WO2021035503A1 - 一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用 - Google Patents

一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用 Download PDF

Info

Publication number
WO2021035503A1
WO2021035503A1 PCT/CN2019/102679 CN2019102679W WO2021035503A1 WO 2021035503 A1 WO2021035503 A1 WO 2021035503A1 CN 2019102679 W CN2019102679 W CN 2019102679W WO 2021035503 A1 WO2021035503 A1 WO 2021035503A1
Authority
WO
WIPO (PCT)
Prior art keywords
freeze
luminescent bacteria
protective agent
dried
toxicity
Prior art date
Application number
PCT/CN2019/102679
Other languages
English (en)
French (fr)
Inventor
方俊
张琳琳
刘刚
蒋红梅
刘芬
胡超
Original Assignee
湖南农业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 湖南农业大学 filed Critical 湖南农业大学
Priority to PCT/CN2019/102679 priority Critical patent/WO2021035503A1/zh
Priority to CN201980006903.1A priority patent/CN111566199A/zh
Publication of WO2021035503A1 publication Critical patent/WO2021035503A1/zh

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/18—Water
    • G01N33/186—Water using one or more living organisms, e.g. a fish
    • G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms

Definitions

  • 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 the online monitoring of comprehensive toxicity of water quality.
  • Luminescent bacteria are bacteria that contain luciferase in the body. Under normal physiological conditions, they can emit blue-green visible "cold light” bacteria with a wavelength in the range of 450-490nm. They are facultative, oxidative and autotrophic bacteria.
  • Luminescent bacteria have several different classification methods, such as traditional, systematic, and numerical classification. So far, the species of luminescent bacteria found worldwide are 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 lucidum, Photobacterium luminescens, Vibrio fischeri, and Vibrio Qinghai. Vibrio fischeri is the standard strain adopted by ISO, and Vibrio Qinghai is a unique freshwater bacteria in my country.
  • Pollutants mainly inhibit bacterial luminescence through the following two ways: directly inhibit the activity of luciferase involved in the luminescence reaction; inhibit the metabolic process related to the luminescence reaction in the cell, any toxic substance that can disrupt the respiration, growth, and metabolism of the luminescence bacteria , Can determine the toxicity of toxic substances according to the change of luminous intensity.
  • the main sensitive poisons are organic pollutants and heavy metals.
  • the change of the luminous intensity is measured by a biophotometer, which is the basic basis why luminous bacteria can be used to detect toxic and harmful substances in the environment.
  • Luminescent bacteria have emerged in the 1970s.
  • the American Beckman company has developed a bioluminescence photometer, the Microtox system, which uses freeze-dried powder of natural luminescent bacteria and can be tested after resuscitation. The results obtained are comparable to those of fish.
  • the results of the 96-hour acute toxicity test were similar. Since then, luminescent bacteria toxicity detection technology has become popular around the world, and environmental monitoring and research institutions in various countries have adopted this method to quickly test the biological toxicity of environmental samples.
  • Water source water quality online monitoring, monitoring and early warning system includes the law of water source water quality change, the selection of early warning parameters, the selection of online instruments and system integration.
  • Online water quality toxicity monitoring technology is the basis of water quality early warning. Online water toxicity monitoring technology is to fully automate and continuous the laboratory water toxicity analysis process, that is, sampling, reagent preparation, pretreatment, reaction, and calculation. In this way, the requirements for the activity of the recipient object are more stringent, and it is necessary to automatically and continuously provide high-activity indicator organisms. Otherwise, the monitoring signal may be unstable and the monitoring result is unreliable, which directly affects the performance, accuracy and precision of the online monitoring instrument. Therefore, the stability maintenance of the biological activity of the luminescent bacteria is one of the core technologies of the water toxicity online monitor.
  • Vibrio fischeri is often used in water toxicity online monitoring instruments by the formation of bacterial liquid, immobilized bacterial film or freeze-dried powder. Because of the long shelf life of freeze-dried powder, it is easy to transport and use, and is the most commonly used storage method.
  • the protective agent technology and formula commonly used in the research on the protective agent of luminescent bacteria freeze-dried powder in China are low in protection efficiency.
  • the freeze-dried powder prepared from the freeze-dried powder protective agent is recovered and placed in a 4 °C strain storage cup for refrigeration ,
