The profiles of bioactive compounds (including phenolics and flavonoids in free and bound fractions, anthocyanins, proanthocyanidins, vitamin E, and γ-oryzanol) of outer and inner rice bran from six colored rice samples collected from local markets were investigated. Proanthocyanidins could only be detected in red rice bran but not in black rice bran. The free fraction of the extracts dominated the total phenolics (72-92%) and the total flavonoids (72-96%) of colored rice bran. Most of the phenolic acids (83-97%) in colored rice bran were present in the bound form. Protocatechualdehyde was identified for the first time in the bound fraction of red rice bran by high performance liquid chromatography-photodiode array/electrospray ionization tandem mass spectrometry. The antioxidative activities of the free fraction of the colored rice bran were attributed to the proanthocyanidins in red colored rice and anthocyanins in black rice, while that of the bound fraction was mainly due to the phenolic acids.

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Reverse-phase solid-phase extraction (SPE) is regularly used for separating and purifying food-derived oligosaccharides and peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, the diversity in physicochemical properties of peptides may prevent the complete separation of the two types of analytes. Peptides present in the oligosaccharide fraction not only interfere with glycomics analysis but also escape peptidomics analysis. This work evaluated different SPE approaches for improving LC-MS/MS analysis of both oligosaccharides and peptides through testing on peptide standards and a food sample of commercial interest (proteolyzed almond extract). Compared with conventional reverse-phase SPE, mixed-mode SPE (reverse-phase/strong cation exchange) was more effective in retaining small/hydrophilic peptides and capturing them in the high-organic fraction and thus allowed the identification of more oligosaccharides and dipeptides in the proteolyzed almond extract, with satisfactory MS/MS confirmation. Overall, mixed-mode SPE emerged as the ideal method for simultaneously improving the identification of food-derived oligosaccharides and small peptides using LC-MS/MS analysis.

This study reveals that unexpected degradation of food oligosaccharides can occur during conventional glycomics workflows, including sample preparation and analysis by liquid chromatography-mass spectrometry (LC-MS). With the present investigation, we aim to alert the scientific community of the susceptibility of specific glycosidic linkages to degradation induced by heat and acid. Key standard oligosaccharides representing the major types found in foods (3′-sialyllactose and 6′-sialyl-N-acetyllactosamine for milk, raffinose and stachyose for legumes) were selected as model systems and underwent each of the following treatments independently: (1) labeled with the derivatizing agent 1-aminopyrene-3,6,8-trisulfonic (APTS) (followed by analysis with a capillary electrophoresis system coupled with a fluorescence detector), (2) dried from an acetonitrile-water mixture containing 0.1% trifluoroacetic acid, and (3) injected into an LC-MS system. We demonstrated that both raffinose and stachyose degraded during APTS-labeling by the acid in the labeling reagents. We also discovered that during centrifugal evaporation at 37 °C, all of the four nonderivatized oligosaccharides tested were partially degraded. Additionally, when the LC-MS eluent contained 0.1% formic acid, 3′-sialyllactose, raffinose, and stachyose underwent extensive in-source fragmentation during analysis. Lastly, we identified a simple strategy that can reduce the probability of incorrect oligosaccharide identification resulting from extensive in-source fragmentation.

N-Glycans are structurally similar to human milk oligosaccharides, the gold standard prebiotics for infants. Bovine milk N-glycans released by endo-β-N-acetylglucosaminidase (EndoBI-1) were shown to have similar prebiotic selectivity as human milk oligosaccharides, explaining the interest for N-glycan recovery for use as prebiotics. Industrial thermal treatments such as hightemperature short-time (HTST) and ultra-high-temperature (UHT) might favor the enzymatic deglycosylation of N-glycans through promoting protein denaturation. We investigated the effects of HTST (72 °C for 15 s) and UHT (135 °C for 3 s) on N-glycan release from bovine colostrum glycoproteins by nonimmobilized and amino-immobilized EndoBI-1. A total of 104 N-glycans including isomers/anomers were identified by high-resolution mass spectrometry. In both EndoBI-1 forms, HTST increased the release of N-glycans; however, the impact of UHT on releasing N-glycans was comparable to the nonthermal treatment. Although the amino-immobilized enzyme similarly released neutral N-glycans as the free form, it released fewer sialylated and fucosylated Nglycans.