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Method for high fermentation yield of recombinant proline aminopeptidase and preparation of debittered rice peptide

A technology of proline aminopeptidase and rice peptide, which is applied in the fields of enzyme preparations, food additives, and fermentation technology, and can solve problems such as influence fields

Active Publication Date: 2018-03-06
湖北跃海生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the deep processing of rice protein, its hydrolyzate often presents a strong bitter taste, which affects its application fields

Method used

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  • Method for high fermentation yield of recombinant proline aminopeptidase and preparation of debittered rice peptide
  • Method for high fermentation yield of recombinant proline aminopeptidase and preparation of debittered rice peptide
  • Method for high fermentation yield of recombinant proline aminopeptidase and preparation of debittered rice peptide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Fermentation and cultivation of recombinant Bacillus subtilis under different rotating speed conditions

[0056] The recombinant Bacillus subtilis stored in a glycerol tube at -40°C was inserted into the seed medium, and cultured on a rotary constant temperature shaker with a speed of 220r / min and a culture temperature of 37°C for 20 hours. Insert the seed liquid into a 5L fermenter equipped with 3L fermentation medium with an inoculum of 5%, feed sterile air during the fermentation process, the ventilation rate is 1.5vvm, the fermentation temperature is 37°C, pH7.0, stirring speed Set 200rpm, 300rpm, 400rpm, 500rpm respectively, and the fermentation time is 36h. During the fermentation process, samples were taken every 2 hours, and the dry weight of the bacteria, the activity of extracellular proline aminopeptidase and intracellular proline aminopeptidase were measured.

[0057] The results showed that the proline aminopeptidase activity increased from 38.0...

Embodiment 2

[0058] Embodiment 2: fermentation culture under the guidance of fermentation kinetics

[0059] Insert the seed liquid into a 5L fermenter with 3L fermentation medium at an inoculum amount of 5%, feed sterile air during the fermentation process, the ventilation rate is 1.5vvm, and the fermentation conditions: the speed control is 0-6h 200rpm, 6 -12h is 400rpm, 12-28h is 500rpm, 28h to the end of fermentation is 400rpm; pH regulation is 0-12h without controlling pH, 12-16h is pH7.0, 16-28hpH is not controlled, and pH7.0 after 28h; temperature The regulation is 8h before fermentation at 40°C, 35°C for 8-12h, and 33°C from 12h to the end of fermentation. During the fermentation process, samples were taken every 4 hours to measure the dry weight of the bacteria, the activity of extracellular proline aminopeptidase and intracellular proline aminopeptidase, etc.

[0060] The results showed that the fermentation strategy under kinetic analysis could significantly increase the yield o...

Embodiment 3

[0061] Embodiment 3: the effect of recombinant subtilis proline aminopeptidase in rice proteolysis

[0062] Prepare a 5% rice protein solution with pure water buffer, adjust the pH to 9.0 with 2M NaOH solution, keep it warm in a water bath at 90°C for 30 minutes, after cooling, add 15000U / g rice protein alkaline protease, react at 50°C for 4 hours, and boil for 15 minutes in a water bath After cooling, continue to add 3000U / g rice protein leucine aminopeptidase powder, react at 50°C for 2h, boil water bath for 15min, add 200U / g rice protein recombinant proline aminopeptidase enzyme solution after cooling, 50°C Reaction for 2 hours, boiling water bath for 15 minutes, cooling, the hydrolyzate mainly exists in the form of small peptides and free amino acids, the content of small peptides below 180Da reaches 44.70%, fully hydrolyzed the exposed N-terminal proline residues, free proline The acid content is 0.149 mg / mL, which is 1064.3 times the content of unhydrolyzed free proline....

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Abstract

The invention discloses a method for high fermentation yield of recombinant proline aminopeptidase and preparation of debittered rice peptide and belongs to the field of fermentation technology, enzyme preparations and food additives. The invention determines a fermentation method for high yield of proline aminopeptidase by recombinant bacillus subtilis by utilizing fermentation kinetics analysis,so that the yield of the proline aminopeptidase is improved to 174.8 U / mL. The proline aminopeptidase cooperates with alkali protease and leucine aminopeptidase to hydrolyze rice protein, the contentof free amino acid is 27.3 times of that of un-hydrolyzed free amino acid, the content of small peptide below 180 Da in a hydrolysate reaches 44.70 percent, exposed N-terminal proline residues are fully hydrolyzed, and the content of free proline is 1064.3 times of that of un-hydrolyzed free proline, so that the rice protein hydrolysis depth is improved. The method disclosed by the invention hasa good application prospect in the field of food and beverages as well as processing and utilization of food protein resources.

Description

technical field [0001] The invention relates to a high-yield fermented recombinant proline aminopeptidase and a method for preparing debittered rice peptide, belonging to the fields of fermentation technology, enzyme preparations and food additives. Background technique [0002] Microbial fermentation culture optimization strategies often start from the optimization of the medium and the control of the fermentation process. At present, it is not possible to infer and calculate the medium formulation and fermentation process control strategy suitable for a certain microorganism from the basic principles of biochemical reactions. Different microorganisms are suitable for different culture media and fermentation process control strategies. There is no single culture method suitable for the fermentation and cultivation of a variety of microorganisms. Therefore, small fermentation equipment such as shake flasks and glass tanks are required for a specific microorganism. According ...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12P21/06C12R1/125
CPCC12N9/485C12P21/06C12Y304/11009C12Y304/11001C12Y304/11005
Inventor 田亚平王开道李婷婷张大伟周楠迪
Owner 湖北跃海生物科技有限公司
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