  • the luminous intensity of the bacteria drops rapidly during the use of the online toxicity monitoring instrument, resulting in a small number of live bacteria in the dry powder, unstable activity and short use period. This results in unreliable detection results during the use of the online toxicity instrument, and the maintenance period is too short (3-5 days).
  • Most of the research is still in the laboratory stage, and the universal online water toxicity instrument at home and abroad has not yet been realized.
  • the present invention only provides a luminescent bacteria freeze-dried protective agent, freeze-dried powder and its application in water quality toxicity detection in view of the shortcomings of the prior art.
  • Each L of the luminescent bacteria freeze-dried protective agent is composed of the following components: 100-200g skimmed milk, 20-120g sucrose, 10-60g sodium chloride, and the rest is water.
  • each L of the luminescent bacteria freeze-dried protective agent is composed of the following components: skimmed milk 100-150, sucrose 20-60, sodium chloride 10-30, and the rest is water.
  • each L of the luminescent bacteria freeze-dried protective agent is composed of the following components: 140 g of skim milk, 60 g of sucrose, 30 g of sodium chloride, and the rest is water.
  • the luminescent bacteria is Vibrio fischeri.
  • the preparation method of the above-mentioned luminescent bacteria freeze-dried protective agent is to first dissolve defatted with water, then add sodium chloride and sucrose, and stir for 10-20 minutes, preferably 15 minutes, until all the components are dissolved to obtain the luminescent bacteria freeze-dried protective agent .
  • the luminescent bacteria freeze-dried powder is prepared by adding the above-mentioned luminescent bacteria freeze-dried protective agent to the bacterial liquid of the luminescent bacteria. Specifically, it is prepared by suspending the luminescent bacteria freeze-dried protective agent and the bacteria liquid of the luminescent bacteria in a volume ratio of 1:3 to 1:6. Preferably, the volume ratio of the luminescent bacteria freeze-dried protective agent to the luminescent bacteria liquid is 1:5.
  • the above-mentioned luminescent bacteria freeze-dried protective agent or luminescent bacteria freeze-dried powder can be used as a detection agent for online monitoring of comprehensive toxicity of water quality.
  • the online water quality toxicity monitoring is to use the water quality online toxicity instrument to perform toxicity monitoring on the water quality.
  • the raw materials of the luminescent bacteria freeze-dried protective agent provided by the invention are skimmed milk, sucrose and sodium chloride.
  • Skim milk is a basic protective agent, which can promote the sublimation of freeze-dried samples to form a heat-resistant skeleton to block heat conduction and heat radiation, and it is easy to obtain homogeneous products, expand the mutual distance between cells, and protect the bacteria through the form of wrapping.
  • the luminescence rate of the bacteria is still low overall, so sugar alcohols must be added to improve the survival rate of Vibrio fischeri.
  • the sucrose molecule contains hydroxyl groups.
  • Vibrio fischeri is a marine bacterium, which has certain requirements for the concentration of sodium chloride, and 3% sodium chloride is conducive to the luminescence of Vibrio fischeri.
  • Another object of the present invention is the application of the above freeze-dried protective agent in the preparation of Vibrio fischeri freeze-dried powder.
  • the freeze-dried Vibrio fischeri powder prepared by the freeze-dried protective agent is used in an online water quality comprehensive toxicity analysis instrument. Improve the efficiency of instrument operation and maintenance, reduce costs, and generate economic benefits.
  • Dissolve and mix the luminescent bacteria dry powder with 3% sodium chloride connect it to a liquid culture medium, culture at 200 rpm at 25°C for 20 hours, centrifuge at 4°C at 10000 rpm for 15 minutes, discard the supernatant, and collect the bacteria.
  • the luminescent bacteria freeze-dried protective agent of the present invention is prepared; when preparing, the skim milk is first dissolved in water, then sodium chloride and sucrose are added, and the mixture is stirred for 10-20 minutes, preferably 15 minutes, until all is dissolved.
  • the bacteria solution After resuscitating the bacteria, the bacteria solution is obtained, and the luminescent bacteria freeze-dried protective agent and the bacteria solution of the luminescent bacteria are evenly suspended in a volume ratio (V/V) from 1:2 to 1:7, and then dispensed into small glass bottles; Pre-freeze the aliquoted mixture at -80°C for 4 hours, put it in a freeze dryer, and freeze-dry in a vacuum for 24 hours to obtain a luminescent bacteria freeze-dried powder.
  • V/V volume ratio
