CN105875699B - Improve the method for flour processing quality using recombination wheat endoplasmic reticulum thiol-oxide reductase - Google Patents
Improve the method for flour processing quality using recombination wheat endoplasmic reticulum thiol-oxide reductase Download PDFInfo
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- CN105875699B CN105875699B CN201610422861.8A CN201610422861A CN105875699B CN 105875699 B CN105875699 B CN 105875699B CN 201610422861 A CN201610422861 A CN 201610422861A CN 105875699 B CN105875699 B CN 105875699B
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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
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- 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
Abstract
A kind of method that the present invention discloses application recombination wheat endoplasmic reticulum thiol-oxide reductase improvement flour processing quality, belongs to genetic engineering and cereal science field.This method comprises: prokaryotic expression and having purified recombination wheat endoplasmic reticulum thiol-oxide reductase, the recombinase and flour, sugar, salt (NaCl), vegetable oil, yeast powder and water are stirred evenly, after the segmented weighing of the dough of formation, forming and provocation, bake to obtain bread products.The recombination wheat endoplasmic reticulum thiol-oxide reductase pitch-based sphere is 0.05%~0.2% (w/w, flour-based).Recombination wheat endoplasmic reticulum thiol-oxide reductase of the invention can significantly improve the processing quality of flour, improve the baking qualities such as specific volume, ratio of height to diameter, elasticity and the hardness of bread, and improved effect is suitable with chemical gluten-strengthening agent azodicarbonamide.Therefore, recombination wheat endoplasmic reticulum thiol-oxide reductase may replace chemical gluten-strengthening agent azodicarbonamide for Flour product processing industry.
Description
Technical field
The invention belongs to genetic engineerings and cereal science field, and in particular to a kind of application recombination wheat endoplasmic reticulum sulfydryl oxygen
Changing reductase improves the method for flour processing quality.
Background technique
Wheat is one of world's Three major grain crops, and the total output of China's wheat occupies first of the world, accounts for Gross World Product
1/6th, but restricted by kind, weather, soil etc., the wheat of China's production based on low muscle wheat, is suitable for steamed bun mostly in
Head, cake but the production for not being suitable for bread.However, as the improvement of people's living standards, year by year to the consumption demand of bread
Rise, it is low-quality in low muscle wheat constrain the development of the Flour product such as China's bread.In addition, wheat growth environment, pest and disease damage
Cause the processing quality of flour irregular with postharvest handling.
Determine that flour processing quality is mainly mucedin network, and intermolecular disulfide bond is the base to form the network structure
Plinth.In recent years, Flour product processing industry promotes the flour improver of intermolecular disulfide bond formation to improve Flour product by addition
Processing quality.In a very long time, cheap, efficient " chemistry " flour improver such as potassium bromate, azodicarbonamide etc. exists
Flour product processing industry is used widely.However, " chemistry " modifying agent bring food safety hazards are gradually taken seriously and demonstrate,prove
It is real, as bromate is found to have carcinogenic and genetoxic.Therefore, novel flour improver is found as Flour product processing neck
The developing direction in domain, and safe and efficient, green biological enzyme formulation meets this development trend.
Endoplasmic reticulum thiol-oxide reductase (the wheat endoplasmic reticulum of one of wheat endogenous enzyme
Oxidoreductin 1, wEro1) in wheat participate in new polypeptide chain disulfide bond biosynthesis.WEro1 belongs to sulfydryl oxygen
Change enzyme family member, and thiol oxidase regards as GRAS (Generally Recognized as by U.S.'s food officina (FDA)
Safe, it is considered that safety).Currently, improving Flour product processing quality using wEro1 as biological flour improver both at home and abroad
Research report seldom, mentions in the patent (JP, 2011-177059) that only Japanese scholars Takano Katsumi was delivered in 2011
And Escherichia coli recombination aleuronat disulfide bond isomerase is in the collaboration of wEro1 and FAD (flavin adenine dinucleotide (FAD))
Under can be improved bread.However, the patent does not probe into influence of the wEro1 to bread, and complicated addition individually
Component and cost problem seriously constrain the practical application of wEro1.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of wheat endoplasmic reticulum mercapto
Base oxidoreducing enzyme is improving the application in flour processing quality.
