$Pyrococcus$ $furiosus$ amylase as a Candidate Sterilisation Time-Temperature Integrator

Abstract

Thermal treatment is the most common method used by industry to ensure food is safe for consumption and to increase storage life. To ensure safety, food is often over processed, which can significantly affect its nutritional value as well as taste and flavour attributes. In this study a candidate sterilisation time-temperature integrator (TTI) from the hyperthermophilic Pyrococcus furiosus $\alpha$amylase is investigated. Reliability and accuracy of the TTIs was determined by exposure to various isothermal and non-isothermal industrially relevant temperature profiles. The integrated temperature history obtained by the TTIs correlated generally well with the data obtained from thermocouples installed, although the error increased with hold time of heat treatment. The work showed that the TTIs can be used reliably over a range (3-25 minutes at 121°C) which is relevant for conditions of thermal sterilisation. This was measured by developing a new assay technique for assaying the activity of hyperthermophilic $\alpha$-amylase within the food industry. The assay was calibrated against more laboratory relevant assays and computational models. The kinetics and mechanism of thermal denaturation of Pyrococcus furiosus $\alpha$-amylase was determined through FT-IR, DSC and CD techniques. It was found that through thermal denaturation after the melting temperature (Tm), the enzyme unfolded by first order kinetics from a $\alpha$-helical structure, through $\alpha$-sheet structure to aggregation of the enzyme