JP5370970B2 - Wheat processed product modifier and wheat processed product manufacturing method - Google Patents

Description

  The present invention relates to a modified wheat processed product and a method for producing the processed wheat product.

  Wheat flour is processed into various foods, such as bread, noodles, and confectionery, by adding water and kneading to form a dough exhibiting a unique viscoelasticity not found in other cereals. The peculiar physical property of this dough is due to the property of gluten formed by hydration of gliadin and glutenin, which are storage proteins peculiar to wheat, and it is said that the property depends on the amount of these proteins in the flour.

  It is considered that various bonds and intermolecular interactions are involved in the formation of gluten, and in particular, the formation of cross-links between protein molecules by disulfide bonds (SS bonds) is considered to be most important. In order to form SS bonds under non-heating conditions such as when the dough is kneaded, it is presumed that some oxidation mechanism exists. A series of studies by the present inventors suggests the involvement of protein disulfide isomerase (hereinafter referred to as “PDI”), which is an enzyme that catalyzes SS bond formation in flour dough formation.

  However, PDI becomes less active after reaching the peak of activity during the flowering of wheat, and is present only in trace amounts in wheat seeds. In addition to the complexity of the process of extracting PDI from wheat, the process itself is unstable and easily deactivated, so it takes a lot of cost and time to acquire PDI.

  In order to solve such a problem, for example, International Publication No. WO 98/35049 discloses that yeast is transformed with a gene encoding protein disulfide isomerase derived from yeast, and the pH of the medium of the transformed host cells is reduced. A method for producing yeast PDI has been proposed in which protein disulfide isomerase is secreted in an active state outside the host cell by culturing in the vicinity of gender (Patent Document 1).

  Oxidized PDI is considered to be involved in the expression of physical properties of gluten by forming an SS bond in the protein, but itself becomes reduced PDI by this catalytic reaction. In order for SS bond formation to proceed smoothly in the flour dough, it is necessary to regenerate reduced PDI into oxidized PDI. As an enzyme involved in this regeneration, endoplasmic reticulum oxidoreductase 1 (Endoplasmic Reticulum Oxidorductase 1: hereinafter referred to as “Ero1”) is known. However, Ero1 has been reported in humans and yeasts so far. Although it is only rice in higher plants, there is no report of Ero1 in wheat.

International Publication WO98 / 35049

  The present invention obtains recombinant Ero1 (hereinafter referred to as “rEro1”) from genetic information in order to analyze the action of wheat PDI on flour dough formation and bread-making properties, and improves gluten formation brought about by the regeneration of PDI by rEro1. It aims at providing the manufacturing technology of the processed wheat product which applied.

  The present invention is based on such findings. Recombinant Endoplasmic Reticulum Oxidoreductase 1: Recombinant Endoplasmic Reticulum Oxidoreductase 1 produced from a transformed cell transformed with a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 1. The present invention provides a processed wheat product modifier comprising, as main components, rEro1), protein disulfide isomerase (PDI), and flavin adenine dinucleotide (FAD).

  The present invention also relates to a recombinant endoplasmic reticulum oxidoreductase 1 (rEro1) produced from a transformed cell transformed with a polynucleotide having the base sequence represented by SEQ ID NO: 1, and a protein disulfide isomerase (PDI). And a processed wheat product comprising a flavin adenine dinucleotide (FAD) and a wheat processed product modifier added to the flour to provide a dough.

  According to the modified wheat processed product and the method for producing a processed wheat product of the present invention, it is possible to increase the amount of SS bond formation in the flour dough, so that the processed wheat product is improved in quality by increasing the amount of gluten formation. Can improve the quality. In addition, since the amount of gluten formation is small and it can be modified to suitable flour even when using small flour that has not been suitable as wheat for processed wheat products so far, the applicable range of flour can be expanded. It becomes possible.

It is a figure for demonstrating the bridge | crosslinking formation between protein molecules by a disulfide bond (SS bond), and the catalytic reaction concerned with it. It is a flowchart which shows the outline of the process of acquiring recombinant wheat Ero1. It is an electrophoresis pattern which shows insolubilization of full length rEro1 expression protein. It is a figure which shows the elution pattern of the column chromatography of a transformant. It is the electrophoresis pattern of SDS-PAGE of the unadsorbed fraction of FIG. 4, a 10 mM imidazole fraction, and a 50 mM imidazole fraction. It is a figure which shows the result of having investigated the activity of rEro1 by fading of FAD. The changes in the concentration of O ₂ is a graph showing the results of examining the activity of REro1. It is a figure which shows the manufacturing method of bread, and the order of a test. It is a figure which shows the result of having compared the specific volume of the bread after baking. It is a figure which shows the result of the sensory evaluation of the bread after baking. It is a figure which shows the result of having examined SS bond formation ability in wheat protein by PDI and rEro1.

