CN111566199A - Luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of luminescent bacterium freeze-drying protective agent and freeze-dried powder in water quality comprehensive toxicity online monitoring - Google Patents
Luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of luminescent bacterium freeze-drying protective agent and freeze-dried powder in water quality comprehensive toxicity online monitoring Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
Abstract
A luminescent bacteria freeze-drying protective agent, freeze-dried powder and application thereof in water quality comprehensive toxicity on-line monitoring. Each L of the luminous bacteria freeze-drying protective agent consists of the following components: 100-200 g of skim milk, 20-120 g of cane sugar, 10-60 g of sodium chloride and the balance of water. The luminous bacteria freeze-dried powder is prepared by adding a luminous bacteria freeze-drying protective agent into a luminous bacteria liquid. The luminescent bacterium freeze-drying protective agent or luminescent bacterium freeze-drying powder can be used as a detection preparation for online water quality toxicity monitoring. The luminous bacteria freeze-dried powder is placed in a strain storage cup at 4 ℃ for refrigeration after recovery, can be used for an online toxicity monitoring instrument for 15d, can replace an imported freeze-drying protective agent, reduces the operation cost of reagent consumables of the online toxicity monitoring instrument, improves the continuous stable operation of the online toxicity monitoring instrument, and plays a role in biological toxicity early warning for water quality safety.
Description
Technical Field
The invention relates to the technical field of environmental biological monitoring, in particular to a luminous bacteria freeze-drying protective agent, freeze-dried powder and application thereof in water quality comprehensive toxicity online monitoring.
Background
The luminous bacteria are bacteria containing own fluorescent enzyme in vivo, can emit 'cold light' bacteria with blue-green visible light wavelength within 450-490 nm under normal physiological conditions, and are facultative aerobic chemoautotrophic bacteria.
There are several different methods for classifying luminescent bacteria, including traditional, systematic, numerical classification, etc. The types of luminescent bacteria found worldwide to date are roughly classified into the following types: bacillus, Vibrio and Shewanella. At present, several kinds of luminous bacteria commonly used at home and abroad are: photobacterium leiognathi, Photobacterium brightens, Vibrio fischeri, and Vibrio qinghaiensis. Vibrio fischeri is a standard strain adopted by ISO, and Vibrio qinghaiensis is a unique freshwater bacterium in China.
Contaminants inhibit bacterial luminescence primarily through two pathways: directly inhibiting the activity of the fluorescent enzyme participating in the luminescence reaction; the method can inhibit the metabolic process related to the luminous reaction in the cell, and can determine the toxicity of the toxic substance according to the change of luminous intensity when the toxic substance can destroy the respiration, growth and metabolism of the luminous bacteria. The main sensitive poisons are organic pollutants and heavy metals. The change of the luminous intensity is measured by a biological photometer, which is the basic basis why the luminous bacteria can be used to detect toxic and harmful substances in the environment.
As the research on the potential influence of the compound pollution of the aquatic ecosystem increasingly depends on toxicity experiments, a short-term, economic and rapid test system needs to be developed to replace the traditional long-term toxicity experiments. The luminous bacteria have been established in the last 70 th century, and the American Beckman company develops a bioluminescence photometer, namely a Microtox system, wherein the system uses freeze-dried powder of natural luminous bacteria, and can be tested after recovery, and the obtained result is similar to the 96-hour acute toxicity test result of fish. From this luminous bacteria toxicity detection technology, it is popular worldwide, and environmental monitoring and research institutions of various countries adopt this method to rapidly test the biological toxicity of environmental samples.
The water source water quality on-line monitoring and early warning system comprises a water source water quality change rule, early warning parameter selection, on-line instrument selection and system integration. The on-line water quality toxicity monitoring technology is the basis of water quality early warning. The on-line water toxicity monitoring technology is characterized in that the laboratory water toxicity analysis process, namely the processes of sampling, reagent preparation, pretreatment, reaction, calculation and the like, is completely automated and continuous. Thus, the requirement for the activity of the subject is more stringent, and an indicator organism with high activity needs to be provided automatically and continuously. Otherwise, the monitoring signal may be unstable, the monitoring result is unreliable, and the performance, accuracy and precision of the on-line monitoring instrument are directly affected. Therefore, the stability maintenance of the biological activity of the luminous bacteria is one of the core technologies of the water toxicity online monitor.