  • the luminescent bacteria freeze-dried protective agent of the present invention effectively increases the amount of viable bacteria of the luminescent bacteria freeze-dried powder, so that it can reach 1.5*10 9 CFU/g, the freeze-dried powder can be recovered within 5 minutes, and the amount of photons reaches a relative It is stable and can be used continuously for about 15 days with the online water quality comprehensive toxicity analysis instrument. It is beneficial to the production research of luminescent bacteria freeze-dried powder and its application in environmental monitoring, and effectively improves its economic benefits.
  • the present invention provides a freeze-dried protective agent that can effectively improve the survival rate, biological activity stability and sensitivity of the freeze-dried Vibrio fasciflorus powder.
  • the freeze-dried powder prepared from the freeze-dried protective agent is resuscitated and placed in 4
  • the bacteria storage cup can be refrigerated at °C, which can be used for online toxicity monitoring instruments for 15 days, which can replace imported freeze-dried protective agents, reduce the operating cost of reagents and consumables for online toxicity monitoring instruments at home and abroad, and improve the continuous stability of the national groundwater station online toxicity monitoring instruments Operation, play a real biological toxicity early warning role for the country's water quality safety.
  • Figure 1 is a graph showing the results of recovery time and luminous intensity of freeze-dried powder of luminescent bacteria
  • Figure 2 is a linear fitting diagram of standard poisons
  • Figure 3 is a graph showing the sensitivity change of 15-day continuous use of dry bacteria powder when applied to an online toxicity monitoring instrument
  • Figure 4 is a graph showing changes in toxicity stability of multiple batches of freeze-dried powder test reference water.
  • Example 1 The bacterial content of luminescent bacteria prepared with different proportions of protective agents
  • the Vibrio fischeri (standard bacteria of the luminescent bacteria) is cultured and produced, and finally the protective agent listed in Table 1 is added, freeze-dried, and stored at low temperature -80°C in the refrigerator When taking it out, use 3% sodium chloride to resuscitate, carry out 10-fold gradient dilution, plate culture and count to get the number of Vibrio fischeri in each different protective agent ratio.
  • the blank control uses 14g skimmed milk dissolved in 100mL In sterile water, a protective agent was prepared as a control group.
  • Group Skim milk g
  • Sucrose g
  • Sodium chloride g
  • Bacteria content CFU/g
  • the lyophilized powder of Vibrio fischeri prepared by using the lyophilized protective agent of the present invention has a bacterial content greater than that of the control group, and group 4 is the best ratio of the protective agent.
  • the protective agent formula of group 3 in Example 1 was used in the preparation of Vibrio fischeri lyophilized powder, and the volume ratio of the lyophilized protective agent was 1:1, 1:2, 1:3, 1:4, 1: 5, 1:6, 1:7 (Table 2) were added to the Vibrio fischeri bacteria solution, and the bacteria content of the freeze-dried powder after adding different amounts of protective agent was measured.
  • the detailed operation steps were the same as in Example 1.
  • a bioluminescence tester was used to determine the amount of bacteria and initial luminescence in different volume ratios.
  • the positive quality control EC50 value of the freeze-dried powder obtained from the curve in Figure 2 is 5.05mg/L, which is better than the EC50 value (20mg/L) of the positive quality control of imported dry powder. It indicates that the dry powder is used in the field at home and abroad after recovery. On the online toxicity analyzer, when toxic substances are polluted in the water body, the instrument must first send out an alarm instruction and quickly start the emergency plan.
  • Example 5 Applying to the online toxicity monitoring instrument, the sensitivity change of dry bacteria powder after 15 days of continuous use
  • Vibrio fischeri lyophilized powder prepared with the best lyophilized protective agent formula is refrigerated and transported to the water station where the toxic online analysis instrument is installed. After hydration and recovery, it is put into the instrument strain storage cup, stirred and cultivated for 10 minutes to prepare 20 mg/ L's zinc sulfate heptahydrate positive quality control standard solution was tested, and the test was performed twice a day, and the average value of the two times was taken for 15 consecutive days. The test results are shown in Figure 3.
  • the positive quality control data applied to the on-site biological toxicity analysis instrument of the on-site water station the positive quality control of 20mg/L zinc sulfate heptahydrate was tested continuously for 15 days, and the toxicity value was ⁇ 60%, which met the online luminous bacteria method.
  • the use requirements of the biological toxicity analyzer are that the lyophilized powder of Vibrio fischeri prepared by the best protective agent of the present invention has high sensitivity and good stability.
  • Example 6 Changes in toxicity stability of multiple batches of freeze-dried powder test reference water