Another object of the present invention is to provide a kind of application recombination wheat endoplasmic reticulum thiol-oxide reductases to improve flour
The method of processing quality.This method is that a new way is started in Flour product improvement in China's.
The purpose of the invention is achieved by the following technical solution:
The present invention, which provides a kind of wheat endoplasmic reticulum thiol-oxide reductase, is improving the application in flour processing quality.
Specifically, wheat endoplasmic reticulum thiol-oxide reductase is improving the application in dough and bread.
Wheat endoplasmic reticulum thiol-oxide reductase need to be only added in flour can reach enhancing flour quality characteristic, improve
The purpose of baking quality of bread.
A kind of method that application recombination wheat endoplasmic reticulum thiol-oxide reductase improves flour processing quality, will recombinate wheat
Endoplasmic reticulum thiol-oxide reductase, flour, sugar, salt (NaCl), vegetable oil, yeast powder and water stir evenly, the dough warp of formation
After segmentation weighing, forming and provocation, bake to obtain bread products.Wherein, each component additive amount are as follows: flour 95~105
Parts by weight, 0.8~1.5 parts by weight of yeast powder, 1~2 parts by weight of salt, 55~65 parts by weight of water, sugared 5~8 parts by weight, vegetable oil 1
~2 parts by weight.
The recombination wheat endoplasmic reticulum thiol-oxide reductase pitch-based sphere is 0.05%~0.2% (w/w, flour
Base).
The method that the application recombination wheat endoplasmic reticulum thiol-oxide reductase improves flour processing quality, including it is as follows
Specific steps:
(1) material: 95~105 parts by weight of flour, 0.8~1.5 parts by weight of yeast powder, 1~2 parts by weight of salt, water 55~65
Parts by weight, sugared 5~8 parts by weight, 1~2 parts by weight of vegetable oil, recombination wheat endoplasmic reticulum thiol-oxide reductase 0.05%~
0.2% (w/w, flour-based)；
(2) and face: above-mentioned material being mixed, 10~15min is stirred with the speed of 150~180r/min, is formed it into
Even dough；
(3) form: by with good dough be divided into 40~60 parts by weight/, rub with the hands circle, shape sabot；
(4) it ferments: the provocation disk equipped with dough being placed in provocation in proofing box, temperature is controlled at 30~35 DEG C, relatively wet
Degree 75%~85%, 60~80min of fermentation time；
(5) bake: by the dough after provocation, baking 10~15min, oven temperature is 180~200 DEG C, obtain bread at
Product.
The preparation method of the recombination wheat endoplasmic reticulum thiol-oxide reductase, using coli expression system table
It reaches, and using column chromatography method purifying recombination wEro1 albumen；FAD is added to pure according to the molar ratio of wEro1:FAD=1:1~5
In the recombination wEro1 protein solution of change, biologically active recombination wheat endoplasmic reticulum thiol-oxide reductase is obtained.Including such as
Lower step:
(1) full genome synthetic technology is utilized, gene order (the SEQ ID of wheat endoplasmic reticulum thiol-oxide reductase has been synthesized
NO:1), and it is connected on vector plasmid pET-28a (+), restriction enzyme site is NdeI and XhoI, has constructed wheat endoplasmic reticulum mercapto
Base oxidoreducing enzyme recombinant expression plasmid pET-28a-wero1；
(2) recombinant expression plasmid pET-28a-wero1 is transferred in e. coli bl21 (DE3) competent cell, is obtained
To recombinant expression bacterium；Recombinant expression bacterium is inoculated into the culture of LB culture medium to OD600Reach 0.6~0.8, in 16~37 DEG C of conditions
Lower addition 0.2~1.0mM IPTG induction recombinant protein collects thallus after expressing 8~12 hours；
(3) supernatant is collected by centrifugation after ice-bath ultrasonic is broken in the thallus collected, and purifies recombination wEro1 using Ni-NTA
Albumen；
(4) low according to the molar ratio addition FAD of wEro1:FAD=1:1~5 into the recombination wEro1 protein solution of purifying
Temperature is incubated overnight, and extra FAD is removed through desalting processing, obtains biologically active recombination wheat endoplasmic reticulum sulfhydryl oxidase also
Protoenzyme.After recombinant protein concentration, cold storage is spare in -20 DEG C.
