Development of a new prototypal natural whey starter production system to study biodiversity and technological features fluctuations during back-slopping practice used in Parmigiano Reggiano PDO cheese production: penicillin G as a case study

Natural whey starter (NWS) is an undefined and variable microbial community of starter lactic acid bacteria (SLAB), traditionally produced by back-slopping and used in the manufacture of several hard-cooked Italian cheeses, including Parmigiano Reggiano PDO. Despite its importance, the parameters influencing the microbial composition and technological performance of NWS remain poorly investigated, largely due to the lack of controlled studies that do not interfere with actual cheese production. Although the Parmigiano Reggiano PDO regulations strictly forbid the use of selected starters and require NWS to be obtained exclusively through spontaneous whey fermentation following curd extraction, we developed a freeze-dried prototypical natural starter culture (NSC) system that simulates, under laboratory conditions, the back-slopping process used in Parmigiano Reggiano dairies to ferment sweet whey into NWS. This system (referred to as NSC0) effectively replicated fresh NWS and was successfully propagated through three consecutive rounds of laboratory-scale cheese-making. Using this validated system, we demonstrated that raw cow milk spiked with penicillin G at or below the maximum residue limits (MRL) negatively impacted NSC fermentative performance during the first round of caseification and significantly reduced microbial counts during the second round, suggesting a cumulative inhibitory effect of penicillin G, non-observable in a single back-slopping cycle. As SLAB declined, spoilage yeasts increased. qPCR analysis revealed that 2 and 4 ppb penicillin G impaired NWS biodiversity by inhibiting Lactobacillus delbrueckii and streptococci, whereas Lactobacillus helveticus remained unaffected. The validated NSC system developed in this study offers a robust platform for future investigations into the abiotic and biotic factors affecting NWS, advancing our ability to predict the behavior of undefined starter cultures under realistic cheese-making conditions.