Project's information

Project's title Isolation and selection of bacteriocin-producing bacteria from marine environment showing anti-Vibrio activity for aquaculture application
Project’s code VAST02.05/20-21
Research hosting institution Nha Trang Institute of Technology Research and Application
Project leader’s name Huynh Hoang Nhu Khanh, PhD
Project duration 01/01/2020 - 31/12/2021
Project’s budget 600 million VND
Classify Excellent
Goal and objectives of the project

The study aims to obtain some marine bacteria showing the antiVibrio activity and ability to produce bacteriocin and also purify and characterise 3 bacteriocins with antiVibro activity.

Main results

Theoretical results:
The isolation of marine bacteria followed by a screening of bacteria for anti-Vibrio activity resulted in a collection of 30 putative bacteria with anti-Vibrio activity. 16S rRNA sequencing of 16 out of 30 isolates confirmed all of them + to be Bacillus species with different strain variants across two sample types with diversity in species and bioactivity. Additionally, a three-step procedure was performed to purify two anti-Vibrio bacteriocins (compound BD1-HPLC-1 and compound CL3-HPLC-1) presented in the 24 hours-old culture of Bacillus NTBD1 and Bacillus NTCL3. The procedure includes (i) solvent extraction of bacteriocin from cells, (ii) hydrophobic interaction chromatography and (iii) reverse-phase HPLC. And, a two-step procedure was applied to purify an anti-Vibrio lipopeptide produced by Bacillus sp. NTBD1 after 48 hour-old cultivation, including acid precipitation, followed by methanol extraction and HPLC. Two HPLC-based compounds (BD1-HPLC-1, and CL3-HPLC-1) showed a single band on Tricine SDS PAGE, and the HPLC based BD1-HPLC-2 showed 01 peak with isoforms with a molecular weight of ~1 kDa which was identified by MALDI-TOF MS.  
Characterisation of these 3 purified compounds found high thermal stability as the anti-Vibrio activity was remained after thermal treatment at 80°C for 15 minutes, pH stability of 3 – 9.  In addition, two bacteriocins (CL3-HPLC-1 and BD1-HPLC-1) had a similar molecular weight of 2-5 kDa; while the BD1-HPLC-2 had a molecular weight of ~ 1 kDa. Additionally, these peptides had similarly broad antimicrobial spectra against a range of both Gram-negative bacteria and Gram-positive bacteria.
Applied results:
These isolated marine bacteria with anti-Vibrio activity are promising probiotic sources, and sources for extracting antimicrobial compounds for further application. In addition, three purified bacteriocins can be served as promising antimicrobial compounds and natural & safe food preservatives for application in aquaculture, the food industry and pharmaceuticals in the future.

Novelty and actuality and scientific meaningfulness of the results

The project was conducted to evaluate the bacterial communities in two sea regions (Nha Trang and Phu Quy) for anti-Vibrio activity. This study provides the evidence of sponge-associated bacteria with anti-Vibrio activity from Phu Quy sea region which has been previously unknown. In addition, this study provides the  purification and characterisation studying of bacteriocins with anti-Vibrio activity produced by marine bacteria when the majority of current studies are carried out with the only objective of evaluating the marine bacteria for antimicrobial activity.  
The project is significant because the strains with antiVibrio activity obtained from the project can be used as probiotics for aquaculture uses. Also, these three bacteriocins with significant anti-Vibrio found in this study can be used as safe antimicrobial and food preservatives for reducing antibiotic uses and developing sustainable aquaculture.

Products of the project

- Published articles: 01 article published in Microorganisms (SCI-E) and 01 article published in Vietnam Journal of Marine Science and Technology (VAST2).
- Specific products:

  • 01 set of marine bacterial strains containing 30 strains of marine microorganisms capable of bacteriocin biosynthesis, of which 16 are identified by 16s rRNA gene (Currently kept at the NhaTrang Institute of Technology Research and Application).
  • 03 Bacteriocin with purity ≥ 50% and their specification profile.

- It is necessary to continue to carry out further studies on the structural characteristics of the obtained bacteriocins, in order to orient their applications.
- Continue to optimize the process of obtaining and purifying bacteriocins from 2 strains of marine bacteria B. amyloliquefacines NTBD1 and B. flexus NTCL3 to obtain peptides with higher content for in vivo applications.
- A number of antibacterial compounds derived from marine microorganisms isolated from sponge samples need to be studied extensively, in order to search for new biologically active substances in terms of structure or function, application orientation and effectively exploit marine microbial resources.


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