WO2017215216A1 - 应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法 - Google Patents

应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法 Download PDF

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WO2017215216A1
WO2017215216A1 PCT/CN2016/110019 CN2016110019W WO2017215216A1 WO 2017215216 A1 WO2017215216 A1 WO 2017215216A1 CN 2016110019 W CN2016110019 W CN 2016110019W WO 2017215216 A1 WO2017215216 A1 WO 2017215216A1
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recombinant
wheat
endoplasmic reticulum
weight
parts
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胡松青
刘光
侯轶
黄滟波
李琳
张喜梅
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华南理工大学
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    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/06—Products with modified nutritive value, e.g. with modified starch content
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

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  • the invention belongs to the fields of genetic engineering and cereal science, and particularly relates to a method for improving the quality of flour processing by using a recombinant wheat endoplasmic reticulum thiol reductase.
  • Wheat is one of the world's three major food crops. China's total wheat output ranks first in the world, accounting for one-sixth of the world's total output. However, due to the constraints of varieties, climate, soil, etc., most of the wheat produced in China is medium and low. Wheat-based, suitable for taro, cake but not for bread making. However, with the improvement of people's living standards, the demand for bread consumption has increased year by year, and low-quality medium-low gluten wheat has restricted the development of bread and other facial products in China. In addition, wheat growth environment, pests and diseases and post-harvest treatment lead to uneven processing quality of flour.
  • the quality of flour processing is mainly the gluten protein network, and the intermolecular disulfide bond is the basis for forming the network structure.
  • the noodle product processing industry has improved the processing quality of noodle products by adding a flour improver that promotes the formation of intermolecular disulfide bonds.
  • a flour improver that promotes the formation of intermolecular disulfide bonds.
  • cheap and efficient "chemical" flour improvers such as potassium bromate and azoformamide have been widely used in the surface processing industry.
  • food safety hazards brought about by "chemical” improvers have gradually been valued and confirmed, such as bromate found to be carcinogenic and genotoxic. Therefore, the search for new flour improvers has become the development direction in the field of pasta processing, and safe, efficient and green biological enzyme preparations are in line with this development trend.
  • wEro1 The endoplasmic reticulum oxidoreductin (wEro1), one of the endogenous enzymes in wheat, is involved in the biosynthesis of the nascent peptide disulfide bond in wheat.
  • wEro1 is a member of the sulfhydryl oxidase family, and thiol oxidase is recognized by the US Food and Drug Administration (FDA) as GRAS (Generally Recognized as Safe).
  • FDA US Food and Drug Administration
  • GRAS Generally Recognized as Safe
  • the primary object of the present invention is to provide a wheat endoplasmic reticulum thioredoxase for improving the quality of flour processing.
  • Another object of the present invention is to provide a method for improving the quality of flour processing by using a recombinant wheat endoplasmic reticulum thiol reductase. This method opens up a new way for the improvement of pasta products in China.
  • the invention provides a wheat endoplasmic reticulum thiol reductase for improving the quality of flour processing.
  • the dough is divided and weighed, formed and proofed, it is baked to obtain a finished bread.
  • the components are added in an amount of 95 to 105 parts by weight of flour, 0.8 to 1.5 parts by weight of yeast powder, 1 to 2 parts by weight of salt, 55 to 65 parts by weight of water, 5 to 8 parts by weight of sugar, and 1 to 8 parts by weight of vegetable oil. 2 parts by weight.
  • the recombinant wheat endoplasmic reticulum thiol-reductase is added at a level of 0.05% to 0.2% (w/w, flour base).
  • the method for using the recombinant wheat endoplasmic reticulum sulfhydryl oxidoreductase to improve the quality of flour processing comprises the following specific steps:
  • Material 95 to 105 parts by weight of flour, 0.8 to 1.5 parts by weight of yeast powder, 1 to 2 parts by weight of salt, 55 to 65 parts by weight of water, 5 to 8 parts by weight of sugar, 1 to 2 parts by weight of vegetable oil, and recombinant wheat Endoplasmic reticulum thiol reductase 0.05% to 0.2% (w/w, flour base);
  • Baking The dough after the proofing is baked for 10 to 15 minutes, and the temperature of the oven is 180 to 200 ° C to obtain a finished bread product.
