Identification of the Peptide PyroQ-βCasein_194–209 as a Highly Specific and Sensitive Marker to Differentiate between Ultrahigh-Temperature Processed (UHT) Milk and Mildly Heated Milk

Abstract

In this study, a new approach was introduced to identify marker peptides that reflect the thermal treatment of commercial milk samples and differentiate ultrahigh-temperature processed (UHT) milk from mildly heated milk. Peptide profiles of training set samples, pasteurized (n = 20), extended shelf life (n = 29), and UHT (n = 29) milk, were recorded by MALDI-TOF-MS after StageTip microextraction. As marker candidates, 13 peptides were selected, and their cutoff levels were defined. The quality of the cutoff levels was then tested with a blind test set. Thus, the peptide m/z 1701.0, which was identified as pyroQ-βcasein, could ideally differentiate UHT milk from mildly heated milk with an accuracy of 100%. Due to its high reliability and sensitivity, this peptide may be applied in routine analysis to monitor thermal processing of milk. An additional heating experiment showed that the marker peptide candidates are formed during milk processing by endogenous enzymes and selective thermal cleavage.

Design of a Prediction Model for the Differentiation of Pasteurized Milk from Heated ESL Milk by Peptide Profiling

“…All milk samples were immediately refrigerated after purchase, defatted by centrifugation (twice at 1100 × g and 4 °C for 60 min) and removal of the upper fat layer, aliquoted and frozen at −80 °C prior to analysis. Possible residual fat traces were removed during the subsequent extraction of the milk peptide fraction by stop‐and‐go‐extraction tips (StageTip) according to Dalabasmaz et al . and did not interfere with peptide analysis.…”

“…Peptide profiling by MALDI‐TOF‐MS and relative quantification of the MALDI spectra based on the internal standard peptide m / z 1589.9 were carried out as reported previously by Dalabasmaz et al . Briefly, the matrix consisted of 4‐chloro‐α‐cyanocinnamic acid and the samples were spotted by the sandwich method.…”

“…For peptide sequencing, fragmentation spectra were obtained by enhanced product ion (EPI) scans with a scan rate of 10 000 Da s −1 , with scan range and CE depending on the peptide mass of the precursor ion. Calculation of CE and de novo sequencing of the marker peptides was performed as previously described . At least three EPI experiments were run simultaneously using three different CE values (CE −5, CE, CE +5) and the best fragmentation spectra were selected for peptide sequencing.…”

“…Both the food industry and public health authorities require reliable and sensitive analytical methods to control the production and monitor quality and authenticity of processed milk. In a previous study, UHT could be differentiated from ESL/pasteurized milk by one single peptide marker, whereas the differentiation of ESL and traditionally pasteurized milk was more challenging. The purpose of the present study was to define marker peptides reflecting even minimal differences in the heat load, thus enabling the differentiation of pasteurized milk from ESL milk.…”

“…These processes can lead to the release or degradation of peptides. Accordingly, specific peptides were suggested as markers reflecting the heat load of different milk types, that is, raw, pasteurized, UHT, and powdered milk 13a,17a,18 . In contrast to traditional methods, milk authentication by peptide profile analysis has the potential to characterize different parameters in one run, such as species, heat treatment, or fermentation procedure.…”