Understanding the Effects of Lactose Hydrolysis Modeling on the Main Oligosaccharides in Goat Milk Whey Permeate.

Enzymatic hydrolysis of lactose is a crucial step to improve the efficiency and selectivity of membrane-based separations toward the recovery of milk oligosaccharides free from simple sugars. Response surface methodology was used to investigate the effects temperature (25.9 to 54.1 °C) and amount of enzyme (0.17 to 0.32% w/w) at 1, 2, and 4 h of reaction on the efficiency of lactose hydrolysis by Aspergillus oryzae β-galactosidase, preservation of major goat whey oligosaccharides, and on the de-novo formation of oligosaccharides. Lactose hydrolysis above 99% was achieved at 1, 2, and 4 h, not being significantly affected by temperature and amount of enzyme within the tested conditions. Formation of 4 Hexose (Hex) and 4 Hex 1 Hex and an increased de-novo formation of 2 Hex 1 N-Acetyl-Neuraminic Acid (NeuAc) and 2 Hex 1 N-Glycolylneuraminic acid (NeuGc) was observed in all treatments. Overall, processing conditions using temperatures ≤40 °C and enzyme concentration ≤0.25% resulted in higher preservation/formation of goat whey oligosaccharides

Discovery of Novel High-Molecular Weight Oligosaccharides Containing N‑Acetylhexosamine in Bovine Colostrum Whey Permeate Hydrolyzed with Aspergillus oryzae β‑Galactosidase

Bovine milk oligosaccharides (BMOs) that resemble human milk oligosaccharides are found in whey permeate, indicating that dairy streams can be used as a potential source of bioactive oligosaccharides. Recovery of oligosaccharides from whey permeate is hindered by their low abundance and high concentration of lactose. In the present work, lactose in bovine colostrum whey permeate was hydrolyzed by Aspergillus oryzae β-galactosidase to facilitate subsequent monosaccharide removal by membrane separation. Chromatographic separation coupled with high-resolution mass spectrometry revealed β-galactosidase degradation of several β-linkage-containing BMOs and production of novel oligosaccharides that ranged in size from 5 to 11 monosaccharide units containing several galactose repeating units and N-acetylhexosamine at their reducing ends. Optimization of BMO hydrolysis and separation methodology could generate high amounts of hetero-oligosaccharides for improved recovery of potentially biotherapeutic oligosaccharides

Food Bioactive Compounds against Diseases of the 21st Century

Editorial.Peer Reviewe

Recent advances in immobilization strategies for glycosidases.

Glycans play important biological roles in cell-to-cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large-scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo-β-N-acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI-1) that cleaves N-N'-diacetyl chitobiose moieties found in the N-linked glycan (N-glycan) core of high mannose, hybrid, and complex N-glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N-glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state-of-the-art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:104-112, 2017

From a Single-Stage to a Two-Stage Countercurrent Extraction of Lipids and Proteins from Full-Fat Chickpea Flour: Maximizing Process Extractability and Economic Feasibility

The mainstream adoption of chickpea proteins and lipids requires a thorough understanding of the impact of critical extraction parameters (enzyme use, reaction time, and solids-to-liquid ratio—SLR) and modes of extraction (single-stage extraction—SSE and countercurrent extraction—CCE) on the simultaneous extraction of lipids and proteins from full-fat chickpea flour and economic process feasibility. A kinetics study revealed that 68.5% oil and 87% protein extraction yields can be achieved using 0.5% protease at pH 9.0, 50 °C, 60 min, and 1:10 SLR, highlighting the role of proteolysis and an adequate incubation time on overall extractability. An increased gradient concentration between the matrix and aqueous media solutes at a lower SLR (1:15), and reduced slurry viscosity increased oil and protein extractability to 80 and 91%, respectively. The high-water usage in the SSE was addressed by the development of a two-stage CCE that reduced water usage by 47% while increasing oil and protein extractability to ~96%. Higher extractability and reduced water usage in the two-stage CCE resulted in a higher net gross profit, thus outweighing its higher operating costs. The results presented herein further widen the scope of bioprocessing standards for full-fat chickpea flour and add to the elucidation of the impact of key processing conditions on the extractability and economic feasibility of the production of chickpea ingredients for subsequent food/nutraceutical applications

Coupling Mass Spectrometry-Based "Omic" Sciences with Bioguided Processing to Unravel Milk's Hidden Bioactivities.

Many of milk's functional molecules could not be discovered until the right concordance of novel separation and analytical technologies were developed and applied. Many health-promoting components still await discovery due to technical challenges in their identification, isolation and testing. As new analytical technologies are assembled, new functional milk molecules will be discovered. Bovine milk is a source of a wide array of known bioactive compounds from a variety of molecular classes, including free glycans, lipids, glycolipids, peptides, proteins, glycoproteins, stem cells and microRNA. Because milk is such a complex mixture, when analyzed without fractionation or purification, many components mask the analytical signal of others, so some components cannot be detected. Modern analytics allow for the discovery and characterization of hundreds of novel milk compounds with high-resolution and high-accuracy. Liquid chromatography paired with electrospray ionization allows the separation of peptides, glycans and glycolipids for improved mass spectrometric detection. Target proteins and glycoproteins can now be purified from intact milk or other dairy streams by chromatography in order to better characterize these proteins for new bioactivities. The combination of advanced analytics with the new engineering capabilities will allow for high molecular resolution and separation techniques that can be scaled-up to semi-industrial and industrial scale for translation of lab-based discoveries. Bioguided analysis and design of dairy processing side streams will result in the transformation of waste into isolated functional ingredients to add value to dietary products

Coupling Mass Spectrometry-Based "Omic" Sciences with Bioguided Processing to Unravel Milk's Hidden Bioactivities.

