Cryopreservation and resuscitation methods of microorganisms for monitoring toxicity and their application in food and water quality monitoring

Abstract

The invention belongs to the field of biotechnology and discloses a method for freezing and resuscitating microorganisms for monitoring toxicity, so as to obtain freeze-dried powder with long storage period, stable performance and rapid resuscitation, which can be applied to the detection of harmful substances in food and water quality.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a freezing and resuscitation method of microorganisms for monitoring toxicity and an application thereof in food and water quality monitoring. Background Art

[0002] Vibrio harveyi is a luminous Gram-negative bacterium in the ocean. The bacteria are arc-shaped or short rod-shaped, with lateral flagella, and are halophilic. Vibrio harveyi can survive independently in aquatic environments and can also serve as a normal parasite of marine animals. Vibrio harveyi does not grow in salt-free culture media, but grows well on salt-containing nutrient agar plates. When grown on TCBS plates, the colonies are yellow and round, and opaque; on ordinary agar media, they are off-white and round. It is widely distributed in aquatic environments and is an important pathogen that causes marine bioluminescent vibriosis.

[0003] The luminescent bacteria method used in environmental monitoring uses a sensitive photoelectric measurement system to measure the effect of poisons on the luminescence intensity of luminescent bacteria. The concentration of the poison is linearly negatively correlated with the luminescence intensity of the bacteria, that is, the stronger the toxicity of the toxic substance, the more obvious the luminescence inhibition. Marine luminescent bacteria are used in marine ecological environment monitoring with the characteristics of low cost, simplicity and convenience, and have attracted much attention from scientific researchers and have developed rapidly in recent years. Marine luminescent bacteria can be divided into five categories according to the degree of similarity of their DNA, including Vibrio harveyi, Vibrio fischeri, Photobacterium, Vibrio hanedi and Vibrio vulnificus. In the application of marine luminescent bacteria at home and abroad, Photobacterium luminescens has been studied the most in environmental pollution detection, while other strains have been less studied. There are differences in biological properties between different strains, and the conditions for cultivation and rejuvenation are very different, so it is difficult to have a reference role.

[0004] The luminescent bacteria in the luminescent bacteria method usually appear in the form of bacterial liquid and freeze-dried powder in monitoring. The preservation of bacterial liquid is difficult, and when conducting detection, the luminescent bacteria are generally centrifuged to remove the culture medium before use, which is time-consuming, and the centrifugation process will have a certain impact on the activity of the bacteria. Freeze-dried powder is made after low-temperature drying. It can be transported for a short time at room temperature and has a long shelf life. It can be stored for 3-9 months at -20°C, but it has defects such as weak luminescence stability after recovery and decreased luminescence efficiency. The freeze-dried powder of luminescent bacteria has higher requirements than the freeze-dried powder of general strains, and needs to have the following characteristics: low strain mortality and high vitality; can be quickly revived to a high vitality state; the components in the resuscitation solution are as simple and controllable as possible to avoid affecting the test results.

[0005] How to freeze and activate the luminescent bacteria is a key step in the determination. For the method of using relative luminescence intensity to detect the toxicity of substances, the requirements for luminescence intensity are relatively strict. It is necessary to ensure that the luminescence intensity remains relatively stable within a certain period of time and to extend its service life as much as possible. At present, there are few studies on the freezing and activation of Vibrio harveyi, and only a few literatures report on the cultivation of Vibrio harveyi.

[0006] Reference 1: Research on screening the optimal culture medium for luminescent marine bacteria, Wu Zirong et al., Journal of East China Normal University; five luminescent bacteria including Photobacterium luminescens and Vibrio harveyi were cultured under different culture medium conditions with oscillation, and samples were taken at different times of culture to measure the luminescence intensity; the experimental results showed that each of the five luminescent bacteria tested had a culture medium that was most favorable for their own luminescence, and the requirements for the composition of the culture medium were also different.

[0007] Reference 2: Screening and control effect study of Listeria monocytogenes quorum sensing inhibitors, Wu Yaqian, Guangdong Ocean University, cultured Vibrio harveyi in LB liquid culture medium at 37℃ shaking incubator.

