CN101407797A - Method for producing cellulose, xylanase, glucanase and pectic enzyme by submerged fermentation of Trichoderma reesei - Google Patents

Abstract

The invention discloses a method for producing cellulose, xylanase, dextranase and pectase through the fermentation of a Trichoderma reesei liquid. In the invention, the Trichoderma reesei liquid is selected as a strain; microcrystalline cellulose, bran and other culturing preparations including monopotassium phosphate, sodium citrate and ammonium sulphate are selected as inducers for preparing the cellulose, the xylanase, the dextranase and the pectase through fermentation. fermenting enzyme activity: 1600Mu/ml of cellulose, 1400Mu/ml of dextranase, 500Mu/ml of xylanase, and 500Mu/ml of pectase, which can simultaneously exert the effects for decomposing the cellulose and hemicellulose components of a plant cytoderm under the conditions of the pH value of 3.5-5 and the temperature of 30-50 DEG C. The method is applied to extracting feed and Chinese traditional medicines as well as destroying the plant cytoderm for leading the effective components to be released.

Description

The method of trichoderma reesei liquid fermentative production cellulase, zytase, dextranase and polygalacturonase

Technical field

The present invention relates to a kind of microbial fermentation technology field, be specifically related to a kind of liquid Trichodermareesei method of production of cellulose enzyme, zytase, dextranase and polygalacturonase simultaneously.

Background technology

Domesticly now mostly be to utilize koning trichoderma solid fermentation production of cellulose enzyme, except that the enzyme component that contains needs, also can contain raw material waste residue, fermentation thalline and the assorted bacterium of fermentation in the leavened prod, can have influence on the effect of product on feed.And the bacterial classification of domestic employing liquid fermenting or technology, it mainly is the product that produces a kind of enzyme, except that indivedual through strain improvements and process reform simultaneously fermentative production cellulase and the zytase, do not have relevant can while fermentative production cellulase, the report and the technology of zytase, dextranase and polygalacturonase.But the broken wall of each plant decomposes, and all needs the combined action of these four kinds of enzymes, contains these four kinds of enzymes simultaneously to discharge effective raw material for the plant broken wall very necessary so produce a kind of product.

Summary of the invention

The object of the present invention is to provide the method for a kind of trichoderma reesei liquid fermentative production cellulase, zytase, dextranase and polygalacturonase.

It is Trichodermareesei that the present invention selects bacterial classification for use, and inductor is selected Microcrystalline Cellulose and wheat bran for use, and other cultivates preparation is potassium primary phosphate, Trisodium Citrate and ammonium sulfate, and fermentation makes cellulase, zytase, dextranase and polygalacturonase.

Specific embodiment of the present invention is:

Get the seed tank culture base: the 0.5-3% Microcrystalline Cellulose, the 0.5-3% wheat bran, the 0.3-1% potassium primary phosphate, the 0.1-0.8% Trisodium Citrate, 0.2-1% ammonium sulfate, surplus is a water, the pH value is between 4.6-5.3.

Secondary jar substratum: the 0.5-3% Microcrystalline Cellulose, the 0.5-3% wheat bran, the 0.3-1% potassium primary phosphate, the 0.1-0.8% Trisodium Citrate, 0.2-1% ammonium sulfate, surplus is a water, the pH value is between 4.6-5.3.

Fermentation tank culture medium: the 2-5% Microcrystalline Cellulose, the 1-4% wheat bran, the 0.3-1% potassium primary phosphate, the 0.1-0.8% Trisodium Citrate, 0.2-1% ammonium sulfate, surplus is a water, sterilization back pH value is between 4.6-5.3.

Under aseptic technique, the inclined-plane seed is inserted, 121 ℃ of sterilizations 30 minutes and being cooled in 30 ℃ the seeding tank down, be 1: 0.2 in ventilation, tank pressure 0.05Mpa cultivated 30-48 hour under 30 ℃ ± 1 ℃ the condition; Being transferred to 121 ℃ through the culture transferring pipeline and sterilizing 30 minutes and be cooled in 30 ℃ ± 1 ℃ secondary jar, is 1: 0.2 in ventilation, and tank pressure 0.05Mpa cultivated about 24 hours under the condition that temperature is 30 ℃ ± 1 ℃; Being transferred to 121 ℃ through the culture transferring pipeline sterilized 30 minutes and was cooled in 30 ℃ ± 1 ℃ fermentor tank, ventilation 1: 0.5, tank pressure 0.05Mpa, cultivated 100-120 hour under the condition that temperature is 30 ℃ ± 1 ℃, put into flocculation jar adding super-cell through the blowing pipeline, carry out press filtration at plate-and-frame filter press, clear liquid is squeezed into circulation tank and is entered the ultra-filtration membrane thickener and carry out ultrafiltration and concentration, the good work in-process of ultrafiltration and concentration is squeezed into buffer tank carry out the scraper plate vacuum concentration again and promptly make cellulase, zytase, dextranase and polygalacturonase.

Can produce cellulase, dextranase, zytase and four kinds of enzymes of polygalacturonase simultaneously through experiment discovery Trichodermareesei, and fermenting enzyme is lived: cellulase 1600u/ml, dextranase 1400u/ml, zytase 500u/ml, polygalacturonase 500u/ml.Various enzymes can be brought into play simultaneously under the temperature 30-50 ℃ of condition and render a service fiber and half fibre composition that decomposes plant cell wall at pH value 3.5-5.The present invention is applied on feed and the traditional Chinese medicine extraction, destroys plant cell wall, and effective constituent is released.This product is the prozyme of cellulase, dextranase, zytase, polygalacturonase.