Many of milk's functional molecules could not be discovered until the right concordance of novel separation and analytical technologies were developed and applied. Many health-promoting components still await discovery due to technical challenges in their identification, isolation and testing. As new analytical technologies are assembled, new functional milk molecules will be discovered. Bovine milk is a source of a wide array of known bioactive compounds from a variety of molecular classes, including free glycans, lipids, glycolipids, peptides, proteins, glycoproteins, stem cells and microRNA. Because milk is such a complex mixture, when analyzed without fractionation or purification, many components mask the analytical signal of others, so some components cannot be detected. Modern analytics allow for the discovery and characterization of hundreds of novel milk compounds with high-resolution and high-accuracy. Liquid chromatography paired with electrospray ionization allows the separation of peptides, glycans and glycolipids for improved mass spectrometric detection. Target proteins and glycoproteins can now be purified from intact milk or other dairy streams by chromatography in order to better characterize these proteins for new bioactivities. The combination of advanced analytics with the new engineering capabilities will allow for high molecular resolution and separation techniques that can be scaled-up to semi-industrial and industrial scale for translation of lab-based discoveries. Bioguided analysis and design of dairy processing side streams will result in the transformation of waste into isolated functional ingredients to add value to dietary products

Triacylglycerols are preferentially oxidized over free fatty acids in heated soybean oil.

In oil, free fatty acids (FFAs) are thought compared the efficiency of hydrolysis wto be the preferred substrate for lipid oxidation although triacylglycerols (TAGs) are the predominant lipid class. We determined the preferential oxidation substrate (TAGs versus FFAs) in soybean oil heated at 100 °C for 24 h, after validating a method for quantifying esterified and free lipid oxidation products (i.e., oxylipins) with mass-spectrometry. Reaction velocities and turnover (velocity per unit substrate) of FFA, and free and TAG-bound (esterified) oxylipins were determined. FFA hydrolysis rate and turnover were orders of magnitude greater (16-4217 fold) than that of esterified and free oxylipin formation. The velocity and turnover of TAG-bound oxylipins was significantly greater than free oxylipins by 282- and 3-fold, respectively. The results suggest that during heating, TAGs are preferentially oxidized over FFAs, despite the rapid hydrolysis and availability of individual FFAs as substrates for oxidation. TAG-bound oxylipins may serve as better markers of lipid oxidation

Revitalizing Unfermented Cabernet Sauvignon Pomace Using an Eco-Friendly, Two-Stage Countercurrent Process: Role of pH on the Extractability of Bioactive Phenolics

As the major byproduct of the winemaking industry, grape pomace remains an untapped source of valuable bioactive phenolic compounds. This study elucidated the optimal aqueous extraction parameters for maximizing phenolic extractability, while avoiding the use of harsh conventional solvents and limiting water usage, from Cabernet Sauvignon grape pomace in which the red grape was processed for white wine. In the single-stage aqueous extraction process (AEP), the concurrent impact of pH (2.64–9.36), solids-to-liquid ratio (SLR, g pomace/mL water) (1:50–1:5), and temperature (41.6–58.4 °C) on the total phenolic content (TPC) of Cabernet Sauvignon pomace was evaluated alongside a kinetic study (15–90 min). Optimal single-stage extraction conditions (pH 9.36, 1:50 SLR, 50 °C, 75 min) guided the development of a two-stage countercurrent extraction process (pH 9.36, 1:10 SLR, 50 °C, 75 min) to further reduce water consumption without compromising overall extractability. The countercurrent process reduced fresh water usage by 80%, increased the TPC of the extracts by 18%, and improved the in vitro antioxidant activities (ABTS and ORAC) of the extracts. Untargeted metabolomics enabled the identification of a diverse pool of phenolics, especially flavonol glycosides, associated with grape pomace, while further phenolic quantitation detected improvements in the release of commonly bound phenolics such as ferulic acid, p-coumaric acid, syringic acid, and protocatechuic acid in alkaline extracts compared to the ethanolic extract. This investigation provides an efficient, eco-friendly extraction strategy suitable for applications in functional food, beverage, nutraceutical, and cosmetic industries

From solvent extraction to the concurrent extraction of lipids and proteins from green coffee: An eco-friendly approach to improve process feasibility

The production of green coffee oil by mechanical pressing of green coffee beans has been precluded by low extraction yields, which generates a protein-rich byproduct (cake) containing variable amounts of lipids. Subsequent utilization of the cake requires the removal of the residual cake oil by solvent extraction. An eco-friendly extraction strategy, using water, enzymes, and mechanical treatments, was evaluated to concurrently extract lipids and proteins from green coffee flour, without the use of harsh solvents. Among the enzymatic treatments evaluated, the use of 0.5% alkaline protease led to higher protein (62.2%) and oil (47.7%) extractability in a shorter time (30 min). This enzymatic treatment was optimized with respect to solids-to-liquid ratio (SLR) (1:17.5-1:7) and concentration of enzyme (0.1-0.9% w/w). Although optimum extraction conditions (1:17.5 SLR and 0.1% enzyme) achieved high protein (70%) and oil (48%) extractability and reduced enzyme use by 80%, a higher water usage was required. Therefore, a two-stage countercurrent extraction was developed to reduce water usage in the process. The countercurrent extraction strategy not only reduced the amount of water used in the process by 60% but promoted higher protein (72%) and oil (58%) extractability, compared with the single-stage process (62.2 and 47.7%, respectively)