Optimization of C22 bacteriophage lyophilization factors using response surface methodology

Name: Nathida Manbua

Organization : King Mongkut's Institute of Technology Ladkrabang. School of Engineering

Email : 63601050@kmitl.ac.th

Name: Teeraporn Suteewong

Organization : King Mongkut's Institute of Technology Ladkrabang. School of Engineering

Email : teeraporn.su@kmitl.ac.th

Name: Udom Sae-Ueng

Organization : National Science and Technology Development Agency (NSTDA). National Center for Genetic Engineering and Biotechnology (BIOTEC)

Email : udom.sae@biotec.or.th

keyword: Response Surface Methodology

LCSH: Bacteriophages

LCSH: Lyophilization

LCSH: Plaque assay technique

Abstract: Bacteriophages (phages) are viruses that infect specific bacteria. Phages have been used to prevent pathogenic bacteria causing diseases in crops as a natural biocontrol alternative. Phages in liquid form impose practical difficulties in storage and transportation in remote agricultural areas. Phage has been preserved in dried form through lyophilization (freeze drying) to overcome these difficulties. Typically, many variations of lyophilization factors must be performed to achieve the optimal condition to retain phage infectivity quantified by phage titer. Response Surface Methodology (RSM) has been applied since it enables fewer trials, which saves resources, time, and labor. In this study, we optimized the factors involved in the lyophilization of C22 phage to maximize phage titer using RSM, specifically the Box-Behnken Design (BBD) method. C22 phage is capable of lysing Ralstonia solanacearum, causing bacterial wilt disease and, thus presenting a potential biocontrol agent. The factors studied were the concentration of sucrose, the concentration of trehalose, the ionic strength of the buffer, and the phage volume ratio, which have been demonstrated to influence phage retention during lyophilization. The response was measured by a plaque assay, which yields phage titers indicating phage survival during the lyophilization. The BBD method was used to find the optimal combination of four factors that resulted in the highest phage remaining titer. The plaque assay was performed in order to confirm the optimal condition retrieved from the BBD method. The optimal condition is as follows: 0.49 M sucrose, 0 M trehalose, 0.24 M ionic strength of the buffer, and 300 microliters of phage volume ratio. The interaction term between the trehalose concentration and phage volume ratio (BD) was more significant than the independent phage volume ratio (D). The highest remaining phage titer was 93.39%, identified with a response optimizer. The finding of this work will improve the lyophilization process, which promotes the effective utilization of phages in biocontrol

King Mongkut's University of Technology North Bangkok. Central Library

Address: BANGKOK

Email: library@kmutnb.ac.th

Created: 2022

Modified: 2022-12-13

Issued: 2022-12-13

BibliograpyCitation : In Naresuan University. The 31st Thai Institute of Chemical Engineering and Applied Chemistry Conference (TiCHE 2022) (pp.140-151). Phitsanulok : Naresuan University

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