can be added to the bread dough, and there is no difference in the dough's cohesion and mixing time. The so-called improvement in physical properties refers to the consistency and homogeneity of the dough in the mixing, dividing, and shaping steps compared to the active gluten addition zone. Specifically, in the mixing step, the dough's cohesion, elasticity, smoothness, gloss, and surface film tightness are equal and homogeneous; in the dividing step, its adhesion to the workbench, softness, extensibility, elasticity, and dough compactness are equal and homogeneous; and in the shaping step, its relaxation and softness are equal and homogeneous. The so-called increase in volume refers to the absence of difference in specific volume (mL/g) after baking compared to the active gluten addition zone. The so-called improved texture refers to the fact that, compared with the active gluten addition area, the soft texture, the degree of ease of chewing, the resilience, and the degree of clumping are equal and homogeneous.

In this invention, the specific volume is determined using the rapeseed substitution method. The rapeseed substitution method involves preparing a container one size larger than the test sample, filling it with rapeseed, and smoothing the top surface. The rapeseed is temporarily removed from the container, bread is placed inside, the rapeseed is then returned to the container, and the top surface is smoothed again. The volume of the overflowing rapeseed is measured using a graduated cylinder. This volume of rapeseed is equivalent to the volume of the test sample.

In this invention, the so-called resilient quality in the texture improvement effect refers to a property evaluated by sensory evaluation, which is the characteristic of bread attempting to restore its original volume after being compressed and broken by teeth when chewed in the mouth. [Example]

The present invention will be illustrated by the following examples. The present invention is not limited thereto.

—Preparation Method of Bread Improver— The formula for the bread improver is shown in Table 1. Weigh each raw material and mix them thoroughly using a mixer or similar equipment to prepare Bread Improver 1 and Bread Improver 2. Bread Improver 1 uses the elastin-degrading enzyme of this invention, and Bread Improver 2 uses Orientase 5N. Orientase 5N is a protease used in protein processing, peptide production, seasoning production, miso and soy sauce production, and confectionery production. It is also used as a degrading enzyme for V-type collagen components in Japanese Patent Application Publication No. 2023-024185. Since the amount of this protease added is different from the elastin-degrading enzyme of this invention, it is adjusted to be equal in weight to the elastin-degrading enzyme of this invention. These bread improvers are weighed according to the bread recipe and added together with powdered raw materials such as wheat flour, and supplied to the mixing stage of the bread-making process.

[Table 1]

—Incorporation of Round Bread (1)— To compare whether the bread improver 1 listed in Table 1 can function as a substitute for active gluten, the effect of its addition to round bread was investigated. The raw materials and formula of the round bread are shown in Table 2. The values in the table are set to 100 for the amount of cereal flour contained in the raw materials, and the other raw materials are expressed as a percentage of baking (to flour). Comparison area 1 is a test area with 2% active gluten added to the flour, compared with comparison area 2 (no active gluten and bread improver added) to confirm the effect of gluten addition. In Embodiment 1, 1% of the 2% active gluten in the flour was replaced with bread improver 1. When adding 1% active gluten, bread improver 1 was used at 1/10 of its original amount, resulting in an addition of 0.1% active gluten. In Embodiment 2, the entire 2% active gluten in the flour was replaced with bread improver 1. When adding 2% active gluten, bread improver 1 was used at 1/10 of its original amount, resulting in an addition of 0.2% active gluten. The water system shows that the amount added was fine-tuned by kneading the dough within 11 minutes of starting the mixing process, allowing for adjustments based on the desired dough consistency.

[Table 2]

―Round Bread (1) Bread Making Steps― Table 3 shows the bread making steps for round bread. Weigh the raw materials based on Table 2 and mix them using a longitudinal mixer. The mixing conditions shown in Table 3 are the generally recommended conditions for comparison zone 2 (without added active gluten and bread improvers). L represents low speed, ML represents low-medium speed, and MH represents high-medium speed. The numbers are in minutes representing the time for each mixing speed. A downward arrow ↓ indicates the addition of fat. During the mixing process, observe the properties of the bread dough and determine the optimal water absorption within the first 11 minutes of mixing. Next, while observing the properties of the bread dough, fine-tuning the mixing time was made. Finally, the kneading temperature was adjusted to 28°C, and a 60-minute floor time was allowed for initial proofing. After the floor time, the bread dough was divided into 60g portions and allowed to rest for 25 minutes. The dough was then rolled using a forming machine, and the length of each portion as it rolled through the curling net onto the pressing plate was measured. A second proofing was then performed for 60 minutes (38°C, 85% relative humidity), followed by baking. The baked bread was cooled at room temperature and provided the next day for sensory evaluation and volumetric analysis.