  • a biologically active recombinant wheat endoplasmic reticulum thiol reductase was obtained. Including the following steps:
  • the recombinant expression plasmid pET-28a-wero1 was transfected into E. coli BL21 (DE3) competent cells to obtain recombinant expression bacteria; the recombinant expression bacteria were inoculated into LB medium until the OD 600 reached 0.6-0.8. The cells were induced by adding 0.2-1.0 mM IPTG at 16-37 ° C for 8-12 hours.
  • the amino acid sequence of the wheat endoplasmic reticulum thiol reductase is shown in SEQ ID NO: 2.
  • the mechanism of the present invention is that the recombinant wheat endoplasmic reticulum thiol-reductase is obtained by prokaryotic expression, and the addition of the enzyme can effectively enhance the flour powder characteristics and improve the specific volume, height-diameter ratio, elasticity and hardness of the bread.
  • the quality has achieved the same improvement effect as the chemical strong arsenic azoformamide (ADA), and it can be used as a new biological flour improver instead of ADA for the surface product processing industry.
  • ADA chemical strong arsenic azoformamide
  • the present invention has the following advantages and effects:
  • the present invention provides for the first time a method for improving the quality of flour processing by using a recombinant wheat endoplasmic reticulum thiol reductase.
  • the recombinant enzyme can effectively enhance the flour powder characteristics, improve the breadth, specific diameter ratio, elasticity and hardness of the bread, and achieves a comparative improvement with the chemical strong arsenic azoformamide (ADA), which can become A new bio-flour improver that replaces ADA for the pasta processing industry.
  • ADA chemical strong arsenic azoformamide
  • the invention adopts the method of prokaryotic expression to obtain the bioactive recombinant endoplasmic reticulum thiol-reductase of wheat, which has the characteristics of high expression, simple purification, easy amplification and suitable for industrial application.
  • Figure 1 is a double restriction enzyme digestion of recombinant expression plasmid pET-28a-wero1; Lane M: DL5000 DNA Marker; Lane 1: double-cut fragment, white underlined for the gene fragment of interest.
  • Figure 2 is the expression and solubility analysis of recombinant wEro1; wherein, lane M: molecular weight Marker; lane 1: uninduced bacterial solution; lane 2: induced bacterial solution; lane 3: cell disrupted solution; The cell disrupted supernatant; the white arrow points to the recombinant wEro1 protein.
  • Figure 3 is an SDS-PAGE analysis of recombinant wEro1 after affinity chromatography; lane 1, molecular weight Marker; lane 2: cell disruption; lane 3: unbound protein; lanes 4-8: eluate different tube collection group Minute.
  • Figure 4 is the enzymatic properties of recombinant wEro1.
  • Figure 5 is a comparison of the effects of recombinant wEro1 and azocarboxamide on the texture parameters of bread; among them, different letters indicate significant differences (P ⁇ 0.05).
  • the flour used in the examples of the present invention was purchased from the grain market; the restriction enzymes NdeI and XhoI were purchased from Thermo Fisher Scientific; the pET-28a (+) plasmid was purchased from Bao Bioengineering (Dalian) Co., Ltd.; Escherichia coli BL21 (DE3) was purchased from Novagen; the whole gene sequence synthesis was completed by Guangzhou Aiji Biotechnology Co., Ltd.
  • the wheat endoplasmic reticulum thiol reductase gene (SEQ ID NO: 1) was synthesized in Guangzhou Ai Ke Biotechnology Co., Ltd. and ligated into the cloning vector pGSI. 1 ng of recombinant cloning plasmid was taken and subjected to NdeI and XhoI restriction endonuclease reaction (37 ° C, 30 min), and then subjected to agarose gel electrophoresis and purification of wheat endoplasmic reticulum thioredoxase gene fragment with cohesive ends.
  • the gene fragment 20 ⁇ g was mixed with 5 ⁇ g of the plasmid pET-28a (+) digested with the same restriction enzyme, and ligated at 20 ° C for 1 h under the action of T4 DNA ligase. After ligation, the product was transferred into E. coli DH5 ⁇ competent state, and cultured on LB solid medium containing kanamycin for 12 to 16 hours. The plasmid was extracted from the monoclonal colony with sufficient morphology, and the recombinant plasmid was identified by double enzyme digestion. According to the electrophoresis results, the extracted plasmid contained the target gene fragment (Fig. 1).
