SYBR Green Real-Time PCR Assay for Detection of the Indicator Bacillus cereus to Validate Thermal Processing of Tender Coconut Water

Abstract

The purpose of this study is to establish thermal processing validation of tender coconut water by using SYBR green Real time PCR assay for direct detection of the biological indicator bacteria, B. cereus, a frequent cross contaminant in packaged tender coconut water. Nucleotide sequences coding for the hemolytic enterotoxin hemolysin BL (Hbl) widely associated with B. cereus food poisoning was used as the target gene for the real time PCR assay. Specific amplification was observed with B. cereus when the specificity of the assay was examined using other closely related spore forming food spoilage Bacilli. The present real-time PCR assay provides a detection of limit (LOD) of 103 CFU/ml of tender coconut water. The thermal resistance of B. cereus (ATCC 14579) was found that D95 2.46 min and Z value 10.45°C in tender coconut water. To validate the thermal processing conditions, B. cereus was artificially contaminated into coconut water, which was thermally processed and checked with real-time PCR assay to detect the survival of B. cereus in tender coconut water bottles without pre-enrichment. The bacterial load enumerated by the real-time PCR assay correlated with the time consuming standard conventional culture method. The thermal processing of tender coconut water packed in polypropylene bottles was validated with 6D concept and minimum 18 min of process time is required to achieve sterilization value (F-value) of 15. The product was analyzed for its physical, chemical properties and commercial sterility after the validated processing conditions and products were acceptable for 6 months without preservative