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用。每L发光细菌冻干保护剂由如下组分组成：脱脂乳100—200g，蔗糖20—120g，氯化钠10—60g，其余为水。发光细菌冻干粉由发光细菌冻干保护剂加入发光细菌的菌液中制备而成。该发光细菌冻干保护剂或发光细菌冻干粉可作为检测制剂用于在线水质毒性监测中。该发光细菌冻干粉复苏后置于4℃菌种储存杯冷藏，可供在线毒性监测仪器使用15d，能够替代进口冻干保护剂，降低在线毒性监测仪器的试剂耗材运行成本，提高在线毒性监测仪器的持续稳定运行，为水质安全起到生物毒性预警作用。

Description

一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用 技术领域
本发明涉及环境生物监测技术领域，具体涉及一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用。
背景技术
发光细菌是体内自身含荧光酶的细菌，在正常的生理条件下，可发出波长在450—490nm范围内蓝绿色可见的“冷光”细菌，是兼性好氧化能自养型细菌。
发光细菌有传统、系统、数值分类等几种不同的分类方法。迄今为止，全世界范围内发现的发光细菌种类，大致分为以下种类：杆菌属、弧菌属和希瓦氏菌属。目前国内外常用的几种发光细菌为：鳆鱼发光杆菌、明亮发光杆菌、费氏弧菌，青海弧菌。费氏弧菌是ISO采用的标准菌株，青海弧菌是我国特有的淡水菌。
污染物主要通过以下两个途径来抑制细菌发光：直接抑制参与发光反应的荧光酶的活性；抑制细胞内与发光反应有关的代谢过程，凡是能破坏发光细菌呼吸，生长，新陈代谢的任何时有毒物质，都可以根据发光强度的变化来测定有毒物质毒性的大小。其主要的敏感毒物为有机污染物和重金属两类。发光强度的变化通过生物测光仪测定出来，这就是为什么可以利用发光细菌来检测环境中有毒、有害物质的基本依据。
由于研究水生生态系统复合污染的潜在影响越来越依赖毒性实验，需要发展一种短期、经济、快速的测试体系来代替传统的长期毒性实验。发光细菌在上个世纪70年代己经兴起了，美国Beckman公司研制了生物发光光度计，即Microtox系统，该系统使用天然发光菌的冻干粉，复苏后即可测试，所得结果与鱼类的96小时急性毒性试验结果相近。从此发光细菌毒性检测技术在全球范围内流行起来，各国的环境监测和研究机构采用该法快速测试环境样品的生物毒性。
水源水质在线监测监测预警系统包括水源水质变化规律、预警参数的选择、在线仪器的选择和系统集成。在线水质毒性监测技术是水质预警的基础。在线水质毒性监测技术是将实验室水质毒性分析过程，即采样、试剂配制、预处理、反应和计算等过程完全自动化和连续化。这样对受式对象的活性要求更严格，需要自动连续的提供高活性的指示生物。否则，就可能出现监测信号不稳定，监测结果不可靠，直接影响在线监测仪器的性能、准确度及精密度。所以，发光细菌生物活性的稳定性保持是水质毒性在线监测仪的核心技术之一。
费氏弧菌常以菌液，固定化菌膜或冻干粉的形成应用在水质毒性在线监测仪器中，因冻干粉保质期长，便于运输、使用方便，是最常用的保存方法。目前，关于国内在发光细菌冻干粉保护剂的研究中常用的保护剂工艺、配方保护效率低，由冻干粉保护剂制备而成的冻干粉复苏后置于4℃菌种储存杯冷藏，供在线毒性监测仪器使用的过程中细菌的发光强度迅速下降，导致干粉中活菌数量较小，活性不稳定、使用周期短。这就造成了在线毒性仪器使用过程中检测结果不可靠，维护周期过短(3—5天)。大部分研究仍停留在实验室阶段，尚未实现国内外在线水质毒性仪器的通用。
发明内容
本发明只在针对现有技术的不足，提供一种发光细菌冻干保护剂、冻干粉及其在水质毒性检测中的应用。
为了实现上述目的，本发明提供的技术方案为：
每L所述发光细菌冻干保护剂由如下组分组成：脱脂乳100—200g，蔗糖20—120g，氯化钠10—60g，其余为水。