The amino acid sequence of the wheat endoplasmic reticulum thiol-oxide reductase is as shown in SEQ ID NO:2.
Mechanism of the invention is: the present invention obtains recombination wheat endoplasmic reticulum thiol-oxide reductase by prokaryotic expression,
Flour quality characteristic can effectively be enhanced by individually adding the enzyme, improve the bakings product such as specific volume, ratio of height to diameter, elasticity and hardness of bread
Matter, reached with the comparable improvement of chemical gluten-strengthening agent azodicarbonamide (ADA), can become substitution ADA new bio face
Flour modifying agent is used for Flour product processing industry.
The present invention compared with the existing technology, have following advantages and effects
(1) present invention provides a kind of application recombination wheat endoplasmic reticulum thiol-oxide reductase improvement flour processing product for the first time
The method of matter.The recombinase can effectively enhance flour quality characteristic, improve the baking such as specific volume, ratio of height to diameter, elasticity and hardness of bread
Quality is roasted, the comparable improvement with chemical gluten-strengthening agent azodicarbonamide (ADA) has been reached, the novel of substitution ADA can be become
Biological flour improver is used for Flour product processing industry.
(2) present invention application recombination wheat endoplasmic reticulum thiol-oxide reductase improves the method for bread baking properties, only needs
The recombinase is individually added in bread making process, is not required to add other additives again, simplifies production process, reduce life
Produce cost.
(3) method that the present invention uses prokaryotic expression, obtains biologically active recombination wheat endoplasmic reticulum sulfydryl oxygen
Change reductase, has the features such as expression quantity is high, and simple purification is easy to amplify, and is suitble to industrial applications.
Detailed description of the invention
Fig. 1 is the double digestion identification of recombinant expression plasmid pET-28a-wero1；Wherein, swimming lane M:DL5000DNA
Marker；Swimming lane 1: double digestion segment, white underscore are purpose genetic fragment.
Fig. 2 is expression and the soluble analysis for recombinating wEro1；Wherein, swimming lane M: molecular weight Marker；Swimming lane 1: it does not induce
Bacterium solution；Swimming lane 2: bacterium solution after induction；Swimming lane 3: clasmatosis liquid precipitate；Swimming lane 4: clasmatosis liquid supernatant；White arrow is signified
Place is recombination wEro1 albumen.
Fig. 3 is the SDS-PAGE analysis of recombination wEro1 after affinity chromatography；Wherein, swimming lane 1: molecular weight Marker；Swimming lane 2:
Clasmatosis liquid；Swimming lane 3: unbonded albumen；Swimming lane 4~8: eluent difference pipe collects component.
Fig. 4 is the zymologic property for recombinating wEro1.
Fig. 5 is to recombinate the influence of wEro1 and azodicarbonamide to bread texture parameter to compare；Wherein, different letters indicate
Significant difference (P < 0.05).
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
For not specifically specified technological parameter, routine techniques progress can refer to.Face used in the embodiment of the present invention
Powder is purchased from grain market；Restriction enzyme NdeI and XhoI are purchased from Thermo Fisher Scientific company；pET-28a
(+) plasmid is purchased from precious bioengineering (Dalian) Co., Ltd；E. coli bl21 (DE3) is purchased from Novagen company；Full genome sequence
Column synthesis is completed by Guangzhou Ai Ji Bioisystech Co., Ltd.
Embodiment 1 recombinates wheat endoplasmic reticulum thiol-oxide reductase expression vector establishment
Wheat endoplasmic reticulum thiol-oxide reductase gene (SEQ ID NO:1) is synthesized in the limited public affairs of Guangzhou Ai Ji biotechnology
Department, and be connected on cloning vector pGSI.1ng recombinant clone plasmid is taken, through NdeI and XhoI restriction enzyme reaction (37
DEG C, 30min) after, it carries out agarose gel electrophoresis and purifies the wheat endoplasmic reticulum thiol-oxide reductase base for having cohesive end
Because of segment.Plasmid pET-28a (+) mixing by 20 μ g genetic fragments with 5 μ g through identical restriction enzyme digestion, in T4DNA ligase
In 20 DEG C of connection 1h under effect.Product is transferred in bacillus coli DH 5 alpha competence after connection, and it is solid to be coated on the LB containing kanamycins
12~16h is cultivated on body culture medium.The full monoclonal colonies of picking form extract plasmid, carry out double digestion mirror to recombinant plasmid
It is fixed, confirm that the plasmid of extraction contains target gene fragment (Fig. 1) according to electrophoresis result.
