Elucidating the Molecular Basis of Distinct Cheese Flavours Associated with an Adjunct Culture

Abstract

Previous studies had shown that the distinct, desirable, but atypical flavours of Cheddar cheese manufactured with the adjunct Lb. helveticus NZ1 did not correlate with unusual levels of typical flavour compounds. Cheeses made with the NZ1 adjunct were described as sweet, savoury (i.e., roast meat, marmite), fruity, and more mature but less bitter than standard control cheeses manufactured solely with mesophilic starter. This highlighted the possibility that novel flavour compounds and pathways could be responsible for the flavour differentiation observed. Elucidation of such compounds could provide opportunities for the delivery of novel flavours into fermented dairy products, as well as enabling the precise control for the delivery of such flavours.

Cheeses were manufactured with a range of Lb. helveticus cultures. Multivariate analysis of the sensory profiles of the ripening cheeses was used to assign the cultures to either an NZ1-like flavour group or a reduced-NZ1-type flavour group. The levels of all free amino acids were higher in the NZ1-like cheeses than in the cheeses with a reduced-NZ1-type flavour profile at any given point in ripening, but the branched chain amino acids (valine, leucine, and isoleucine) and glutamic acid, proline and lysine were the most influential for discriminating cheeses of the two flavour groups. No correlations between flavour profiles and volatile compounds were apparent, despite the distinct differences in the aroma of cheeses assigned to the two flavour groups, confirming the previous observations.

To identify proteins that were uniquely associated with adjuncts producing the NZ1-like cheese flavours, the acidic and basic proteomes of the adjuncts assigned to the two flavour groups were compared using two-dimensional difference fluorescence gel electrophoresis. Several candidate proteins possibly responsible for the production of cheese flavours associated with Lb. helveticus NZ1 were identified.

Three proteins from the acidic proteome were differentially abundant (p≤0.01). Cystathionine β-synthase, involved in cysteine-methionine inter-conversion, was present in lower abundance (-5.39 fold; p=0.0065) in NZ1-like adjuncts compared with the reduced-NZ1-type adjuncts. The gene for this protein also showed homology to cysteine synthase. In contrast, a general stress protein from the Gls24 family of proteins, was present in higher abundance (14.83 fold; p=0.0065) in NZ1-like adjuncts. The third protein, which resolved in gel spot #1459, was also differentially abundant (-4.68 fold; p=0.0095), but low abundance of this protein precluded identification.

Multivariate analysis indicated that the proteins cystathionine β-synthase/cysteine synthase and the unidentified protein in gel spot #1459 might be co-regulated. In addition, an ATP-dependent endopeptidase proteolytic subunit, clpP, clustered with these proteins and was present in lower abundance (-3.46 fold; p=0.046) in NZ1-like adjuncts compared with the reduced NZ1-type-adjuncts.

These findings suggest that elevated proteolysis and/or the differential regulation of several proteins (including stress-related proteins and those involved in the metabolism of sulphur-containing amino acids), may account for the production of the distinct cheese flavours associated with the adjunct Lb. helveticus NZ1.