LU101961B1 - Freeze-dried protecting agent and freeze-dried powder of the luminescent bacteria and their application in water quality comprehensive toxicity on-line monitoring - Google Patents

Freeze-dried protecting agent and freeze-dried powder of the luminescent bacteria and their application in water quality comprehensive toxicity on-line monitoring Download PDF

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LU101961B1
LU101961B1 LU101961A LU101961A LU101961B1 LU 101961 B1 LU101961 B1 LU 101961B1 LU 101961 A LU101961 A LU 101961A LU 101961 A LU101961 A LU 101961A LU 101961 B1 LU101961 B1 LU 101961B1
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freeze
dried
luminescent bacteria
protecting agent
bacteria
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LU101961A
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German (de)
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Jun Fang
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Univ Hunan Agricultural
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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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Abstract

A freeze-dried protecting agent of luminescent bacteria, freeze-dried powder of the luminescent bacteria and their application in water quality comprehensive toxicity on-line monitoring are provided. The freeze-dried protecting agent of the luminescent bacteria per liter consists of: 100-200 g of skimmed milk, 20-120 g of sucrose, 10-60 g of sodium chloride, and water. The freeze-dried powder of the luminescent bacteria is prepared by adding the freeze-dried protecting agent of the luminescent bacteria to the bacterial broth of the luminescent bacteria. The freeze-dried protecting agent of the luminescent bacteria or the freeze-dried powder of the luminescent bacteria mentioned above can be used as a preparation in water quality toxicity on-line monitoring. The freeze-dried powder of the luminescent bacteria of the present invention after resuscitation can be refrigerated in a 4°C bacteria storage vessel, and can be used by the water quality toxicity on-line monitoring instrument for 15 days. The freeze-dried powder of the luminescent bacteria of the present invention can replace the freeze-dried protecting agents imported from other countries, can reduce the cost of reagent consumables for water quality toxicity on-line monitoring instruments in China and other countries, and can improve the continuous and stable operation of the water quality toxicity on-line monitoring instrument in the water quality monitoring stations in China, thereby improving the early warning of biological toxicity for national water quality safety.

Description

LU101961 ||
TECHNICAL FIELD > The present invention relates to the technical field of environmental biological . monitoring, and more particularly, to a freeze-dried protecting agent of luminescent . bacteria, freeze-dried powder of the luminescent bacteria and their application in water | quality comprehensive toxicity on-line monitoring. | Luminescent bacteria contain luciferase and are regarded as "cold light" bacteria, a © facultative autotrophic aerobe.
Luminescent bacteria are capable of emitting blue-green | visible light within the range of 450-490 nm under normal physiological conditions. . There are several different classification methods for classifying luminescent | bacteria, such as traditional, systematic, numerical classification methods and so on.
Until | recently, the luminescent bacteria found all over the world can be roughly divided into the | following categories: Bacillus, Vibrio and Shewanella.
At present, several kinds of | commonly used luminescent bacteria in China and other countries include: | Photobacterium Leiognathi, Photobacterium Phosphoreum, Vibrio fischeri, Vibrio | Qinghaiensis.
Vibrio fischeri is the standard bacterial strain used in International | Standardization Organization (ISO), and Vibrio Qinghai is a freshwater bacteria endemic | Pollutants inhibit bacterial luminescence mainly by two ways.
The first way is to | directly inhibit the activity of the luciferase participating in the luminescence reaction; and | the second way is to inhibit the metabolic processes related to the luminescence reaction | in the cell.
The toxicity of any toxic substance capable of disrupting the breathing, growth | and metabolism of luminescent bacteria can be measured according to the change in . luminescent intensity.
The sensitive toxic substances include organic pollutants and heavy | metals.
The change in luminescent intensity is measured by a biological photometer.
