JP2010051196A - Modifier for wheat-processed product, and method of manufacturing the wheat-processed product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology of applying a PDI, derived from a transformed cell, to a wheat-processed product by clarifying the difference between a PDI derived from wheat and the PDI derived from the transformed cell. <P>SOLUTION: A modifier for the wheat-processed product is predominantly composed of a recombinant protein disulfide isomerase produced from the transformed cell transformed by a polynucleotide consisting of a base sequence of SEQ ID No.1. In a method of manufacturing the wheat-processed product, dough is formed by adding the modifier to wheat flour. <P>COPYRIGHT: (C)2010,JPO&INPIT

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-heated conditions such as when the dough is mixed, it is presumed that some oxidation mechanism exists, but there are few reports on the mechanism of SS bond formation in the dough.

  In recent years, the presence of protein disulfide isomerase (hereinafter referred to as “PDI”) has become known as an enzyme that catalyzes the formation of SS bonds. Since PDI has the function of forming SS bonds in the oxidized state and cross-linking and polymerizing proteins, it is considered that PDI in wheat is involved in gluten 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 great deal of cost and time to acquire PDI.

In order to solve such problems, 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 transformed host cells are adjusted to a medium pH. A method for producing yeast PDI has been proposed in which protein disulfide isomerase is secreted in an active state to the outside of the host cell by culturing in the vicinity of gender (Patent Document 1).
International Publication WO98 / 35049

  The above publication describes that PDI can be applied to improve the substance of protein-containing foods such as bread dough, ham and sausage, marine products, tofu, etc., but specifically PDI is applied to protein-containing foods There is no. In addition, the characteristics of PDI vary depending on its origin, and the characteristics of PDI required vary depending on the type of food. Therefore, the use of PDI needs to be examined individually.

  Therefore, an object of the present invention is to clarify the difference between wheat-derived PDI and transformed cell-derived PDI, and to provide a technique for applying transformed cell-derived PDI to processed wheat products.

  The present inventors independently produced a recombinant wheat PDI (hereinafter referred to as “rPDI”), and as a result of investigating the involvement of such rPDI in gluten formation and bread-making properties, rPDI has different characteristics from wheat PDI. Gained knowledge that it is advantageous for the production of processed wheat products.

  The present invention is based on such findings, and is an improvement of processed wheat products mainly composed of recombinant protein disulfide isomerase produced from a transformed cell transformed with a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1. An agent is provided.

  The present invention also provides a dough by adding to a wheat flour a modifying agent composed mainly of recombinant protein disulfide isomerase produced from a transformed cell transformed with a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1. A method for producing a processed wheat product is provided.

  According to the modified wheat processed product and the method for producing a processed wheat product of the present invention, it is possible to improve the quality of the processed wheat product, and to use a flour that has not been suitable as wheat for the processed wheat product so far. Even if it is a case, since it can be modified to a suitable flour, the applicable range of the flour can be expanded.

  A preferred embodiment of the present invention will be described. The modifying agent for processed wheat products of the present invention contains, as a main component, recombinant protein disulfide isomerase (rPDI) produced from a transformed cell transformed with a polynucleotide having the base sequence represented by SEQ ID NO: 1.

  In the present invention, the “processed wheat product” refers to a food mainly composed of wheat flour, such as bread such as bread, confectionery bread and yeast donut; noodles such as Chinese noodles, udon, buckwheat and pasta; And confectionery such as cookies; skins such as dumplings, shumai, and spring rolls.

The method for synthesizing rPDI is, for example, as follows. Germination and rooting are performed by cultivating wheat at 28 ° C. for 48 hours, the wheat root is picked, and mRNA is extracted from the wheat root using RNeasy Plant Mini Kit (manufactured by QIAGEN). Preparation of cDNA from mRNA is performed using PrimeSTAR ^™ RT-PCR Kit (TaKaRa).

  The prepared cDNA is PCR-amplified using primers designed to amplify the wheat PDI open reading frame (ORF) based on the two types of wheat PDI gene information obtained from the Japan DNA Data Bank.

  The amplified wheat PDI ORF is inserted into the expression vector pET21a (Novagen) and transformed into the expression host BL21 (DE3) by the heat shock method. LB or ampicillin medium is used as a culture medium for the transformant and cultured at 37 ° C. until O.D.600 reaches 0.6 or more. Thereafter, 0.2 mM IPTG is added and expression is induced at 20 ° C. for 16 hours.

  Thereafter, the cells are collected by centrifugation, and the cell pellet is suspended with 20 mM Tris-HCl (pH 7.4) containing 100 mM NaCl in 1/10 volume of the culture solution. This is lysed under ice cooling. After centrifuging the solution, the obtained supernatant is added to a TALON Metal Affinity Resin (TaKaRa) column (0.25 cm × 5 cm) equilibrated with lysis buffer at a flow rate of 0.5 ml / min.