[0008] Document 3: CN110590918A discloses that Vibrio harveyi ATCC BB-170 is inoculated into LB broth medium at a 1% inoculum amount (v / v), cultured at 30°C for 24 hours, and after two passages, inoculated into LB broth medium at a 1.0% inoculum amount (v / v), cultured at 30°C for 12 hours to achieve cell proliferation culture.

[0009] However, the above literature does not disclose the effects of specific freezing and resuscitation methods on the proliferation activity and luminescence intensity of the strain Vibrio harveyi. There are some reports on the frozen powder of other luminous bacteria in the prior art. For example:

[0010] Document 4: Research on the rapid detection method of water quality toxicity of luminescent bacteria based on the quorum sensing system, Wang Lin, master's thesis of Chongqing University, records: In order to improve the recovery effect of Vibrio fischeri freeze-dried powder, the composite cryoprotectant of freeze-dried powder was studied, and it was found that the composite cryoprotectant made of 15% skim milk, 5% glycerol, and 12.5% ​​trehalose dissolved in water can effectively enable the bacteria to survive after the freeze-drying process. Under the protection of this cryoprotectant, the freeze-dried powder can be stored at -20℃ for at least 6 months; the recovery time is shortened from the traditional 15-30min to 5min, greatly improving the monitoring efficiency. However, there are differences in physical and chemical properties between different strains. The inventor applied the composite cryoprotectant to Vibrio harveyi, and its freeze-dried powder can be stored at -20℃ for only 3 months, and the survival rate of the recovered cells is only 40-50%, and the luminescent activity recovery time takes more than 20min, which cannot meet the actual needs.

[0011] Document 5: CN 111979140 A discloses that a luminescent bacillus solution and a freeze-drying protective agent (weight parts: 10% skim milk powder, 8% sucrose, 1% sodium glutamate, and the solvent is sterile water) are mixed in a volume ratio of 1:1 and placed in a vial; the mixture is cooled to -70°C within 60 minutes, frozen for 12 hours, and then placed at -40°C for vacuum freeze drying to obtain a freeze-dried powder, which is finally stored at -20°C. However, when the above freezing method is used to freeze Vibrio harveyi, the freeze-dried powder can be stored at -20°C for less than 6 months, and the survival rate of the revived cells is about 50%, which also cannot meet the requirements of industrial application. Summary of the invention

[0012] In view of the shortcomings of existing technologies, it is necessary to develop a specific freezing method for Vibrio harveyi in order to obtain freeze-dried powder with a long storage period, stable performance, and rapid recovery, which can be used in the detection of harmful substances in food and water quality.

[0013] The present invention is achieved through the following technical solutions.

[0014] A method for freezing and thawing microorganisms for monitoring toxicity comprises the following steps:

[0015] 1) After activation, the Vibrio harveyi strain was inoculated into LB liquid medium and cultured to a density of (1-10)×10 8 cfu / ml, then centrifuge in a high-speed centrifuge, remove the supernatant, collect the bacteria, add freezing solution at a volume ratio of 1-2:1-2, mix well, and dispense into freezing tubes, 1 ml per tube, pre-freeze at -50°C for 0.5-2h, put into a vacuum freeze dryer, dry for 1-10h to obtain lyophilized powder, seal the tube, and store at -20°C for a long time;

[0016] 2) Add 5-10 ml of NaCl aqueous solution to each vial of freeze-dried powder and shake gently to completely dissolve and revive it.

[0017] As a specific embodiment,

[0018] The freezing solution comprises skim milk, urea and sodium hyaluronate, and the solvent is water.

[0019] In a more specific embodiment,

[0020] The freezing solution includes 10% skim milk + 5% urea + 5% sodium hyaluronate, the above being the mass ratio, and the solvent is water.

[0021] In a preferred embodiment,

[0022] The components of the freezing solution are: 10% skim milk + 5% urea + 5% sodium hyaluronate, the above is the mass ratio, and the solvent is water.

[0023] In a more preferred embodiment,

[0024] The freezing solution comprises the following components: 10% skim milk + 5% urea + 5% sodium hyaluronate + 0-0.01% lanthanum nitrate, the above being the mass ratios, and the solvent being water.