That | is the reason why luminescent bacteria can be used to detect toxic and harmful substances | in the environment. ;
As the study of the potential effects of combined pollution of aquatic ecosystems È depends more and more on toxicity experiments, it is necessary to develop a short-term, . economical and rapid testing system to replace the traditional long-term toxicity | experiments.
The study of luminescent bacteria has sprung up in the 1970s.
Beckman | Company of the United States developed a bioluminescence photometer, namely Microtox | system.
In the Microtox system, freeze-dried powder of natural luminescent bacteria after . resuscitation is used to test toxic substances.
The results obtained are similar to the results | of the 96-hour acute toxicity test of fish.
Henceforward, the toxicity detection technology | of luminescent bacteria has become popular all over the world, and environmental | monitoring and research institutions in various countries have used this method to quickly | test the biological toxicity of environmental samples. |
The water source quality on-line monitoring and early warning system includes the | regularity of quality change of the water source, the selection of early warning parameters, ; the selection of on-line monitoring instruments and system integration.
Water quality . toxicity on-line monitoring technology is the basis of the water quality early warning | system.
The water quality toxicity on-line monitoring technology is used to fully automate . and continue the processes of laboratory water quality toxicity analysis, that is, sampling, . reagent preparation, pretreatment, reaction and calculation.
In this way, the requirements | of the activity of tested objects are stricter, and it is necessary to automatically and | continuously provide indicator organisms with high activity.
Otherwise, monitoring | signals are unstable and monitoring results are unreliable, which directly affect the | performance, accuracy and precision of the on-line monitoring instruments.
Therefore, | maintaining the stability of the biological activity of luminescent bacteria is one of the | core technologies of the on-line water toxicity monitoring instruments. | Vibrio fischeri is typically used in the water quality toxicity on-line monitoring | instrument by the formation of bacterial broth, immobilized bacterial film or freeze-dried | powder.
Because the freeze-dried powder has a long shelf life and is easy to transport and | use, it is the most commonly used preservation method.
At present, the protection . efficiency of the protecting agent technology and formula commonly used in the research | of the freeze-dried powder of the luminescent bacteria in China is low.
The freeze-dried | powder prepared by using the freeze-dried protecting agent is refrigerated in a 4°C bacteria | storage vessel after resuscitation.
The luminescent intensity of bacteria sharply decreases | in the toxicity on-line monitoring instrument during use, resulting in a small number of | active bacteria preserved in the dried powder, unstable activity and short service cycle. | The test results of the toxicity on-line monitoring instruments are unreliable and the | instruments have to be maintained in 3-5 days.
This is an excessively short time period. . As a result, most of the research is still laboratory-based research, and the general use of | water quality toxicity on-line monitoring instruments in China and other countries has not | been realized.
In view of the shortcomings of the prior art, the present invention provides a freeze- | dried protecting agent of luminescent bacteria, freeze-dried powder of the luminescent | bacteria and their application in water quality toxicity detection. | In order to achieve the above objective, the present invention adopts the following | technical solution. .
The freeze-dried protecting agent of the luminescent bacteria per liter consists of: 100- ; 200 g of skimmed milk, 20-120 g of sucrose, 10-60 g of sodium chloride, and water. .
