In-situ enriching method and screening method of pullulanase indigenous strains

An in situ enrichment, pullulanase technology, applied in biochemical equipment and methods, determination/inspection of bacteria, microorganisms, etc. Easy to operate, clear purpose, reliable effect

Active Publication Date: 2018-01-05

HENAN UNIV OF SCI & TECH

2 Cites 0 Cited by

AI-Extracted Technical Summary

Problems solved by technology

The traditional enrichment method of pullulanase-producing indigenous strains can generally isolate a small number of strains, but the existing problems are also obvious. These deficiencies are concentrated in: (1) The isolation method is too programmed and single, and the isolated strains The repetition rate is extremely high, the diversity is poor, and there are few innovative research results; (2) Enrichment culture for a certain period of time before isolation culture is obviously more targeted than no enrichment, and the effect is slightly better, but in vitro enrichment also has The enriched environment of the sample still has natural defects that are fundame...

Abstract

The invention relates to an in-situ enriching method and a screening method of pullulanase indigenous strains. The in-situ enriching method comprises the following steps: burying a matrix to which amylopectin is adhered into soil; enriching; then taking out the matrix; and collecting soil on the surface of the matrix to obtain the strain-enriched soil. The screening method comprises the followingsteps: uniformly mixing the strain-enriched soil and sterile water; centrifuging; performing gradient dilution on an upper suspension; coating a flat separating plate with the gradient diluting solution; culturing; and then identifying. The enriching method has the major characteristic that amylopectin is used as a primary inducing substrate for in-situ enriching the pullulanase indigenous strainsin soil, so that the quantity of pullulanase indigenous populations can be increased in an original ecological environment, and particularly some weak populations with pullulanase activity can be certainly proliferated, and as a result, the detection rate of the target strains can be increased.

Application Domain

BacteriaMicrobiological testing/measurement +1

Technology Topic

PopulationPullulanase activity +7

Image

Examples

Experimental program(2)
Comparison scheme(2)
Effect test(2)

Example Embodiment

[0026] Example 1

[0027] The in situ enrichment method of the indigenous pullulanase-producing strain in this embodiment includes:

[0028] A. Substrate handling

[0029] Take 5g of sterilized absorbent cotton in the ultra-clean workbench and put it into a sterilized beaker containing 15g of pullulan and 50mL of sterile water, and turn the absorbent cotton to make the pullulan infiltrate and adhere to the absorbent cotton as much as possible. Absorbent cotton blocks are kept sterile.

[0030] B. In-situ enrichment in soil

[0031] Dig a 5cm deep pit in the sampling environment soil (the wasteland soil on both sides of the Luohe River in the suburbs of Luoyang), sprinkle 100mL sterile water in the pit, take out the absorbent cotton block processed in step A, and arrange the cotton block into a rectangle ( subject to no breakage), lay flat on the bottom of the hole, and cover the absorbent cotton with the in-situ soil when digging the hole. After 8 days of in-situ enrichment, the absorbent cotton block was carefully taken out, and the excess soil was shaken off, and the soil on the surface of the absorbent cotton was collected in an ultra-clean workbench as a soil sample for dilution plate culture.

[0032] In the present embodiment, the method for screening indigenous pullulanase-producing strains includes:

[0033] Weigh 5g of the above-mentioned enriched soil into an Erlenmeyer flask containing 45mL sterile water and a small amount of glass beads, shake at 150r/min for 30min at room temperature, let stand for 5min, take the upper layer of bacterial suspension for 10-fold gradient dilution, take 10 -4 、10 -5 、10 -6 Gradient dilutions were applied to separate plates (with pullulan as a substrate) for culture. Incubate at 30°C for 48 hours. Add Lushi's iodine solution dropwise to detect whether the colonies have hydrolysis circles, and those with hydrolysis circles are strains capable of producing pullulanase.

[0034] The formula of the medium in the separation plate is: glutinous rice starch 10-12g/L, peptone 8g/L, yeast powder 2g/L, MgSO 4 ·7H 2 O 0.5g/L, K 2 HPO 4 1g/L, agar 18g/L, pH 7.0.

