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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.

Scope: Advanced glycation endproducts (AGEs) are suspected to stimulate inflammatory signaling pathways in target tissues via activation of the receptor for AGEs. Endotoxins are generally recognized as potential contamination of AGE preparations and stimulate biological actions that are very similar as or identical to those induced by AGEs. Methods and results: In our study, we used glycolaldehyde-modified β-lactoglobulin preparations as model AGEs and employed two methods to remove endotoxin using either affinity columns or extraction with Triton X-114 (TX-114). Affinity column-purified AGEs retained their ability to stimulate inflammatory signaling as measured by mRNA expression of inflammatory cytokines in the human lung epithelial cell line Beas2b. However, glycolaldehyde-modified AGEs purified by extraction with TX-114 did not show any stimulation of mRNA expression of inflammatory cytokines. The presence of a cell stimulating endotoxin-like activity was demonstrated in the detergent phase after extraction with TX-114, thus indicating that not AGEs but a lipophilic contamination was responsible for the stimulation of inflammatory signaling. Conclusion: Our results demonstrate that glycolaldehyde-modified AGEs are unable to induce inflammatory signaling in receptor for AGE-expressing cells. The observed cell-activating activity can be ascribed to an endotoxin-like lipophilic contamination present in AGE preparations and affinity column purification was insufficient to remove this contamination.

Advanced glycation endproducts (AGEs) and their precursor dicarbonyls are generally perceived as having adverse health effects. They are also considered to be initiators and promoters of disease and aging. However, proof for a causal relationship is lacking. On the other hand, it is known that AGEs and melanoidins possess beneficial properties, such as antioxidant and metal-chelating activities. Furthermore, some AGEs may stimulate the cellular detoxification system, generally known as the phase II drug metabolizing system. We show here that several reactive dicarbonyl intermediates have the capability to stimulate the cellular phase II detoxification systems in both a reporter cell line and primary rat hepatocytes. In addition, we demonstrate that dicarbonyls can attenuate the inflammatory signaling induced by tumor necrosis factor-alpha in a reporter cell system.

Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.