Preferably, the freeze-dried protecting agent of the luminescent bacteria per liter | consists of: 100-150 g of the skimmed milk, 20-60 g of the sucrose, 10-30 g of the sodium | chloride, and water. | Preferably, the freeze-dried protecting agent of the luminescent bacteria per liter | consists of: 140 g of the skimmed milk, 60 g of the sucrose, 30 g of the sodium chloride, . and water. | Preferably, the luminescent bacteria are Vibrio fischeri. | A method for preparing the freeze-dried protecting agent of the luminescent bacteria | includes: dissolving the skimmed milk with water, then adding the sodium chloride and | the sucrose, stirring for 10-20 min (preferably 15 min) until all the components are | dissolved to obtain the freeze-dried protecting agent of the luminescent bacteria. | The freeze-dried powder of the luminescent bacteria is prepared by adding the freeze- | dried protecting agent of the luminescent bacteria to a bacterial broth of the luminescent | bacteria. Specifically, the freeze-dried powder of the luminescent bacteria is prepared by . mixing the freeze-dried protecting agent ofthe luminescent bacteria and the bacterial broth | of the luminescent bacteria according to the volume ratio of from 1:3 to 1:6. Preferably, | the volume ratio of the freeze-dried protecting agent of the luminescent bacteria to the | bacterial broth of the luminescent bacteria is 1:5. | The freeze-dried protecting agent of the luminescent bacteria or the freeze-dried | powder of the luminescent bacteria mentioned above can be used as a preparation in water . quality comprehensive toxicity on-line monitoring system. The water quality toxicity on- | line monitoring system employs the water quality toxicity on-line monitoring instrument to monitor the water quality. |
LU101961 |.
The present invention is further described as follows. .
The raw materials of the freeze-dried protecting agent of the luminescent bacteria of | the present invention includes skimmed milk, sucrose and sodium chloride. The skimmed | milk is a basic protecting agent, which can promote the sublimation of freeze-dried | samples to form a heat-resistant skeleton for blocking heat conduction and thermal | radiation. Furthermore, the skimmed milk is easy to obtain homogeneous products, expand . the distance between cells, and protect the bacteria by wrapping. When the skimmed milk | is separately used as the protecting agent, the luminescent rate of the bacteria is still low, | so sugar alcohols need to be added to improve the survival rate of the Vibrio fischeri. | Sucrose molecules contain hydroxyl groups, which can act with phosphate groups in | phospholipids of a membrane of the bacteria or polar groups of protein of the bacteria to | form hydrogen bonds in the freezing and drying process, so that a "hydration layer" is | formed on the cell surface. The "hydration layer" is configured to maintain the integrity of | the structure and function of the membrane and the protein and also to rehydrate cells or | repair damaged cells quickly. In addition, Vibrio fischeri is a marine bacteria, which | requires a certain concentration of a sodium chloride solution, and thus a sodium chloride | solution with a concentration of 3% is added to promote the luminescence of Vibrio | fischeri. | Another objective of the present invention is to apply the freeze-dried protecting agent | to the preparation of Vibrio fischeri freeze-dried powder. The Vibrio fischeri freeze-dried | powder prepared by using the freeze-dried protecting agent is applied in the water quality | comprehensive toxicity on-line analysis instrument to improve the operation and | maintenance efficiency of the instrument, reduce the cost and produce economic benefits. | The specific application method is as follows. . The dried powder of the luminescent bacteria is dissolved in the sodium chloride | solution with a concentration of 3% and mixed evenly, added to the liquid medium, | cultured at 25°C and 200rmp for 20 h, and centrifugated at 4°C and 10000rmp for 15 min.
È The supernatant is discarded, and the bacteria are collected. | The freeze-dried protecting agent of the luminescent bacteria of the present invention | is prepared as follows: the skimmed milk is dissolved in water, then sodium chloride and | sucrose are added, and are stirred for 10-20 min (preferably 15 min) until all the | components are dissolved. | The bacterial broth is obtained after the bacteria are resuscitated.
The freeze-dried | protecting agent of the luminescent bacteria and the bacterial broth of the luminescent | bacteria are mixed and suspended evenly according to the volume ratio (V/V) from 1:2 to | 1:7, and are then sub-packed into small glass bottles.
The sub-packed mixture is pre-frozen | at -80°C for 4 h, and then put into a freeze-dryer.
In the freeze-dryer, the sub-packed | mixture is vacuum and freeze-dried for 24 h to obtain the freeze-dried powder of the | luminescent bacteria. . The freeze-dried protecting agent of the luminescent bacteria of the present invention | effectively increases the amount of the active bacteria in the freeze-dried powder of the | luminescent bacteria, reaching up to 1.5*10° CFU/g.