Example Embodiment

[0035] Example 2

[0036] The in situ enrichment method of the indigenous pullulanase-producing strain in this embodiment includes:

[0037] A. Substrate handling

[0038] Take 5g of sterilized absorbent cotton in the ultra-clean workbench and put it into a sterilized beaker containing 20g of pullulan and 50mL of sterile water, and turn the absorbent cotton to make the pullulan infiltrate and adhere to the absorbent cotton as much as possible. Absorbent cotton blocks are kept sterile.

[0039] B. In-situ enrichment in soil

[0040]Dig a 10cm deep pit in the sampling environment soil (corn field soil in the suburbs of Luoyang), sprinkle 150mL sterile water in the pit, take out the absorbent cotton block processed in step A, and arrange the cotton block into a rectangle ( subject to no breakage), lay flat on the bottom of the hole, and cover the absorbent cotton with the in-situ soil when digging the hole. After 12 days of in-situ enrichment, the absorbent cotton block was carefully taken out, and the excess soil was shaken off. Then, the soil on the surface of the absorbent cotton was collected in an ultra-clean workbench as a soil sample for dilution plate culture.

[0041] In the present embodiment, the method for screening indigenous pullulanase-producing strains includes:

[0042] Weigh 5g of enriched soil and put it into an Erlenmeyer flask filled with 45mL sterile water and a small amount of glass beads, shake at 180r/min for 30min at room temperature, let it stand for 10min, take the upper bacterial suspension for 10-fold gradient dilution, and take 10 -4 、10 -5 、10 -6 Gradient dilutions were applied to separate plates (with pullulan as a substrate) for culture. Incubate at 30°C for 48 hours. Add Lushi's iodine solution dropwise to detect whether the colonies have hydrolysis circles, and those with hydrolysis circles are strains capable of producing pullulanase.

[0043] The formula of the medium in the separation plate is: glutinous rice starch 10-12g/L, peptone 8g/L, yeast powder 2g/L, MgSO 4 ·7H 2 O 0.5g/L, K 2 HPO 4 1g/L, agar 18g/L, pH 7.0.

PUM

Description & Claims & Application Information

We can also present the details of the Description, Claims and Application information to help users get a comprehensive understanding of the technical details of the patent, such as background art, summary of invention, brief description of drawings, description of embodiments, and other original content. On the other hand, users can also determine the specific scope of protection of the technology through the list of claims; as well as understand the changes in the life cycle of the technology with the presentation of the patent timeline. Login to view more.

Classification and recommendation of technical efficacy words

clear purpose
Reliable effect

In-situ enriching method and screening method of pullulanase indigenous strains

An in situ enrichment, pullulanase technology, applied in biochemical equipment and methods, determination/inspection of bacteria, microorganisms, etc. Easy to operate, clear purpose, reliable effect

AI-Extracted Technical Summary

Problems solved by technology

Abstract

Image

Examples

Example Embodiment

Example Embodiment

PUM

Description & Claims & Application Information

Similar technology patents

Separation method of soil microorganisms capable of inhibiting wheat ear rot pathogenic microorganisms

Bead pull type driver

Environment-friendly efficient cleaning agent for removing antibiotics and preparation method and application thereof

Mechanical arm and operation method thereof for super-resolution imaging

Plant drink and making method thereof

Preparation method of novel disinfectant

Vehicle-mounted mobile phone fixing device for back row passenger

Vacuum bottle with dripless housing

Classification and recommendation of technical efficacy words

Plant drink and making method thereof

Separation method of soil microorganisms capable of inhibiting wheat ear rot pathogenic microorganisms

Environment-friendly efficient cleaning agent for removing antibiotics and preparation method and application thereof

High-temperature nano-grade anti-carburizing material and coating, and application thereof

Electric storage device

Electrically conductive paste and multilayer ceramic substrate

Fuzzy control-based continuous commutation failure prevention control method for high-voltage direct-current transmission system

Application of beta-carotene in preparation of pig sperm cryoprotectant