  Preferred embodiments of the modified wheat processed product and the method for producing the processed wheat product of the present invention will be described. In the present invention, the “processed wheat product” refers to a food mainly composed of wheat flour, such as breads such as bread, confectionery bread and yeast donuts; noodles such as Chinese noodles, udon, buckwheat and pasta; Sweets such as buns and Chinese buns; gyoza, shumai, spring rolls such as spring rolls, and others: porridges, bamboo rings, etc.

  FIG. 1 is a diagram for explaining the formation of a cross-link between protein molecules by a disulfide bond (SS bond) and the catalytic reaction involved in the cross-linking.

  Wheat protein, which is a substrate for processed wheat products, forms gluten when SH groups are cross-linked and SS-bonded during dough mixing. At that time, oxidized PDI oxidizes the SH group of the substrate to form an SS bond, and itself becomes reduced PDI. However, by itself, the reaction of PDI becomes unilateral, and when the amount of oxidized PDI eventually decreases, SS bonding does not proceed unless oxidized PDI is added.

Therefore, Ero1 is required to catalyze the reaction of oxidizing this reduced PDI and regenerating it into oxidized PDI. In Ero1, oxidized Ero1 oxidizes reduced PDI to become reduced Ero1. As a result, oxidized PDI can again catalyze the SS bond. On the other hand, reduced Ero1 is oxidized Ero1 next reacted with FAD, FAD changes to FADH _2. However, FADH ₂ can be regenerated to FAD by reacting with O ₂ .

  Endoplasmic reticulum oxidoreductase 1 (Endoplasmic Reticulum Oxidoductase 1: Ero1) used as a modifier for processed wheat products of this embodiment is a trait transformed with a polynucleotide comprising the base sequence represented by SEQ ID NO: 1. Recombinant endoplasmic reticulum oxidoreductase 1 (rEro1) produced from transformed cells.

  rEro1 is present on the N-terminal side of natural Ero1 and lacks a highly hydrophobic transmembrane region for attachment to the membrane of the endoplasmic reticulum. Like Ero1 in FIG. It has a catalytic role. In this embodiment, rEro1 cannot be obtained in the soluble fraction even when transformation is performed using the base sequence in which this transmembrane region is present (FIG. 3).

  The PDI used as a modifier for processed wheat products of this embodiment may be PDI extracted from natural products such as wheat or rPDI. As rPDI, for example, rPDI produced from a transformed cell transformed with a polynucleotide having the base sequence represented by SEQ ID NO: 2 can be used. The obtained rPDI has a PDI activity of 200 to 632 U / protein of 1 mg, and is preferable because it is superior in temperature stability and pH stability to natural PDI derived from wheat.

Flavin adenine dinucleotide (FAD) used as a modifier for processed wheat products of this embodiment is a coenzyme type vitamin B ₂ and is involved in the exchange of hydrogen during the redox reaction of rEro1.

  In the method for producing a processed wheat product of this embodiment, the above-described modifier is added to wheat flour to form a dough.

  By forming the dough by adding the modifier to wheat flour, the formation of disulfide bonds in the dough is promoted. For example, in bread, the specific volume increases and a soft mouthfeel bread can be produced.

  In the method for producing a processed wheat product of the present embodiment, the amount of rEro1 added is 2 to 20 times the molar ratio of the PDI, considering the association with PDI in the dough of the processed wheat product Is preferred. It is considered that the more rEro1 is added, the higher the association with PDI is. However, as a result of actually producing a processed wheat product, it is sufficient to add rEro1 in an amount 10 times the molar ratio of PDI. The effect of can be obtained.

1. Expression and acquisition of wheat rPDI and wheat rEro1 (1) Wheat rPDI
For the expression of wheat rPDI, wheat PDI gene information was retrieved from the Japan DNA Data Bank (DDBJ), and two types of wheat PDI gene information were aligned on Bio-Edit. After alignment, primers were prepared on the 3 ′ end side and 5 ′ end side outside the open reading frame (ORF) region of the base sequence represented by SEQ ID NO: 2, and total RNA extracted from wheat root could be reverse transcribed. The PDI ORF was amplified by PCR using cDNA as a template, inserted into the expression vector pET21a, transformed into the expression host E. coli BL21 (DE3), and expressed as a His-Tag fusion protein. Next, the bacterial cells were crushed by ultrasonic treatment, rPDI was extracted, and then purified with a cobalt column.