As a result: successfully constructing recombinant expression carrier pET-28a-wero1.
The inducing expression and purifying of the recombination of embodiment 2 wEro1
1, the inducing expression of wEro1 is recombinated
The recombinant plasmid pET-28a-wero1 built is converted into BL21 (DE3), 3 monoclonal colonies of picking arrive
In LB culture medium containing 50 μ g/mL kanamycins, 37 DEG C, cultivate under the conditions of 200r/m to OD600To 0.6~0.8 or so, it is added
0.2~1mM IPTG 8~12h of inducing expression, inducing temperature range is between 16~37 DEG C.It analyzes, recombinates through SDS-PAGE
WEro1 obtains soluble-expression (Fig. 2).
2, the affinity purification of wEro1 is recombinated
Thallus after collecting expression, is added buffer and is resuspended, and is crushed somatic cells, ultrasound condition using probe type ultrasonic instrument
For 350w, duty ratio 0.4:0.6, ultrasonic 15min.After ultrasound is complete, supernatant is collected after 8000r/m centrifugation 20min.
Supernatant is slowly injected into Ni-NTA affinity column, and destination protein is purified in the way of linear elution, is collected each
Eluting peak component, and utilize SDS-PAGE detection recombinant protein (Fig. 3).
Merge destination protein solution, FAD is added by the molar ratio of wEro1:FAD=1:1~5,4 DEG C of processing overnight, then pass through
Cold storage is spare in -20 DEG C after desalination, concentration.
As a result: recombination wEro1 obtains soluble-expression, obtains high-purity destination protein through a step affinity chromatography.
The zymologic property of the recombination of embodiment 3 wEro1
WEro1 contains a variety of zymologic properties, and the present embodiment selects FAD reduction activation easy to detect.Its principle is wEro1's
Coenzyme F AD can be reduced into FADH under the action of dithiothreitol (DTT) (DTT)2, the latter is at 450nm without light absorption value.Therefore, lead to
It crosses light absorption value at detection 450nm to change, the FAD reduction activation of wEro1 can be probed into.
Enzyme activity determination reaction system is as follows: it is slow that 2 μM of wEro1 and 10 μM of FAD are added to 50mM Tris-HCl (pH8.0)
It rushes in solution, 12.5mM DTT starting reaction is added, measures light absorption value at 450nm.
As a result: as shown in figure 4, wEro1 shows apparent FAD reduction activation after addition DTT.
Embodiment 4 recombinates influence of the wEro1 to flour farinograph property
Characterizing recombination wEro1 using micro farinograph influences flour farinograph property.4g flour is added in powder alms bowl, 63r/m
After premixing 1min, 57% water is added, identical speed has obtained farinograph parameter and Rubus biflorus Buch after persistently stirring 15min.With not
Adding any substance is negative control group, to add azodicarbonamide (ADA) (20 μ g/g) for positive controls, to add recombination
WEro1 albumen (0.1%, w/w, flour-based) is experimental group.
As a result: compared with negative control, after azodicarbonamide and wEro1 is added, Rubus biflorus Buch is changed significantly, dough it is steady
It fixes time and mass number all significantly improves.However, recombination wEro1 is added and stablizes to flour compared to addition azodicarbonamide group
Time and mass number influence bigger (table 1), show to recombinate wEro1 in terms of improving farinograph property better than " chemistry " modifying agent azo
Formamide.
Table 1 recombinates the influence of wEro1 and azodicarbonamide to flour Rubus biflorus Buch and compares
| It is formed time (min) | Stablize time (min) | Weakness degree (FU) | Mass number | |
| Control | 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 |
Note: azodicarbonamide pitch-based sphere is 20 μ g/g, and recombination wEro1 pitch-based sphere is 0.1% (w/w, flour-based).Together
Different letters indicate significant difference (P < 0.05) in one column.