The Vibrio fischeri is usually applied to an online water toxicity monitoring instrument by forming a bacterial liquid, an immobilized biofilm or freeze-dried powder, and the freeze-dried powder is convenient to transport and use due to long shelf life and is the most common storage method. At present, the protective agent process and formula commonly used in the research on the luminescent bacteria freeze-dried powder protective agent at home are low in protective efficiency, the freeze-dried powder prepared from the freeze-dried powder protective agent is put in a strain storage cup for refrigeration at 4 ℃ after being recovered, and the luminous intensity of bacteria is rapidly reduced in the process of being used by an online toxicity monitoring instrument, so that the quantity of viable bacteria in the dry powder is small, the activity is unstable, and the service cycle is short. This results in unreliable detection results and short maintenance period (3-5 days) during the use of the online toxicity instrument. Most researches still stay in the laboratory stage, and the universality of online water quality toxicity instruments at home and abroad is not realized.
Disclosure of Invention
The invention provides a luminescent bacterium freeze-drying protective agent, freeze-dried powder and application thereof in water toxicity detection only aiming at the defects of the prior art.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
each L of the luminous bacteria freeze-drying protective agent consists of the following components: 100-200 g of skim milk, 20-120 g of cane sugar, 10-60 g of sodium chloride and the balance of water.
Preferably, each L of the luminous bacteria freeze-drying protective agent consists of the following components: 100-150 parts of skim milk, 20-60 parts of cane sugar, 10-30 parts of sodium chloride and the balance of water.
More preferably, each L of the luminescent bacteria lyoprotectant consists of the following components: 140g of skim milk, 60g of cane sugar, 30g of sodium chloride and the balance of water.
Preferably, the luminescent bacterium is vibrio fischeri.
The preparation method of the luminescent bacteria freeze-drying protective agent comprises the steps of firstly dissolving degreasing in water, then adding sodium chloride and cane sugar, stirring for 10-20 min, preferably 15min, and obtaining the luminescent bacteria freeze-drying protective agent after all the components are dissolved.
The luminous bacteria freeze-dried powder is prepared by adding the luminous bacteria freeze-dried protective agent into a bacterial liquid of luminous bacteria. Specifically, the luminescent bacterium freeze-drying protective agent and the luminescent bacterium liquid are suspended according to the volume ratio of 1:3 to 1: 6. Preferably, the volume ratio of the luminous bacteria freeze-drying protective agent to the luminous bacteria bacterial liquid is 1: 5.
The luminescent bacterium freeze-drying protective agent or the luminescent bacterium freeze-drying powder can be used as a detection preparation for online monitoring of comprehensive toxicity of water quality. The online water quality toxicity monitoring is to monitor the water quality by using a water quality online toxicity instrument.
The invention is further illustrated below:
the luminescent bacteria freeze-drying protective agent provided by the invention is prepared from skim milk, sucrose and sodium chloride. The skim milk is a basic protective agent, can promote the sublimation of a freeze-dried sample to form a heat-resistant framework to block heat conduction and heat radiation, is easy to obtain a homogeneous product, enlarges the mutual distance between cells, and protects thalli in a wrapping mode. When skim milk is used as a protective agent, the luminous efficiency of bacteria is still low, so that sugar alcohols are required to be added to improve the survival rate of the vibrio fischeri. The sucrose molecules contain hydroxyl groups, and can form hydrogen bonds with phosphate groups in thallus cell membrane phospholipid or thallus protein polar groups in the freezing and drying processes, so that a 'hydration layer' is formed on the cell surface, the structural and functional integrity of cell membranes and proteins is kept, and the cells can be quickly rehydrated or damaged cells can be repaired. In addition, the vibrio fischeri is a marine bacterium which has a certain requirement on the concentration of sodium chloride, and 3 percent of sodium chloride is beneficial to the luminescence of the vibrio fischeri.