(2) Wheat rEro1
FIG. 2 is a flowchart showing an outline of a process for obtaining the recombinant wheat Ero1. Regarding the expression of wheat Ero1, since there was no report of the nucleotide sequence of wheat Ero1, the gene information of human and yeast Ero1 was retrieved from DDBJ and aligned on Bio-Edit. After alignment, a primer was prepared from the common sequence, and the sequences of both ends of wheat Ero1 were revealed by 3′-RACE and 5′-RACE. Further, primers were prepared outside the 3 ′ end and 5 ′ end of the ORF region of wheat Ero1, and the ORF of Ero1 was amplified by PCR using cDNA obtained by reverse transcription of total RNA extracted from wheat root as a template. Then, it was inserted into the expression vector pET21a, transformed into the expression host E. coli BL21 codon Plus (DE3) -RIL, and expressed and purified in the same manner as rPDI. However, the full length rEro1 obtained was not expressed in the soluble fraction (FIG. 3). Therefore, a highly hydrophobic transmembrane region present on the N-terminal side was deleted, and a base sequence represented by SEQ ID NO: 1 was prepared. Then, by expressing the base sequence represented by SEQ ID NO: 1 by the same operation as described above, 49 kDa rEro1 was expressed in the soluble fraction and was successfully obtained (see FIGS. 4 and 5).

activity rEro1 is over time by measuring the discoloration due to reduction of the FAD is a coenzyme of Erol, also FADH ₂ and _{O 2} produced is to measure the concentration reduction of _{O 2} when become reactive to _H 2 _{O 2} Was confirmed. FIG. 6 shows the result of FAD fading, and FIG. 7 shows the result of the change in O ₂ concentration.

As shown in FIG. 6, the activity of rEro1 was confirmed by measuring the yellow discoloration associated with reduction to FADH ₂ by acting on FAD as reduced rEro1 in DTT because Ero1 is a flavin reductase. And confirmed to have activity. Further, as shown in FIG. 7, FADH ₂ that generated measures the _{O 2} consumption amount due to be converted electrons to _{O 2} to donor and _H 2 _{O 2,} is rEro1 concentration dependent manner _{O 2} consumption rate It was also confirmed that it increased.

2. Effect of rPDI and rEro1 on bread-making properties In order to analyze the effect of rPDI and rEro1 on bread-making properties, a medium-powder flour (Nisshin Flour Mill's “Song” (product) Name)) was used as a sample, and a bread-making test was conducted by adding rPDI, rEro1 and FAD. In addition, as a reference example, a medium flour is used, a bread without adding the modifier of the present invention and a strong flour (“Camellia” (trade name) of Nisshin Flour Milling Co., Ltd.) are used, and the modifier of the present invention is used. A bread with no added was also produced. At this time, the amount of pure water added in Reference Example 1 was 137 ml.

  FIG. 8 shows a bread manufacturing method and a test sequence. rPDI is 50 U / wheat flour 200 g (0.25 U / wheat flour 1 g) or 500 U / wheat flour 200 g (2.5 U / wheat flour 1 g), about 4.3 nmol / wheat flour 200 g (0.022 nmol / wheat flour 1 g), protein 243 μg / wheat flour 200 g The equivalent was added. The rPDI was calculated with a specific activity of 205.4 U / mg and a molecular weight of 56600.

  rEro1 is the same mole as rPDI, or 10 times the molar ratio, about 4.3 nmol / wheat flour 200 g or 43 nmol wheat flour 200 g (0.022 nmol or 0.22 nmol) / wheat flour 1 g), protein 208.6 μg or 2086 μg / wheat flour 200 g equivalent was added. In addition, rEro1 was calculated with a molecular weight of 48500.

  FAD was added with 5 μmol / 200 g flour (0.025 U / wheat flour 1 g). In consideration of the association with rEro1 in the dough, an excessive amount of FAD was added.

  The result of comparing the specific volume of the bread after baking is shown in FIG. The rapeseed replacement method was used for the specific volume of the baked bread. The rapeseed replacement method is a method for subtracting the volume of rapeseed when the sample and rapeseed are filled in the same container from the volume of rapeseed packed in a container of a certain capacity to obtain the volume of the sample.