―Results of Round Bread (1)― (A) Comparison and Evaluation Results of Water Absorption The water absorption of the bread dough was compared for “Comparison Zone 1”, “Comparison Zone 2”, “Implementation Zone 1”, and “Implementation Zone 2” as shown in Table 2. Water absorption is the amount of water added to the bread dough during kneading within the first 11 minutes of mixing. The optimal water absorption was determined by evaluating whether the amount of water added significantly affected the mixing operation and kneading time. The results of water absorption, scraping time, time to detach from the bottom, and final MH time for each test zone are shown in Table 4. The term "scraping" refers to the process of scraping the dough off the mixing bowl during the mixing step if the water is not absorbed by the gluten, causing the dough to separate and become too soft. This dough will stick to the sides of the mixing bowl and cannot be held onto the mixing hook, making it impossible to knead. Therefore, the dough is scraped off the mixing bowl with a strainer or spatula to hold it onto the mixing hook. This process requires some effort. Table 4 shows at what point in the mixing step this process was performed. The term "detached from the bottom" refers to the state in which, during the mixing step, the dough becomes a cohesive mass and stabilizes, then is lifted from the mixing bowl by the rotation of the whisk and centrifugal force. Table 4 shows the time points when this state is achieved. "Final MH" refers to the kneading time at MH speed (medium-high speed) after the addition of fat during the mixing step, and this time is adjusted according to the cohesive state of the bread dough. Excessive water absorption is one factor that makes it difficult for the gluten network in bread dough to form. When water is added in quantities exceeding the absorbency of the gluten, the final mixing time (MH) needs to be adjusted appropriately to complete the mixing of the bread dough.

[Table 4]

According to Table 4, the water absorption of Comparison Zone 2 (without active gluten or bread improver) was 66% of the flour, consistent with the water absorption of a typical round bread recipe. On the other hand, the water absorption of Comparison Zone 1 (with active gluten) was 67% of the flour, confirming that the active gluten increased the water absorption. The water absorption of Experiment Zones 1 and 2 (with bread improver 1) was 67% of the flour, the same as in Comparison Zone 1 (with active gluten). This result demonstrates that bread improver 1 and active gluten have equivalent water absorption capacity. The scraping time was consistent across the four test zones, indicating that the process can be performed in the same manner as a typical mixing operation. In terms of bottom detachment time, Comparison Zone 1 was the fastest, Comparison Zone 2 was roughly the same as Implementation Zone 1, while Implementation Zone 2 showed a slight tendency to lengthen. Ultimately, the MH time was consistent across the four test zones, indicating that it could be performed in the same manner as a typical hybrid operation.

The results above show that bread improver 1 can achieve the same "increased water absorption" effect as active gluten. Furthermore, it does not cause any changes to the production line, such as requiring more scraping operations or extending mixing time; bread making can be carried out in the same way, whether active gluten is added or no ingredients are added.

(B) Comparison and Evaluation Results of Improved Physical Properties Figure 1 shows the lengths of the formed round loaves in "Comparison Area 1", "Comparison Area 2", "Implementation Area 1", and "Implementation Area 2". This measured value represents the length of the long side of the bread dough when it is rolled and extruded onto the rolling plate using a forming machine, with n=10. The test method used was Steel's test, <p=0.05. Table 5 shows the observed dough properties during the mixing, dividing, and forming steps, and displays the descriptive results.