[0025] In a more preferred embodiment,

[0026] The components of the freezing solution are: 10% skim milk + 5% urea + 5% sodium hyaluronate + 0.005% lanthanum nitrate, the above ratios are by mass, and the solvent is water.

[0027] In the most preferred embodiment,

[0028] The freezing solution comprises the following components: 10% skim milk + 5% urea + 5% sodium hyaluronate + 0.01% lanthanum nitrate, the above being the mass ratio, and the solvent being water.

[0029] As an important embodiment of the present invention, a freezing solution is disclosed, the specific components of which are: 10% skim milk + 5% urea + 5% sodium hyaluronate + 0.01% lanthanum nitrate, the above being the mass ratio, and the solvent being water.

[0030] As another inventive point of the present invention,

[0031] The present invention also seeks to protect the application of any one of the above-mentioned methods in food water quality monitoring.

[0032] The present invention also claims to protect the use of the above-mentioned freezing solution in the freezing of Vibrio harveyi. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Effect of lanthanum nitrate addition on luminescence intensity. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be described more clearly and completely below in conjunction with the specific embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this invention.

[0035] Example 1

[0036] The formula of LB medium is as follows:

[0037] Tryptone 10g / L

[0038] Yeast extract 5g / L

[0039] Sodium chloride (NaCl) 10g / L

[0040] The pH of the medium was adjusted to 7.5 with NaOH.

[0041] The strain used in the experiment is Vibrio harveyi ATCC 33843.

[0042] After the strain was activated, it was inoculated into LB liquid medium and cultured at 25°C and 180 rpm until the density reached 5×10 8 cfu / ml, then centrifuge at 6000rpm for 3min in a high-speed centrifuge, remove the supernatant, collect the bacteria, add freezing solution in a 1:1 volume ratio, mix well, and dispense into freezing tubes, 1ml per tube, pre-freeze at -50℃ for 1h, then put into a vacuum freeze dryer, dry for 5h to obtain freeze-dried powder, seal the tube, and store at -20℃ for 6 months.

[0043] Resuscitation of freeze-dried powder: Add 10mL of 2% (w / v) NaCl solution to each freeze-dried powder and shake gently to dissolve it completely. After 5 minutes, test the recovery rate, recovery rate = (number of viable bacteria after recovery / number of viable bacteria before freeze-drying) × 100%. The same batch of bacterial liquid was freeze-dried using different freezing solutions (the solvent was water) for comparability. The number of viable bacteria before freeze-drying was controlled at 10.6×10 8 cfu / ml.

[0044] 1. Select the components of the refrigerant and test the influence of a single component.

[0045] Table 1

[0046] Protective agent type Mass fraction % Recovery rate% Skim milk 1 13.7 Skim milk 5 19.8 Skim milk 10 21.3 Trehalose 1 23.6 Trehalose 5 27.1 Trehalose 10 16.4 glycerin 1 7.2 glycerin 5 16.9 glycerin 10 11.5 sucrose 1 13.7 sucrose 5 25.0 sucrose 10 27.1 Mannitol 1 4.1 Mannitol 5 9.0 Mannitol 10 8.4 Urea 1 6.2 Urea 2.5 11.3 Urea 5 13.8 Sodium Hyaluronate 1 9.9 Sodium Hyaluronate 2.5 15.4 Sodium Hyaluronate 5 17.8

[0047] As shown in Table 1 , the protective effects of mannitol and glycerol as single components were relatively poor and were no longer selected for subsequent experiments.

[0048] 2. Select five components and concentrations of skim milk, trehalose, sucrose, urea, and sodium hyaluronate from Table 1 to combine and explore the best protection solution.

[0049] Table 2

[0050]

[0051] Since skim milk contains a large amount of macromolecular protein substances, it can effectively protect bacteria and is a basic substance commonly used in freezing solutions. As shown in Table 2, the protective efficacy of skim milk 10% + urea 5% + sodium hyaluronate 5% can reach 95.5%, the protective efficacy of skim milk 10% + sucrose 10% + urea 5% + sodium hyaluronate 5% is 96.1%, and the protective efficacy of skim milk 10% + trehalose 5% + urea 5% + sodium hyaluronate 5% is 96.8%. The protective effects of the three groups are significantly better than those of other groups. Comparing groups 3-5, groups 4 and 5 added a certain proportion of sucrose and trehalose, which had little effect on the protective efficacy and had little practical application significance. In addition, in order to ensure the accuracy of the subsequent luminescence monitoring results, the components in the freeze-dried powder should be as simple and controllable as possible to avoid affecting the test results. It is not recommended to add sucrose and trehalose.

