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Compound biochemical resuscitation promoting factor composition and applications thereof

A technology of composition and factors, applied in the field of microorganisms

Inactive Publication Date: 2019-03-29
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, improving the cultivability of difficult-to-cultivate microorganisms in the environment by adding biochemical recovery factors is a screening method with great potential for straw-degrading microorganisms. Reports on degrading microorganisms

Method used

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  • Compound biochemical resuscitation promoting factor composition and applications thereof
  • Compound biochemical resuscitation promoting factor composition and applications thereof
  • Compound biochemical resuscitation promoting factor composition and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] This example specifically illustrates the preparation method of the composite biochemical recovery factor composition.

[0030] The specific steps for obtaining the two recombinant proteins are as follows:

[0031] 1) Construction of recombinant bacteria expressing cell biological recovery factors

[0032] The coding genes of Rpf protein MLUT_RS18675 and CHU_0099 were connected to the multiple cloning site of pET28a vector plasmid by one-step cloning method to obtain pET28a-MLUT_RS18675 and pET28a-CHU_0099 recombinant vector plasmids respectively; the recombinant vector plasmids were respectively transformed into E. coli BL21 (DE3) to construct the corresponding recombinant strain E. coli BL21(DE3) pET28a-MLUT_RS18675 and E. coli BL21(DE3) pET28a-CHU_0099. Select a single colony to extract the plasmid for sequencing to confirm that the expression vector is constructed correctly.

[0033] 2) Induced expression of cell biological recovery factors

[0034] Streak...

Embodiment 2

[0043] This example specifically illustrates the application effect of different concentrations of compound biochemical recovery factor compositions in the screening of wheat straw degrading microorganisms in biogas slurry

[0044] The specific operation steps are as follows:

[0045] 1) Screening of wheat straw degrading microbial flora

[0046] Weigh 50 mL of PCS medium components and dilute to 45 mL with pure water. Dry the wheat straw crushed through a 20-mesh sieve at 60°C to constant weight for use.

[0047] Add 1.0 wt% wheat straw (0.5 g) as a substrate, sterilize at 121 °C for 15 minutes, and cool to room temperature for later use. Pass high-purity nitrogen gas (purity 99.999%) into the PCS medium for 2 minutes, then immediately insert 5 mL of fresh biogas slurry as the inoculum, add different concentrations of complex biochemical recovery factor compositions, and continue to pass high-purity nitrogen gas for 2 minutes , closed with a rubber stopper with an aluminum...

Embodiment 3

[0058] This example specifically illustrates the application effect of different compound biochemical recovery factor composition additions in the screening of wheat straw degrading bacteria in different environmental samples

[0059] The specific operation steps are as follows:

[0060] 1) Screening of wheat straw degrading microbial flora

[0061] Weigh 50 mL of PCS medium components and dilute to 45 mL with pure water. Dry the wheat straw crushed through a 20-mesh sieve at 60°C to constant weight for use.

[0062] Add 1.0 wt% wheat straw (0.5 g) as a substrate, sterilize at 121 °C for 15 minutes, and cool to room temperature for later use. Pass high-purity nitrogen gas (purity 99.999%) into the PCS medium for 2 minutes, then immediately insert 5 g of high-temperature compost soil, dairy cow rumen juice and forest soil as inoculum, add different concentrations of complex biochemical recovery factor compositions, and continue Infuse high-purity nitrogen gas for 2 minutes, ...

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Abstract

The invention provides a compound biochemical resuscitation promoting factor composition and applications thereof. The composition components include two Rpf proteins and sodium pyruvate. The two Rpfproteins include a recombinant protein MLUT RS 18675 derived from micrococcus luteus, and a recombinant protein CHU 0099 derived from cythophaga hutchinsonii. The recombinant protein is obtained by heterologous expression of escherichia coli and purification. The straw is added as a substrate and an inoculum into a screening culture medium, the compound biochemical resuscitation promoting factor composition is added at the same time, and the obtained cultured microbial solution is screened straw degradation microbial flora which can be used in the straw degradation process. With the composition and the application thereof provided by the invention, the proportion of viable but not culturable microorganisms can be improved through the activation effect of biological resuscitation promotingfactors and the protection mechanism of the sodium pyruvate, so that high-activity microbial flora can be screened and used for the fields of ecological agriculture, environmental management and energy chemical industry.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a compound biochemical recovery factor composition and its application. Background technique [0002] When the culture conditions (such as temperature, pH, osmotic pressure, oxygen concentration, and nutrient concentration) change, microorganisms will enter a "dormant" state to cope with changes in environmental conditions. This state is called a living but non-culturable state (Viable but not culturable, VBNC), also known as hard-to-cultivate microorganisms. Microorganisms entering the VBNC state cannot be cultured artificially. Studies based on quantitative analysis of 16S rRNA have shown that 90% to 99% of microorganisms in most environments are uncultivable or difficult to cultivate. Microorganisms in the VBNC state can be revived under certain conditions. There are mainly two kinds of revival factors reported so far, namely biological revival factor protein and chemical reviv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/22C12N1/38
CPCC12N1/22C12N1/38
Inventor 孔祥平叶小梅王宇欣朱宁杜静奚永兰靳红梅张敏
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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