Lactobacillus casei expressing methylglyoxal synthase causes browning and heterocyclic amine formation in Parmesan cheese extract.

Undesired browning of Parmesan cheese can occur during the latter period of ripening and cold storage despite the relative absence of reducing sugars and high temperatures typically associated with Maillard browning. Highly reactive α-dicarbonyls such as methylglyoxal (MG) are products and accelerants of Maillard browning chemistry and can result from the microbial metabolism of sugars and AA by lactic acid bacteria. We demonstrate the effects of microbially produced MG in a model Parmesan cheese extract using a strain of Lactobacillus casei 12A engineered for inducible overexpression of MG synthase (mgsA) from Thermoanaerobacterium thermosaccharolyticum HG-8. Maximum induction of plasmid-born mgsA led to 1.6 mM MG formation in Parmesan cheese extract and its distinct discoloration. The accumulation of heterocyclic amines including ß-carboline derivatives arising from mgsA expression were determined by mass spectrometry. Potential MG-contributing reaction mechanisms for the formation of heterocyclic amines are proposed. These findings implicate nonstarter lactic acid bacteria may cause browning and influence nutritional aspects of Parmesan by enzymatic conversion of triosephosphates to MG. Moreover, these findings indicate that the microbial production of MG can lead to the formation of late-stage Maillard reaction products such as melanoidin and ß-carbolines, effectively circumventing the thermal requirement of the early- and intermediate- stage Maillard reaction. Therefore, the identification and control of offending microbiota may prevent late-stage browning of Parmesan. The gene mgsA may serve as a genetic biomarker for cheeses with a propensity to undergo MG-mediated browning.

A profile of sphingolipids and related compounds tentatively identified in yak milk.

This work characterized a fraction of constituents in yak milk within the realm of approximately 1,000 to 3,000 Da using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Eleven samples of yak milk powder from the Sichuan province of China were received by the Department of Food Science, University of Wisconsin-Madison, and stored at room temperature until analysis. Sample preparation involved delipidation and deproteinization of yak milk samples and cold ethanol precipitation. Subsequently, MALDI time-of-flight mass spectrometry was performed in positive ion, reflector mode (AB Sciex TOF/TOF 4800 MALDI; AB Sciex, Foster City, CA). The instrument was first calibrated with the manufacturer's 6-peptide mixture, and each spectrum was internally calibrated using the accurate mass of ACTH Fragment 18-39 standard peptide (protonated mass at m/z 2464.199) present in each sample. Laser power was adjusted for the calibration standards and for each sample so that the signal obtained for the most-abundant ion in each spectrum could be maximized, or kept below ~2×10(4) to preserve spectral quality. Structure and name based on mass were matched using the Metlin metabolite database (https://metlin.scripps.edu/index.php). Results of the current work for yak milk powder showed a large variety of sphingolipid structures with clusters around 1,200, 1,600, and 2,000 Da. The profiling matched several glycosphingolipids, such as gangliosides GA1, GD1a, GD1b, GD3, GM1, GM2, GM3, and GT2 and several other unique moieties, including deaminated neuraminic acid (KDN) oligosaccharides, and fucose containing gangliosides. Matrix preparation and MALDI time-of-flight parameters were important factors established in this work to allow high resolution profiling of complex sphingolipids in yak powder milk.

Identification of endogenous peptides recognized by in vivo or in vitro generated alloreactive cytotoxic T lymphocytes: distinct characteristics correlated with CD8 dependence.

We studied the molecular basis for CD8 independence of in vivo generated (BM3.3) versus CD8 dependence of in vitro sensitized (KB5.C20/Des) alloreactive H-2K(b)-specific cytotoxic T lymphocytes (CTL). Using microcapillary high-performance liquid chromatography fractionation of H-2K(b) eluates, mass spectrometry and CTL reconstitution assays, we determined that BM3.3 and KB5.C20 recognize, respectively, a single peptide (pBM1) expressed on 8,000 H-2K(b) molecules per allogeneic cell, and three distinct peptides (pKB1, 2, 3), each expressed on around 200 H-2K(b) molecules per allogeneic cell. CD8 (in)dependence was intrinsic to the respective TCR/H-2K(b)-peptide interactions. KB5.C20 and BM3.3 TCR illustrate the correlation that appears to exist between CD8 dependence/low affinity and in vitro sensitization as opposed to low dependency on CD8 and high TCR affinity observed after in vivo sensitization. The results suggest that CD8-dependent alloreactive CTL obtained in vitro with high frequency correspond to low-affinity TCR from the MHC-biased TCR repertoire unpurged by negative selection and have implications for cellular immunotherapeutic approaches.