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A method for high-yield fermentation of recombinant proline aminopeptidase and preparation of debittered rice peptide

A proline aminopeptidase, high-yield technology, applied in the fields of food additives, enzyme preparations, fermentation technology, and can solve problems such as impact fields

Active Publication Date: 2020-08-04
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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  • A method for high-yield fermentation of recombinant proline aminopeptidase and preparation of debittered rice peptide
  • A method for high-yield fermentation of recombinant proline aminopeptidase and preparation of debittered rice peptide
  • A method for high-yield fermentation 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 present disclosure discloses methods for high-yield fermentation of recombinant proline aminopeptidase and preparation of debittered rice peptide, belonging to the fields of fermentation technology, enzyme preparation and food additives. The present disclosure utilizes fermentation kinetic analysis to determine the high-yield fermentation method of proline aminopeptidase by recombinant Bacillus subtilis, and improve the yield of proline aminopeptidase to reach 174.8 U / mL. Proline aminopeptidase cooperates with alkaline protease and leucine aminopeptidase to hydrolyze rice protein. The free amino acid content is 27.3 times the unhydrolyzed free amino acid content, and the small peptide content below 180 Da in hydrolysate reaches 44.70%. The exposed N-terminal proline residue is fully hydrolyzed, and the free proline content is 1,064.3 times that of the unhydrolyzed free proline content, which increases the degree of rice protein hydrolysis. The method of the present disclosure has a good application prospect in the fields of foods and beverages and 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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Patent Type & Authority Patents(China)
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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