Method for detecting optimal density and time for bacterium suppression of chitosan

Abstract

The invention discloses a method for detecting optimal density and time for the bacterium suppression of chitosan, comprising the steps of: providing a plurality of solutions, the chitosan densities of which are in graded distribution, wherein each solution includes suppressed bacteria with the same density and a culture medium; standing all the solutions for culturing; in the culturing process, selecting a plurality of time points to measure the OD600 value of each solution and compute the suppression rate of the chitosan so as to obtain optimal density and time for the bacterium suppression of the chitosan. The invention only needs the solutions, the chitosan densities of which are in graded distribution; the suppressed bacteria in the solutions have the same density; after standing and culturing, the bacterium suppression rates of the solutions are measured at the plurality of time points; and the optimal density and time for the bacterium suppression of the chitosan can be obtained through a regression curve or an approximate method.

Description

technical field

[0001] The invention relates to the technical fields of plant protection and microbiology, in particular to a method for detecting the optimal concentration and time of chitosan inhibiting bacteria. Background technique

[0002] Chitosan is the product of chitin after deacetylation treatment. Chitin with a degree of deacetylation greater than 55% is usually called chitosan. Its scientific name is polyglucosamine, and its chemical name is (1,4)-2. -Amino-2-deoxy-β-D-glucose is widely found in the shells of shrimps, crabs and other animals and in the cell walls of algae and fungi.

[0003] Chitosan is a natural substance with a wide antibacterial spectrum, which has different degrees of inhibitory effects on bacteria, fungi and viruses. As early as the late 1880s, scientists discovered that chitosan had an inhibitory effect on Fusarium and the like.

[0004] Ralstonia wilt is a devastating bacterial disease that infects a variety of plants all over the world....

Examples

Embodiment 1

[0020] The preparation of embodiment 1 chitosan

[0021] Chitosan powder (purchased from Sigma company) was dissolved in 1% acetic acid by volume percentage to prepare a chitosan solution with a concentration of 10 mg / mL, adjust the pH to 5.6, and sterilize with an autoclave at 121 ° C for 20 min ,stand-by. At the same time, the acetic acid with a pH of 5.6 and a volume percentage of 1% was sterilized for use.

Embodiment 2

[0022] The preparation of embodiment 2 solanacear bacteria suspension

[0023] The solanacearum Rs-91 used in the present invention is provided by Fujian Academy of Agricultural Sciences. Pick on TZC medium (peptone 10.0g; hydrolyzed casein 1.0g; glucose 5.0g; TTC (2,3,5-triphenyltetrazolium chloride) 0.05g; water 1000mL; pH 7.0-7.2) The grown single colony of R. solanacearum was inoculated in LB medium (yeast extract 5g; peptone 10g; sodium chloride 5g; water 1000mL; pH 7.0-7.2), 30°C, 170r / min shaking culture for 24h.

Embodiment 3

[0024] The mixing of embodiment 3 chitosan and solanacearum bacterium suspension

[0025] Prepare 6 sterilized centrifuge tubes, add 10 μL Ralstonia solanacearum suspension (OD 600 =0.3) and 965 μL of LB medium. At the same time, add 5 μL of 10 mg / mL chitosan solution and 20 μL of prepared acetic acid solution to tube 1, add 10 μL of chitosan solution and 15 μL of acetic acid solution to tube 2, add 15 μL of chitosan solution and 10 μL of acetic acid solution to tube 3 20 μL of chitosan solution and 5 μL of acetic acid solution were added to No. 4 tube, only 25 μL of chitosan solution was added to No. 5 tube, and only 25 μL of acetic acid solution was added to No. 6 tube. Finally, in No. 1-5 mixed cultures, the final concentrations of chitosan reached 0.05, 0.10, 0.15, 0.20 and 0.25 mg / mL respectively, while No. 6 tube was used as a control without adding chitosan. 100 μL of each was added to a 96-well culture plate (in order to reduce errors, no culture was added to the edg...

technical field

[0001] The invention relates to the technical fields of plant protection and microbiology, in particular to a method for detecting the optimal concentration and time of chitosan inhibiting bacteria. Background technique

[0002] Chitosan is the product of chitin after deacetylation treatment. Chitin with a degree of deacetylation greater than 55% is usually called chitosan. Its scientific name is polyglucosamine, and its chemical name is (1,4)-2. -Amino-2-deoxy-β-D-glucose is widely found in the shells of shrimps, crabs and other animals and in the cell walls of algae and fungi.

[0003] Chitosan is a natural substance with a wide antibacterial spectrum, which has different degrees of inhibitory effects on bacteria, fungi and viruses. As early as the late 1880s, scientists discovered that chitosan had an inhibitory effect on Fusarium and the like.

[0004] Ralstonia wilt is a devastating bacterial disease that infects a variety of plants all over the world....

Examples

Embodiment 1

[0020] The preparation of embodiment 1 chitosan

[0021] Chitosan powder (purchased from Sigma company) was dissolved in 1% acetic acid by volume percentage to prepare a chitosan solution with a concentration of 10 mg / mL, adjust the pH to 5.6, and sterilize with an autoclave at 121 ° C for 20 min ,stand-by. At the same time, the acetic acid with a pH of 5.6 and a volume percentage of 1% was sterilized for use.

Embodiment 2

[0022] The preparation of embodiment 2 solanacear bacteria suspension

[0023] The solanacearum Rs-91 used in the present invention is provided by Fujian Academy of Agricultural Sciences. Pick on TZC medium (peptone 10.0g; hydrolyzed casein 1.0g; glucose 5.0g; TTC (2,3,5-triphenyltetrazolium chloride) 0.05g; water 1000mL; pH 7.0-7.2) The grown single colony of R. solanacearum was inoculated in LB medium (yeast extract 5g; peptone 10g; sodium chloride 5g; water 1000mL; pH 7.0-7.2), 30°C, 170r / min shaking culture for 24h.

Embodiment 3

[0024] The mixing of embodiment 3 chitosan and solanacearum bacterium suspension

[0025] Prepare 6 sterilized centrifuge tubes, add 10 μL Ralstonia solanacearum suspension (OD 600 =0.3) and 965 μL of LB medium. At the same time, add 5 μL of 10 mg / mL chitosan solution and 20 μL of prepared acetic acid solution to tube 1, add 10 μL of chitosan solution and 15 μL of acetic acid solution to tube 2, add 15 μL of chitosan solution and 10 μL of acetic acid solution to tube 3 20 μL of chitosan solution and 5 μL of acetic acid solution were added to No. 4 tube, only 25 μL of chitosan solution was added to No. 5 tube, and only 25 μL of acetic acid solution was added to No. 6 tube. Finally, in No. 1-5 mixed cultures, the final concentrations of chitosan reached 0.05, 0.10, 0.15, 0.20 and 0.25 mg / mL respectively, while No. 6 tube was used as a control without adding chitosan. 100 μL of each was added to a 96-well culture plate (in order to reduce errors, no culture was added to the edg...

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