  • the recombinant plasmid pET-28a-wero1 was transformed into BL21 (DE3), and 3 monoclonal colonies were picked into LB medium containing 50 ⁇ g/mL kanamycin and cultured at 37 ° C, 200 r / m. To OD 600 to about 0.6 to 0.8, 0.2 to 1 mM IPTG was added to induce expression for 8 to 12 hours, and the induction temperature ranged from 16 to 37 °C. Recombinant wEro1 was obtained by SDS-PAGE analysis (Fig. 2).
  • the expressed cells were collected, resuspended in a buffer solution, and the cells were disrupted by a probe type ultrasonic instrument, and the ultrasonic condition was 350 W, the duty ratio was 0.4:0.6, and the ultrasonic was 15 min. After the ultrasound was completed, the supernatant was collected by centrifugation at 8000 r/m for 20 min.
  • the supernatant was slowly injected into the Ni-NTA affinity chromatography column, and the target protein was purified by linear elution. The fractions of each peak were collected and the recombinant protein was detected by SDS-PAGE (Fig. 3).
  • RESULTS Recombinant wEro1 was expressed in soluble form, and a high purity target protein was obtained by one-step affinity chromatography.
  • wEro1 contains a variety of enzymatic properties, and this example selects a convenient FAD reducing activity for detection.
  • the principle is that the coenzyme FAD of wEro1 can be reduced to FADH 2 by the action of dithiothreitol (DTT), and the latter has no absorbance at 450 nm. Therefore, the FAD reducing activity of wEro1 can be investigated by detecting the change in absorbance at 450 nm.
  • DTT dithiothreitol
  • the enzyme activity assay reaction system was as follows: 2 ⁇ M wEro1 and 10 ⁇ M FAD were added to a 50 mM Tris-HCl (pH 8.0) buffer solution, and the reaction was initiated by adding 12.5 mM DTT, and the absorbance was measured at 450 nm.
  • the effect of recombinant wEro1 on the flour quality of flour was characterized by a micro-powder analyzer. 4g of flour was added to the whitefly, and after premixing for 63 minutes at 63 r/m, 57% of water was added, and the same speed was continuously stirred for 15 minutes to obtain the powdery curve and the powdery parameters. A negative control group was added without adding any substance, and azocarboxamide (ADA) (20 ⁇ g/g) was added as a positive control group, and recombinant wEro1 protein (0.1%, w/w, flour base) was added as an experimental group.
  • ADA azocarboxamide
  • the method for improving bread baking quality by using the above-mentioned recombinant wheat endoplasmic reticulum thiol-reductase is as follows:
  • Material 95 parts by weight of flour, 0.8 parts by weight of yeast powder (Anqi high active dry yeast, commercially available), 1 part by weight of salt, 55 parts by weight of water, 5 parts by weight of sugar, vegetable oil (Arowana edible blend oil, Commercially available) 1 part by weight of recombinant wheat endoplasmic reticulum thiol oxidoreductase 0.05% (w/w, flour based).
  • Baking The dough after being proofed is baked in an oven for 10 minutes, and the temperature of the oven is 180 ° C to obtain a finished bread.
  • the prepared bread was cooled at room temperature for 2 hours, and physical quality analysis was carried out, and the bread physical parameters were measured for weight, volume, height and width.
  • a negative control group was added without adding any substance, and azozamide (20 ⁇ g/g) was added as a positive control group.
  • Material 105 parts by weight of flour, 1.5 parts by weight of yeast powder (Anqi high active dry yeast, commercially available), 2 parts by weight of salt, 65 parts by weight of water, 8 parts by weight of sugar, vegetable oil (Arowana edible blending oil, Commercially available) 2 parts by weight of recombinant wheat endoplasmic reticulum thiol oxidoreductase 0.2% (w/w, flour based).
  • Baking The dough after being proofed is baked in an oven for 15 minutes, and the temperature of the oven is 200 ° C to obtain a finished bread.
  • the prepared bread was cooled at room temperature for 2 hours, and then the bread texture quality analysis was carried out.
  • the bread was cut into 2 cm/block, and the texture of the bread was measured: p/25 probe, pre-test and test speed 1 mm/s, measured speed 5 mm/s, compression ratio 50%, and down-pressure interval 10 s.
  • a negative control group was added without adding any substance, and azozamide (20 ⁇ g/g) was added as a positive control group.
