MODELING THE EFFECTS OF FREEZING RATES, STORAGE TEMPERATURES AND TIMES ON INACTIVATION OF Vibrio vulnificus
Vibrio vulnificus is a naturally occurring bacterium that is commonly found in raw oysters. When ingested, it can cause fatal septicemia in immune-compromised individuals or in patients with liver disease. Due to consumer preference for raw oysters, post-harvest treatments that reduce this pathogen while preserving the raw oyster’s sensory characteristics are very important. Freezing of oysters can reduce the levels of Vibrio vulnificus and increase their shelf life to more than 12 weeks when stored at -20˚C.
The objective of this study was to develop predictive kinetic models to describe the inactivation of pure cultures of Vibrio vulnificus by freezing temperatures and rates, frozen storage temperatures, and frozen storage times.
In this study, Vibrio vulnificus was diluted in phosphate buffered saline (PBS) to obtain approximately 107 colonies/ml of the bacterium. Three sets of vials were frozen at -10°C, -35°C, and -80°C, and stored at -10°C. Survival of Vibrio vulnificus was followed after freezing and storage at 0, 3, 6 and 9 days. Two other sets were frozen and stored at -35°C and -80°C, and survival was followed after freezing and every week for six weeks of frozen storage. For every treatment, real time-temperature data was obtained by placing thermocouples in blank vials. The predictive model was developed by analyzing the inactivation kinetics with first order, Weibull, and Peleg inactivation models.
Results from the three different freezing temperatures showed that their effect on survival of Vibrio vulnificus is not significantly different (a= 0.05), and on average caused a 1.6 log10 reduction. Frozen storage temperatures had a larger effect on the survival of Vibrio vulnificus. After one week of storage, samples stored at -35°C and -80°C showed 0.06 and 1.10 log10 reductions from the initial storage counts respectively. On the other hand, samples stored at -10°C showed slightly more than 3 log10 reduction regardless of initial freezing temperature.
When analyzing the overall effect of the freezing treatments, an additional 0.75 log reduction was caused by the change of storage temperature, as in the case of samples frozen at -35°C and -80°C that were transferred to storage at -10°C. The combined effect of freezing and one week frozen storage resulted in 1.5, 2.6 and 4.9 log reductions for samples frozen and stored at -80°C, -35°C and -10°C, respectively. The combined effects of freezing, change of storage temperature, and one week of frozen storage at -10°C resulted in 5.94 and 5.6 log reductions for samples frozen at -35°C and -80°C, respectively. Statistical analysis of these results showed that all values were significantly different from each other, except the two that were transferred from -80°C and -35°C to -10°C. These results suggest that inactivation of Vibrio vulnificus may be caused by intracellular formation ice crystals and their subsequent growth (re-crystallization) during frozen storage.
Analysis of kinetic data showed that storage temperature is the critical parameter in survival of Vibrio vulnificus. As recognized by several authors, first order kinetics is not suited for describing the reduction of most bacterial populations, and certainly not Vibrio vulnificus during frozen storage. Peleg inactivation kinetics also failed to describe adequately the Vibrio vulnificus inactivation, regardless of trying to fit the non-temperature dependent n-value to temperature. An adaptation of the Weibull model resulted in a successful description of the survival curves during frozen storage, by fitting the β parameter to temperature through a quadratic equation. A constant describing the inactivation during freezing was added to the mentioned model. The equation was written in the form of: log10Nt = log No – 1.22 – {[ t / 10^(-1.163-0.0466 T)] ^ (0.00025 T2 + 0.01524 T + 0.49325)}.
In conclusion, while the freezing process results in a reduction of Vibrio vulnificus, the rate of inactivation depends on the storage temperature.