Lectin microarray profiling demonstrates equivalent global glycosylation for whey protein ingredients enriched with α-lactalbumin and milk fat globule membrane.

Human milk fat globule membrane (MFGM) and whey proteins are nutritionally and functionally valuable, with many beneficial bioactivities associated with their glycosylation. However glycosylation of milk components other than free milk oligosaccharides are underinvestigated. Whey protein concentrate (WPC) ingredients with various enrichments or depletions are used in infant formula (IF) formulations to contribute to human milk equivalence and bioactivity benefits, but their overall or global glycosylation has not been compared. We compared the global glycosylation of commercial WPC ingredients for use in various IF formulations; two MFGM-enriched WPC ingredients (high fat HF1 and lower fat HF2), an α-lactalbumin-enriched WPC (WPC Lac) which has α-lactalbumin concentration closer to human milk and significantly less ß-lactoglobulin which is not present in human milk, and two base WPC ingredients (WPC 80 and WPC 35) using lectin microarray profiling. WPC Lac and WPC HF1 glycosylation were highly similar to each other and both somewhat similar to WPC 35, while WPC HF2 was more similar to the base WPC 80 ingredient. N-linked glycosylation analysis demonstrated that WPC HF1 and WPC Lac were qualitatively most similar to one another, with WPC 80 and WPC 35 having similar structures, confirming lectin microarray profiling as a valuable method to compare global glycosylation. Thus WPC Lac may be a valuable ingredient for providing equivalent glycosylation to MFGM supplementation.

Reduction of multiple reaction monitoring protein target list using correlation analysisa.

High mass resolution mass spectrometry provides hundreds to thousands of protein identifications per sample, and quantification is typically performed using label-free quantification. However, the gold standard of quantitative proteomics is multiple reaction monitoring (MRM) using triple quadrupole mass spectrometers and stable isotope reference peptides. This raises the question how to reduce a large data set to a small one without losing essential information. Here we present the reduction of such a data set using correlation analysis of bovine dairy ingredients and derived products. We were able to explain the variance in the proteomics data set using only 9 proteins across all major dairy protein classes: caseins, whey, and milk fat globule membrane proteins. We term this method Trinity-MRM. The reproducibility of the protein extraction and Trinity-MRM methods was shown to be below 5% in independent experiments (multi-day single-user and single-day multi-user) using double cream. Further application of this reductionist approach might include screening of large sample cohorts for biologically interesting samples before analysis by high-resolution mass spectrometry or other omics methodologies.

Implications of the Maillard reaction on bovine alpha-lactalbumin and its proteolysis during in vitro infant digestion.

This study investigated the functionality and digestibility of Maillard reaction products (MRPs) of alpha-lactalbumin (α-la), a major whey protein and component of infant formulas. The impact of different carbohydrates (glucose, galactose or galacto-oligosaccharides (GOS)) and heating duration was studied. SDS-PAGE, UV and color measurements monitored reaction extent, which varied between carbohydrates whereby galactose reacted more readily than glucose. Surface hydrophobicity and antioxidant capacity were found to be significantly (p < 0.05) higher following Maillard conjugation, with GOS-based MRPs elevating antioxidant capacity ∼50-fold compared to α-la. In addition, the digestive proteolysis of MRPs was evaluated using an infant in vitro gastro-duodenal model. SDS-PAGE analyses of digesta revealed Maillard conjugation generally increased α-la's susceptibility to proteolysis. Interestingly, GOS-based MRPs presented an optimization challenge, since heating for 12 h delayed proteolysis, while extended heating resulted in the highest susceptibility to proteolysis. Proteomic analyses further demonstrated the differences in enzymatic cleavage patterns and helped identify bioactive peptides rendered bioaccessible during the digestion of α-la or its MRPs. Bioinformatic mining of the proteomic data using PeptideRanker also gave rise to two potentially novel bioactive peptides, FQINNKIW and GINYWLAHKALCS. Finally, antioxidant capacity of luminal contents, measured by DPPH, revealed Maillard conjugation increased the antioxidant capacity of both gastric and duodenal digesta. Overall, this work draws a link between the Maillard reaction, digestive proteolysis and the bioaccessibility of bioactive peptides and antioxidant species in the infant alimentary canal. This could help rationally process infant formulas towards improved nutritional and extra-nutritional benefits.

Antioxidant activity of bovine alpha lactalbumin Maillard products and evaluation of their in vitro gastro-duodenal digestive proteolysis.

Food processing offers various pathways to tailor food functionality and digestibility. This work sought to study the impact of thermally-induced Maillard reaction between bovine alpha-lactalbumin (α-la) and fructose or fructo-oligosacchrides on physicochemical properties, antioxidant capacity and in vitro digestive fate under simulated adult and infant conditions. Colloidal stability (measured by DLS) was decreased as a result of the Maillard glycation, while antioxidant capacity (determined by FRAP) and surface hydrophobicity (H0 measurements) were elevated. Semi-dynamic in vitro digestion of Maillard conjugates revealed a mixed trend as a result of postulated competing effects of glycation on α-la's susceptibility to proteolysis; steric hindrance accompanied by protein unfolding could hinder or promote the availability of enzymatic cleavage sites. Results also showed thermal processing altered the digestive breakdown profile of α-la under infant conditions contrary to negligible effects observed under adult conditions. Evaluation of the antioxidant capacity during digestion (via DPPH assay) revealed that adult digesta possessed increased antioxidant activity throughout the gastric phase compared to infant digesta, whereas infant digesta of conjugates exhibited an increase in antioxidant capacity in the duodenum compared to adult. Moreover, during infant digestion of conjugates, an increase in antioxidant capacity was observed in the later stages of the digestion. Overall, this work demonstrates that controlled thermal processing of bovine α-la could potentially modulate its functionality and digestibility, particularly as it pertains to its ability to interfere with oxidative reactions in the lumen, possibly through the generation of bioactive peptides.

The impact of the Maillard reaction on the in vitro proteolytic breakdown of bovine lactoferrin in adults and infants.

The Maillard reaction has been proposed as a natural pathway to functionalize proteins and modulate their proteolysis. Nevertheless, gaps in understanding the digestive fate of Maillard reaction products (MRPs) still exist, especially regarding bioactive proteins such as lactoferrin (LF). UV absorbance and SDS-PAGE were used to monitor reaction progression under mild thermal processing (60 °C, 79% RH). Dynamic light scattering showed that MRPs had increased colloidal size and turbidity at 3 < pH < 10. FRAP analysis and in vitro digestion experiments demonstrated that MRPs possessed improved antioxidant capacity and higher susceptibility to proteolysis to varying extents under adult conditions compared to infant conditions. Proteomic analyses of MRP digesta revealed altered enzymatic cleavage patterns with no pronounced changes in the formation of known bioactive peptides. These also indicated that MRPs may breakdown in the gastro-intestinal tract to potentially form novel bioactive peptides. Overall, this work highlights that the Maillard reaction could be harnessed to modify the extent of proteolysis and bioactivity of proteins.

Impact of the Maillard reaction on the antioxidant capacity of bovine lactoferrin.

Studies raise the notion that the Maillard reaction (MR) may be harnessed to modify the antioxidant capacity of alimentary proteins. However, little is known about the impact of MR on bioactive proteins. Glucose and fructose were used as model moieties reacting with lactoferrin (LF). UV absorbance and SDS-PAGE analyses were used to monitor MR progression during 36 h of mild thermal processing (60 °C, 79% RH). FTIR and CD did not reveal changes in LF structure; However, dynamic light scattering showed MR increased mean particle sizes and sample turbidity at 3