The invention also aims to provide the application of the freeze-drying protective agent in preparing the vibrio fischeri freeze-dried powder, and the vibrio fischeri freeze-dried powder prepared by the freeze-drying protective agent is applied to a water quality comprehensive toxicity on-line analyzer, so that the operation and maintenance efficiency of the analyzer is improved, the cost is reduced, and the economic benefit is generated.
The specific application mode is as follows:
dissolving the luminous bacteria dry powder with 3% sodium chloride, mixing, inoculating into liquid culture medium, culturing at 25 deg.C 200rmp for 20 hr, centrifuging at 4 deg.C 10000rmp for 15min, discarding supernatant, and collecting thallus.
Preparing the luminescent bacterium freeze-drying protective agent; the preparation method comprises dissolving skimmed milk in water, adding sodium chloride and sucrose, and stirring for 10-20 min, preferably 15min, until all skim milk is dissolved.
Recovering the bacteria to obtain a bacterial liquid, uniformly suspending the bacterial liquid of the luminous bacteria and the freeze-drying protective agent of the luminous bacteria according to the volume ratio (V/V) of 1:2 to 1:7, and subpackaging the mixture into a small glass bottle; pre-freezing the subpackaged mixture at-80 ℃ for 4h, putting into a freeze dryer, and carrying out vacuum freeze drying for 24h to obtain the luminous bacteria freeze-dried powder.
The luminous bacteria freeze-drying protective agent effectively improves the viable bacteria amount of luminous bacteria freeze-dried powder, and can reach 1.5 x 109CFU/g, freeze-dried powder can be in 5minAnd recovering, wherein the photon quantity is relatively stable, and the method can be continuously used for about 15 days by a water quality comprehensive toxicity online analysis instrument. The method is beneficial to the production research of the luminescent bacterium freeze-dried powder and the application in environmental monitoring, and effectively improves the economic benefit.
In a word, the invention provides a freeze-drying protective agent capable of effectively improving the survival rate, the biological activity stability and the sensitivity of the vibrio fischeri freeze-dried powder, freeze-dried powder prepared by the freeze-drying protective agent is recovered and then placed in a strain storage cup at 4 ℃ for refrigeration, and the freeze-dried powder can be used for an online toxicity monitoring instrument for 15 days, can replace the freeze-drying protective agent imported from abroad, reduce the operation cost of reagent consumables of the online toxicity monitoring instrument at home and abroad, improve the continuous and stable operation of the online toxicity monitoring instrument at a national water station, and play a real role in biological toxicity early warning for the national water quality safety.
Drawings
FIG. 1 is a graph showing the results of the recovery time and luminescence intensity of lyophilized powder of luminescent bacteria;
FIG. 2 is a standard poison linear fit plot;
FIG. 3 is a graph showing the sensitivity change of the continuously used 15d dried fungus powder applied to an online toxicity monitoring instrument;
fig. 4 is a graph of the toxicity stability change of the reference water for the multi-batch lyophilized powder test.
Detailed Description
EXAMPLE 1 bacterial content of luminescent bacteria prepared with different proportions of protective agents
Culturing and manufacturing the vibrio fischeri (standard bacteria of luminous bacteria) according to the steps in the 'specific application mode' of the invention, finally adding the protective agent with the numerical value listed in the table 1, freezing and drying, storing at low temperature in a refrigerator at-80 ℃, recovering with 3% sodium chloride when taking out, performing gradient dilution by 10 times, performing plate culture counting to obtain the number of the vibrio fischeri contained in each different protective agent ratio, and preparing the protective agent as a control group by adopting a blank control that 14g of skim milk is dissolved in 100mL of sterile water.