[Table 5]

According to Figure 1, the length of the formed round loaves showed a significant difference between "Comparison Zone 1" and "Comparison Zone 2," but no significant difference was observed between "Comparison Zone 1" and "Implementation Zone 1," or between "Comparison Zone 1" and "Implementation Zone 2." The length of the formed round loaves showed a tendency to shorten due to the addition of active gluten, a tendency also observed when bread improver 1 was added. According to Table 5, during the mixing steps, "Comparison Zone 1" and "Implementation Zone 2" showed similar properties in terms of dough elasticity, dough stretching, and cohesion. In the dividing step, the dough in "Comparison Zone 1" and "Implementation Zone 1" showed similar elasticity and resilience when rounded. In the shaping step, the dough's firmness was the same in "Comparison Zone 1," "Implementation Zone 1," and "Implementation Zone 2," or only slightly different in "Implementation Zone 1." The dough after rolling in all three test zones showed a tensile texture. "Comparison Zone 2" exhibited weak stretching and cohesion in the mixing step, weaker elasticity in the dividing step, and a lack of tension in the dough after rolling in the shaping step—many characteristics different from the other test zones.

Based on the above results, it is believed that bread improver 1 can achieve the same "physical property improvement" as active gluten, and bread making can be carried out in the same way as when using active gluten.

(C) Comparison and Evaluation Results of Volume Increase Effect Table 6 shows the average specific volume of the baked round breads in "Comparison Area 1", "Comparison Area 2", "Implementation Area 1", and "Implementation Area 2". Specific volume was measured by randomly selecting three round breads as a group for each measurement, calculating the specific volume of each group, and performing three measurements. Additionally, Figure 2 shows the measurement results of waist support (n=9). The test method used was the Steel test, <p=0.05. Waist support is the ratio of the bottom width to the side width of the baked round bread (bottom width/side width); the smaller this value, the better the waist support.

[Table 6]

According to Table 6, the specific volume of the baked round bread differed significantly between "Comparison Zone 1" and "Comparison Zone 2," while there was almost no difference between "Comparison Zone 1" and "Implementation Zone 1," and between "Comparison Zone 1" and "Implementation Zone 2." This confirms that the specific volume of the baked round bread increased due to the addition of active gluten. Furthermore, it shows that even with the addition of bread improver 1, a specific volume equivalent to that of active gluten can be obtained. Next, the waist support of the baked round bread was evaluated. As shown in Figure 2, there is a significant difference between "Comparison Area 1" and "Comparison Area 2". However, no significant difference is observed between "Comparison Area 1" and "Implementation Area 1", or between "Comparison Area 1" and "Implementation Area 2". This confirms that adding active gluten can improve lumbar support, indicating that adding bread improver 1 can achieve the same effect.

The results above show that bread improver 1 has the same "volume increase effect" as active gluten.

(D) Comparison and Evaluation Results of Texture Improvement Figure 3 shows the sensory evaluation results in "Comparison Area 1", "Comparison Area 2", "Implementation Area 1", and "Implementation Area 2". The sensory evaluation was conducted on round bread baked the day before. The sensory evaluation items were set as "softness", "ease of chewing", "resilience", and "clumping". "Softness" evaluates the softness of the bread in the mouth, a light, airy feel. "Ease of biting" evaluates the ease with which the bread can be bitten through, and whether it has a rubbery, stretchy quality. "Resilience" evaluates the bread's ability to return to its original volume after being compressed and broken by the teeth while chewing. "Clumping" evaluates the unpleasant feeling of lumps of bread remaining in the mouth after chewing for a while.

Regarding "softness," "ease of chewing," and "clumping," no significant differences were observed between "Comparison Area 1" and the other three test areas. However, among all sensory evaluation items, only "Comparison Area 2" showed a different tendency in evaluation results compared to "Comparison Area 1." This indicates that adding active gluten alters "softness," "ease of chewing," and "clumping." Regarding "resilience," a significant difference was observed between "Comparison Area 1" and "Comparison Area 2," showing that adding active gluten can improve resilience. Since no significant difference was found between "Comparison Area 1" and "Implementation Area 1", and between "Comparison Area 1" and "Implementation Area 2", it indicates that by adding bread improver 1, the same and homogeneous effect as active gluten can be obtained.

