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    Application of industrial treatments to donor human milk: influence of pasteurization treatments, storage temperature, and time on human milk gangliosides
    (Springernature, 2018) Salcedo, Jaime; Karav, Sercan; Le Parc, Annabelle; Cohen, Joshua L.; de Moura Bell, Juliana M. L. N.; Sun, Adam; Lange, Matthew C.
    Donor milk is the best option when mother's own milk is unavailable. Heat treatments are applied to ensure donor milk safety. The effects of heat treatments on milk gangliosides-bioactive compounds with beneficial antibacterial, anti-inflammatory, and prebiotic roles-have not been studied. The most abundant gangliosides in non-homogenized human milk were characterized and quantified by liquid chromatography-mass spectrometry (LC-MS)/MS before and after pasteurization treatments mimicking industrial conditions (63 degrees C/30 min, 72 degrees C/15 s, 127 degrees C/5 s, and 140 degrees C/6 s). Ganglioside stability over a 3-month period was assessed following the storage at 4 and 23 degrees C. Independent of the heat treatment applied, gangliosides were stable after 3 months of storage at 4 or 23 degrees C, with only minor variations in individual ganglioside structures. These findings will help to define the ideal processing and storage conditions for donor milk to maximize the preservation of the structure of bioactive compounds to enhance the health of fragile newborns. Moreover, these results highlight the need for, and provide a basis for, a standardized language enabling biological and food companies, regulatory agencies, and other food stakeholders to both annotate and compute the ways in which production, processing, and storage conditions alter or maintain the nutritive, bioactive, and organoleptic properties of ingredients and foods, as well as the qualitative effects these foods and ingredients may have on conferring phenotype in the consuming organism. Donor milk treatment: key nutrients preserved after pasteurization Donor human milk, the best alternative to mother's own milk, usually needs to be pasteurized before use out of safety concerns. Daniela Barile at University of California Davis, USA, and colleagues studied the effects of heat treatment and storage temperature and time on milk gangliosides, a class of sugar-derived compounds important for neural and brain development of newborns, among other bioactivities. They found that, while there were minor structural changes during mimicked industrial pasteurization processes, gangliosides remain stable for at least three months either in the refrigerator or at room temperature. These results may help standardize the processing protocols and storage conditions for donor milk, and the methods can be extended to other bioactive components.
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    Immobilization of an Endo-?-N-acetylglucosaminidase for the Release of Bioactive N-glycans
    (Mdpi, 2018) Cohen, Joshua L.; Karav, Sercan; Barile, Daniela; Bell, Juliana M. L. N. de Moura
    As more is learned about glycoproteins' roles in human health and disease, the biological functionalities of N-linked glycans are becoming more relevant. Protein deglycosylation allows for the selective release of N-glycans and facilitates glycoproteomic investigation into their roles as prebiotics or anti-pathogenic factors. To increase throughput and enzyme reusability, this work evaluated several immobilization methods for an endo-beta-N-acetylglucosaminidase recently discovered from the commensal Bifidobacterium infantis. Ribonuclease B was used as a model glycoprotein to compare N-glycans released by the free and immobilized enzyme. Amino-based covalent method showed the highest enzyme immobilization. Relative abundance of N-glycans and enzyme activity were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Kinetic evaluation demonstrated that upon immobilization, both V-max and the K-m decreased. Optimal pH values of 5 and 7 were identified for the free and immobilized enzyme, respectively. Although a higher temperature (65 vs. 45 degrees C) favored rapid glycan release, the immobilized enzyme retained over 50% of its original activity after seven use cycles at 45 degrees C. In view of future applications in the dairy industry, we investigated the ability of this enzyme to deglycosylate whey proteins. The immobilized enzyme released a higher abundance of neutral glycans from whey proteins, while the free enzyme released more sialylated glycans, determined by nano-LC Chip Q-ToF MS.
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    Recent advances in immobilization strategies for glycosidases
    (Wiley, 2017) Karav, Sercan; Cohen, Joshua L.; Barile, Daniela; de Moura Bell, Juliana Maria Leite Nobrega
    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--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. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:104-112, 2017