Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics.

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn2+-binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential.

Development in Maillard Reaction and Dehydroalanine Pathway Markers during Storage of UHT Milk Representing Differences in Casein Micelle Size and Sedimentation.

Ultra-high temperature (UHT) processing of milk can result in protein changes during storage; however, the progress of dehydroalanine (DHA) mediated protein cross-linking and Maillard reactions in relation to the sediment formation have not been investigated previously. Liquid chromatography-mass spectrometry, based on multiple reaction monitoring (MRM), was used to absolutely quantify concentrations of furosine, N-ε-(carboxyethyl)lysine (CEL), N-ε-(carboxymethyl)lysine (CML), lanthionine (LAN) and lysinoalanine (LAL) in skim milk and sediment of UHT milk produced from raw milk with either small or large casein micelles. The results showed a higher molar proportion of the advanced stage Maillard reaction products CEL and CML in the sediment, compared to early stage Maillard reaction product furosine, whereas furosine was predominant in the skim milk. Both LAL and LAN increased during storage in the skim milk phase, however only LAL was identified in the sediment. The milk pool with large native casein micelles, known to have a higher percentage of sedimentation, contained higher proportions of furosine, CEL, CML and LAL in the sediment compared to milk with smaller native casein micelles. The study demonstrates the potential contribution of processing-induced protein-protein interactions to sedimentation in UHT milk during storage.

Influence of Lactose on the Maillard Reaction and Dehydroalanine-Mediated Protein Cross-Linking in Casein and Whey.

A liquid chromatography-mass spectrometry method based on multiple reaction monitoring (MRM) was developed for the simultaneous quantification of markers representing two potentially competing pathways, the Maillard reaction and the dehydroalanine pathway. The two pathways involve the same residues in the proteins to some extent, namely, the essential amino acid lysine, as well as free-amino terminals available on proteins and polypeptides, competition between the two pathways in food systems may occur. The developed method comprises the following markers of the Maillard reaction: furosine, N-ε-(carboxyethyl)lysine (CEL) and N-ε-(carboxymethyl)lysine (CML), together with the dehydroalanine reaction pathway markers; lanthionine (LAN) and lysinoalanine (LAL), as well as lysine itself. The validated method was then used for the absolute quantification of heat-induced protein modifications in model systems of micellar casein and whey protein isolates (MCI and WPI, respectively) in the presence or absence of lactose. As expected, the Maillard reaction markers furosine, CEL and CML increased during the applied heat treatment in the presence of lactose, whereas the dehydroalanine markers, LAN and LAL increased with heating in both MCI and WPI, both in the presence and absence of lactose, although at lower levels in the presence of lactose, confirming the competing state of the two pathways.

Green Tea Extract Decreases Arg-Derived Advanced Glycation Endproducts but Not Lys-Derived AGEs in UHT Milk during 1-Year Storage.

Epigallocatechin gallate (EGCG)-enriched green tea extract (GTE) was added to lactose-reduced UHT-treated milk to evaluate its role in perturbing the Maillard reaction and the formation of advanced glycation endproducts (AGEs) during 1-year storage. The UHT processing caused epimerization of EGCG into gallocatechin gallate (GCG). For milk samples with added 0.1% w/v GTE, a EGCG/GCG loss of 26% was found soon after the UHT treatment and the loss increased to 64% after the 1-year of storage. LC-MS/MS analysis revealed the presence of various EGCG/GCG-α-dicarbonyl adducts and EGCG/GCG-hydroxymethylfurfural adducts in milk samples, while EGCG/GCG-amino acid adducts were not detected. Although EGCG/GCG trapped α-dicarbonyl compounds including glyoxal, methylglyoxal, 3-deoxyglucosone/3-deoxygalactosone, and diacetyl, it did not lower their net steady-state concentrations, except of 3-deoxyglucosone. The addition of GTE reduced the formation of Arg-derived AGEs by 2- to 3-fold, but surprisingly enhanced the accumulation of furosine and lysine-derived AGEs [Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine)] by 2-4-fold depending on the concentration of the added GTE and storage time. The present study shows that trapping of α-dicarbonyl compounds by EGCG may not be the major pathway for inhibiting the formation of AGEs in milk.

Limitation of Maillard Reactions in Lactose-Reduced UHT Milk via Enzymatic Conversion of Lactose into Galactooligosaccharides during Production.

