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A luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of the freeze-dried powder in on-line monitoring of comprehensive toxicity of water quality

A technology of freeze-drying protective agent and luminescent bacteria, which is applied to bacteria, test water, measuring devices, etc., can solve the problems of low formula protection efficiency, unreliable test results, short maintenance period, etc., to improve the survival rate and stabilize the biological activity. performance, reduce the operating cost of reagent consumables, and improve the effect of continuous and stable operation

Inactive Publication Date: 2020-08-21
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the protective agent process and formulation protection efficiency commonly used in the research of luminescent bacteria freeze-dried powder protective agent in China is low. The freeze-dried powder prepared from the freeze-dried powder protective agent is placed in a 4°C strain storage cup for refrigeration after recovery. , the luminous intensity of the bacteria drops rapidly during the process of being used for the on-line toxicity monitoring instrument, resulting in a small number of viable bacteria in the dry powder, unstable activity, and short service life
This has resulted in unreliable test results during the use of online toxicity instruments, and the maintenance cycle is too short (3-5 days)
Most of the research is still in the laboratory stage, and the general use of online water quality toxicity instruments at home and abroad has not yet been realized.

Method used

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  • A luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of the freeze-dried powder in on-line monitoring of comprehensive toxicity of water quality
  • A luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of the freeze-dried powder in on-line monitoring of comprehensive toxicity of water quality
  • A luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of the freeze-dried powder in on-line monitoring of comprehensive toxicity of water quality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The bacteria content of the luminescent bacteria prepared by the protective agent of different proportions of embodiment 1

[0031] Cultivate Vibrio fischeri (standard bacterium of luminous bacteria) according to the steps in the "specific application mode" of the present invention, and finally add the protective agent with the values ​​listed in Table 1, freeze-dry, and store at a low temperature in a -80°C refrigerator , resuscitated with 3% sodium chloride when taken out, carried out 10-fold gradient dilution, counted by plate culture, and obtained the number of Vibrio fischeri contained in each different protective agent ratio, and the blank control used 14g of skim milk dissolved in 100mL Protective agent was prepared in sterile water as the control group.

[0032] Table 1

[0033] group Skimmed milk (g) Sucrose (g) Sodium chloride (g) Bacteria content (CFU / g) group 1 8 1 0.5 3.1*10 8 CFU / g

[0034] It can be seen from the results tha...

Embodiment 2

[0035] Determination of the optimal addition amount of embodiment 2 protective agent

[0036] The protective agent formula of Group 3 in Example 1 is used for the production of Vibrio fischeri freeze-dried powder, respectively according to the volume ratio of freeze-dried protective agent 1:1, 1:2, 1:3, 1:4, 1: 5, 1:6, 1:7 (Table 2) were added to the Vibrio fischeri bacteria liquid, and the bacteria content of the freeze-dried powder was measured after adding different amounts of protective agents. The detailed operation steps were the same as in Example 1. At the same time, the bioluminescence tester was used to measure the bacteria content and initial luminescence in different volume ratios.

[0037] Table 2

[0038]

[0039]

[0040] It can be seen from the results that when the volume ratio of the freeze-dried protective agent is 1:5, the prepared freeze-dried powder has a large amount of viable bacteria after resuscitation, and the initial luminescence value is the...

Embodiment 3

[0041] The determination (table 3) of the optimal recovery time of embodiment 3 protective agent

[0042] table 3

[0043] Freeze-dried powder recovery time Best conditioner dry powder brightness Imported online culture dry powder brightness 1min 6270042 0 2min 8001385 0 3min 9268910 0 4min 11009371 1 5min 12105973 3 10min 12369710 20 20min 12200934 236 30min 11983175 1142 60min 11826740 4967 120min 11760143 15672 1d 11056213 4468207

[0044] Through Table 3 and figure 1 It can be seen that the relative luminous intensity mainly reaches more than 12 million after 5 minutes of resuscitation, and the luminous intensity reaches a steady state within 5-30 minutes. The imported dry powder of online culture needs to be cultivated on-line for 1 day before reaching more than 4 million. Through the comparison of the recovery time and the photon brightness after recovery, it can be concluded tha...

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Abstract

The invention discloses a luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of the freeze-dried powder in on-line monitoring of comprehensive toxicity of waterquality. Each L of the luminous bacterium freeze-drying protective agent is composed of the following components: 100-200g of skim milk, 20-120g of cane sugar, 10-60g of sodium chloride and the balance of water. The luminescent bacterium freeze-dried powder is prepared by adding a luminescent bacterium freeze-drying protective agent into a luminescent bacterium solution. The luminescent bacteriumfreeze-drying protective agent or the luminescent bacterium freeze-dried powder can be used as a detection preparation for on-line water quality toxicity monitoring. The resuscitated luminous bacterium freeze-dried powder is refrigerated in a strain storage cup at 4 DEG C, can be used for an online toxicity monitoring instrument for 15 days, can replace an imported freeze-drying protective agent,reduces the operation cost of reagent consumables of the online toxicity monitoring instrument, improves the continuous and stable operation of the online toxicity monitoring instrument, and plays a role in biotoxicity early warning for the safety of water quality.

Description

technical field [0001] The invention relates to the technical field of environmental biological monitoring, in particular to a luminescent bacteria freeze-dried protective agent, freeze-dried powder and its application in online monitoring of comprehensive toxicity of water quality. Background technique [0002] Luminescent bacteria are bacteria that contain luciferase in their body. Under normal physiological conditions, they can emit blue-green visible "cold light" bacteria with wavelengths in the range of 450-490nm. They are facultative aerobic autotrophic bacteria. [0003] There are several different classification methods for luminescent bacteria, including traditional, systematic, and numerical classifications. So far, the species of luminescent bacteria found in the world can be roughly divided into the following species: Bacillus, Vibrio and Shewanella. At present, several kinds of luminescent bacteria commonly used at home and abroad are: Photobacterium chinensis,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/04C12N1/20C12Q1/02G01N33/18C12R1/63
CPCC12N1/04C12N1/20C12Q1/025G01N33/1866
Inventor 方俊张琳琳刘刚蒋红梅刘芬胡超
Owner HUNAN AGRICULTURAL UNIV
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