  • the bread texture results showed that the addition of azocarboxamide or recombinant wEro1 could significantly reduce the hardness and viscosity of the bread and improve the elasticity of the bread, indicating that both can improve the quality of the bread. Moreover, there was no significant difference between the breads to which recombinant wEro1 and azocarboxamide were added (Fig. 5). Therefore, the texture results further indicate that recombinant wEro1 can replace azoformamide and exert a comparable ability to improve bread quality.

Abstract

一种应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法，包括：原核表达并纯化重组小麦内质网巯基氧化还原酶，将该重组酶与面粉、糖、盐、植物油、酵母粉和水搅拌均匀，形成的面团经分割称重、成形以及醒发后，焙烤得到面包成品。所述的重组小麦内质网巯基氧化还原酶添加水平为0.05％～0.2％(w/w，面粉基)。

Description

应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法 技术领域
本发明属于基因工程和谷物科学领域，具体涉及一种应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法。
背景技术
小麦是世界三大粮食作物之一，我国小麦的总产量居世界之首，占世界总产量的六分之一，但受品种、气候、土壤等的制约，我国生产的小麦多以中低筋小麦为主，适用于馒头、蛋糕但不适用于面包的制作。然而，随着人们生活水平的提高，对面包的消费需求逐年上升，低品质的中低筋小麦制约了我国面包等面制品的发展。此外，小麦生长环境、病虫害和采后处理导致面粉的加工品质参差不齐。
决定面粉加工品质主要是面筋蛋白网络，而分子间二硫键是形成该网络结构的基础。近年来，面制品加工行业通过添加促进分子间二硫键形成的面粉改良剂来改善面制品加工品质。在很长一段时间里，廉价、高效的“化学”面粉改良剂如溴酸钾、偶氮甲酰胺等在面制品加工行业得到广泛应用。然而，“化学”改良剂带来的食品安全隐患逐渐被重视和证实，如溴酸盐被发现具有致癌和遗传毒性。因此，寻找新型的面粉改良剂成为面制品加工领域的发展方向，而安全、高效、绿色的生物酶制剂符合这种发展趋势。
小麦内源酶之一的内质网巯基氧化还原酶(wheat endoplasmic reticulum oxidoreductin 1，wEro1)在小麦体内参与新生肽链二硫键的生物合成。wEro1属于巯基氧化酶家族成员，而巯基氧化酶被美国食药局(FDA)认定为GRAS(Generally Recognized as Safe，一般认为安全)。目前，国内外利用wEro1作为生物面粉改良剂改善面制品加工品质的研究报道很少，只有日本学者Takano Katsumi在2011年发表的专利(JP，2011-177059)中提及了大肠杆菌重组小麦蛋白质二硫键异构酶在wEro1和FAD(黄素腺嘌呤二核苷酸)的协同下能够提高面包品质。然而，该专利并未单独探究wEro1对面包品质的影响，且复杂的添加组分以及成本问题严重制约了wEro1的实际应用。
发明内容
为了克服现有技术的缺点与不足，本发明的首要目的在于提供一种小麦内质网巯基氧化还原酶在改善面粉加工品质中的应用。
本发明的另一目的在于提供一种应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法。该方法为我国面制品改良开创一条新途径。
本发明的目的通过下述技术方案实现：
本发明提供一种小麦内质网巯基氧化还原酶在改善面粉加工品质中的应用。
