Liquid fermentation method for producing pectinase

A liquid fermentation and pectinase technology, applied in the field of bioengineering, can solve the problems of limited wide application, high production cost, low enzyme activity, etc., and achieve the effects of increasing fermentation capacity, reducing fermentation cost, and significantly acid resistance.

Active Publication Date: 2020-03-17
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to its late start, low enzyme activity and high production costs, the wide application of this enzyme has been severely limited. At present, it is mainly used in fruit juice production, fruit wine brewing, canning processing, wood preservation, pulp bleaching, degumming of hemp, and prevention and treatment of plant diseases, etc. aspect

Method used

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  • Liquid fermentation method for producing pectinase
  • Liquid fermentation method for producing pectinase
  • Liquid fermentation method for producing pectinase

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Aspergillus niger LD-092 fermentation enzyme production and the extraction method of pectinase produced

[0035] The fermentative enzyme production method of Aspergillus niger LD-092 mainly comprises the following steps:

[0036] Slant culture: Pick a ring of Aspergillus niger LD-092 and inoculate it on a solid slant medium, and culture at a constant temperature of 30°C for 36 hours to obtain first-class seeds;

[0037] Shake flask culture: Take a ring of the first-class seeds and insert them into the seed medium, and cultivate them for 48 hours at a constant temperature of 30°C and a shaker speed of 200r / min to obtain the second-class seed liquid;

[0038] Seed tank culture: put the secondary seed solution into the seed tank culture medium at a ratio of 15% (v / v) of the inoculation amount, and cultivate for 45 hours at a constant temperature of 30°C and a rotational speed of 200r / min;

[0039] Fermentation tank culture: the seed liquid in the seed tank is co...

Embodiment 2

[0050] Embodiment 2 Aspergillus niger LD-092 fermentation performance verification

[0051] Carry out 50L fermenter verification experiment according to the fermentative enzyme production method of embodiment 1 Aspergillus niger LD-092, fermentation cycle 155h, its 7 batches of fermentative enzyme production situation, the pectinase average enzyme activity in the fermented liquid is 82882U / mL, table 1 shows that the strain not only produces high-yield pectinase, but also has remarkable stability in its fermentation performance and the enzymatic activity of pectinase produced by it.

[0052] Table 1 Enzyme production by fermentation of 7 batches of high pectinase-producing strains

[0053]

Embodiment 3

[0054] Embodiment 3 pectinase activity assay method

[0055] (1) Preparation of enzyme liquid: the fermentation liquid was centrifuged at 6000 r / min for 15 min, and the supernatant was collected to be the crude enzyme liquid.

[0056] (2) Determination method: 3,5-dinitrosalicylic acid method (DNS method) is adopted. Take 0.5ml of appropriately diluted crude enzyme solution in a 25mL colorimetric tube, add 2.0ml of 0.4% pectin solution (pH4.0) as a substrate solution, react in a water bath at 45°C for 30min, add DNS solution and boil for 5min, cool down, and constant volume To 25ml, measure the absorbance at 520nm.

[0057] (3) Definition of enzyme activity: under certain conditions, the amount of enzyme needed to decompose pectin to generate 1 μmol galacturonic acid per minute is an enzyme activity unit (U).

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Abstract

The invention, which belongs to the technical field of bioengineering, particularly relates to a method for producing pectinase. According to the method of producing pectinase through liquid fermentation, strain aspergillus niger LD-092 adopted by the method is obtained by carrying out Co60 ray mutagenesis treatment on an aspergillus niger original strain AJ213; and the preservation number of thestrain is CGMCC No.18559. The enzyme activity of pectinase produced by fermentation of the aspergillus niger LD-092 reaches 80000 to 85000U/mL; the optimum pH range of the pectinase obtained by fermentation is 2.5 to 4.0; the pectinase has the optimal action temperature range of 30 to 50 DEG C; the remarkable acid resistance is realized; the pectinase can be preserved for 36h in a PH range of 3.0to 4.0; and the enzyme activity is kept to be over 85%. The pectinase can be applied to the industrial production widely; the industrial application range of pectinase is extended substantially; and the application value of pectinase is improved.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a liquid fermentation method for producing pectinase. Background technique: [0002] Pectinase is a general term for a class of enzymes that can degrade pectin by hydrolysis and cleavage. It is a complex enzyme containing multiple components, mainly including protopectinase, pectin esterase, polygalacturonase and pectin lyase 4 categories. Pectinase was first seen in 1930 and was used to prepare wine and fruit juice. In the 1960s, scientists had a relatively clear understanding of the chemical properties of plant tissue components, so people were able to use pectinase more widely and effectively. [0003] According to reports, more than 40 kinds of microorganisms have been found to produce pectinase in nature, including Aspergillus, Penicillium, Fusarium, Kluyveromyces, and some facultative anaerobic bacteria. At present, pectinase is mostly produced by fer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/18C12N9/26C12N9/88C12R1/685
CPCC12N1/14C12N9/88C12N9/2408C12N9/18C12Y301/01011C12Y302/01015C12Y402/02002C12R2001/685C12N1/145
Inventor 郭庆文钱娟娟王兴吉曹世源张杰王克芬王金余
Owner SHANDONG LONGKETE ENZYME PREPARATION
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