The method of 5 wEro1 of embodiment improvement bread baking properties
Improve the method for bread baking properties, concrete technology stream using above-mentioned recombination wheat endoplasmic reticulum thiol-oxide reductase
Journey is as follows:
(1) material: 95 parts by weight of flour, yeast powder (Angel high activity dried yeast, commercially available) 0.8 parts by weight, 1 weight of salt
Part, 55 parts by weight of water, sugared 5 parts by weight, vegetable oil (Jin Longyu edible blend oil, commercially available) 1 parts by weight, recombination wheat endoplasmic reticulum mercapto
Base oxidoreducing enzyme 0.05% (w/w, flour-based).
(2) and face: above-mentioned material being added to in the alms bowl of face, 10min is stirred with the speed of 150r/min, is formed it into
Even dough.
(3) form: will be placed on balance with good dough, be divided into 40 parts by weight/, on bread forming machine rub with the hands circle,
Shape sabot.
(4) it ferments: the provocation disk equipped with dough is placed in provocation in proofing box, temperature control is at 30 DEG C, relative humidity
75%, fermentation time 60min.
(5) bake: the dough after provocation is put in oven and bakes 10min, oven temperature be 180 DEG C, obtain bread at
Product.
Will bread obtained at room temperature cool down 2h after carry out physical qualities analysis, bread physical parameter measurement weight, volume,
Height and width.Not add any substance as negative control group, to add azodicarbonamide (20 μ g/g) as positive controls.
As shown in table 2, compared to negative control group, after recombination wEro1 is added, the ratio of height to diameter and specific volume of bread are significantly mentioned
Height has increased separately 11.3% and 8.2%.And compared to positive control, the bread of addition recombination wEro1 and azodicarbonamide it
Between do not have the significance of difference.The above result shows that recombination wEro1 has comparable raising therewith compared with azodicarbonamide
Bread ability, therefore the recombinase can substitute chemical gluten-strengthening agent azodicarbonamide, as native flours modifying agent application
In Flour product processing.
Table 2 recombinates the influence of wEro1 and azodicarbonamide to bread physical parameter and compares
| Volume (cm3) | Highly (cm) | Width (cm) | Specific volume (cm3/g) | Ratio of height to diameter | |
| Control | 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 |
Note: azodicarbonamide pitch-based sphere is 20 μ g/g, and recombination wEro1 pitch-based sphere is 0.05% (w/w, flour-based).
Different letters indicate significant difference (P < 0.05) in same row.
The method of 6 wEro1 of embodiment improvement bread baking properties
Improve the method for bread baking properties using above-mentioned recombination wheat endoplasmic reticulum thiol-oxide reductase, difference exists
In using following process flow:
(1) material: 105 parts by weight of flour, yeast powder (Angel high activity dried yeast, commercially available) 1.5 parts by weight, 2 weight of salt
Part, 65 parts by weight of water, sugared 8 parts by weight, vegetable oil (Jin Longyu edible blend oil, commercially available) 2 parts by weight, recombination wheat endoplasmic reticulum mercapto
Base oxidoreducing enzyme 0.2% (w/w, flour-based).
(2) and face: above-mentioned material being added to in the alms bowl of face, 15min is stirred with the speed of 180r/min, is formed it into
Even dough.
(3) form: will be placed on balance with good dough, be divided into 60 parts by weight/, on bread forming machine rub with the hands circle,
Shape sabot.
(4) it ferments: the provocation disk equipped with dough is placed in provocation in proofing box, temperature control is at 35 DEG C, relative humidity
85%, fermentation time 80min.
(5) bake: the dough after provocation is put in oven and bakes 15min, oven temperature be 200 DEG C, obtain bread at
Product.
The analysis of bread texture quality is carried out after bread obtained is cooled down 2h at room temperature.Bread is erected and is cut into 2cm/ block, face
Packet texture testing condition: p/25 probe, before surveying and test speed 1mm/s, speed 5mm/s after survey, compression ratio 50% push interval
10s.Not add any substance as negative control group, to add azodicarbonamide (20 μ g/g) as positive controls.