The freeze-dried powder can | resuscitate in 5 min with the relatively stable quantity of photons and can be used . continuously for about 15 days by the water quality comprehensive toxicity on-line | analysis instrument.
Therefore, the present invention is beneficial to the production and | research of the freeze-dried powder of the luminescent bacteria and its application in | environmental monitoring, which effectively improves the economic benefit. | In short, the present invention provides a freeze-dried protecting agent capable of | effectively improving the survival rate, biological activity, stability and sensitivity of the | Vibrio fischeri freeze-dried powder.
The freeze-dried powder prepared by using the freeze- | dried protecting agent after resuscitation can be refrigerated in a 4°C bacteria storage | vessel, and can be used by water quality toxicity on-line monitoring instruments for 15 |
LU101961 | days.
The freeze-dried powder of the luminescent bacteria of the present invention can | replace the freeze-dried protecting agents imported from other countries, can reduce the | cost of reagent consumables for water quality toxicity on-line monitoring instruments in | China and other countries, and can improve the continuous and stable operation of the | water quality toxicity on-line monitoring instrument in the water quality monitoring . stations in China, thereby improving the early warning of biological toxicity for the | national water quality safety. |
FIG. 1 is a line chart showing the results of resuscitation time and luminescent | intensity of the freeze-dried powder of the luminescent bacteria. . FIG. 2 is a standard linear fitting diagram of toxicity. . FIG. 3 is a line chart showing the change of sensitivity of freeze-dried powder used . continuously for 15 days on a toxicity on-line monitoring instrument. | FIG. 4 is a line chart showing the change of toxicity stability of reference water tested | by multiple batches of freeze-dried powder test. | Embodiment 1: The number of luminescent bacteria prepared by protecting agents | according to different ratios . Vibrio fischeri (standard bacteria of the luminescent bacteria) is cultured and prepared . according to the steps in the above specific application method of the present invention, | and then the protecting agent of the ratios listed in Table 1 is added to obtain a mixture. ) The mixture is freeze-dried, and is stored at -80°C in a refrigerator.
When the mixture is | taken out, the sodium chloride solution with a concentration of 3% is adopted to perform resuscitation, and the mixture is subjected to 10 times gradient dilution and plate culture | to obtain the number of Vibrio fischeri in each group of mixtures with protecting agents . of different ratios. The control group is set as a protecting agent formed by dissolving 14 | g of the skimmed milk in 100mL of sterile water. . Table 1 | ; ; Number of | Skimmed | Sucrose Sodium i | Group type ; | luminescent bacteria |.
milk (g) (2 chloride (g) | As can be seen from the results of the Table 1, the number of luminescent bacteria of | the Vibrio fischeri freeze-dried powder prepared by using the freeze-dried protecting agent | of the present invention is higher than the number of luminescent bacteria of the control | group, and Experimental group 4 shows the optimal ratio of the protecting agent. ; Embodiment 2: Determination of the optimal amount of protecting agent .
The protecting agent formula of Experimental group 3 in Embodiment 1 is used in | the preparation of the Vibrio fischeri freeze-dried powder. The freeze-dried protecting , agent and the bacterial broth of the Vibrio fischeri are mixed respectively according to the . volume ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7 (Table 2). After different amounts of | the freeze-dried protecting agent are respectively added to the bacterial broth of the Vibrio . fischeri, the number of luminescent bacteria of the freeze-dried powder is measured. The ; detailed operation steps are the same as Embodiment 1. Meanwhile, the number of | luminescent bacteria and the initial luminescent value of the freeze-dried powder with | different volume ratio are measured by bioluminescence photometer. . Table 2 | Volume ratio of | ; Number of luminescent _ ; | freeze-dried protecting ; Initial luminescent value | . bacteria (CFU/g) | agent to bacterial broth |
1.5%10° CFU/g 12869710 | As can be seen from the results of Table 2, when the volume ratio of the freeze-dried . protecting agent to the bacterial broth is 1:5, the resuscitated freeze-dried powder has the | highest amount of active bacteria and the highest initial luminescence value, which is | beneficial to the sensitive response to toxic substances in water and can prolong its use | cycle in the instrument. |
Embodiment 3: Determination of optimal resuscitation time of protecting agent (Table 3) . Table 3 ‘ Luminance of dried ‘ ‘ CL Luminance of imported dried] .
