Method for detecting form and metabolic activity of filamentous fungi

A technology of metabolic activity and detection method, which is applied in the field of detection of mycelia morphology and metabolic activity, can solve the problems affecting the synthesis of cell metabolites, and achieve the effect of convenient and fast detection, simple operation and few steps

Active Publication Date: 2014-09-10
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem with spheroid growth is that the mycelium inside the spheroid will autolyse due to nutrient deficiency, which will affect cell metabolism and product synthesis

Method used

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  • Method for detecting form and metabolic activity of filamentous fungi
  • Method for detecting form and metabolic activity of filamentous fungi
  • Method for detecting form and metabolic activity of filamentous fungi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] With Penicillium chrysogenum as the starting strain, follow the steps below:

[0030] As the starting bacteria, follow the steps below:

[0031] A. First prepare the seed solution in a shake flask, then inoculate the seed solution in a 250mL shake flask with 30mL fermentation medium, the medium components are corn steep liquor (50% dry weight) 46.5g / L, lactose 130g / L, calcium carbonate 10g / L, potassium dihydrogen phosphate 4 g / L, ammonium sulfate 4.5 g / L, sodium sulfate 1.5 g / L, corn oil 4 drops, ammonium phenylacetate (10%) 2 drops, at pH 5.8 , under the condition of 25°C, cultivate at a stirring speed of 220r / min;

[0032] B. After culturing for 72 hours, absorb 100 μL of fermentation broth, filter and wash with a 300-mesh sieve, and remove the interference of colored impurities in the fermentation broth;

[0033] C. After filtering, put the hyphae in a small centrifuge tube, add physiological saline to dilute to 1mL, draw 100μL and spread it evenly on the glass sl...

Embodiment 2

[0041] Using a strain of Aspergillus niger preserved in the laboratory as the starting bacterium, follow the steps below:

[0042] A. Put 50 mL of fermentation medium into a 250 mL Erlenmeyer flask, and insert 2 mL of spore suspension (5×107 spores). The medium components are: glucose 100g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium carbonate 50g / L. Cultivate at 30°C with a stirring speed of 180r / min;

[0043] B. After culturing for 48 hours, draw 100 μL of fermentation broth, filter and wash with a 300-mesh sieve, and remove the interference of colored impurities in the fermentation broth;

[0044] C. After filtering, place the mycelium in a small centrifuge tube, add physiological saline to dilute to 1 mL, draw 100 μL and evenly spread it on the glass slide;

[0045] D. Add 200 μL of methylene blue staining solution dropwise, and stain for 10 minutes;

[0046] The preparation method of the methylene blue staining solut...

Embodiment 3

[0058] A strain of Rhizopus oryzae preserved in the laboratory is used as the starting bacterium, and the following steps are followed successively:

[0059] A, first carry out the preparation of seed liquid in shake flask, then seed liquid is inoculated in the 3.7L fermentor that 2.5L fermentation medium is housed, fermentation temperature 37 degrees Celsius, stirring speed 200rpm, medium composition is: glucose 150g / L, sulfuric acid 3g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.75g / L, zinc sulfate 0.2g / L, calcium carbonate 50g / L.

[0060] B. After culturing for 48 hours, draw 100 μL of fermentation broth, filter and wash with a 300-mesh sieve, and remove the interference of colored impurities in the fermentation broth;

[0061] C. After filtering, put the hyphae in a small centrifuge tube, add physiological saline to dilute to 1mL, draw 100μL and spread it evenly on the glass slide ( image 3 a);

[0062] D. Add 200 μL of modified methylen...

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Abstract

The invention discloses a method for detecting form and metabolic activity of filamentous fungi. The method comprises the following steps: (1) inoculating filamentous fungi and carrying out fermenting culture; (2) taking 100-150muL of fermentation liquor when culturing to 48-72 hours, filtering by using a mesh sieve of 300-400 meshes and rinsing to remove interference of colored impurities in the fermentation liquor; (3) putting filtered hypha into a small centrifuge tube, adding normal saline to dilute into 1-2mL, and sucking up 100-150muL of solution to evenly coat on a glass slide; (4) dropwise adding 200-250muL of improved methylene blue staining fluid and staining for 10-15 minutes; (5) dropwise adding 100-150muL of improved carbolic acid fuchsin staining fluid and staining for 5-8 minutes; (6) rinsing the slide by distilled water, and observing by a cover plate. Observation of morphology is more convenient by staining means; meanwhile, the activity difference of thalli growing in different areas at one stage can be obviously distinguished, and the physiological metabolic activity is broadly understood.

Description

technical field [0001] The invention relates to a method for real-time monitoring of the fermentation process of filamentous bacteria, in particular to a method for detecting the mycelium morphology and metabolic activity of filamentous bacteria. Background technique [0002] Humans have a long history of using fungi, but it was not until after World War I, with the rise of liquid fermentation technology, that filamentous fungal fermentation gradually flourished in the industrial field. Until now, filamentous fungi have been widely used in the fermentation industry for antibiotics (penicillin, cephalosporin, etc.), organic acids (citric acid, gluconic acid, fumaric acid, etc.) and enzyme preparations (amylase, pectinase, cellulase, etc.) etc.), these products are in great demand in the fields of medicine, food and other industries. [0003] During the fermentation process, the morphological changes of filamentous bacteria are complex, showing completely different morphologi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04
Inventor 郑之明王鹏胡以华顾有林王辉王丽刘红霞
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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