具体地，小麦内质网巯基氧化还原酶在改善面团以及面包品质中的应用。
仅需在面粉中添加小麦内质网巯基氧化还原酶即能达到增强面粉粉质特性，改善面包烘焙品质的目的。
一种应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法，将重组小麦内质网巯基氧化还原酶、面粉、糖、盐(NaCl)、植物油、酵母粉和水搅拌均匀，形成的面团经分割称重、成形以及醒发后，焙烤得到面包成品。其中，所述各组分添加量为：面粉95～105重量份，酵母粉0.8～1.5重量份，盐1～2重量份，水55～65重量份，糖5～8重量份，植物油1～2重量份。
所述的重组小麦内质网巯基氧化还原酶添加水平为0.05％～0.2％(w/w，面粉基)。
所述的应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法，包括如下具体步骤：
(1)材料：面粉95～105重量份，酵母粉0.8～1.5重量份，盐1～2重量份，水55～65重量份，糖5～8重量份，植物油1～2重量份，重组小麦内质网巯基氧化还原酶0.05％～0.2％(w/w，面粉基)；
(2)和面：将上述材料混合，以150～180r/min的速度搅拌10～15min，使其形成均匀的面团；
(3)成型：将和好的面团分割成40～60重量份/个，搓圆，成形装盘；
(4)发酵：将装有面团的醒发盘置于醒发箱中醒发，温度控制在30～35℃，相对湿度75％～85％，发酵时间60～80min；
(5)烘焙：将醒发后的面团，烘焙10～15min，烤箱温度为180～200℃，得到面包成品。
所述的重组小麦内质网巯基氧化还原酶的制备方法，采用大肠杆菌表达体系表达，并采用柱层析方法纯化重组wEro1蛋白；按照wEro1:FAD＝1:1～5的摩尔比添加FAD到纯化的重组wEro1蛋白溶液中，获得具有生物活性的重组小麦内质网巯基氧化还原酶。包括如下步骤：
(1)利用全基因合成技术，合成了小麦内质网巯基氧化还原酶的基因序列(SEQ ID NO：1)，并连接至载体质粒pET-28a(+)上，酶切位点为NdeI和XhoI，构建出了小麦内质网巯基氧化还原酶重组表达质粒pET-28a-wero1；
(2)将重组表达质粒pET-28a-wero1转入到大肠杆菌BL21(DE3)感受态细胞中，得到重组表达菌；将重组表达菌接种到LB培养基培养至OD600达到0.6～0.8，在16～37℃条件下添加0.2～1.0mM IPTG诱导重组蛋白表达8～12小时后收集菌体；
(3)收集的菌体经冰浴超声破碎后，离心收集上清液，利用Ni-NTA纯化重组wEro1蛋白；
(4)按照wEro1:FAD＝1:1～5的摩尔比添加FAD到纯化的重组wEro1蛋白溶液中，低温孵育过夜，经脱盐处理去除多余的FAD，获得具有生物活性的重组小麦内质网巯基氧化还原酶。重组蛋白浓缩处理后，冻藏于-20℃备用。
所述的小麦内质网巯基氧化还原酶的氨基酸序列如SEQ ID NO：2所示。
本发明的机理是：本发明通过原核表达获得了重组小麦内质网巯基氧化还原酶，单独添加该酶能有效增强面粉粉质特性，改善面包的比容、高径比、弹性和硬度等烘焙品质，达到了与化学强筋剂偶氮甲酰胺(ADA)相当的改善效果，可成为替代ADA的新型生物面粉改良剂，用于面制品加工行业。
本发明相对于现有技术，具有如下的优点及效果：
(1)本发明首次提供了一种应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法。该重组酶能有效增强面粉粉质特性，改善面包的比容、高径比、弹性和硬度等烘焙品质，达到了与化学强筋剂偶氮甲酰胺(ADA)的相当的改善效果，可成为替代ADA的新型生物面粉改良剂，用于面制品加工行业。
(2)本发明应用重组小麦内质网巯基氧化还原酶改善面包焙烤品质的方法，只需在面包制作过程单独添加该重组酶，不需再添加其它的添加剂，简化了生产工序，降低了生产成本。
(3)本发明采用原核表达的方法，获得了具有生物活性的重组小麦内质网巯基氧化还原酶，具备表达量高，纯化简便，易于放大，适合工业化应用等特点。
附图说明
图1是重组表达质粒pET-28a-wero1的双酶切鉴定；其中，泳道M：DL5000DNA Marker；泳道1：双酶切片段，白色下划线为目的基因片段。
