Challenges and Opportunities for Micro-Extraction Techniques to Accurately Monitor Cheese Volatilome Evolution During Ripening and Promptly Detect Spoilage

Food quality has molecular foundation in the food metabolome (i.e., the complete set of primary and specialized metabolites together with volatiles generated by endogenous or exogenous phenomena – i.e., the volatilome). Modern “omics” disciplines, and their investigation approaches, have a great potential in the context of food quality objectification offering the possibility to define pattern of markers with strong correlation to specific phenomena. Moreover, food quality is a dynamic characteristic that relates firstly to primary ingredients composition and stability, and evolves along processing stages and during shelf-life/storage. The contribution focuses on sorptive-based (micro-)extraction strategies capable of capturing cheese volatilome during ripening with emphasis on those components strongly correlated to positive sensory attributes (i.e., key-aroma compounds and potent odorants) and to spoilage induced by bacteria and yeasts grow. The challenging aspect of on-site sampling at ripening cellars is further complicated by the need to work in sterile conditions to avoid contamination and ensure cheese microbial integrity. Cheese samples at 90-days ripening stage were made available by a local producer (i.e., Beppino Occelli Agrinatura S.r.l.) from Cuneo Province, Italy. Cheese samples were divided in two classes: “good” and “spoiled” as resulted by farmer inspection before sampling session. Volatiles and semi-volatiles sampling was therefore conducted in parallel on the surface of good and spoiled cheese wheels directly in cellars at 10°C (± 2°C) by static headspace with conventional solid phase micro extraction devices – HS-SPME and different polymers/materials (e.g., PDMS, CAR/PDMS/DVB, PDMS/DVB) or by SPME arrows™ (Restek Srl, Cernusco, Milan, Italy). On site sampling is illustrated in Figure 1A. Direct contact sampling was also done on cheese surface by sorptive-tape extraction – STE with PDMS tapes. Volatiles were then analyzed by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-ToF MS). The contour plot of a good cheese sample volatilome is shown in Figure 1B with some enlarged areas in Fig. 1C and 1D. Spoilage fingerprint is dominated by several aromatic compounds likely correlated with aerobic bacteria metabolism; other informative classes with an up-regulation in the spoiled cheese volatilome are primary and secondary alcohols, linear aldehydes and hydroxylated short-chain fatty acids. The higher amount of sorbent/adsorbent material in SPME arrow™ enables a further enrichment of semi-volatiles while with STE with PDMS sorptive material highly-lipophilic compounds (e.g., higher LogP values) achieve higher concentration factors (CFs). The validation of spoilage markers was conducted by sampling cheese portions in a laboratory environment by HS-SPME with optimized parameters (HS volume, sampling temperature and time). Results confirmed that on-site sampling in sterile conditions can effectively detect spoilage volatiles patterns and by combining complementary approaches (HS and direct contact) with different sorptive materials, ripening and spoilage phenomena are more comprehensively explained.