  As a result, compared to the specific volume of bread (Reference Example 2) produced with medium-strength flour, bread with rEro1 added 4.3 nmol (same molar ratio as rPDI 50U) and FAD 5 μmol in addition to rPDI 50 U / wheat flour 200 g (Example 1) is an increase of about 2.1%, rPDI 50 U / wheat flour 200 g, rEro1 43.0 nmol (10 times the molar ratio of rPDI 50 U) and FAD 5 μmol bread (Example 2) Is about 7.4% increase, rPDI 500U / wheat flour 200g and rEro1 43.0 nmol (10 times the molar ratio of rPDI 50U) and FAD 5 μmol bread (Example 3) is about 5.0% Increased.

  From this, it was speculated that the coexistence with rPDI, rEro1 and FAD complemented the regeneration mechanism of reduced rPDI to the oxidized form, and the SS bond formation of wheat flour gluten proceeded.

  A sensory test was conducted with 10 healthy adults as panelists. Except for the comprehensive evaluation, the control (Reference Example 2) is made 3 points subjectively by the 5-point evaluation method, 5 points for the superior one, 4 points for the slightly better one, 3 points equivalent to the control, Slightly inferior ones were given 2 points and inferior ones were given 1 point. Comprehensive evaluation was performed by the ranking method, and was scored as 1st place 5 points, 2nd place 4 points, 3rd place 3 points, 4th place 2 points, 5th place 1 point.

  The results are shown in Table 1 and FIG. In Table 1 and FIG. 10, “Control” means bread (Reference Example 2) manufactured with medium-strength flour, “Comparative Example 1” means bread manufactured by adding 50 U of rPDI / 200 g of flour, “Comparative Example 2” means a bread produced by adding rPDI 50U / wheat flour 200 g and rEro1 43.0 nmol (10 times the molar ratio of rPDI 50U). As shown in Table 1 and FIG. 10, the bread of Example 2 obtained high evaluation for all items, indicating that it was a good bread.

3. Analysis of SS bond-forming ability by wheat PDI and rEro1 In order to analyze SS bond-forming ability in wheat protein by PDI and rEro1, crude PDI enzyme solution (PDI activity 5.0 U) prepared from wheat grains (Haruyutaka; produced in Hokkaido) REro1 (2.0 nmol) and FAD (20.0 nmol) were added to a protein concentration of 5.3 mg / ml and 0.2 nmol as PDI, and the mixture was heated at 37 ° C. for 3 hours, and then the amount of SH in the test solution was changed. Was measured by the NBD-Cl method, and the SS bond formation amount was calculated from the SH group decrease amount.

  The results are shown in FIG. As shown in FIG. 11, with the crude enzyme solution alone, the SS bond formation amount was 0.16 μmol / protein g, but when rEro1 and FAD were added thereto, the SS bond formation amount was 1.7 μmol / protein g. Increased about 11 times. From this, it became clear that the presence of PDI, rEro1, and FAD promotes the formation of SS bonds in wheat protein.

Claims (6)

Recombinant endoplasmic reticulum oxidoreductase 1 (rEro1) produced from a transformed cell transformed with a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1,
Protein disulfide isomerase (PDI);
Flavin adenine dinucleotide (FAD);
A modified product for processed wheat products.   The protein disulfide isomerase (PDI) is a recombinant protein disulfide isomerase (rPDI) produced from a transformed cell transformed with a polynucleotide having the base sequence represented by SEQ ID NO: 2. A modifier for processed wheat products.   The modifier for processed wheat products according to claim 1 or 2, wherein the processed wheat products are breads.   Recombinant end-plasmic reticulum oxidoreductase 1 (rEro1), protein disulfide isomerase (PDI), and flavin adenine dinucleotide produced from a transformed cell transformed with a polynucleotide having the base sequence represented by SEQ ID NO: 1. A method for producing a processed wheat product, wherein a dough is formed by adding a flour processed product modifier comprising (FAD) as a main component to wheat flour.   The protein disulfide isomerase (PDI) is a recombinant protein disulfide isomerase (rPDI) produced from a transformed cell transformed with a polynucleotide having the base sequence represented by SEQ ID NO: 2. Manufacturing method of processed wheat products.   The method for producing a processed wheat product according to claim 4 or 5, wherein the processed wheat product is bread.