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Method for improving submerged fermentation level of trichoderma reesei cellulase liquid

A liquid submerged fermentation, Trichoderma reesei technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems that are not suitable for large-scale industrial production, expensive sophorose, low enzyme production level, etc. , to achieve the effect of improving the liquid submerged fermentation level of Trichoderma reesei cellulase, prolonging the stable period of bacterial growth, and simple operation process

Inactive Publication Date: 2013-03-13
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason is that the existing cellulase batch fermentation bacteria have a short stable growth period, long fermentation cycle, low enzyme production level and high production cost, making it difficult to realize industrial application
In response to these problems, Chinese patents 200910091968.9, 201010040047.2, etc. reported that the method of feeding culture and controlling dissolved oxygen was used to improve the level of cellulase produced by Trichoderma reesei, but they all added efficient induction of cellulase in their feed medium Sophorose, and the addition amount is relatively high, sophorose is expensive and not suitable for large-scale industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The Trichoderma reesei CICC 13052 strain was inoculated on a PDA slant and cultured in a constant temperature incubator at 30°C for 6 days. Then the activated bacterial strain on the PDA slant medium was prepared to a concentration of 2×10 7 The spore suspension per mL was inoculated in fresh seed medium according to the inoculation amount of 3%, and cultured on a shaker at 28° C. with a rotation speed of 180 rpm. Wherein, the composition of the seed medium is: 1% microcrystalline cellulose, 1.5% corn steep liquor, 1% glucose, and the pH is adjusted to 4.8.

[0017] The seed liquid after culturing for 24 hours was inoculated into fresh fermentation medium according to the inoculum amount of 5% (the liquid volume in a 5L fermentation tank was 2.5L) for cultivation. The pH and dissolved oxygen in the fermenter are not controlled. After 24 hours, the pH of the fermentation broth is controlled to maintain at 4.8. After 24 to 84 hours, the dissolved oxygen in the fermentati...

Embodiment 2

[0020] The Trichoderma reesei CICC 13052 strain was inoculated on a PDA slant and cultured in a constant temperature incubator at 28°C for 7 days. Then the activated bacterial strain on the PDA slant medium was prepared to a concentration of 10 8 Each / mL spore suspension was inoculated in fresh seed medium according to the inoculation amount of 3%, and cultured on a shaker at 28° C. with a rotation speed of 200 rpm. Wherein, the composition of the seed medium is: 2% microcrystalline cellulose, 1.5% corn steep liquor, 1% glucose, and the pH is adjusted to 4.5.

[0021] Inoculate the seed liquid after culturing for 28 hours into a fresh fermentation medium (5L fermentation tank liquid volume is 2.5L) according to 5% inoculum amount for cultivation. The segmentation control and feeding strategy are as follows: from the beginning of fermentation to 36 hours The pH and dissolved oxygen in the fermenter are not controlled. After 36 hours, the pH of the fermentation broth is control...

Embodiment 3

[0024] The Trichoderma reesei CICC 13052 strain was inoculated on a PDA slant and cultured in a constant temperature incubator at 30°C for 7 days. Then the activated bacterial strain on the PDA slant medium was prepared to a concentration of 10 8 Each / mL spore suspension was inoculated in fresh seed medium according to the inoculation amount of 3% or 2.5%, and cultured on a shaker at 28°C with a rotation speed of 200 rpm. Wherein, the composition of the seed medium is: 2% microcrystalline cellulose, 2% corn steep liquor, 1% glucose, and the pH is adjusted to 4.8.

[0025] Inoculate the seed liquid after cultivating for 30 hours into a fresh fermentation medium (2.5L in a 5L fermentation tank) according to the inoculum size of 5% or 4.5%. The pH and dissolved oxygen in the fermenter were not controlled for 28 hours. After 28 hours, the pH of the fermented liquid was controlled to maintain at 4.9. The dissolved oxygen in the fermented liquid was controlled at 35 to 55% by adjus...

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Abstract

The invention provides a method for improving a submerged fermentation level of trichoderma reesei cellulase liquid. The method provided by the invention comprises the following steps of: first-grade seed culturing: preparing a spore suspension solution from activated trichoderma reesei CICC 13052 bacterial strain on a PDA (Potato-Dextrose-Agar) slant culture medium and inoculating the spore suspension solution into a fresh seed culture medium according to the inoculating amount of 3% to be cultured; and fermenting and culturing: inoculating the first-grade seeds in a logarithm growing periodinto a fresh fermenting culture medium to be cultured and controlling the pH values and dissolved oxygen in a pot of the fermenting process by sections to maintain the pH values and dissolved oxygen of the different periods at the different levels, and carrying out feed supplement and controlled culturing. By utilizing the method provided by the invention, the liquid is fermented for 168 hours; the enzyme activity of the fermenting liquid is measured by utilizing an international standard method which is recommended by the IUPAC (International Union of Pure and Applied Chemistry); and the enzyme activity of filter paper is 14.2 IU / mL and is improved by 67.1% compared with the interval fermenting enzyme activity level (8.5 IU / mL) before the process is optimized.

Description

technical field [0001] The invention relates to a fermentation method of Trichoderma reesei cellulase, in particular to a method capable of improving the liquid submerged fermentation level of Trichoderma reesei cellulase. Background technique [0002] Cellulose is the most abundant polysaccharide on the earth, and it is also the most potential renewable resource in nature. Its effective utilization is a hot spot that people pay attention to at present. As a multi-component enzyme system that degrades cellulose to generate glucose, cellulase has the advantages of high specificity, mild reaction conditions and less environmental pollution. Therefore, in order to make better use of cellulose substances, the research and application of cellulase has attracted more and more attention from scholars at home and abroad. [0003] Cellulase has been widely used in many fields such as textile, food fermentation, feed, deep processing of agricultural and sideline products, papermaking...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12R1/885
Inventor 陈树林马立娟李晨贾文娣武改红
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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