Bread texture the result shows that, add azodicarbonamide or recombination wEro1 can significantly reduce bread hardness and glutinous
Property, bread elasticity is improved, shows that the two can improve the quality of bread.Moreover, the face of addition recombination wEro1 and azodicarbonamide
Do not have the significance of difference (Fig. 5) between packet.Therefore, texture result further demonstrates that, recombination wEro1 can substitute azo formyl
Amine plays comparable improvement bread ability therewith.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (8)
1. wheat endoplasmic reticulum thiol-oxide reductase is improving the application in flour processing quality, it is characterised in that: only need to be in face
Wheat endoplasmic reticulum thiol-oxide reductase is added in powder can reach enhancing flour quality characteristic, improve the mesh of baking quality of bread
's.
2. a kind of method that application recombination wheat endoplasmic reticulum thiol-oxide reductase improves flour processing quality, it is characterised in that:
Recombination wheat endoplasmic reticulum thiol-oxide reductase, flour, sugar, salt, vegetable oil, yeast powder and water are stirred evenly, the face of formation
After the segmented weighing of group, forming and provocation, bake to obtain bread products；
Wherein, the recombination wheat endoplasmic reticulum thiol-oxide reductase pitch-based sphere is 0.05%~0.2%w/ of flour-based
w。
3. according to the method described in claim 2, it is characterized by: each component additive amount are as follows: 95~105 weight of flour
Part, 0.8~1.5 parts by weight of yeast powder, 1~2 parts by weight of salt, 55~65 parts by weight of water, sugared 5~8 parts by weight, 1~2 weight of vegetable oil
Measure part.
4. according to the method described in claim 3, it is characterized by:
The method that the application recombination wheat endoplasmic reticulum thiol-oxide reductase improves flour processing quality, including it is following specific
Step:
(1) material: 95~105 parts by weight of flour, 0.8~1.5 parts by weight of yeast powder, 1~2 parts by weight of salt, 55~65 weight of water
Part, sugared 5~8 parts by weight, 1~2 parts by weight of vegetable oil recombinate wheat endoplasmic reticulum thiol-oxide reductase 0.05%~0.2%；
(2) and face: above-mentioned material being mixed, 10~15min is stirred with the speed of 150~180r/min, is formed it into uniform
Dough；
(3) form: by with good dough be divided into 40~60 parts by weight/, rub with the hands circle, shape sabot；
(4) it ferments: the provocation disk equipped with dough is placed in provocation in proofing box, temperature control is at 30~35 DEG C, relative humidity
75%~85%, 60~80min of fermentation time；
(5) it bakes: by the dough after provocation, baking 10~15min, oven temperature is 180~200 DEG C, obtains bread products.
5. application according to claim 1, it is characterised in that:
The amino acid sequence of the wheat endoplasmic reticulum thiol-oxide reductase is as shown in SEQ ID NO:2.
6. application according to claim 1, it is characterised in that:
The nucleotide sequence of the wheat endoplasmic reticulum thiol-oxide reductase is as shown in SEQ ID NO:1.
7. according to the described in any item methods of claim 2~4, it is characterised in that:
The recombination wheat endoplasmic reticulum thiol-oxide reductase is expressed using coli expression system, and uses column chromatography side
Method purifying recombination wEro1 albumen；FAD is added to the recombination wEro1 albumen purified according to the molar ratio of wEro1:FAD=1:1~5
In solution, biologically active recombination wheat endoplasmic reticulum thiol-oxide reductase is obtained.
8. according to the method described in claim 7, it is characterized by:
The preparation method of the recombination wheat endoplasmic reticulum thiol-oxide reductase, includes the following steps:
(1) full genome synthetic technology is utilized, has synthesized the gene order of wheat endoplasmic reticulum thiol-oxide reductase, and be connected to load
On constitution grain pET-28a (+), restriction enzyme site is NdeI and XhoI, has constructed the recombination of wheat endoplasmic reticulum thiol-oxide reductase
Expression plasmid pET-28a-wero1；
(2) recombinant expression plasmid pET-28a-wero1 is transferred in e. coli bl21 (DE3) competent cell, obtains weight
Group expression bacterium；Recombinant expression bacterium is inoculated into the culture of LB culture medium to OD600Reach 0.6~0.8, adds under the conditions of 16~37 DEG C
Thallus is collected after adding 0.2~1.0mM IPTG induction recombinant protein to express 8~12 hours；
(3) supernatant is collected by centrifugation after ice-bath ultrasonic is broken in the thallus collected, and purifies recombination wEro1 albumen using Ni-NTA；
(4) according to the molar ratio addition FAD of wEro1:FAD=1:1~5 into the recombination wEro1 protein solution of purifying, low temperature is incubated
It educates overnight, extra FAD is removed through desalting processing, obtain biologically active recombination wheat endoplasmic reticulum sulfhydryl oxidase reduction
Enzyme.