Resuscitation time of, powder prepared by | | | | powder through on-line |, freeze-dried powder |the optimal protecting | culture | agent | As can be seen from Table 3 and FIG. 1, after the resuscitation of 5 min, the relative | luminescent intensity of the freeze-dried powder prepared by using the optimal protecting | agent substantially reaches more than 12 million and reaches a stable state in 5-30 min. | The relative luminescent intensity of the imported freeze-dried powder reaches more than | 4 million after on-line culture for one day before. According to the comparison of the | resuscitation time and the photon luminance after resuscitation, the resuscitation time of | the Vibrio fischeri freeze-dried powder prepared by using the optimal protecting agent of | the present invention is greatly shortened, and its luminance is higher than the luminance | of the imported dried powder.
Therefore, the dried powder of the present invention is | conveniently used in the on-site on-line monitoring instrument, and reduces the operation U and maintenance cost of the instrument. | Embodiment 4: Sensitivity test of freeze-dried powder | Positive control solutions (zinc sulfate heptahydrate) with different concentrations are Ë measured to obtain the corresponding luminescence inhibition rate of the Vibrio fischeri. . Taking the logarithm of the concentration of the positive control solution as the x- | coordinate and the corresponding relative inhibition rate as the y-coordinate to draw a | curve, the data with the inhibition rate in the range of 20-80% are linearly fitted by the | least square method to obtain the fitted equation y=alogx-+b and R? value, where x denotes | the concentration of the zinc sulfate heptahydrate and y denotes the relative inhibition rate. | The condition of y=50% is substituted into the fitted equation to calculate the | corresponding x value. | The EC50 value of the positive control of the freeze-dried powder of the present | invention calculated by the curve in FIG. 2 is 5.05mg / L, which is larger than the EC50 | value (20mg/L) of the positive control of the dried powder imported from other countries. . It shows that the dried powder of the present invention can be used in the water quality . | toxicity on-site on-line analysis instrument in China and other countries after resuscitation. | When toxic substance pollutes the water, the instrument issues alarm instructions as first | time and then the emergency plan is quickly started. |
Embodiment 5: Sensitivity change of the dried bacterial powder during use continuously | for 15 days on a water quality toxicity on-line monitoring instrument | The Vibrio fischeri freeze-dried powder prepared by the optimal freeze-dried | protecting agent formula is refrigerated and transported to the water station provided with . a water quality toxicity on-site on-line analysis instrument. After hydration and | resuscitation, the freeze-dried powder is put into the bacteria storage vessel of the | instrument. The freeze-dried powder is then stirred and cultured for 10 minutes. The | positive control standard solution of the zinc sulfate heptahydrate of 20mg/L is prepared | to test the sensitivity twice a day. The average value of two times is taken. The test . continues for 15 days, and the test results are shown in FIG. 3. | According to the positive control data on the biological toxicity on-line analysis instrument of the on-site water station, the positive control of the zinc sulfate heptahydrate | of 20mg/L is tested continuously for 15 days, and the average toxicity values are all less | than 60%, which meets the requirement of the biological toxicity on-line analysis L instrument. Therefore, the Vibrio fischeri freeze-dried powder prepared by the optimal . freeze-dried protecting agent formula of the present invention has high sensitivity and ; good stability. | Embodiment 6: Changes in toxicity stability of reference water tested by multiple batches | of freeze-dried powder .