图2是重组wEro1的表达及可溶性分析；其中，泳道M：分子量Marker；泳道1：未诱导菌液；泳道2：诱导后菌液；泳道3：细胞破碎液沉淀；泳道4： 细胞破碎液上清；白色箭头所指处为重组wEro1蛋白。
图3是亲和层析后重组wEro1的SDS-PAGE分析；其中，泳道1：分子量Marker；泳道2：细胞破碎液；泳道3：未结合蛋白；泳道4～8：洗脱液不同管收集组分。
图4是重组wEro1的酶学性质。
图5是重组wEro1和偶氮甲酰胺对面包质构参数的影响比较；其中，不同字母表示差异显著(P＜0.05)。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
对于未特别注明的工艺参数，可参照常规技术进行。本发明的实施例中使用的面粉购于粮食市场；限制性内切酶NdeI和XhoI购于Thermo Fisher Scientific公司；pET-28a(+)质粒购于宝生物工程(大连)有限公司；大肠杆菌BL21(DE3)购于Novagen公司；全基因序列合成由广州艾基生物技术有限公司完成。
实施例1重组小麦内质网巯基氧化还原酶表达载体构建
小麦内质网巯基氧化还原酶基因(SEQ ID NO：1)合成于广州艾基生物技术有限公司，并连接至克隆载体pGSI上。取1ng重组克隆质粒，经NdeI和XhoI限制性内切酶反应(37℃，30min)后，进行琼脂糖凝胶电泳并纯化带有粘性末端的小麦内质网巯基氧化还原酶基因片段。将20μg基因片段与5μg经相同限制酶酶切的质粒pET-28a(+)混合，在T4DNA连接酶作用下于20℃连接1h。连接后产物转入大肠杆菌DH5α感受态中，涂布于含卡那霉素的LB固体培养基上培养12～16h。挑取形态饱满的单克隆菌落提取质粒，对重组质粒进行双酶切鉴定，根据电泳结果证实了提取的质粒含有目的基因片段(图1)。
结果：成功构建了重组表达载体pET-28a-wero1。
实施例2重组wEro1的诱导表达及纯化
1、重组wEro1的诱导表达
将构建好的重组质粒pET-28a-wero1转化至BL21(DE3)中，挑取3颗单克隆菌落到含50μg/mL卡那霉素的LB培养基中，37℃，200r/m条件下培养至OD600到0.6～0.8左右，加入0.2～1mM IPTG诱导表达8～12h，诱导温度范围在16～37℃之间。经SDS-PAGE分析，重组wEro1获得可溶表达(图2)。
2、重组wEro1的亲和纯化
收集表达后的菌体，加入缓冲液重悬，利用探头式超声仪破碎菌体细胞，超声条件为350w，占空比0.4:0.6，超声15min。超声完全后，8000r/m离心20min后收集上清液。
上清液缓慢注入Ni-NTA亲和层析柱中，利用线性洗脱方式纯化目的蛋白，收集各流出峰组分，并利用SDS-PAGE检测重组蛋白(图3)。
合并目的蛋白溶液，按wEro1:FAD＝1:1～5的摩尔比加入FAD，4℃处理过夜，再经脱盐、浓缩后冻藏于-20℃备用。
结果：重组wEro1获得可溶表达，经一步亲和层析获得高纯度目的蛋白。
实施例3重组wEro1的酶学性质
wEro1含多种酶学性质，本实施例选择检测方便的FAD还原活性。其原理为wEro1的辅酶FAD在二硫苏糖醇(DTT)的作用下能够还原成FADH2，后者在450nm处无吸光值。因此，通过检测450nm处吸光值变化，可以探究wEro1的FAD还原活性。
酶活测定反应体系如下：2μM wEro1和10μM FAD加入到50mM Tris-HCl(pH8.0)缓冲溶液中，加入12.5mM DTT启动反应，450nm处测定吸光值。
结果：如图4所示，添加DTT后，wEro1表现出了明显的FAD还原活性。
实施例4重组wEro1对面粉粉质特性的影响
采用微量粉质仪表征重组wEro1对面粉粉质特性影响。4g面粉加入粉钵中，63r/m预混1min后，加入57％的水，相同速度持续搅拌15min后得到了粉质曲线和粉质参数。以不添加任何物质为阴性对照组，以添加偶氮甲酰胺(ADA)(20μg/g)为阳性对照组，以添加重组wEro1蛋白(0.1％，w/w，面粉基)的为实验组。
结果：与阴性对照相比，加入偶氮甲酰胺和wEro1后，粉质参数变化显著，面团的稳定时间和质量数都显著提高。然而，相比于添加偶氮甲酰胺组，加入重组wEro1对面粉稳定时间和质量数影响更大(表1)，表明重组wEro1在提高粉质特性方面优于“化学”改良剂偶氮甲酰胺。