优选地，每L所述发光细菌冻干保护剂由如下组分组成：脱脂乳100—150，蔗糖20—60，氯化钠10—30，其余为水。
更优选地，每L所述发光细菌冻干保护剂由如下组分组成：脱脂乳140g，蔗糖60g，氯化钠30g，其余为水。
优选地，所述发光细菌为费氏弧菌。
上述发光细菌冻干保护剂的制备方法是先将脱脂加水溶解，然后加入氯化钠和蔗糖，搅拌10—20min，优选为15min，直到各组分全部溶解后，即得发光细菌冻干保护剂。
所述发光细菌冻干粉由上述发光细菌冻干保护剂加入发光细菌的菌液中制备而成。具体而言，是将发光细菌冻干保护剂与发光细菌的菌液按照体积比1:3至1:6混悬配制而成。优选地，所述发光细菌冻干保护剂与发光细菌的菌液的体积比为1:5。
上述发光细菌冻干保护剂或发光细菌冻干粉可作为检测制剂用于水质综合毒性在线监测中。所述在线水质毒性监测是利用水质在线毒性仪器对水质进行毒性监测。
下面对本发明作进一步说明：
本发明提供的发光细菌冻干保护剂原料为脱脂乳、蔗糖和氯化钠。脱脂乳为一种基础保护剂，它可以促进冻干样品的升华形成耐热骨架阻断热传导和热辐射，并易取得均质产品，扩大细胞间的相互距离，通过包裹形式保护菌体。单纯的以脱脂乳为保护剂时，细菌的发光率总体仍然较低，因此还需增加糖醇类物质来提高费氏弧菌的存活率。蔗糖分子中 含有羟基，在冷冻和干燥过程中，可与菌体细胞膜磷脂中的磷酸基团或菌体蛋白质极性基团形成氢键，使细胞表面形成一层“水合层”，保持细胞膜和蛋白质结构与功能的完整性，也有利于细胞较快复水或修复受损细胞。另外，费氏弧菌是海洋菌，其对氯化钠浓度有一定的要求，3％的氯化钠有利于费氏弧菌发光。
本发明的另一个目的是上述冻干保护剂在制备费氏弧菌冻干粉中的应用，通过该冻干保护剂制得的费氏弧菌冻干粉用到水质综合毒性在线分析仪器，提高仪器的运维效率，降低成本，产生经济效益。
具体应用的方式如下：
将发光细菌干粉用3％的氯化钠溶解混匀，接入到液体培养基中，25℃200rmp培养20h,4℃，10000rmp离心15min,弃去上清液，收集菌体。
制备本发明所述的发光细菌冻干保护剂；制备时先将脱脂乳加水溶解，后加入氯化钠和蔗糖，搅拌10—20min，优选为15min，直到全部溶解为止。
将菌体复苏后得到菌液，按发光细菌冻干保护剂与发光细菌的菌液按体积比(V/V)1:2至1:7混悬均匀后，分装到小玻璃瓶中；将分装好的混合物于-80℃预冻4h，放入冻干机中，真空冷冻干燥24h，即得发光细菌冻干粉。
本发明所述的发光细菌冻干保护剂有效提高了发光细菌冻干粉的活菌量，使其可以达到1.5*10 9CFU/g,冻干粉可以在5min钟内复苏，光子量达到相对稳定，可供水质综合毒性在线分析仪器连续使用15天左右。有利于发光细菌冻干粉的生产研究以及在环境监测中的应用，有效提高了其经济效益。
总之，本发明提供了一种能有效提高发费氏弧菌冻干粉存活率和生物活性稳定性、灵敏度的冻干保护剂，由该冻干保护剂制备的冻干粉复苏后置于4℃菌种储存杯冷藏，可供在线毒性监测仪器使用15d，能够替代国外进口冻干保护剂，降低国内外在线毒性监测仪器的试剂耗材运行成本，提高国家地水站在线毒性监测仪器的持续稳定运行，为国家的水质安全起到真正意义上的生物毒性预警作用。
附图说明
图1是发光细菌的冻干粉复苏时间与发光强度结果图；
图2是标准毒物线性拟合图；
图3是应用到在线毒性性监测仪器上连续使用15d菌干粉灵敏度变化图；
图4是制作的多批次冻干粉测试参比水的毒性稳定性变化图。
具体实施方式
实施例1 不同配比的保护剂制备的发光菌的含菌量
按本发明中“具体应用方式”中的步骤对费氏弧菌(发光细菌的标准菌)进行培养制作，最后加入表1中所列数值的保护剂，冷冻干燥，低温储存-80℃冰箱中，取出时用3％的氯化钠复苏，进行10倍梯度稀释，平板培养计数，得出每个不同保护剂配比所含费氏弧菌的数量，空白对照采用14g的脱脂乳溶于100mL无菌水中，制成保护剂为对照组。
表1
组别 脱脂乳(g) 蔗糖(g) 氯化钠(g) 含菌量(CFU/g)
