The aim of this study was to identify a specific indicator of freshness, allowing routine distinction between freshly squeezed Citrus juices (FSCJs) and FSCJ-like products having undergone mild pasteurisation, based on the quantification of the native enzyme activities pectin methylesterase (PE) and peroxidase (POD).
For the validation of the determination methods, enzyme activities were reduced by thermal treatment. Heat treatment was performed using the Actijoule unit of a tubular heater at a flow rate of 60 L/h. Freshly squeezed orange juices (FSOJs) were continuously heated on a pilot plant scale at six different temperatures (42-92 °C) for a residence time of 12 s, followed by countercurrent flow cooling to ambient temperature using tap water. The cloud stability and residual activities of PE and POD were monitored at 4 °C over a storage up to 62 days. Microbial activity was insignificant due to good hygienic practice, thus ensuring that the enzyme activities detected were of fruit origin. The juices processed at temperatures >= 62 °C were characterized by minor residual activities. When exposed to temperatures = 72 °C. Unlike POD, total PE activity proved to be an indicator of freshness with universal applicability to Citrus juices derived from orange, mandarin, and lemon or blends thereof. Freshly squeezed juices can analytically be distinguished from cold-stored, minimally processed products that dispose of an almost completely inactivated thermo-labile PE fraction, and thus extended shelf life.
The method developed for freshness authentication of FSCJs was evaluated for routine application. Standard test conditions allowed reliable discrimination between FSCJ and chilled Citrus juices that had comparative advantages due to their longer shelf life. 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 (spiking). The average of the mean activities determined by 3 analysts with intra-assay precision <= 8.4 proved reproducibility of the method. FSCJ batches were unambiguously distinguished from four commercial chilled juices, because the activities detected for the latter were by far below the limit of detection (LOD), and thus confirmed mild preservation as indicated by labelling.
Consequently, regarding analyses of samples with unknown processing history, PE was suggested as an indicator enzyme for the freshness of FSCJs, allowing their unambiguous distinction from minimally heat-processed juices.