TABLE 1
| Group of | Skim milk (g) | Sucrose (g) | Sodium chloride (g) | Bacterial content (CFU/g) |
| |
8 | 1 | 0.5 | 3.1*108CFU/ |
| Group | ||||
| 2 | 10 | 2 | 1 | 3.4*108CFU/ |
| Group | ||||
| 3 | 12 | 4 | 2 | 6.7*108CFU/ |
| Group | ||||
| 4 | 14 | 6 | 3 | 1.5*109CFU/ |
| Group | ||||
| 5 | 16 | 8 | 4 | 4.3*108CFU/ |
| Group | ||||
| 6 | 18 | 10 | 5 | 6.1*107CFU/ |
| Group | ||||
| 7 | 20 | 12 | 6 | 3.8*107CFU/ |
| Group | ||||
| 8 | 22 | 14 | 8 | 3.0*107CFU/ |
| Control group | ||||
| 14 | 2.9*107CFU/g |
From the results, the bacteria content of the vibrio fischeri freeze-dried powder prepared by using the freeze-drying protective agent is larger than that of the control group, and the group 4 is the optimal proportion of the protective agent.
EXAMPLE 2 determination of optimum addition amount of protective agent
The protective agent formula of group 3 in example 1 is used for preparing Vibrio fischeri freeze-dried powder, and the volume ratio of the freeze-drying protective agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 (Table 2) were added to the Vibrio fischeri bacterial liquid, the bacterial content of the lyophilized powder after adding different amounts of protective agent was measured, and the detailed procedure was the same as in example 1. Meanwhile, the bacteria content and the initial luminescence quantity of different volume ratios are measured by a bioluminescence tester.
TABLE 2
The results show that when the volume ratio of the freeze-drying protective agent is 1:5, the prepared freeze-dried powder has more viable bacteria after resuscitation, and the initial luminous value is highest, so that the sensitivity response to toxic substances in water is facilitated, and the service cycle of the freeze-drying protective agent on an instrument is prolonged.
EXAMPLE 3 determination of optimal Resuscitation time for protective Agents (Table 3)
TABLE 3
| Time for recovery of freeze-dried powder | Optimum dry powder brightness of protectant | Brightness of imported online cultured dry powder |
| 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 |
As can be seen from Table 3 and FIG. 1, the relative luminous intensity after 5min of resuscitation mainly reaches 1200 or more ten thousand, the luminous intensity reaches a stable state within 5-30 min, and the dry bacteria powder cultured on line at the inlet reaches 400 or more ten thousand after being cultured for 1 day by an on-line instrument. Compared with the recovery time and the photon brightness after recovery, the recovery time of the Vibrio fischeri freeze-dried powder prepared by the optimal protective agent is greatly shortened, and the brightness of the Vibrio fischeri freeze-dried powder is higher than that of the imported dry powder. The dry powder is convenient to use on-site on-line monitoring instruments, and the operation maintenance amount and the operation cost of the instruments are reduced.
Example 4 sensitivity testing of lyophilized powder
Measuring positive quality control liquid (zinc sulfate heptahydrate) with different concentrations to obtain corresponding luminescence inhibition rate of vibrio fischeri, wherein logarithm of positive quality control concentration is an abscissa, a graph is drawn by taking corresponding relative inhibition rate as an ordinate, and linear fitting is performed on data with the inhibition rate within a range of 20-80% by adopting a least square method to obtain fitting equation y which is alogx + b and R2 values, wherein x is the concentration of zinc sulfate heptahydrate, and y is the relative inhibition rate; and substituting the fitting equation with y being 50%, and calculating the corresponding x value.
The EC50 value of the prepared freeze-dried powder with positive quality control calculated by the curve in figure 2 is 5.05mg/L, and is superior to the EC50 value (20mg/L) of the dry powder with positive quality control entered abroad, which indicates that the dry powder is used on an on-line toxicity analyzer at home and abroad after being recovered, and when toxic substance pollution occurs in water, the analyzer firstly sends out an alarm instruction, and an emergency plan is quickly started.
Example 5 application to an on-line toxicity monitoring apparatus with continuous use of 15-day Dry powder sensitivity Change
The Vibrio fischeri freeze-dried powder prepared by the optimal freeze-drying protective agent formula is refrigerated and transported to a water station with a toxicity online analysis instrument on site, the Vibrio fischeri freeze-dried powder is placed into an instrument strain storage cup after hydration recovery, stirred and cultured for 10min, 20mg/L zinc sulfate heptahydrate positive quality control standard solution is prepared, testing is carried out, 2 times of testing are carried out every day, the average value of the two times is taken, the testing is carried out for 15 days continuously, and the testing result is shown in figure 3.
