Improved stability of live attenuated vaccine gdhA derivative Pasteurella multocida B:2 by freeze drying method for use as animal vaccine.

The efficacy of attenuated strain of gdhA derivative Pasteurella multocida B:2 mutant as a live vaccine to control haemorrhagic septicaemia (HS) disease in cattle and buffaloes has been demonstrated. In order to use P. multocida B:2 mutant as a commercial product, it is essential to optimise its formulation for high viability and stability of the live cells. The effectiveness of freeze-drying process using different protective agent formulations for improving cells viability was explored. Sugar and nitrogen compounds were used as protective agents in freeze-drying and the capability of these compounds in maintaining the viability of mutant P. multocida B:2 during subsequent storage was investigated. A complete loss in viability of freeze-dried mutant P. multocida B:2 was monthly observed until 6-12 months of storage at -30 °C, 4 °C and 27 °C when nitrogen compound or no protective agent was added. Trehalose and sucrose showed significantly high survival rate of 93-95% immediately after freeze-drying and the viability was retained during the subsequent storage at -30 °C and 4 °C. A smooth cell surface without any cell-wall damage was observed for the cells formulated with trehalose under scanning electron micrograph. This study presented a freeze-drying process generating a dried live attenuated vaccine formulation with high stability for commercial applications.

Evaluation of the Estimation Capability of Response Surface Methodology and Artificial Neural Network for the Optimization of Bacteriocin-Like Inhibitory Substances Production by Lactococcus lactis Gh1.

Bacteriocin-like inhibitory substances (BLIS) produced by Lactococcus lactis Gh1 had shown antimicrobial activity against Listeria monocytogenes ATCC 15313. Brain Heart Infusion (BHI) broth is used for the cultivation and enumeration of lactic acid bacteria, but there is a need to improve the current medium composition for enhancement of BLIS production, and one of the approaches is to model the optimization process and identify the most appropriate medium formulation. Response surface methodology (RSM) and artificial neural network (ANN) models were employed in this study. In medium optimization, ANN (R2 = 0.98) methodology provided better estimation point and data fitting as compared to RSM (R2 = 0.79). In ANN, the optimal medium consisted of 35.38 g/L soytone, 16 g/L fructose, 3.25 g/L sodium chloride (NaCl) and 5.40 g/L disodium phosphate (Na2HPO4). BLIS production in optimal medium (717.13 ± 0.76 AU/mL) was about 1.40-fold higher than that obtained in nonoptimised (520.56 ± 3.37 AU/mL) medium. BLIS production was further improved by about 1.18 times higher in 2 L stirred tank bioreactor (787.40 ± 1.30 AU/mL) as compared to that obtained in 250 mL shake flask (665.28 ± 14.22 AU/mL) using the optimised medium.

Growth Enhancement of Probiotic Pediococcus acidilactici by Extractive Fermentation of Lactic Acid Exploiting Anion-Exchange Resin.

Fermentation employing lactic acid bacteria (LAB) often suffers end-product inhibition which reduces the cell growth rate and the production of metabolite. The utility of adsorbent resins for in situ lactic acid removal to enhance the cultivation performance of probiotic, Pediococcus acidilactici was studied. Weak base anion-exchange resin, Amberlite IRA 67 gave the highest maximum uptake capacity of lactic acid based on Langmuir adsorption isotherm (0.996 g lactic acid/g wet resin) compared to the other tested anion-exchange resins (Amberlite IRA 410, Amberlite IRA 400, Duolite A7 and Bowex MSA). The application of Amberlite IRA 67 improved the growth of P. acidilactici about 67 times compared to the control fermentation without resin addition. Nevertheless, the in situ addition of dispersed resin in the culture created shear stress by resins collision and caused direct shear force to the cells. The growth of P. acidilactici in the integrated bioreactor-internal column system containing anion-exchange resin was further improved by 1.4 times over that obtained in the bioreactor containing dispersed resin. The improvement of the P. acidilactici growth indicated that extractive fermentation using solid phase is an effective approach for reducing by-product inhibition and increasing product titer.

Strategies for improving production performance of probiotic Pediococcus acidilactici viable cell by overcoming lactic acid inhibition.

Lactic acid bacteria are industrially important microorganisms recognized for fermentative ability mostly in their probiotic benefits as well as lactic acid production for various applications. Fermentation conditions such as concentration of initial glucose in the culture, concentration of lactic acid accumulated in the culture, types of pH control strategy, types of aeration mode and different agitation speed had influenced the cultivation performance of batch fermentation of Pediococcus acidilactici. The maximum viable cell concentration obtained in constant fed-batch fermentation at a feeding rate of 0.015 L/h was 6.1 times higher with 1.6 times reduction in lactic acid accumulation compared to batch fermentation. Anion exchange resin, IRA 67 was found to have the highest selectivity towards lactic acid compared to other components studied. Fed-batch fermentation of P. acidilactici coupled with lactic acid removal system using IRA 67 resin showed 55.5 and 9.1 times of improvement in maximum viable cell concentration compared to fermentation without resin for batch and fed-batch mode respectively. The improvement of the P. acidilactici growth in the constant fed-batch fermentation indicated the use of minimal and simple process control equipment is an effective approach for reducing by-product inhibition. Further improvement in the cultivation performance of P. acidilactici in fed-bath fermentation with in situ addition of anion-exchange resin significantly helped to enhance the growth of P. acidilactici by reducing the inhibitory effect of lactic acid and thus increasing probiotic production.