As described above, based on the results of (A) to (II) regarding round bread (1), it was confirmed that adding active gluten to the "Comparison Zone 2" of unadded round bread increased water absorption, improved physical properties, increased volume, and improved texture. Furthermore, in the "Implementation Zone 1" where both active gluten and bread improver 1 were used, or in the "Implementation Zone 2" where bread improver 1 replaced the entire amount of active gluten, no significant difference was observed in the effect of adding active gluten compared to the "Comparison Zone 1" where active gluten was added. However, significant differences or a different tendency were observed compared to the "Comparison Zone 2" where no active gluten was added. This result shows that bread improver 1 has the same and similar additive effect as active gluten, and is a bread improver that can replace active gluten.

—Incorporation of Round Bread (2)— To compare whether bread improver 1 and bread improver 2 listed in Table 1 could function as substitutes for active gluten, the effects of their addition to round bread were investigated. The difference between bread improver 1 and bread improver 2 lies in the type of protease they contain; both are proteases that can be used in bread making. The raw materials and formulation of the round bread are shown in Table 7. The bread-making and evaluation methods were performed in the same manner as for round bread (1).

[Table 7]

—Bread-making steps for round bread (2)— The same bread-making steps as for round bread (1) are used.

―Results of Round Bread (2)― (A) Comparison and Evaluation Results of Water Absorption The water absorption of the bread dough was compared against "Comparison Area 1", "Implementation Area 1", and "Comparison Area 3" as shown in Table 7. This was performed in the same manner as for round bread (1), and the results are shown in Table 8.

[Table 8]

According to Table 8, the water absorption of Comparison Zone 1, which added active gluten, was 68% of the flour, confirming that active gluten increases water absorption. The water absorption of Comparison Zone 1, which added bread improver 1, and Comparison Zone 3, which added bread improver 2, was also 68% of the flour, the same amount as Comparison Zone 1 with active gluten. This result demonstrates that bread improvers 1 and 2 have the same water absorption capacity as active gluten. Furthermore, there were no differences in the scraping time, detachment time from the bottom, and final MH time among the three test zones, indicating that the process can be carried out in the same way as normal mixing operations.

(B) Comparison and Evaluation Results of Improved Physical Properties Figure 4 shows the lengths of the shaped round loaves in "Comparison Area 1", "Implementation Area 1", and "Comparison Area 3". The measurement and testing methods were the same as for the round loaves (1). Table 9 shows the observed dough properties during the mixing, dividing, and shaping steps, and displays the descriptive results.

[Table 9]

According to Figure 4, no significant difference was observed in the length of the formed round bread between "Comparison Area 1" and "Implementation Area 1," but a significant difference was observed between "Comparison Area 1" and "Comparison Area 3." The addition of bread improver 2 showed that it made the formed round bread longer than in other test areas. According to Table 9, during the mixing step, "Comparison Area 1" yielded a dough with strong elasticity, a smooth surface, and a glossy texture; however, "Comparison Area 3" yielded a dough with weak elasticity and a sticky surface. In the dividing step, the dough in "Comparison Zone 1" and "Implementation Zone 1" exhibit similar elasticity and resilience when rounded. In contrast, the dough in "Comparison Zone 3" has weak elasticity and is difficult to feel when rounded. In the shaping step, the dough in "Comparison Zone 1" and "Implementation Zone 1" has the same firmness, or only a slight difference in firmness compared to "Implementation Zone 1." The dough after rolling exhibits a tensile texture. On the other hand, the dough in "Comparison Zone 3" lacks tension after rolling and tends to sag. The above observations show that in the "comparison zone 3", the dough has weak stretching and cohesion in the mixing step, weak elasticity in the dividing step, and lacks tension after being rolled in the forming step, exhibiting many characteristics different from other test zones.

The above results show that the effects of adding bread improver 2 and active gluten are different, so it cannot replace active gluten.

(C) Comparison and Evaluation Results of Volume Increase Effect Table 10 shows the average specific volume of the baked round breads in "Comparison Area 1", "Implementation Area 1", and "Comparison Area 3". The measurement method was the same as that for round bread (1). Furthermore, Figure 4 shows the measurement results of waist support (n=9). The testing method was the same as that for round bread (1).

[Table 10]

According to Table 10, the specific volume of the baked round bread was almost the same between "Comparison Zone 1" and "Implementation Zone 1", and between "Comparison Zone 1" and "Comparison Zone 3". However, as shown in Figure 4, there was a significant difference in the waist support of the baked round bread between "Comparison Zone 1" and "Comparison Zone 3". Although the specific volume of "Comparison Zone 3" increased in the same way as "Comparison Zone 1" and "Implementation Zone 1", its waist support was worse than that of "Comparison Zone 1". Based on this result, bread improver 2 did not show the same or similar effect as active gluten.