According to the positive quality control data applied to the on-line biological toxicity analyzer of the on-site water station, 20mg/L zinc sulfate heptahydrate is continuously tested for 15 days, the toxicity value is equal to or larger than 60%, the use requirement of the on-line biological toxicity analyzer adopting the luminous bacteria method is met, and the Vibrio fischeri freeze-dried powder prepared by adopting the optimal protective agent has high sensitivity and good stability.
Example 6 Multi-batch Freeze-dried powder test for toxicity stability Change in reference Water
Preparing 6 batches of Vibrio fischeri freeze-dried powder by using an optimal freeze-drying protective agent formula, dissolving and recovering 3% of sodium chloride for 10min, adding 2mL of 3% of sodium chloride into a detection pool, adding 3 parallel samples in each batch, adding 50uL of recovered samples in different batches, shaking up, putting into a toxicity detector, reading, manually calculating toxicity results, and showing toxicity value results of each batch in figure 4.
As can be seen from the graph 4, the toxicity values of the first-class water tested by 6 batches of freeze-dried powder prepared by the optimal protective agent fluctuate within +/-5%, so that the use requirements of toxicity instruments are met.
Claims (10)
1. The photobacteria freeze-drying protective agent is characterized in that each L of the photobacteria freeze-drying protective agent consists of the following components: 100-200 g of skim milk, 20-120 g of cane sugar, 10-60 g of sodium chloride and the balance of water.
2. The photobacteria lyoprotectant of claim 1 wherein each L of said photobacteria lyoprotectant consists of: 100-150 parts of skim milk, 20-60 parts of cane sugar, 10-30 parts of sodium chloride and the balance of water.
3. The photobacteria lyoprotectant of claim 2, wherein each L of said photobacteria lyoprotectant consists of: 140g of skim milk, 60g of cane sugar, 30g of sodium chloride and the balance of water.
4. The photoprotective bacteria cryoprotectant of claim 1, wherein the photobacterium is vibrio fischeri.
5. The method for preparing the freeze-drying protective agent for the luminous bacteria as claimed in any one of claims 1 to 4, wherein the method comprises the steps of firstly dissolving skim milk in water, then adding sodium chloride and cane sugar, and stirring for 10-20 min until all the components are dissolved, thereby obtaining the freeze-drying protective agent for the luminous bacteria.
6. A luminescent bacterium freeze-dried powder, which is characterized in that the luminescent bacterium freeze-dried powder is prepared by adding the luminescent bacterium freeze-drying protective agent of any one of claims 1 to 4 into a bacterial liquid of luminescent bacteria.
7. The lyophilized powder of luminescent bacteria of claim 6, which is prepared by suspending the lyophilized protectant of luminescent bacteria of any one of claims 1 to 4 and a bacterial solution of luminescent bacteria according to a volume ratio of 1:3 to 1: 6.
8. The lyophilized powder of luminescent bacteria of claim 7, which is prepared by suspending the lyophilized protectant of luminescent bacteria of any one of claims 1 to 4 and bacterial liquid of luminescent bacteria according to a volume ratio of 1: 5.
9. Use of the luminescent bacterium freeze-drying protective agent according to any one of claims 1 to 4 or the luminescent bacterium freeze-drying powder according to any one of claims 6 to 8 as a detection preparation in on-line monitoring of water quality comprehensive toxicity.
10. The use of claim 9, wherein the online monitoring of water quality comprehensive toxicity is the toxicity monitoring of water quality by using a water quality online toxicity instrument.
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| CN112358988B (en) * | 2020-11-10 | 2022-09-20 | 广西壮族自治区农业科学院 | Lactobacillus plantarum LDVS008 strain and application thereof |
| CN115786204A (en) * | 2022-12-01 | 2023-03-14 | 中国水产科学研究院黄海水产研究所 | Vacuum freeze-drying protective agent suitable for vibrios and freeze-drying method |
| CN115786204B (en) * | 2022-12-01 | 2023-08-15 | 中国水产科学研究院黄海水产研究所 | Vacuum freeze-drying protective agent and freeze-drying method suitable for vibrio |
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