Lactose-hydrolyzed (LH) ultrahigh temperature (UHT) processed milk is more prone to Maillard reactions and formation of advanced glycation end products (AGEs) during processing and storage than conventional (CON) UHT milk because of the presence of free galactose and glucose. Commercially available ß-d-galactosidases with transgalactosylating activity can incorporate galactose into galactooligosaccharides (GOSs) and potentially limit Maillard reactions in this lactose-reduced GOS-containing milk. The aim of this study was to examine the extent of Maillard reactions in a lactose-reduced GOS milk compared to LH and CON milk after UHT processing. The GOS milk had significant lower levels of lysine- and arginine-derived AGEs compared to LH milk, while their concentrations were similar to those found in CON milk. The total concentration of measured Arg-derived AGEs was similar to the total concentration of Lys-derived AGEs in the three types of milk, indicating that Arg is an important source of AGEs in milks. Interestingly, the GOS milk generated threefold higher concentrations (up to 330 ± 6 µM) of 3-deoxyglucosone (3-DG, a C6 α-dicarbonyl). These results demonstrate that GOS milk could be a potential alternative for LH milk for lactose-intolerant individuals, although further studies are needed to understand the increased formation of 3-DG in GOS-containing milk.

Quantitation of α-Dicarbonyls and Advanced Glycation Endproducts in Conventional and Lactose-Hydrolyzed Ultrahigh Temperature Milk during 1 Year of Storage.

A comprehensive quantitative characterization of Maillard reaction products was carried out for conventional (CON) and lactose-hydrolyzed (LH) ultrahigh temperature (UHT) milk during storage at 20, 30, and 40 °C for 1 year. The accumulation of 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal) in LH-UHT milk ranged from 20-fold (at 20 °C) to 44-fold (at 40 °C) higher than that in CON-UHT milk. High temperature storage (40 °C) significantly accelerated the accumulation of 3-DG, 3-DGal, and 5-hydroxymethyl furfural but not the majority of the analyzed advanced glycation endproducts (AGEs). The concentrations of major AGEs including N-ε-carboxymethyllysine (CML), N-ε-carboxyethyllysine (CEL), methylglyoxal-hydroimidazolone isomers (MG-H1/H3), glyoxal-hydroimidazolone isomers (G-H1/H3), and G-H2 detected in CON milk during storage were in the range 12-700, 1-14, 8-45, 4-13, and 1-30 µM, respectively, while they were 30-570, 2-88, 17-150, 9-20, and 5-34 µM, respectively, in LH milk. Pyrraline, S-(carboxymethyl)cysteine (CMC), and glyoxal-lysine dimer were detected in lower levels, while MG-H2, methylglyoxal-lysine dimer, argpyrimidine, glyoxal-lysine-amide, glycolic acid-lysine-amide, and pentosidine were not detected in any of the milk samples. This work demonstrates for the first time that five of the analyzed AGEs (CML, CEL, MG-H1/H3, G-H1/H3, and G-H2) could be selected as markers for evaluation of the extent of the Maillard reaction in UHT milk. These results contribute to a better understanding of how Maillard reactions progress during storage of UHT milk and can be used to develop strategies to inhibit Maillard reactions in LH milk.

Green Tea Polyphenols Decrease Strecker Aldehydes and Bind to Proteins in Lactose-Hydrolyzed UHT Milk.

The effect of epigallocatechin gallate enriched green tea extract (GTE) on flavor, Maillard reactions and protein modifications in lactose-hydrolyzed (LH) ultrahigh temperature (UHT) processed milk was examined during storage at 40 °C for up to 42 days. Addition of GTE inhibited the formation of Strecker aldehydes by up to 95% compared to control milk, and the effect was similar when GTE was added either before or after UHT treatment. Release of free amino acids, caused by proteolysis, during storage was also decreased in GTE-added milk either before or after UHT treatment compared to control milk. Binding of polyphenols to milk proteins was observed in both fresh and stored milk samples. The inhibition of Strecker aldehyde formation by GTE may be explained by two different mechanisms; inhibition of proteolysis during storage by GTE or binding of amino acids and proteins to the GTE polyphenols.

Plasmin activity in UHT milk: relationship between proteolysis, age gelation, and bitterness.

Plasmin, the major indigenous protease in milk, is linked to quality defects in dairy products. The specificity of plasmin on caseins has previously been studied using purified caseins and in the indigenous peptide profile of milk. We investigated the specificity and proteolytic pathway of plasmin in directly heated UHT milk (>150 °C for <0.2 s) during 14 weeks of storage at 20 °C in relation to age gelation and bitter peptides. Sixty-six peptides from αS- and ß-caseins could be attributed to plasmin activity during the storage period, of which 23 were potentially bitter. Plasmin exhibited the highest affinity for the hydrophilic regions in the caseins that most probably were exposed to the serum phase and the least affinity for hydrophobic or phosphorylated regions. The proteolytic pattern observed suggests that plasmin destabilizes the casein micelle by hydrolyzing casein-casein and casein-calcium phosphate interaction sites, which may subsequently cause age gelation in UHT milk.