组1 8 1 0.5 3.1*10 8CFU/g
组2 10 2 1 3.4*10 8CFU/g
组3 12 4 2 6.7*10 8CFU/g
组4 14 6 3 1.5*10 9CFU/g
组5 16 8 4 4.3*10 8CFU/g
组6 18 10 5 6.1*10 7CFU/g
组7 20 12 6 3.8*10 7CFU/g
组8 22 14 8 3.0*10 7CFU/g
对照组 14     2.9*10 7CFU/g
从结果可以看出，使用本发明的冻干保护剂制备的费氏弧菌冻干粉的含菌量均大于对照组的含菌量，组4为保护剂最佳配比。
实施例2 保护剂最佳添加量的确定
将实施例1中组3的保护剂配方用于费氏弧菌冻干粉的制作，分别按冻干保护剂体积比为1:1，1:2，1:3，1:4，1：5，1:6，1:7(表2)添加到费氏弧菌菌液中，测添加不同量的保护剂后冻干粉的含菌量，详细操作步骤同实施例1。同时用生物发光测试仪测定不同体积比的含菌量、初始发光量。
表2
Figure PCTCN2019102679-appb-000001
Figure PCTCN2019102679-appb-000002
从结果可以看出，采用冻干保护剂的体积比为1:5时，制得的冻干粉复苏后的活菌量较多，且初始发光值最高，有利于对水体中有毒物的灵敏度响应，延长其在仪器上的使用周期。
实施例3 保护剂最佳复苏时间的确定(表3)
表3
冻干粉复苏时间 最佳护剂干粉光亮度 进口在线培养干粉光亮度
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
通过表3和图1可以看出，复苏5min后相对发光强主要达到1200多万，发光强度在5—30min内达到稳定状态，进口在线培养的菌干粉需在线仪器培养1天后才达到400多万。通过复苏时间和复苏后的光子亮度较，可得出，采用本发明最佳保护剂制行出的费氏弧菌冻干粉复苏时间大大缩短，且光亮度高于进口干粉。这有利于干粉在现场在线监测仪器上的便捷使用，降低仪器的运行维护量和运行成本。
实施例4 冻干粉的灵敏度测试
测量不同浓度的阳性质控液(七水合硫酸锌)，得到对应的费氏弧菌的发光抑制率，阳性质控浓度对数为横坐标，以其对应的相对抑制率为纵坐标作图，采用最小二乘法将抑制率处于20—80％范围内的数据进行线性拟合，得到拟合方程y＝alogx+b及R2值，其中x为七水合硫酸锌浓度，y为相对抑制率；将y＝50％代入拟合方程中，计算出对应的x值。
通过图2曲线计算所得所制冻干粉的阳性质控EC50值为5.05mg/L,优于国外进干粉阳性质控的EC50值(20mg/L)表明该干粉复苏后用于现场国内外的在线毒性分析仪上，当水体中发生有毒物质污染时，仪器要先发出报警指示，迅速启动应急预案。
实施例5 应用到在线毒性监测仪器上连续使用15天菌干粉灵敏度变化
将最佳冻干保护剂配方制备的费氏弧菌冻干粉冷藏运输到现场安有毒性在线分析仪器的水站，水合复苏后放入仪器菌种储存杯中，搅拌培养10min，配制20mg/L的七水合硫酸锌阳性质控标准溶液，进行测试，每天测试2次，取两次均值，连续测试15d，测试结果见图3。
通过应用到现场水站生物毒性在线分析仪器上的阳性质控数据来看，连续测试15天20mg/L的七水合硫酸锌阳性质控，毒性值均≧60％，满足了发光细菌法的在线生物毒性分析仪的使用要求，采用本发明最佳保护剂制备的费氏弧菌冻干粉灵敏度较高，且稳定性较好。
实施例6 多批次冻干粉测试参比水的毒性稳定性变化
用最佳冻干保护剂配方制备6批次费氏弧菌冻干粉，采用3％的氯化钠溶解复苏10min,取2mL3％的氯化钠加入到检测池中，每批次3个平行样品，加入50uL复苏后的不同批次样品，摇匀，放入毒性检测仪中，读值，人工计算毒性结果，各批次毒性值结果见图4。
通过图4可得出，采用最佳保护剂制备的6批次冻干粉，测试一级水的毒性值均在±5％内波动，满足毒性仪器的使用要求。