  After thoroughly washing unadsorbed protein with the same buffer, the adsorbed protein is eluted with 20 mM Tris-HCl (pH 8.0) containing 100 mM NaCl and 40 mM imidazole.

  The obtained rPDI has PDI activity of 357-632U / protein 1mg, and its temperature stability and pH stability are superior to natural rPDI derived from wheat, so it should be used as a modifier for processed wheat products. Can do.

  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 and isopeptide bonds in the dough is promoted. For example, in bread, the specific volume increases and a soft mouthfeel bread can be produced. it can.

  The amount of the modifier to be added is not particularly limited because it can be appropriately set according to the purpose of the processed wheat product, but is preferably 0.2 U to 5.0 U with respect to 1 g of wheat flour, for example. The dough becomes softer and the specific volume increases as the amount of the modifier added increases. Therefore, when producing a processed wheat product that requires a soft texture, the amount of the modifier added should be increased.

  r As an indication of the amount of PDI added, there is a PDI activity amount of strong powder. That is, the PDI activity amount of strong flour is generally 3.0 to 5.0 U / wheat flour 1 g. Therefore, when producing processed wheat products using medium flour with PDI activity of about 1.5 U / wheat flour 1 g, the PDI activity amount of strong flour It is sufficient to add an amount equivalent to.

1. Acquisition of Recombinant PDI Wheat PDI gene information was retrieved and acquired from Japan DNA Data Bank (DDBJ), and two types of wheat PDI gene information were aligned on Bio-Edit. After alignment, primers can be created outside the 3 'end and 5' end of the Open Reading Frame (ORF) region of the nucleotide sequence represented by SEQ ID NO: 1, and total RNA extracted from wheat root can be reverse-transcribed. The PDI ORF was amplified by PCR using the obtained 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 to extract rPDI, and then purified with a cobalt column. When the activity of SS bond cleavage and SS bond formation was measured using this purified enzyme, both activities were confirmed.

2. Comparison of Properties of Wheat Grain PDI and rPDI (1) For the rPDI obtained in 1 above, the optimum temperature, optimum pH, temperature stability, pH stability, and molecular weight were measured. Moreover, it evaluated similarly about the natural PDI derived from wheat flour. PDI derived from wheat flour was extracted from the enzyme using the method of Ken Kainuma (1998) using Haruyutaka (produced in Hokkaido in 2007). Weigh 4 g of whole wheat grains, crush them with a multi-bead shocker (MB601U (S), manufactured by Yasui Machinery Co., Ltd.), and buffer A (50 mM Tris-HCl (pH 8.0), 5 mM DTT, 50 mM NaCl, 10 mM EDTA). , 2 mM PMSF, Triton X-100) 16 ml was added and homogenized with a Waring blender. This was centrifuged, the resulting supernatant was saturated with ammonium sulfate 20%, and the supernatant obtained by centrifugation was buffered with buffer B (50 mM Tris-HCl ( Dialyzed overnight at pH 8.0)). What was obtained by centrifuging after dialysis was used (indicated as “wheat grain PDI” in Table 1).

  The optimum temperature was measured by using a potassium phosphate buffer (pH 7.5) as the buffer and measuring the enzyme activity at an enzyme reaction temperature of 10 to 50 ° C. The optimum pH was measured by measuring enzyme activity using a potassium phosphate buffer solution (pH 5 to 8) and a phosphate buffer solution (pH 8 to 11). The temperature stability was measured by measuring enzyme activity after warming wheat grain PDI for 1 hour at 10-70 ° C and then rPDI for 3 hours to determine the temperature at which PDI activity remained at 80% or more. . pH stability: Wheat grain PDI is mixed with 10 mM EDTA, 50 mM NaCl, 50 mM Tris-HCl (pH 4-10) at a ratio of 1: 1, and left at 4 ° C. for 4 hours, then rPDI is 4 ° C. for 8 hours. After standing, enzyme activity was measured by reducing activity against insulin (described in detail below), and the pH at which PDI activity remained at 80% or more was determined. The molecular weight was subjected to SDS-PAGE and then analyzed with a gel proanalyzer (Nippon Roper).

PDI activity was measured by the method using insulin of Johanna. L. and Arne. H (1979, 1990). Collect 0.5 ml of 0.4 M potassium phosphate buffer (pH 7.5), 0.5 ml of 8 mM EDTA, 0.1 ml of insulin solution, 0.58 ml of distilled water in a test tube, add 0.02 ml of enzyme solution, and then add 3.3 ml of DTT 0.2 ml. Was added to make 2.0 ml, and the enzyme reaction was carried out at 25 ° C. for 40 minutes. The turbidity of the resulting insulin was measured for absorbance at an absorption wavelength of 650 nm. The enzyme titer was set to 1 U at a pH of 7.5 at 25 ° C. in the presence of DTT and 0.5 mg / ml insulin A ₆₅₀ was changed by 0.01 per minute.