Embodiment

Get the seed tank culture base: 1% Microcrystalline Cellulose, 1% wheat bran, 0.5% potassium primary phosphate, 0.25% Trisodium Citrate, 0.5% ammonium sulfate, water 96.75%, sterilization back pH value is between 4.6-5.3.Secondary jar substratum: 1% Microcrystalline Cellulose, 1% wheat bran, 0.5% potassium primary phosphate, 0.25% Trisodium Citrate, 0.5% ammonium sulfate, water 96.75%, sterilization back pH value is between 4.6-5.3.Fermentation tank culture medium: 3% Microcrystalline Cellulose, 3% wheat bran, 0.5% potassium primary phosphate, 0.25% Trisodium Citrate, 0.5% ammonium sulfate, water 92.75%, sterilization back pH value is between 4.6-5.3.

Under aseptic technique, the Trichodermareesei bacterial classification that potato slope is cultivated is linked into 121 ℃ of sterilizations 30 minutes and being cooled in 30 ℃ the 700 liter seeding tanks down, constantly feeding sterile air, ventilation is 1: 0.2, tank pressure 0.05Mpa, cultivated 30-48 hour under 30 ℃ ± 1 ℃ the condition, through chemical examination detect ripe this after standard, change over to 121 ℃ of sterilizations 30 minutes and be cooled in 30 ℃ ± 1 ℃ secondary jar that contains the 4T substratum through the culture transferring pipeline after the sterilization, ventilation 1: 0.2, tank pressure 0.05Mpa, cultivated about 24 hours under the condition that temperature is 30 ℃ ± 1 ℃, through chemical examination detect ripe this after standard, change over to 121 ℃ of sterilizations 30 minutes and be cooled in 30 ℃ ± 1 ℃ fermentor tank that contains the 10T substratum through the culture transferring pipeline after the sterilization, ventilation 1: 0.5, tank pressure 0.05Mpa, cultivated 100-120 hour under the condition that temperature is 30 ℃ ± 1 ℃, reach put jar standard after, flocculation jar after the blowing pipeline after the sterilization is put into sterilization adds super-cell, carrying out press filtration through clean plate-and-frame filter press later, muddy liquid backflow flocculation jar, clear liquid is squeezed into circulation tank and is entered the ultra-filtration membrane thickener and carry out ultrafiltration and concentration, the good work in-process of ultrafiltration and concentration is squeezed into buffer tank carry out scraper plate vacuum concentration to the vigor that requires again and can make cellulase, zytase, dextranase and polygalacturonase.

Claims (2)

1, the method for a kind of trichoderma reesei liquid fermentative production cellulase, zytase, dextranase and polygalacturonase, it is characterized in that: selecting bacterial classification for use is Trichodermareesei, inductor is selected Microcrystalline Cellulose and wheat bran for use, other cultivates preparation is potassium primary phosphate, Trisodium Citrate and ammonium sulfate, and fermentation makes cellulase, zytase, dextranase and polygalacturonase.

2, the method for trichoderma reesei liquid fermentative production cellulase, zytase, dextranase and polygalacturonase as claimed in claim is characterized in that:

Get the seed tank culture base: the 0.5-3% Microcrystalline Cellulose, the 0.5-3% wheat bran, the 0.3-1% potassium primary phosphate, the 0.1-0.8% Trisodium Citrate, 0.2-1% ammonium sulfate, surplus is a water, the pH value is between 4.6-5.3;

Secondary jar substratum: the 0.5-3% Microcrystalline Cellulose, the 0.5-3% wheat bran, the 0.3-1% potassium primary phosphate, the 0.1-0.8% Trisodium Citrate, 0.2-1% ammonium sulfate, surplus is a water, the pH value is between 4.6-5.3;

Fermentation tank culture medium: the 2-5% Microcrystalline Cellulose, the 1-4% wheat bran, the 0.3-1% potassium primary phosphate, the 0.1-0.8% Trisodium Citrate, 0.2-1% ammonium sulfate, surplus is a water, the pH value is between 4.6-5.3;

Under aseptic technique, the inclined-plane seed is inserted, 121 ℃ of sterilizations 30 minutes and being cooled in 30 ℃ the seeding tank down, be 1: 0.2 in ventilation, tank pressure 0.05Mpa cultivated 30-48 hour under 30 ℃ ± 1 ℃ the condition; Being transferred to 121 ℃ through the culture transferring pipeline and sterilizing 30 minutes and be cooled in 30 ℃ ± 1 ℃ secondary jar, is 1: 0.2 in ventilation, and tank pressure 0.05Mpa cultivated about 24 hours under the condition that temperature is 30 ℃ ± 1 ℃; Being transferred to 121 ℃ through the culture transferring pipeline sterilized 30 minutes and was cooled in 30 ℃ ± 1 ℃ fermentor tank, ventilation 1: 0.5, tank pressure 0.05Mpa, cultivated 100-120 hour under the condition that temperature is 30 ℃ ± 1 ℃, put into flocculation jar adding super-cell through the blowing pipeline, carry out press filtration at plate-and-frame filter press, clear liquid is squeezed into circulation tank and is entered the ultra-filtration membrane thickener and carry out ultrafiltration and concentration, the good work in-process of ultrafiltration and concentration is squeezed into buffer tank carry out the scraper plate vacuum concentration again and promptly make cellulase, zytase, dextranase and polygalacturonase.