Claims (10)

  1. 一种发光细菌冻干保护剂，其特征在于，每L所述发光细菌冻干保护剂由如下组分组成：脱脂乳100—200g，蔗糖20—120g，氯化钠10—60g，其余为水。
  2. 如权利要求1所述的发光细菌冻干保护剂，其特征在于，每L所述发光细菌冻干保护剂由如下组分组成：脱脂乳100—150，蔗糖20—60，氯化钠10—30，其余为水。
  3. 如权利要求2所述的发光细菌冻干保护剂，其特征在于，每L所述发光细菌冻干保护剂由如下组分组成：脱脂乳140g，蔗糖60g，氯化钠30g，其余为水。
  4. 如权利要求1所述的发光细菌冻干保护剂，其特征在于，所述发光细菌为费氏弧菌。
  5. 如权利要求1至4任一项所述发光细菌冻干保护剂的制备方法，其特征在于，所述方法是先将脱脂乳加水溶解，然后加入氯化钠和蔗糖，搅拌10—20min，直到各组分全部溶解后，即得发光细菌冻干保护剂。
  6. 一种发光细菌冻干粉，其特征在于，所述发光细菌冻干粉由权利要求1至4任一项所述的发光细菌冻干保护剂加入发光细菌的菌液中制备而成。
  7. 如权利要求6所述的发光细菌冻干粉，其特征在于，所述发光细菌冻干粉是将权利要求1至4任一项所述的发光细菌冻干保护剂与发光细菌的菌液按照体积比1:3至1:6混悬配制而成。
  8. 如权利要求7所述的发光细菌冻干粉，其特征在于，所述发光细菌冻干粉是将权利要求1至4任一项所述的发光细菌冻干保护剂与发光细菌的菌液按照体积比1:5混悬配制而成。
  9. 如权利要求1至4任一项所述的发光细菌冻干保护剂或权利要求6至8任一项所述的发光细菌冻干粉作为检测制剂在水质综合毒性在线监测中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述水质综合毒性在线监测是利用水质在线毒性仪器对水质进行毒性监测。
PCT/CN2019/102679 2019-08-27 2019-08-27 一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用 WO2021035503A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2019/102679 WO2021035503A1 (zh) 2019-08-27 2019-08-27 一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用
CN201980006903.1A CN111566199A (zh) 2019-08-27 2019-08-27 一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/102679 WO2021035503A1 (zh) 2019-08-27 2019-08-27 一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用

Publications (1)

Publication Number Publication Date
WO2021035503A1 true WO2021035503A1 (zh) 2021-03-04

Family

ID=72074003

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/102679 WO2021035503A1 (zh) 2019-08-27 2019-08-27 一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用

Country Status (2)

Country Link
CN (1) CN111566199A (zh)
WO (1) WO2021035503A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117487793A (zh) * 2023-10-31 2024-02-02 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) 一种野外水质现场检测的联合急性毒性测试方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112358988B (zh) * 2020-11-10 2022-09-20 广西壮族自治区农业科学院 植物乳杆菌菌株ldvs008及其应用
CN115786204B (zh) * 2022-12-01 2023-08-15 中国水产科学研究院黄海水产研究所 一种适用于弧菌的真空冷冻干燥保护剂与冻干方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040038825A1 (en) * 2001-08-10 2004-02-26 Leland Jarrod Ethan Water soluble uv-protective coatings for biological pesticides and process for making same
CN101822288A (zh) * 2010-04-02 2010-09-08 浙江大学 海洋酵母Rhodosporidium paludigenum Fell&Tallman的真空冷冻干燥制品的制备方法
CN103940789A (zh) * 2013-01-17 2014-07-23 刘星海 水毒性检测中发光细菌荧光行为的规范和数值解析
CN104762209A (zh) * 2015-03-30 2015-07-08 华东理工大学 明亮发光杆菌冻干粉及其制备方法
CN106546579A (zh) * 2016-10-28 2017-03-29 华南理工大学 一种有机溶剂提高发光细菌检测毒性物质敏感度的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102465167A (zh) * 2010-11-10 2012-05-23 中国科学院生态环境研究中心 一种发光细菌的快速、高通量急性毒性测试方法
CN102250771A (zh) * 2011-05-31 2011-11-23 重庆大学 一种基于信号分子支撑的快速发光菌剂
CN103468572B (zh) * 2013-09-25 2016-07-06 北京尚洋东方环境科技有限公司 一种发光菌的冻干保护剂
CN108102982B (zh) * 2018-02-07 2021-06-01 山东出入境检验检疫局检验检疫技术中心 麦氏弧菌的真空冷冻干燥保护剂及其保藏方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040038825A1 (en) * 2001-08-10 2004-02-26 Leland Jarrod Ethan Water soluble uv-protective coatings for biological pesticides and process for making same
CN101822288A (zh) * 2010-04-02 2010-09-08 浙江大学 海洋酵母Rhodosporidium paludigenum Fell&Tallman的真空冷冻干燥制品的制备方法
CN103940789A (zh) * 2013-01-17 2014-07-23 刘星海 水毒性检测中发光细菌荧光行为的规范和数值解析
CN104762209A (zh) * 2015-03-30 2015-07-08 华东理工大学 明亮发光杆菌冻干粉及其制备方法
CN106546579A (zh) * 2016-10-28 2017-03-29 华南理工大学 一种有机溶剂提高发光细菌检测毒性物质敏感度的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHENG XIAOYAN, CAI QIANG, PENG HUIMIN, SHENG JIANWU, PENG FANGYI, LIU RUI, CHEN LUJUN: "Control of the Survival and Activity of Luminescent Bacteria", CHINESE JOURNAL OF APPLIED AND ENVIRONMENTAL BIOLOGY, vol. 17, no. 2, 25 April 2011 (2011-04-25), pages 264 - 267, XP055786596, ISSN: 1006-687X, DOI: 10.3724/SP.J.1145.2011.00264 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117487793A (zh) * 2023-10-31 2024-02-02 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) 一种野外水质现场检测的联合急性毒性测试方法

