This work aimed to determine the formation over time of 3-methylbutanal and 3-methylbutan-1-ol recognized as malty during the manufacture of Raclette-type cheese and the fermention of reconstituted skim milk, and filter-sterilized MRS broth. Using dynamic headspace-vacuum transfer in trap extraction followed by gas chromatography coupled with mass spectrometry–olfactometry (DHS-VTT-GC-MS-O) as a screening method for the malty compounds, five compounds (2-methylpropanal, 2- and 3-methylbutanal, and 2- and 3-methylbutan-1-ol) were identified as potential compounds causing the malty aroma in starter culture development and Raclette-type cheeses. 

Focus on compounds having a predominant sensorial effect (3-methylbutanal and 3-methylbutan-1-ol), spikings of leucine, 13C-labeled leucine, α-ketoisocaproic acid, and α-ketoglutaric acid provided a better understanding of their formation pathway. This study highlighted the discrepancies in the formation of 3-methylbutanal and 3-methylbutan-1-ol between the growth media; namely, despite the presence of free leucine available in MRS and the addition of an excess, no increase of the target compounds was observed. The concentration of these compounds in MRS increased only when α-ketoglutaric acid or α-ketoisocaproic acid was added, and a preference for the pathway to α-hydroxyisocaproic acid instead of 3-methylbutanal was shown. In addition, a formation of 3-methylbutanal when the bacteria were not yet active was observed when spiking α-ketoisocaproic acid, which potentially indicates that this part of the metabolism could take place extracellularly. These results could potentially unveil other, not-yet-identified reactants, directly influencing the production of compounds responsible for the malty aroma in Raclette cheese.

Malty aroma has been studied considerably in several food matrices, such as beers, spirits, and dairy products. This aroma has been mainly associated with branched-chain aldehydes, 2-methylpropanal, 2-methybutanal, and 3-methylbutanal, derived from degradation of valine, isoleucine, and leucine, respectively. The formation of the branched chain aldehydes starts intracellularly first from the transamination of the former branched chain amino acids (AAs), which then leads to the formation of the corresponding intermediary α-ketoacids. From then, different pathways can lead to the coupling to Coenzyme A, to the formation of their corresponding α-hydroxyacids, or to the direct or indirect decarboxylation of the α-ketoacids to the aldehydes.
During the development of a new starter culture for Swiss Raclette-type cheese (a semi-hard cheese often ripened over at least three months and manufactured using pasteurized, thermized, or raw milk), such a malty undesirable off-flavor was detected, which had not yet been described in this type of cheese. This was first perceived through sensorial analysis and later analytically. This off-flavor was found to be unpleasant when the cheeses were tasted at room temperature or melted, which is a common way of tasting cheese throughout Switzerland. Since this off-flavor could be attributed to the use of a starter culture, the focus on the source of formation was put on these cultures. Starter cultures are usually not single-strain cultures but rather consist of a mix of one or more strain(s) of several species.