表1重组wEro1和偶氮甲酰胺对面粉粉质参数的影响比较
  形成时间(min) 稳定时间(min) 弱化度(FU) 质量数
对照 1.00±0.00a 1.65±0.07c 127.45±3.46a 51.50±4.52c
ADA 1.00±0.10a 4.70±0.15b 105.50±5.25b 57.30±2.86b
wEro1 1.15±0.07a 7.05±0.49a 95.00±7.07c 62.85±1.20a
注：偶氮甲酰胺添加水平为20μg/g，重组wEro1添加水平为0.1％(w/w，面粉基)。同一列中不同字母表示差异显著(P＜0.05)。
实施例5 wEro1改善面包焙烤品质的方法
利用上述重组小麦内质网巯基氧化还原酶改善面包焙烤品质的方法，具体工艺流程如下：
(1)材料：面粉95重量份，酵母粉(安琪高活性干酵母，市售)0.8重量份，盐1重量份，水55重量份，糖5重量份，植物油(金龙鱼食用调和油，市售)1重量份，重组小麦内质网巯基氧化还原酶0.05％(w/w，面粉基)。
(2)和面：将上述材料加入到和面钵中，以150r/min的速度搅拌10min，使其形成均匀的面团。
(3)成型：将和好的面团置于天平上，分割成40重量份/个，在面包成型机上搓圆，成形装盘。
(4)发酵：将装有面团的醒发盘置于醒发箱中醒发，温度控制在30℃，相对湿度75％，发酵时间60min。
(5)烘焙：将醒发后的面团放于烤箱中烘焙10min，烤箱温度为180℃，得到面包成品。
将制得的面包室温下冷却2h后进行物理品质分析，面包物理参数测定重量、体积、高度和宽度。以不添加任何物质为阴性对照组，以添加偶氮甲酰胺(20μg/g)为阳性对照组。
如表2所示，相比于阴性对照组，加入重组wEro1后，面包的高径比和比容显著提高，分别增加了11.3％和8.2％。而相比于阳性对照，添加重组wEro1和偶氮甲酰胺的面包之间不具有差异显著性。以上结果表明，与偶氮甲酰胺相比，重组wEro1具有与之相当的提高面包品质能力，因此该重组酶可以替代化学强筋剂偶氮甲酰胺，作为天然面粉改良剂应用于面制品加工中。
表2重组wEro1和偶氮甲酰胺对面包物理参数的影响比较
  体积(cm3) 高度(cm) 宽度(cm) 比容(cm3/g) 高径比
对照 129.67±4.51a 3.90±0.10a 7.40±0.10a 2.56±0.09a 0.53±0.01a
ADA 138.56±3.23b 4.23±0.15b 7.40±0.12a 2.74±0.08b 0.58±0.10b
wEro1 139.33±2.31b 4.37±0.06b 7.40±0.17a 2.77±0.05b 0.59±0.02b
注：偶氮甲酰胺添加水平为20μg/g，重组wEro1添加水平为0.05％(w/w，面粉基)。同一列中不同字母表示差异显著(P＜0.05)。
实施例6 wEro1改善面包焙烤品质的方法
利用上述重组小麦内质网巯基氧化还原酶改善面包焙烤品质的方法，不同之处在于，采用以下工艺流程：
(1)材料：面粉105重量份，酵母粉(安琪高活性干酵母，市售)1.5重量份，盐2重量份，水65重量份，糖8重量份，植物油(金龙鱼食用调和油，市售)2重量份，重组小麦内质网巯基氧化还原酶0.2％(w/w，面粉基)。
(2)和面：将上述材料加入到和面钵中，以180r/min的速度搅拌15min，使其形成均匀的面团。
(3)成型：将和好的面团置于天平上，分割成60重量份/个，在面包成型机上搓圆，成形装盘。
(4)发酵：将装有面团的醒发盘置于醒发箱中醒发，温度控制在35℃，相对湿度85％，发酵时间80min。
(5)烘焙：将醒发后的面团放于烤箱中烘焙15min，烤箱温度为200℃，得到面包成品。
将制得的面包室温下冷却2h后进行面包质构品质分析。将面包竖切成2cm/块，面包质构测定条件：p/25探头，测前和测试速度1mm/s，测后速度5mm/s，压缩比50％，下压间隔10s。以不添加任何物质为阴性对照组，以添加偶氮甲酰胺(20μg/g)为阳性对照组。
面包质构结果表明，添加偶氮甲酰胺或重组wEro1都能够显著降低面包硬度和黏性，提高面包弹性，表明两者都能提高面包的质量。而且，添加重组wEro1和偶氮甲酰胺的面包之间不具有差异显著性(图5)。因此，质构结果进一步表明，重组wEro1能够替代偶氮甲酰胺，发挥与之相当的改善面包品质能力。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (9)