Also Published As

Publication number Publication date
CN111566199A (zh) 2020-08-21

Similar Documents

Publication Publication Date Title
WO2021035503A1 (zh) 一种发光细菌冻干保护剂、冻干粉及其在水质综合毒性在线监测中的应用
Mille-Lindblom et al. Ergosterol as a measure of living fungal biomass: persistence in environmental samples after fungal death
Brookes et al. The adenylate energy charge of the soil microbial biomass
Stewart Nitrogen Turnover in Marine and Brackish Habitats II. Use of 15N in Measuring Nitrogen Fixation in the Field
Sadler et al. The function of acetate in photosynthesis by green bacteria
Gómez et al. Modification of the electron transport system (ETS) method for routine measurements of respiratory rates of zooplankton
Li et al. Development of an ATP luminescence-based method for assimilable organic carbon determination in reclaimed water
Jozić et al. Is TBX agar a suitable medium for monitoring Escherichia coli in bathing water using the membrane filtration method?
CN102127588B (zh) 一种定量检测水质毒性的方法
Harris et al. Succinic and lactic dehydrogenase activity in myocardial homogenates from animals at high and low altitude
Zhang et al. Single‐cell rapid identification, in situ viability and vitality profiling, and genome‐based source‐tracking for probiotics products
Walters et al. Microbial substrate storage in activated sludge
CN107988327A (zh) 志贺氏菌干粉化lamp快速检测试剂盒
LU101961B1 (en) Freeze-dried protecting agent and freeze-dried powder of the luminescent bacteria and their application in water quality comprehensive toxicity on-line monitoring
Paciello et al. Strengths and weaknesses in the determination of Saccharomyces cerevisiae cell viability by ATP-based bioluminescence assay
Jin et al. Effect of storage conditions on the stability and fermentability of enzymatic lignocellulosic hydrolysate
WO2022141942A1 (zh) 含有特异性分子靶标的蜡样芽胞杆菌标准菌株及其检测和应用
Fan et al. Microbial quality assessment methods for fresh-cut fruits and vegetables
McMahon et al. Evaluation of VIDAS® Salmonella (SLM) immunoassay method with rappaport-vassiliadis (RV) medium for detection of Salmonella in foods: Collaborative study
CN112198150B (zh) 生物发光法监测污染水体的方法
Takahashi et al. Insufficiency of copper ion homeostasis causes freeze-thaw injury of yeast cells as revealed by indirect gene expression analysis
Xie et al. Persistence of E. coli O157: H7 in urban recreational waters from Spring and Autumn: a comparison analysis
CN106554989A (zh) 一种用于检测乳品中β-内酰胺酶的试剂盒及其检测方法
Rakotonirainy et al. Development of a new procedure based on the energy charge measurement using ATP bioluminescence assay for the detection of living mould from graphic documents
Rodkey et al. A rapid test for distinguishing human from cow's milk based upon a difference in their xanthine oxidase content

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19943024

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19943024

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 19943024

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 13/10/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 19943024

Country of ref document: EP

Kind code of ref document: A1