[0052] 3. The effect of adding trace lanthanum nitrate to the freezing solution on the luminescence intensity of Vibrio harveyi. The relative luminescence intensity of the revived Vibrio harveyi was detected using RS9901 bioluminescence instrument (Shanghai Rongsheng Biology).

[0053] 0, 0.005, 0.01, and 0.02 (mass %) lanthanum nitrate were added to the formula of skim milk 10% + urea 5% + sodium hyaluronate 5%, and the relative luminescence intensity was monitored at different time points. Figure 1 As shown in the figure, 0.01% lanthanum nitrate can restore the luminescence intensity of Vibrio harveyi within 5 minutes, achieving the purpose of rapid detection, while the experimental group without lanthanum nitrate recovery is slow, and it can only reach a stable luminescence intensity after 20 minutes. 0.005% restores the luminescence intensity of Vibrio harveyi after 10 minutes, and basically remains stable; 0.02% added group recovery is slow, and it can only reach a stable luminescence intensity after 20 minutes. The possible reason is that high concentration of lanthanum nitrate has an instantaneous inhibitory effect on Vibrio harveyi, which can only be relieved after a period of time. Analysis of the above possible reasons: lanthanum nitrate quickly stimulates Vibrio harveyi to restore normal metabolism, increases the expression level of catalase, degrades hydrogen peroxide produced in the metabolic process into oxygen and water, and avoids inhibition of bacterial activity; and it may also stimulate Vibrio harveyi to produce and release specific signal stimulating molecules, enhance the induction between bacteria, and make the bacteria quickly restore vitality.

[0054] In addition, we also applied lanthanum nitrate to the freezing solution of luminous bacteria such as Photobacterium luminescens, Vibrio fischeri and Photobacterium brevis. We did not observe that lanthanum nitrate had a positive promoting effect on the rapid recovery of the luminous ability of the above-mentioned luminous bacteria. The specific mechanism is still unclear and needs further study.

[0055] Although the above describes the specific implementation of the present invention in combination with the embodiments, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for freezing and resuscitating the toxicity-monitoring microorganism Vibrio harveyi, characterized in that: The method comprises the following steps: 1) After activation, Vibrio harveyi was inoculated into LB liquid medium and cultured to a density of (1-10)×10 8 cfu / ml, then centrifuge in a high-speed centrifuge, remove the supernatant, collect the bacteria, add freezing solution at a volume ratio of 1-2:1-2, mix well, and dispense into freezing tubes, 1 ml per tube, pre-freeze at -50°C for 0.5-2h, put into a vacuum freeze dryer, dry for 1-10h to obtain lyophilized powder, seal the tube, and store at -20°C for a long time; 2) Add 5-10 ml of NaCl aqueous solution to each vial of freeze-dried powder and shake gently to completely dissolve and revive it; The freezing solution components include: 10% skim milk + 5% urea + 5% sodium hyaluronate, the above is the mass ratio, and the solvent is water.

2. The method according to claim 1, characterized in that The components of the freezing solution are: 10% skim milk + 5% urea + 5% sodium hyaluronate, the above is the mass ratio, and the solvent is water.

3. The method according to claim 1, characterized in that The freezing solution comprises the following components: 10% skim milk + 5% urea + 5% sodium hyaluronate + 0.01% lanthanum nitrate, the above being the mass ratio, and the solvent being water.

4. A freezing solution for Vibrio harveyi, characterized in that: The freezing solution comprises the following components: 10% skim milk + 5% urea + 5% sodium hyaluronate + 0.01% lanthanum nitrate, the above being the mass ratio, and the solvent being water.

5. Use of the freezing solution according to claim 4 in freezing Vibrio harveyi.

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