The effect of X-ray irradiation on volatile profile of Robiola cheese

TThe effect of X-ray irradiation on volatile profile of Robiola cheese Rosalia Zianni1, Michele Tomaiuolo1, Maria Campaniello1, Annalisa Mentana1, Marco Iammarino1, Diego Centonze2, Carmen Palermo3 1 Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Laboratorio Nazionale di Riferimento per il trattamento degli alimenti e dei loro ingredienti con radiazioni ionizzanti, Via Manfredonia, 20 - 71121 Foggia, Italy 2 Università di Foggia, Dipartimento di Scienze Mediche e Chirurgiche, Via Napoli 25 - 71122 Foggia, Italy 3 Università di Foggia, Dipartimento di Medicina Clinica e Sperimentale, Via Napoli 25 - 71122 Foggia, Italy Summary: HS-SPME/GC-MS conditions, optimized by means of experimental design, were used to analyze the volatile profile of Robiola cheese irradiated by X-ray at different dose levels. The evaluation of dose levels over time, performed using a chemometric approach, showed that volatile profiles of irradiated Robiola were more stable than non-irradiated. Keywords: HS-SPME/GC-MS, Chemometric analysis, Food irradiation 1 Introduction X-ray is a non-thermal technology that is applied to foods to remove and reduce pathogens, improving hygiene and promoting shelf-life extension [1]. Irradiation induces chemical transformations in the composition of volatile organic compounds (VOCs) by primary and secondary radiolysis effects [2]. In this study, X-ray irradiation was applied to Robiola cheese produced with cow milk and the modifications in volatile profiles were investigated. Furthermore, the assessment of dose levels over time, was evaluated during the shelf life on the label and beyond, to study the stability of treated cheese through VOCs analysis. Headspace solid-phase microextraction (HS-SPME) technique coupled with gas chromatography mass (GC-MS) was used to extract, isolate and enrich the volatile fraction from the sample matrix. Composite Experimental Design (CCD) and Response Surface Methodology were used for the optimization of the HS-SPME method. Three parameters were selected as independent variables: extraction temperature, extraction time and sample amount. The influence of key parameters was evaluated on the total area and total number of VOCs. The optimized HS-SPME conditions were used to analyze non-irradiated (NI) and X-ray irradiated Robiola samples. The collected data were elaborated by Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) to discriminate the potential variation of volatile profiles induced by irradiation treatment and/or shelf-life. 2 Experimental The X-ray radiation treatment was performed using a low-energy X-ray irradiator (RS-2400, Radsource Inc., Texas, USA). VOCs were analyzed using a 6890N gas chromatograph (Little Falls, DE, USA) coupled by an Agilent 5975 mass selective detector, equipped with a Gerstel MPS auto-sampler (Gerstel, Baltimore, MD, USA). A CCD was implemented considering the selected independent variables assessing them at three different experimental levels. Samples of Robiola packed in plastic boxes of 100 g were exposed to irradiation. X-ray irradiated samples at three dose levels of 1.0, 2.0 and 3.0 kGy and NI samples were analyzed in the optimized HS-SPME conditions. All samples were analyzed at one month before expiration (T0), at expiration (T1) and one month after expiration (T2). Specifically, for irradiated samples, T0, T1 and T2 also corresponded immediately, one month and two months after irradiation treatment. Chemometric analyses including PCA and PLS-DA were performed using free software R version 4.1.1. 3 Results The optimum HS-SPME conditions were estimated by means of the desirability function method, used for simultaneous optimization of the multiple responses. The optimized combination of extraction temperature, extraction time and sample amount, corresponded to the following values: 30 °C, 75 min and 4.8 g. A total of 12 classes of compounds, including alcohols, aldehydes, aromatic compounds, branched-chain alkanes, alkenes, free fatty acids, esters, ketones, methyl esters, oxygen-containing compounds, straight-chain alkanes, and sulphur-containing compounds were identified. PCA was carried out to discriminate the variation of volatile profile between X-ray irradiated and non-irradiated samples, also evaluating the influence of dose level and time effect. The score plot of PCA, reported in Figure 1, shows how three different and separated clouds formed for NI samples, for each time level (T0, T1, T2). For the irradiated samples the separation for time level (T0, T1, T2) is less evident. This means that irradiated samples resulted stable in the time with NI samples, also two months after the treatment and beyond expiration data. The Variable Importance in Projection (VIP) was used for identifying and assessing the discriminant volatiles from the volatolomic dataset [3]. The selected VOCs were used for PLS-DA and this discriminant approach was applied in double cross validation scheme [3]. The results highlighted the strong discriminating capacity of the PLS-DA algorithm in distinguishing irradiated samples from non-irradiated ones, also considering time influence. 4 Conclusions The effect of HS-SPME parameters in terms of extraction temperature, extraction time and sample amount were optimized to obtain the maximum total area and number of VOCs of X-ray irradiated Robiola samples. The best parameters of HS-SPME were used to investigate NI samples and the irradiated ones at three different dose levels. VOCs profiles were studied considering the dose levels over time and the expiration date. Some classes of compounds as alkenes, esters, ketones, methyl esters, straight-chain alkanes, and sulphur-containing compounds increased in irradiated samples, due to oxidation mechanisms induced by the irradiation treatment, as already reported for other matrices [2,4]. X-ray irradiated Robiola showed a different volatile profile than NI samples, but minor changes were also highlighted even after the expiration date and after two months of treatment. X-ray irradiate Robiola had more rich and stable VOCs composition for a long time. Instead, the NI samples reported a different VOCs composition in relation to storage time, a symptom of degradation after shelf life. The classification by PLS-DA as a discriminant approach was effective in the indication of irradiation treatment and the dose levels over time too. Fig. 1. PCA score plots of the VOCs identified in non-irradiated (A) and in irradiated samples (B) at T0 (blue), T1 (red) and T2 (green). References 1. Z.B. Zehi et al.; Current Pharmaceutical Biotechnology 21, 10 (2020), pp 919-926. 2. R. Zianni et al.; Food Chemistry 423 (2023), pp 136239. 3. E. Szymańska et al., Metabolomics, 8 (2012), pp 3-16. 4. U. Bliznyuk et al.; Separations 9, 227 (2022), pp 1-10. This work is supported by funding from the Ministero della Salute, Project code GR-2018-12367064.