Effects of protease-assisted aqueous extraction on almond protein profile, digestibility, and antigenicity.

Almonds (Prunus dulcis) are one of the most consumed tree nuts worldwide and have been recognized as a healthy and nutritious food. Nevertheless, almonds are also a source of allergenic proteins that can trigger several mild to life-threatening allergic reactions. The effects of selected extraction conditions (aqueous vs. protease-assisted aqueous extraction) on the protein profile determined by proteomics analysis of excised SDS-PAGE gel bands, in vitro protein digestibility, and immunoreactivity of almond protein extracts, were evaluated. Proteolysis altered almond protein sequential and conformational characteristics thus affecting digestibility and antigenicity. Proteomics analysis revealed that enzymatic extraction resulted in the reduction of allergen proteins and epitopes. While complete hydrolysis of Prunin 1 and 2 α-chain was observed, Prunin 1 and 2 ß-chains were more resistant to hydrolysis. Protein in vitro digestibility increased from 79.1 to 88.5% after proteolysis, as determined by a static digestion model. The degree of hydrolysis (DH) and peptide content of enzymatically extracted proteins during gastric and duodenal digestion were significantly higher than the ones from unhydrolyzed proteins. Proteolysis resulted in a 75% reduction in almond protein immunoreactivity as determined by a sandwich enzyme-linked immunosorbent assay and a reduction in IgE and IgG reactivities using human sera. The present study shows that moderated hydrolysis (7% DH) using protease can be used as a strategy to improve almond protein digestibility and reduce antigenicity. This study's findings could further enhance the potential use of almond protein hydrolysates in the formulation of hypoallergenic food products with improved nutritional quality and safety.

Early detection of lipid oxidation in infant milk formula by measuring free oxylipins-Comparison with hydroperoxide value and thiobarbituric acid reactive substance methods.

Infant milk formula was used as a model food to compare the sensitivity of thiobarbituric acid reactive substances (TBARS) and hydroperoxide methods to UPLC-MS/MS oxylipin analysis for detecting early lipid oxidation. Two different infant milk formulas were tested during 21 days of storage at 4°C. Formulas 1 and 2 contained canola oil and canola oil + 1% docosahexaenoic acid ethyl ester, respectively. Formulas were sampled up to 21 days of storage. Formula 2 had higher peroxide values than Formula 1 across all time points. However, no significant differences over time in TBARS and peroxide values in either formula were observed. Several oxylipins increased in both formulas starting on day 7 (linoleic acid and alpha-linolenic acid-derived oxylipins in Formula 1 and DHA-derived oxylipins in Formula 2). These results indicate that free oxylipins are effective in detecting early lipid oxidation and distinguishing between formulations containing different fatty acids. PRACTICAL APPLICATION: We have recently shown that primary oxidation products known as oxylipins can be measured in their free form by ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to detect early lipid oxidation. However, a head-to-head comparison of the sensitivity of this approach to conventional spectrophotometric methods has not been evaluated. Our results indicate that free oxylipin measurements are better than conventional methods in detecting early lipid oxidation in milk infant formula  distinguishing between different formulations.

A complete workflow for discovering small bioactive peptides in foods by LC-MS/MS: A case study on almonds.

Identification of bioactive peptides is an increasingly important target for food chemists, particularly in consideration of the widespread application of proteolytic enzymes in food processing. Because the characterization of small peptides by LC-MS/MS is challenging, we optimized a dimethyl labeling technique to facilitate small peptide identification, using almond proteins as a model. The method was validated by comparing the MS/MS spectra of standards and almond-derived peptides in their nonderivatized and derivatized forms. Signal enhancement of a1 ions was proved to effectively aid in the full-length sequencing of small peptides. We further validated this method using two industrially-relevant protein-rich extracts from almond flour: 1737 medium-sized peptides (5-39 amino acids) and 843 small peptides (2-4 amino acids) were identified. The use of an online bioactive peptide database, complemented by the existing literature, allowed the discovery of 208 small bioactive peptides, whereas for medium-sized peptides, only one was reported being bioactive.

Effects of enzyme-assisted extraction on the profile and bioaccessibility of isoflavones from soybean flour.

