Validation and application of a standardized procedure for evaluating freshness of Citrus juices based on pectin methylesterase activity quantitation

A method developed for freshness authentication of freshly squeezed Citrus juices (FSCJ) was evaluated for routine application. It involved titrimetric assessment of pectin methylesterase (PE) activity after enzyme extraction from pulp-standardized juice samples. Standard test conditions enabled reliable discrimination between FSCJ and chilled Citrus juices that had comparative advantages due to extended shelf life. Unlike the latter, FSCJ always displayed PE activities in the linear range between the limit of identification (LOI, 0.42 units g-1 of juice) and the maximum activity found for FSCJ (1.94 units -1), equivalent to 0.0035-0.016 units during titration. However, for model samples having activities < LOI due to production by respective dilution of FSCJ, the responses abruptly fell to unspecific levels below the limit of detection (LOD, 0.21 units g-1). Accuracy was substantiated by 100 -106 % recovery for model juices with PE activities of 0.87 -1.22 units g-1 resulting from FSCJ dilution or PE standard addition, but it was lower (76 - 80 %) near LOI. The average of the mean activities, which were detected by 3 analysts with intraassay precision ≤ 8.4 %, varied with relative standard deviations of 8.2 % for FSCJ and 3.9 % for a sample of the same juice diluted to 60 % (w/w), thus proving reproducibility. FSCJ batches were unambiguously distinguished from four commercial chilled juices, because the activities detected for the latter were by far ≤ LOD and thus confirmed labeled mild preservation.