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Preparation of two lytic vibrio harveyi bacteriophage preparations, and application thereof

A Harvey Vibrio and phage technology, applied in the field of bioengineering, can solve problems such as drug residues, water pollution, food safety, etc.

Inactive Publication Date: 2012-02-22
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] However, with the long-term improper use of disinfectants and antibiotics, many disadvantages have emerged: (1) causing pathogenic bacteria to develop drug resistance, so that the effect of antibiotics is reduced or even ineffective; (2) causing serious pollution of water bodies and causing micro-ecological imbalances in the water environment (3) Drug residues are produced in the sea cucumber body, which affects the quality and safety of the product and endangers human health
It can be seen that traditional antibiotic drugs not only have little effect, but also produce drug residues, pollute the water environment, and cause serious food safety problems

Method used

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  • Preparation of two lytic vibrio harveyi bacteriophage preparations, and application thereof
  • Preparation of two lytic vibrio harveyi bacteriophage preparations, and application thereof
  • Preparation of two lytic vibrio harveyi bacteriophage preparations, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the separation and preparation of phage

[0033] (1) Preparation of host bacteria

[0034] Take out the Vibrio Harvey which has been identified and inoculated in 2216E solid medium and stored in a refrigerator at 4°C for standby, culture overnight at 37°C, pick a single colony, inoculate in 2216E liquid medium, and culture at 37°C for 6 hours with shaking Later, it is used to isolate phage.

[0035] (2) Water samples and their treatment

[0036] Take 50ml of wastewater and pretreat the water sample: add CaCl 2 , MgCl 2 , so that the final concentration is 1mmol / L, and the effect is about 10min. The purpose is to make the phage more easily adsorbed on the surface of the pathogenic bacteria.

[0037] (3) Phage isolation

[0038] Centrifuge the above-mentioned pretreated water sample with a centrifuge at 10,000 rpm for 5 minutes to collect the supernatant; filter the supernatant through a 0.22 μm disposable filter membrane to remove bacteria, impurities ...

Embodiment 2

[0053] Embodiment 2, PEG 8000 precipitation phage particle

[0054] (1) Take 20 mL of the overnight cultured host bacteria solution, transfer it to 1 L of liquid 2216E, and culture it with shaking at 37°C until the initial logarithmic growth stage, about 12 hours;

[0055] (2) Add phage liquid so that the multiplicity of infection (the ratio of the number of infected phages to the number of host bacteria) is about 0.01, and continue shaking culture at 37°C for 24 hours, and the rotation speed is 150rpm;

[0056] (3) Centrifuge the above mixture at 10,000 rpm at 4°C for 10 min to remove bacterial debris, then measure the supernatant, add DNase I and RNase A to the final concentration of 1 μg / mL, and incubate at room temperature for 30 min;

[0057] (4) Add 29.2 g of solid NaCl to 500 ml of culture, stir to dissolve it, and then place the culture in an ice bath for 1 hour. Adding NaCl can promote the sedimentation of bacterial fragments and miscellaneous proteins, which is also ...

Embodiment 3

[0064] Embodiment 3, in vitro antibacterial experiment

[0065] The experiment was set up as 4 groups, and 4 50ml centrifuge tubes were taken and marked as A, B, C, and D respectively; A was the negative control group, B was the antibiotic group, and C was the phage group. Add 40ml 2216E liquid culture medium, 1ml bacterial solution (concentration is 10 8 PFU / ml), 1ml PBS; add 40ml 2216E liquid culture medium, 1ml bacterial solution (concentration is 10 8 PFU / ml), 1ml kanamycin sulfate (concentration is 4mg / ml); C also added 40ml 2216E liquid medium, 1ml bacterial liquid, 1ml phage suspension, respectively 500μl vp-1 and 500μl vp-2, Potency is 1×10 8 pfu / ml; D is a blank control, only 40ml of 2216E medium was added. After shaking for 10 minutes, put it into a microplate reader to measure OD 600 Record the value; put it in a shaker at 37°C and cultivate it, take it out every half hour to measure the OD 600 value and record it. by OD 600 The value is the ordinate, and the...

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Abstract

The present invention relates to preparation of two lytic vibrio harveyi bacteriophage preparations, and an application thereof. A purpose of the present invention is to reduce the number of target pathogenic bacteria in the stichopus japonicus body, the stichopus japonicus culture environment and the stichopus japonicus processing products. Host bacteria of the bacteriophage are vibrio harveyi. The two separated bacteriophages of vp-1 and vp-2 provide strong lysis for the vibrio harveyi, and provide protection effects for the stichopus japonicus and the stichopus japonicus products. The preparation of the present invention is a novel green biological disinfection preparation. According to the present invention, the bacteriophage is purified, amplified, cultured and further enriched on a large-scale; because the lytic bacteriophage specifically kills the drug-resistant vibrio harveyi, the problems of the antibiotics resistance due to abuse of antibiotics, antibiotics residue in animal bodies, and the like are prevented; the preparation of the present invention can be prepared into a spray solution or an eluting solution, and provides purification and protection effects for the sick stichopus japonicus, and the aquaculture water environment; the preparation of the present invention does not have any toxic and side-effects, such that the food-derived pollution is reduced, the security effect is provided for the food safety.

Description

technical field [0001] The invention relates to two strains of bacteriophage and the application thereof, and a method for reducing bacteria in an aquaculture system and product processing. Specifically, the present invention provides pathogenic bacteria for controlling sea cucumbers, sea cucumber culture systems (such as culture ponds, feed, etc.) and food processing of sea cucumbers, especially can significantly kill Vibrio harveyi, which belongs to field of bioengineering. Background technique [0002] However, with the long-term improper use of disinfectants and antibiotics, many disadvantages have emerged: (1) causing pathogenic bacteria to develop drug resistance, so that the effect of antibiotics is reduced or even ineffective; (2) causing serious pollution of water bodies and causing micro-ecological imbalances in the water environment (3) Drug residues are produced in the sea cucumber body, which affects the quality and safety of the product and endangers human hea...

Claims

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Application Information

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IPC IPC(8): A23K1/17A01K61/00C12N7/00C12N7/02A23K20/195
CPCY02A40/81
Inventor 徐永平谭德猛金礼吉李晓宇孙涛郭嘉楠杨冉升赵鹏飞
Owner DALIAN UNIV OF TECH
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