Natural whey starters (NWS) are undefined bacterial communities produced daily from whey of the previous cheese-making round, by application of high temperature. As a result, in any dairy plant, NWS are continuously evolving, undefined mixtures of several strains and/or species of lactic acid bacteria, whose composition and performance strongly depend on the selective pressure acting during incubation. While NWS is critical to assure consistency to cheese-making process, little is known about the composition, functional features, and plant-to-plant fluctuations. Here, we integrated 16S rRNA metabarcoding and culture-dependent methods to profile bacterial communities of 10 NWS sampled in the production area of Parmigiano Reggiano cheese. 16S rRNA metabarcoding analysis revealed two main NWS community types, namely NWS type-H and NWS type-D. Lactobacillus helveticus was more abundant in NWS type-H, whilst Lactobacillus delbrueckii/St. thermophilus in NWS type-D, respectively. Based on the prediction of metagenome functions, NWS type-H samples were enriched in functional pathways related to galactose catabolism and purine metabolism, while NWS type-D in pathways related to aromatic and branched chain amino acid biosynthesis, which are flavor compound precursors. Culture-dependent approaches revealed low cultivability of individual colonies as axenic cultures and high genetic diversity in the pool of cultivable survivors. Co-culturing experiments showed that fermentative performance decreases by reducing the bacterial complexity of inoculum, suggesting that biotic interactions and cross-feeding relationships could take place in NWS communities, assuring phenotypic robustness. Even though our data cannot directly predict these ecological interactions, this study provides the basis for experiments targeted at understanding how selective regime affects composition, bacterial interaction, and fermentative performance in NWS.

Cheese milk "maturation" during natural creaming in large flat vat is a critical step in the production of Parmigiano-Reggiano cheese. Thanks to an improved management, during the last years, a generalized reduction of mesophilic microbial counts has occurred, which did not favour an adequate increase in milk acidity during natural creaming. For this reason, some cheesemakers have introduced the practice to add a rate of natural whey starter in active fermentation in the evening milk to favourize its "maturation". The aim of this work was to verify the effects of this practice on the characteristics of Parmigiano-Reggiano milk and on some chemical, microbiological and sensory properties of the ripened cheese. Thirty-six cheesemaking trials were carried out in a dairy: 12 with 0.2% of young whey starter addition, 12 with 0.4% and 12 as control. The addition of young whey starter improved the coagulation properties of the cheese milk and did not affect the normal succession of thermophilic and mesophilic lactic acid bacteria during the cheese ripening. The chemical composition of the ripened cheeses was not affected by the practice of whey starter addition. From the sensory point of view, the addition of natural whey starter improved the compliance scores and decreased the defects, as eyes and cracks. This work confirmed the empirical observations of some cheesemakers. The adoption of this technique has considerable technological importance, improving the coagulation properties of the milk and the sensory characteristics of the cheese.

Brine soaking is one of the most important steps in the production of Parmigiano Reggiano cheese, since it determines the amount of salt in the final product. Reduction in salt in Parmigiano Reggiano cheese might be important for improving its nutritional profile, but it could affect the manufacturing processes by altering proteolysis and consequently the product quality. In this study, for the first time, salt reduction was explored at the industrial level on real cheese samples manufactured in a local dairy. In particular, 20 wheels were produced with conventional (18 days, 10 wheels) and shorter (12 days, 10 wheels) brining steps. In every group, wheels were studied at two different ripening times, 15 and 30 months. A shorter brining time resulted in an average 12% decrease in salt content. A full characterization of free amino acids and peptides was performed by LC-MS on all samples. Free amino acids and peptides, as expected, increased with ripening, due to proteolysis, with samples having low salt content showing a slightly faster increase when compared to standard ones, hinting to a slightly accelerated proteolytic process. Nonetheless, low-salt and conventional cheeses shared similar sensory profiles at both ripening times.

Lipolysis of PDO Parmigiano Reggiano cheese from different dairies was studied to evaluate the effect of sodium chloride (NaCl) reduction on the lipidic fraction. The total and individual free fatty acid and diglyceride contents were determined for two groups of samples, normal NaCl content (1.50% p/p) and reduced NaCl content (1.37% p/p). In addition, fat, water, NaCl, and cholesterol contents were also determined. The NaCl content was 9.01% higher in the normal NaCl group than in the reduced NaCl group. The most abundant fatty acid was palmitic acid, followed by oleic, myristic, and stearic acid, which altogether accounted for approximately 75% of the total content. Cheese with a normal NaCl content presented concentrations of C8:0 and C18:0 higher than those of reduced NaCl samples, while the latter showed a higher proportion of C10:0, C14:1c, and C16:0. The total free fatty acid and diglyceride contents were higher in the reduced NaCl samples, so a 9.01% reduction in the concentration of this component could accelerate the lipolysis process. On the contrary, the free fatty acid composition profile was similar in both groups.

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