  As a result, wheat grain PDI and rPDI were both 63 kDa, optimum temperature 35 ° C., and optimum pH 8.5, and there was no particular difference regarding these points, but the temperature stability was 10 to 30 for wheat grain PDI. It was found that rPDI was 10-40 ° C, while pH stability was 7-9 for wheat grain PDI, whereas rPDI was 6-9 for pH stability.

  Generally, the bread dough at the end of the main kneading has a slightly acidic pH (about pH 6.5). Therefore, it can be said that the use of rPDI having pH stability of 6 to 9 is advantageous for the formation of disulfide bonds in the dough.

  In addition, during fermentation of bread dough, temperatures above 30 ° C may last for several hours depending on the surrounding environment. Therefore, it is considered that by using rPDI having a temperature stability of 10 to 40 ° C., the PDI activity is maintained for a long time and the formation of disulfide bonds in the dough can be improved.

(2) The PDI activity of rPDI obtained in 1 above and PDI derived from wheat flour obtained in (1) above was measured. The measurement method was the above-described method using insulin of Johanna. L. and Arne. H (1979, 1990). The enzyme titer was set to 1 U at a pH of 7.5 at 25 ° C. in the presence of DTT and 0.5 mg / ml insulin A ₆₅₀ was changed by 0.01 per minute. The results are shown in Table 2.

  As shown in Table 2, it was found that the PDI activity of rPDI was significantly higher than that of wheat-derived PDI. The PDI activity of rPDI per mg of protein is about 371 times in specific activity, which corresponds to the PDI activity of 129 g of flour.

3. Breadmaking test (1) Bread production
The effects of rPDI addition on bread making properties were investigated. rPDI was used by adding to flour. As wheat flour, medium strength flour (Australian Standard White: ASW) having low bread-making property and strong flour (Dark Northern Spring: DNS) having excellent bread-making property were used.

  Add flour and rPDI with the composition shown in Table 3, add water, sugar, salt, yeast and shortening, yeast, knead, ferment for 90 minutes at 27 ° C and 75% humidity, then degas. And further fermented for 30 minutes under the same conditions. After completion of fermentation, the dough was divided into 40 g and formed into a spherical shape, placed in a stainless steel container (diameter 50 mm), fermented at 38 ° C. and 85% humidity for 60 minutes, baked in an oven at 190 ° C. for 15 minutes, A sample was prepared after cooling at room temperature for 3 hours.

  Specific volume and hardness were evaluated about the manufactured bread. The specific volume was calculated by measuring the volume by the rapeseed substitution method and dividing by the weight. As for the hardness, the fired bread was cut into 25 mm × 25 mm × 25 mm, then subjected to a compression test with a plunger having a diameter of 21 mm, and a load at 70% compression was measured. The results are shown in FIG.

  As shown in FIG. 1, bread produced using medium flour (ASW) had a relatively small specific volume and a relatively hard hardness. On the other hand, bread produced by adding rPDI to medium-strength flour (ASW) has intermediate properties between bread produced using medium-strength flour (ASW) and bread produced using strong flour (DNS). Was. From this result, it was found that rPDI is useful as a modifier for improving bread-making properties.

(2) Examination of the amount of rPDI added
In order to examine how the difference in the amount of rPDI added affects the quality of bread, bread was produced using the same production method as in (1) above, except that only the amount of rPDI added was changed. And about the manufactured bread, the specific volume and hardness were evaluated by the method described in said (1). The results are shown in Table 4.

  As shown in Table 4, the bread dough became softer and the specific volume tended to increase as the amount of rPDI added increased. From this result, it was found that a processed wheat product having a desired quality can be produced by adjusting the amount of rPDI added according to the purpose of the processed wheat product.

It is a figure which shows the base sequence of recombinant protein disulfide isomerase (rPDI). It is a figure which shows the result of having examined the influence which it has on the bread-making property by the difference in wheat flour.

Claims (2)

  A modified wheat processed product comprising a recombinant protein disulfide isomerase as a main component produced from a transformed cell transformed with a polynucleotide having the base sequence represented by SEQ ID NO: 1.   Processed wheat product that forms a dough by adding to a wheat flour a modifier mainly composed of recombinant protein disulfide isomerase produced from a transformed cell transformed with a polynucleotide having the base sequence represented by SEQ ID NO: 1. Manufacturing method.