Based on the above results, it can be seen that bread improver 2 cannot achieve the same "volume increase effect" as active gluten.

(D) Comparison and Evaluation Results of Texture Improvement Figure 5 shows the sensory evaluation results in "Comparison Area 1", "Implementation Area 1", and "Comparison Area 3". The evaluation method was the same as that used for the round bread (1).

No significant differences were observed in any of the sensory evaluation items between "Comparison Area 1" and "Implementation Area 1". Significant differences were observed in "Resilience" and "Ease of Chewing" between "Comparison Area 1" and "Comparison Area 3". These results indicate that bread improver 2 cannot achieve the same or similar effects as active gluten in terms of "Resilience" and "Ease of Chewing".

As mentioned above, based on the results of (A) to (II) regarding round bread (2), the "comparison zone 3" using bread improver 2 and the "comparison zone 1" with added active gluten were compared. Many significant differences were observed in the evaluation items, indicating that it could not achieve the same or similar effects as active gluten.

These results clearly show that bread improver 1 has the same and similar additive effect as active gluten, and that it is necessary to include soy protein, phospholipase, and elastin-degrading enzymes composed of polypeptides with specific amino acid sequences.

[Figure 1] Figure 1 shows the results of evaluating the physical property improvement effect based on the length of the shaped bread dough in the evaluation system: Bread roll (1). Comparison area 1 and comparison area 2 show significant differences, while comparison area 1 does not show significant differences with implementation areas 1 and 2. [Figure 2] Figure 2 shows the results of evaluating the volume increase effect based on the quality of waist support in the evaluation system: Bread roll (1). Comparison area 1 and comparison area 2 show significant differences, while other combinations (comparison area 1 with implementation area 1, comparison area 1 with implementation area 2) do not show significant differences. [Figure 3] Figure 3 shows the results of evaluating the texture improvement effect using sensory evaluation in the evaluation system: round bread (1). Comparison area 1 and comparison area 2 showed a significant difference in reproducibility, while there were no significant differences between the other combinations (comparison area 1 and implementation area 1, and each item of comparison area 1 and implementation area 2). [Figure 4] Figure 4 shows the results of evaluating the physical property improvement effect using the length of the shaped bread dough in the evaluation system: round bread (2). Comparison area 1 and comparison area 3 showed a significant difference, while there were no significant differences between the other combinations (comparison area 1 and implementation area 1). [Figure 5] Figure 5 shows the results of evaluating the volume increase effect based on the quality of waist support in the evaluation system: round bread (2). Comparison area 1 and comparison area 3 showed significant differences, while other combinations (comparison area 1 and implementation area 1) showed no significant differences. [Figure 6] Figure 6 shows the results of evaluating the texture improvement effect using sensory evaluation in the evaluation system: round bread (2). Comparison area 1 and comparison area 3 showed significant differences in ease of biting and resilience, while other combinations (items of comparison area 1 and implementation area 1) showed no significant differences.

TWI905696B_113110925_SEQL.xml

Claims (3)

A bread improver comprising: a raw material containing soy protein, phospholipase, and an elastin-degrading enzyme; the phospholipase activity per 100 parts by weight of the bread improver is 5000U to 50000U, and the elastin-degrading enzyme activity is 1U to 1000U; the elastin-degrading enzyme is a polypeptide having the characteristics of (a) to (c) the following: (a) a polypeptide containing an amino acid sequence that has at least 80% sequence identity with an amino acid sequence selected from sequence number 1 and has elastin-degrading activity; (b) a polypeptide derived from the genus *Streptococcus*; and (c) a polypeptide with an optimal reaction pH of 7.0 to 11.0 and an optimal reaction temperature of 50 to 80°C. For example, in the bread improver of Request 1, the phospholipase activity of the bread improver per 100 parts by weight is more than 10,000 U and less than 20,000 U. A method for improving bread by replacing 1 part by weight of gluten with 0.01 to 10 parts by weight of a bread improver as described in claim 1 or 2.