Six batches of Vibrio fischeri freeze-dried powder are prepared by the optimal freeze- | dried protecting agent formula, and are dissolved and resuscitated by the sodium chloride . solution with a concentration of 3% for 10 minutes. 2 mL of sodium chloride solution with | the concentration of 3% is added to the detection pool. Each batch contains three parallel | samples. 50uL of different batches of samples after resuscitation are added to the detection | pool, mixed evenly, and put into the toxicity tester. The values are read, and the toxicity .
LU101961 | results are calculated manually.
The toxicity values of each batch are shown in FIG. 4. . As can be seen from FIG. 4, the toxicity values of the tested primary water of the six | batches of freeze-dried powder prepared by the optimal protecting agent formula fluctuates . within +5%, which can meet the requirements of toxicity monitoring instruments.

Claims (10)

LU101961 ;
1. A freeze-dried protecting agent of luminescent bacteria, characterized in that, the | freeze-dried protecting agent of the luminescent bacteria per liter consists of: 100-200 g | of skimmed milk, 20-120 g of sucrose, 10-60 g of sodium chloride, and water. ,
2. The freeze-dried protecting agent of the luminescent bacteria of claim 1, | characterized in that, the freeze-dried protecting agent of the luminescent bacteria per liter . consists of: 100-150 g of the skimmed milk, 20-60 g of the sucrose, 10-30 g of the sodium f chloride, and water. |
3. The freeze-dried protecting agent of the luminescent bacteria of claim 2, . characterized in that, the freeze-dried protecting agent of the luminescent bacteria per liter : consists oË 140 g of the skimmed milk, 60 g of the sucrose, 30 g of the sodium chloride, | and water. |
4. The freeze-dried protecting agent of the luminescent bacteria of claim 1, ; characterized in that, the luminescent bacteria are Vibrio fischeri. '
5. Amethod for preparing the freeze-dried protecting agent of the luminescent bacteria .
of any one of claims 1-4, comprising: dissolving the skimmed milk with water, then adding . the sodium chloride and the sucrose, stirring for 10-20 min until all the components are | dissolved to obtain the freeze-dried protecting agent of the luminescent bacteria. :
6. A freeze-dried powder of the luminescent bacteria, characterized in that, the freeze- | dried powder of the luminescent bacteria is prepared by adding the freeze-dried protecting | agent of the luminescent bacteria of any one of claims 1-4 to a bacterial broth of the Ë luminescent bacteria. |
7. The freeze-dried powder of the luminescent bacteria of claim 6, characterized in | that, the freeze-dried powder of the luminescent bacteria is prepared by mixing and | suspending the freeze-dried protecting agent of the luminescent bacteria of any one of |
LU101961 | claims 1-4 and the bacterial broth of the luminescent bacteria according to a volume ratio | of from 1:3 to 1:6. |
8. The freeze-dried powder of the luminescent bacteria of claim 7, characterized in . that, the freeze-dried powder of the luminescent bacteria is prepared by mixing and | suspending the freeze-dried protecting agent of the luminescent bacteria of any one of | claims 1-4 and the bacterial broth of the luminescent bacteria according to a volume ratio | of 1:5. |
9. An application, characterized in that, the application takes the freeze-dried | protecting agent of the luminescent bacteria of any one of claims 1-4 or the freeze-dried | powder of the luminescent bacteria of any one of claims 6-8 as a preparation in water | quality comprehensive toxicity on-line monitoring. |
10. The application of claim 9, characterized in that, a water quality toxicity on-line | monitoring instrument is adopted in the water quality toxicity on-line monitoring to | monitor water quality toxicity. |
LU101961A 2019-08-27 2019-08-27 Freeze-dried protecting agent and freeze-dried powder of the luminescent bacteria and their application in water quality comprehensive toxicity on-line monitoring LU101961B1 (en)

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Application Number Priority Date Filing Date Title
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