  1. 小麦内质网巯基氧化还原酶在改善面粉加工品质中的应用。
  2. 根据权利要求1所述的应用，其特征在于：仅需在面粉中添加小麦内质网巯基氧化还原酶即能达到增强面粉粉质特性，改善面包烘焙品质的目的。
  3. 一种应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法，其特征在于：将重组小麦内质网巯基氧化还原酶、面粉、糖、盐、植物油、酵母粉和水搅拌均匀，形成的面团经分割称重、成形以及醒发后，焙烤得到面包成品；
    其中，所述的重组小麦内质网巯基氧化还原酶添加水平为面粉基的0.05％～0.2％w/w。
  4. 根据权利要求3所述的方法，其特征在于：所述各组分添加量为：面粉95～105重量份，酵母粉0.8～1.5重量份，盐1～2重量份，水55～65重量份，糖5～8重量份，植物油1～2重量份。
  5. 根据权利要求4所述的方法，其特征在于：
    所述的应用重组小麦内质网巯基氧化还原酶改善面粉加工品质的方法，包括如下具体步骤：
    (1)材料：面粉95～105重量份，酵母粉0.8～1.5重量份，盐1～2重量份，水55～65重量份，糖5～8重量份，植物油1～2重量份，重组小麦内质网巯基氧化还原酶0.05％～0.2％；
    (2)和面：将上述材料混合，以150～180r/min的速度搅拌10～15min，使其形成均匀的面团；
    (3)成型：将和好的面团分割成40～60重量份/个，搓圆，成形装盘；
    (4)发酵：将装有面团的醒发盘置于醒发箱中醒发，温度控制在30～35℃，相对湿度75％～85％，发酵时间60～80min；
    (5)烘焙：将醒发后的面团，烘焙10～15min，烤箱温度为180～200℃，得到面包成品。
  6. 根据权利要求1或2所述的应用，其特征在于：
    所述的小麦内质网巯基氧化还原酶的氨基酸序列如SEQ ID NO：2所示。
  7. 根据权利要求1或2所述的应用，其特征在于：
    所述的小麦内质网巯基氧化还原酶的核苷酸序列如SEQ ID NO：1所示。
  8. 根据权利要求3～5任一项所述的方法，其特征在于：
    所述的重组小麦内质网巯基氧化还原酶采用大肠杆菌表达体系表达，并采用柱层析方法纯化重组wEro1蛋白；按照wEro1:FAD＝1:1～5的摩尔比添加FAD到纯化的重组wEro1蛋白溶液中，获得具有生物活性的重组小麦内质网巯基氧化还原酶。
  9. 根据权利要求8所述的方法，其特征在于：
    所述的重组小麦内质网巯基氧化还原酶的制备方法，包括如下步骤：
    (1)利用全基因合成技术，合成了小麦内质网巯基氧化还原酶的基因序列，并连接至载体质粒pET-28a(+)上，酶切位点为NdeI和XhoI，构建出了小麦内质网巯基氧化还原酶重组表达质粒pET-28a-wero1；
    (2)将重组表达质粒pET-28a-wero1转入到大肠杆菌BL21(DE3)感受态细胞中，得到重组表达菌；将重组表达菌接种到LB培养基培养至OD600达到0.6～0.8，在16～37℃条件下添加0.2～1.0mM IPTG诱导重组蛋白表达8～12小时后收集菌体；
    (3)收集的菌体经冰浴超声破碎后，离心收集上清液，利用Ni-NTA纯化重组wEro1蛋白；
    (4)按照wEro1:FAD＝1:1～5的摩尔比添加FAD到纯化的重组wEro1蛋白溶液中，低温孵育过夜，经脱盐处理去除多余的FAD，获得具有生物活性的重组小麦内质网巯基氧化还原酶。
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CN110946275A (zh) * 2019-12-03 2020-04-03 广东省农业科学院蚕业与农产品加工研究所 小麦静息巯基氧化酶在改善乳剂型特医食品稳定性中的应用
CN110946275B (zh) * 2019-12-03 2022-12-02 广东省农业科学院蚕业与农产品加工研究所 小麦静息巯基氧化酶在改善乳剂型特医食品稳定性中的应用

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