Application of bacillus halophilus xylanase in improvement of flour processing quality

A technology of lactobacillus and xylanase, which is applied in the fields of genetic engineering and grain science to achieve the effects of improving quality, simplifying purification and improving microstructure

Pending Publication Date: 2022-05-10
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lactobacillus salinus xylanase is a potential enzyme preparation for flour improvement. At present, the research on this enzyme m...

Method used

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  • Application of bacillus halophilus xylanase in improvement of flour processing quality
  • Application of bacillus halophilus xylanase in improvement of flour processing quality
  • Application of bacillus halophilus xylanase in improvement of flour processing quality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Xylanase gene codon optimization and cloning

[0043] Using the whole gene synthesis technology, the whole gene sequence of Pichia pastoris codon-optimized Halolactibacillus miurensis xylanase was synthesized. Wherein, the amino acid sequence of the xylanase from Lactobacillus salinus is shown in SEQ ID NO.1, and the base sequence encoding the xylanase from Lactobacillus salinus is shown in SEQ ID NO.2. Afterwards, the xylanase sequence of Lactobacillus salinus and the carrier plasmid pPICZαA were double-digested with EcoRI and NotI, and ligated with T4 DNase, and the ligated product was transformed into Escherichia coli DH5α competent cells. Pick a single colony, then extract the plasmid for electrophoresis detection, and store the plasmid at -20°C. Then EcoRI and NotI were used to digest and detect the target fragment ( figure 1 ), and then send the plasmid to the company for sequencing. The correctly sequenced plasmid is the codon-optimized recombinant ex...

Embodiment 2

[0044] Induced expression and purification of embodiment 2 xylanase

[0045] 1. Induced expression

[0046]The recombinant expression vector pPICZαA-hxyl obtained in Example 1 was linearized with SacI, and transformed into the host cell Pichia pastoris X33 by electroporation. Perform colony PCR verification on the single colony on the transformed resistance screening plate to ensure the integration of the exogenous gene. Inoculate the screened recombinant Pichia pastoris in 10mL BMGY liquid medium, shake and culture overnight at 30°C, 250rpm; after centrifugation at 3000g at room temperature for 2min, collect the bacteria, transfer it to BMMY medium, and transfer Bacteria solution OD 600 0.5-1.0; 30°C, 250rpm to continue culturing for 24-144h, during which samples were taken every 24h and methanol was added to the medium to 1.0% v / v. After the culture was completed, solid-liquid separation was performed to obtain the fermentation supernatant. SDS-PAGE analysis showed that t...

Embodiment 3

[0049] Embodiment 3 xylanase enzymatic properties assay

[0050] 1. Determination of Enzyme Activity

[0051] (1) Draw a standard curve

[0052] Accurately weigh 0.5g of xylose, and adjust the volume to 100mL with a pH7.0 phosphate buffer solution to obtain a solution of 5mg / mL xylose. Take seven 2mL EP tubes, add 0.00mL, 0.01mL, 0.02mL, 0.03mL, 0.04mL, 0.05mL, 0.10mL xylose standard solution, and add distilled water to 0.5mL. Take 0.2mL xylose diluent respectively in 2mL EP tubes, add 0.30mL DNS reagent and shake well, immediately place in boiling water for 5min to develop color. After cooling, take 200 μL in a microtiter plate, measure its absorbance value at 540 nm, and draw a standard curve with the xylose content as the abscissa and the absorbance as the ordinate.

[0053] DNS reagent: Dissolve 6.3g of 3,5-dinitrosalicylic acid (DNS) in 400mL of distilled water, gradually add 21g of sodium hydroxide, sequentially add 185g of potassium sodium tartrate tetrahydrate, 5.0g...

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Abstract

The invention discloses an application of bacillus halophilus xylanase in improving the processing quality of flour, and belongs to the field of genetic engineering and cereal science. According to the invention, a eukaryotic expression method is adopted to obtain the recombinant bacillus halophilus xylanase with biological activity, and the recombinant bacillus halophilus xylanase has the characteristics of high expression quantity, simplicity and convenience in purification, easiness in amplification, suitability for industrial application and the like. According to the method for improving the processing quality of the flour by independently adding the bacillus halolacticus xylanase, the microstructure of dough can be improved by directly adding the bacillus halolacticus xylanase, the specific volume of whole-wheat bread is remarkably increased, the hardness and chewiness of the whole-wheat bread are reduced, and the quality of the whole-wheat bread is improved; the flour improver is a potential flour improver.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and grain science, and in particular relates to a Pichia pastoris recombinant Lactobacillus salinum xylanase and a preparation method thereof, and the use of the Pichia pastoris recombinant Lactobacillus salinum xylanase in improving flour processing quality Applications. Background technique [0002] Noodle products are the traditional staple food in northern China. Due to the limitations of wheat varieties and geographical factors, flour improvers are often needed when making noodle products. Traditional chemical additives, such as potassium bromate and azodicarbonamide, have been banned from being used in flour due to potential safety hazards. Searching for and developing natural and harmless flour-improving enzyme preparations is gradually entering people's field of vision. [0003] Xylan is the main component of hemicellulose in plant cells. Wheat flour contains about 2% to 3% arabinoxyl...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/81C12N1/19A21D8/04C12R1/84
CPCC12N9/248C12N15/815A21D8/042C12N2800/22
Inventor 韩双艳张亚萍赵风光林影郑穗平
Owner SOUTH CHINA UNIV OF TECH
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