Insights on the bacterial composition of Parmigiano Reggiano Natural Whey Starter by a culture-dependent and 16S rRNA metabarcoding portrait

Abstract

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.

Cultivable microbial diversity, peptide profiles, and bio-functional properties in Parmigiano Reggiano cheese

“…Other reasons for the huge diversity in NWS microbial composition can be related to technological parameters that have changed over space and time, such as temperature-decreasing curves and milk quality. We also consider that NWS microbiota is hard to be cultivated out of the whey environment ( Fornasari et al, 2006 ; Sola et al, 2022 ). One of the reasons could be the presence of peptides and growth factors in whey, which are lacking in microbiological media.…”

“…Accordingly, M17 supplemented with SSW mimics a better NWS environment than M17 medium and significantly improves the recovery of streptococci. Another reason for the low cultivability could be the cross-feeding interactions existing in the NWS community, which hamper the microbial growth of axenic cultures ( Sola et al, 2022 ). We cannot exclude that the low cultivability of NWS microbiota could distort the species abundance detected by a culturable approach.…”

“…Natural whey starter samples were analyzed for pH and titratable acidity as previously reported ( Sola et al, 2022 ). Concerning cheese samples, after removing 15 mm of the rind, cheese cores were finely shredded and immediately analyzed for moisture, NaCl, protein, and fat contents, according to Lolli et al (2021) .…”

“…All PCRs were carried out using a thermal cycler T100 (Bio-Rad, Hercules, CA, United States) in a final volume of 20 μL containing DreamTaq Green Buffer 1 × 2 mmol [L of MgCl 2 , 200 μmol/L of each dNTP, 50 ng of DNA template, 0.5 U DreamTaq Green DNA polymerase (5 U/μL), and 0.5 μmol/L of each primer in multiplex PCR and 1 μmol/L of each primer in pentaplex PCR, respectively]. When required, 16S rRNA gene PCR amplification and ARDRA analysis with diagnostic endonucleases, such as Mse I, EcoR I, and Hha I, were carried out for NWS and cheese isolates, respectively ( Sola et al, 2022 ).…”

“…The microbiota inhabiting raw milk is rapidly replaced by NWS homofermentative and thermophilic starter lactic acid bacteria (SLAB), such as Lactobacillus helveticus , Lactobacillus delbrueckii subsp. lactis , and Streptococcus thermophilus ( Rossetti et al, 2008 ; Bottari et al, 2010 ; Bertani et al, 2020 ; Sola et al, 2022 ). After curd brining, SLAB rapidly depleted in favor of non-starter lactic acid bacteria (NSLAB), mainly Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus ), Lacticaseibacillus paracasei (formerly Lactobacillus paracasei ), and Lacticaseibacillus casei (formerly Lactobacillus casei ) ( Coppola et al, 2000 ; Neviani et al, 2009 ; Solieri et al, 2012 ; Gatti et al, 2014 ).…”