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| CN201610422861.8A CN105875699B (en) | 2016-06-13 | 2016-06-13 | Improve the method for flour processing quality using recombination wheat endoplasmic reticulum thiol-oxide reductase |
| PCT/CN2016/110019 WO2017215216A1 (en) | 2016-06-13 | 2016-12-15 | Method of applying recombinant wheat endoplasmic reticulum thiol oxidoreductin to improve flour processing quality |
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| CN201610422861.8A CN105875699B (en) | 2016-06-13 | 2016-06-13 | Improve the method for flour processing quality using recombination wheat endoplasmic reticulum thiol-oxide reductase |
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| CN105875699B (en) * | 2016-06-13 | 2019-10-18 | 华南理工大学 | Improve the method for flour processing quality using recombination wheat endoplasmic reticulum thiol-oxide reductase |
| CN109006264B (en) * | 2018-06-17 | 2023-04-25 | 河北省农林科学院旱作农业研究所 | Dry-hot air resistant identification method for new wheat variety and special temperature control shed |
| CN112790213B (en) * | 2019-10-28 | 2022-11-11 | 广东省农业科学院蚕业与农产品加工研究所 | Application of resting sulfhydryl oxidase of rice in improving flour processing quality |
| CN110946275B (en) * | 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 | Application of wheat resting sulfhydryl oxidase in improving stability of emulsion type special medical food |
| CN114947046B (en) * | 2022-05-12 | 2023-06-16 | 华南理工大学 | Use of poly-3-hydroxybutyrate as flour or flour product additive |
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| WO1999007727A1 (en) * | 1997-08-12 | 1999-02-18 | Massachusetts Institute Of Technology | Eukaryotic disulfide bond-forming proteins and related molecules and methods |
| JP2011177059A (en) * | 2010-02-26 | 2011-09-15 | Tokyo Univ Of Agriculture | Modifier for wheat-processed product and method for producing wheat-processed product |
| CN102911888A (en) * | 2012-07-06 | 2013-02-06 | 江南大学 | Pichia pastoris coexpression chaperonin genetic engineering bacterium for secreting lipase and application of pichia pastoris coexpression chaperonin genetic engineering bacterium |
| CN103300083A (en) * | 2013-05-15 | 2013-09-18 | 南京农业大学 | Method for improving bread baking characteristic by using recombination lipoxygenase |
| CN103436516A (en) * | 2013-08-23 | 2013-12-11 | 华南理工大学 | Wheat protein disulfide isomerase produced by utilizing escherichia coli prokaryotic expression system, as well as method and application of wheat protein disulfide isomerase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999007727A1 (en) * | 1997-08-12 | 1999-02-18 | Massachusetts Institute Of Technology | Eukaryotic disulfide bond-forming proteins and related molecules and methods |
| JP2011177059A (en) * | 2010-02-26 | 2011-09-15 | Tokyo Univ Of Agriculture | Modifier for wheat-processed product and method for producing wheat-processed product |
| CN102911888A (en) * | 2012-07-06 | 2013-02-06 | 江南大学 | Pichia pastoris coexpression chaperonin genetic engineering bacterium for secreting lipase and application of pichia pastoris coexpression chaperonin genetic engineering bacterium |
| CN103300083A (en) * | 2013-05-15 | 2013-09-18 | 南京农业大学 | Method for improving bread baking characteristic by using recombination lipoxygenase |
| CN103436516A (en) * | 2013-08-23 | 2013-12-11 | 华南理工大学 | Wheat protein disulfide isomerase produced by utilizing escherichia coli prokaryotic expression system, as well as method and application of wheat protein disulfide isomerase |
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