The effects of enzymatic extraction strategies on extractability, bioconversion, and bioaccessibility of biologically active isoflavone aglycones, total phenolic content, and antioxidant activity of aqueous extracts from full-fat soy flour were evaluated. Protease, tannase, and cellulase enzymes were used individually or in combination. Except for the protease treatment, all enzymatic treatments increased the extraction of biologically active isoflavones (daidzein and genistein) compared with the control. The use of a mixture of protease, tannase, and cellulase resulted in increased extractability and/or bioconversion of aglycones from soy flour, indicating a synergistic effect amongst the enzymes. Daidzein and genistein concentrations increased from 29.0 to 158.2 µg/g and from 27.0 to 156.5 µg/g (compared to the control), respectively. Furthermore, enzymatic extraction followed by in vitro gastrointestinal digestion significantly increased the bioaccessibility of isoflavone aglycones, total phenolic content (by 22-45%), and antioxidant activity (by 15-22%) of the extracts. These results demonstrate that enzyme selection is an efficient strategy to maximize the extraction, bioconversion, and bioaccessibility of bioactive isoflavones from soy flour, which could contribute to health benefits associated with the consumption of soy-rich products.

Method optimization of oxylipin hydrolysis in nonprocessed bovine milk indicates that the majority of oxylipins are esterified.

The oxidation of polyunsaturated fatty acids produces bioactive primary oxidation products known as oxylipins. In many biological matrices, the majority of oxylipins are bound (i.e. esterified), and a relatively small proportion (<10%) exists in the free form. The present study tested whether this extends to bovine milk following method evaluation of various extraction and base hydrolysis protocols for measuring bound oxylipins. Free (unbound) oxylipins were also measured. Folch extraction followed by sodium carbonate hydrolysis in the presence of methanol containing 0.1% of acetic acid and 0.1% of butylated hydroxytoluene resulted in greater oxylipin concentrations and better surrogate standard recoveries compared to other methods that did not involve Folch extraction or the addition of methanol with hydrolysis base. Sodium hydroxide was better than sodium carbonate in hydrolyzing bound oxylipins under the same conditions. Milk analysis of oxylipins with mass-spectrometry following Folch extraction and sodium hydroxide hydrolysis revealed that 95% of oxylipins in bovine milk were esterified. Most of the detected oxylipins were derived from linoleic acid, which accounted for 92 and 88% of oxylipins in the free and esterified pools, respectively. These results demonstrate that the majority of bovine milk oxylipins are bound, and that linoleic-acid derived metabolites are the most abundant oxylipin species in free and bound lipid pools. Additional studies are needed to understand the role of different oxylipin pools in both calf and human nutrition. PRACTICAL APPLICATION: A method involving Folch lipid extraction and sodium hydroxide hydrolysis was validated for esterified oxylipin measurements in bovine milk. Application of the method revealed that the majority (∼95%) of oxylipins in bovine milk were bound. Linoleic-acid derived oxylipins were the most abundant species in both bound and free milk fractions (88-92%). The results highlight the presence of a new pool of oxidized lipids in milk, potentially involved in modifying its sensory and nutritional properties.

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.

Effects of industrial heat treatments on bovine milk oxylipins and conventional markers of lipid oxidation.

The effects of industrial heat treatments of raw bovine milk subjected to Batch Pasteurization (BP), High Temperature Short Time (HTST) and Ultra High Temperature (UHT) on the formation of primary (hydroperoxide content and oxylipins) and secondary lipid oxidation products (thiobarbituric acid reactive species -TBARS) were evaluated. Total fatty acid content, percent of free fatty acids (FFA), and total antioxidant capacity (TAC) were also measured. Except for a 30% reduction in capric acid (C10:0) after UHT compared to BP, no significant differences in total fatty acid concentrations were detected amongst the heat treatments. Compared to raw bovine milk, no statistically significant effects of heat treatment were observed on percent FFA (0.29-0.31%), hydroperoxide concentration (0.0558-0.0624 mmol L-1), and TBARS values (13.4-18.9 µg MDA kg-1). HTST and UHT led to significant reductions (50-65%) in linoleic and alpha-linolenic acid oxidized metabolites compared with raw milk and batch pasteurized milk. Compared to raw milk (2943.7 µmol of TEAC L-1), TAC was significantly reduced by all heat treatments (2245 - 2393 µmol of TEAC L-1), although no statistically significant differences were observed amongst the treatments. The results demonstrate that heat processing reduces milk oxylipin content and antioxidant capacity and that oxylipin and TAC measurements provide a new sensitive approach to assess the impact of milk processing on lipid oxidation. The nutritional, shelf life and sensory implications of reduced oxylipins in HTST and UHT processed bovine milk merit further investigation.

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria.

UNLABELLED: Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-ß-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides. IMPORTANCE: It has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated N-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of B. infantis, which is a key infant gut microbe. The selectivity of consumption of individual N-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip-quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that B. infantis can consume the range of glycan structures released from whey protein concentrate.