Effects of wet heat treatment on the germination of individual spores of Clostridium perfringens.

摘要

Aim: To analyse the effect of wet heat treatment on nutrient and non-nutrient germination of individual spores of Clostridium perfringens. Methods and Results: Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat-treated spores of Cl. perfringens with various germinants. When incubated in water at 90-100掳C for 10-30 min, more than 90% of spores were inactivated but 50-80% retained their Ca 2+-dipicolinic acid (CaDPA). The wet heat-treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640-1680 cm -1 (amide I) and 1230-1340 cm -1 (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat-treated spores that retained CaDPA germinated with KCl or l-asparagine, but wet heat treatment increased values of Tlag, Delta Trelease and Delta Tlys, during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl. perfringens spores lacking the essential cortex-lytic enzyme (CLE), SleC, exhibited longer Tlag and Delta Trelease values during KCl germination than wild-type spores and germinated poorly with CaDPA. Wet heat-treated wild-type spores germinating with CaDPA or dodecylamine exhibited increased Tlag, Delta Trelease and Delta Tlys values, as did wet heat-treated sleC spores germinating with dodecylamine. Conclusions: (i) Some proteins important in Cl. perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat. Significance and Impact of the Study: This study provides information on the germination of individual Cl. perfringens spores and improves the understanding of effects of wet heat treatment on spores.