Chemical and Biomolecular Engineering - Research Publications

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    Synthesis of N-Acetyllactosamine and N-Acetyllactosamine-Based Bioactives
    Alavijeh, MK ; Meyer, AS ; Gras, SL ; Kentish, SE (AMER CHEMICAL SOC, 2021-06-21)
    N-Acetyllactosamine (LacNAc) or more specifically β-d-galactopyranosyl-1,4-N-acetyl-d-glucosamine is a unique acyl-amino sugar and a key structural unit in human milk oligosaccharides, an antigen component of many glycoproteins, and an antiviral active component for the development of effective drugs against viruses. LacNAc is useful itself and as a basic building block for producing various bioactive oligosaccharides, notably because this synthesis may be used to add value to dairy lactose. Despite a significant amount of information in the literature on the benefits, structures, and types of different LacNAc-derived oligosaccharides, knowledge about their effective synthesis for large-scale production is still in its infancy. This work provides a comprehensive analysis of existing production strategies for LacNAc and important LacNAc-based structures, including sialylated LacNAc as well as poly- and oligo-LacNAc. We conclude that direct extraction from milk is too complex, while chemical synthesis is also impractical at an industrial scale. Microbial routes have application when multiple step reactions are needed, but the major route to large-scale biochemical production will likely lie with enzymatic routes, particularly those using β-galactosidases (for LacNAc synthesis), sialidases (for sialylated LacNAc synthesis), and β-N-acetylhexosaminidases (for oligo-LacNAc synthesis). Glycosyltransferases, especially for the biosynthesis of extended complex LacNAc structures, could also play a major role in the future. In these cases, immobilization of the enzyme can increase stability and reduce cost. Processing parameters, such as substrate concentration and purity, acceptor/donor ratio, water activity, and temperature, can affect product selectivity and yield. More work is needed to optimize these reaction parameters and in the development of robust, thermally stable enzymes to facilitate commercial production of these important bioactive substances.
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    Effect of rennet on the composition, proteolysis and microstructure of reduced-fat Cheddar cheese during ripening
    Soodam, K ; Ong, L ; Powell, IB ; Kentish, SE ; Gras, SL (SPRINGER FRANCE, 2015-09-01)
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    Improving beta-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization
    Alavijeh, MK ; Meyer, AS ; Gras, SL ; Kentish, SE (AMER CHEMICAL SOC, 2020-11-02)
    The biotransformation of lactose into gut-bioactive glycans catalyzed by β-galactosidase can give economic value to lactose-rich side streams generated in the food or the dairy industry. Herein, we study the immobilization of the commercially used β-galactosidase from Bacillus circulans onto silica particles using an enzyme immobilization technology involving a cross-linked layer-by-layer encapsulation method. The immobilized β-galactosidase was used for the synthesis of N-acetyllactosamine (LacNAc) as an important precursor for numerous bioactive compounds and a prebiotic in itself. Techniques including molecular analysis, enzyme activity determination, secondary structure analysis, thermodynamic characterization, and the determination of thermal and operational stability were conducted to characterize the immobilized enzyme. Changes in the activity of the enzyme after immobilization were attributed to possible changes in electrostatic, covalent, and protein-protein interactions. Immobilization significantly improved the enzymatic LacNAc yield compared to the free enzyme. In turn, this improved the economics and the sustainability of the process. The immobilized enzyme encapsulated in multilayer films was significantly more stable in the presence of divalent cations and its thermostability also substantially increased with the thermal denaturation activation energy increasing from 53 to 294 kJ mol-1. The immobilized enzyme was successfully reused in eight consecutive reaction cycles with no significant reduction in the LacNAc yield. The improved transgalactosylation yield and productivity, higher stability, and reusability obtained with this immobilization method provide new opportunities for industrial applications.
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    Heat induced denaturation, aggregation and gelation of almond proteins in skim and full fat almond milk
    Devnani, B ; Ong, L ; Kentish, S ; Gras, S (Elsevier BV, 2020-09-30)
    The effect of thermal treatment (45-95 ⁰C for 30 minutes) on the structure of almond milk proteins was assessed, as the unfolding and association of these proteins in response to heat is not well understood. Above 55 ⁰C, protein surface hydrophobicity and particle size increased and alpha helical structure decreased, reducing the stability of skim or full fat milk. Fractal protein clusters were observed at 65-75 ⁰C and weakly flocculated gels with a continuous protein network occurred at 85-95 ⁰C, resulting in gels with high water holding capacity and a strength similar to dairy gels. The presence of almond fat increased gel strength but led to a more heterogenous microstructure, which may be improved by homogenisation. Elasticity could also be increased with protein concentration. This study improves our understanding of the heat stability of almond milk proteins and indicates their potential as a gelling ingredient for vegan and vegetarian products.
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    The role of cations in regulating reaction pathways driven by Bacillus circulans β-galactosidase
    Karimi Alavijeh, M ; Meyer, AS ; Gras, S ; Kentish, SE (Elsevier, 2020-09-01)
    A β-galactosidase (EC 3.2.1.23) from Bacillus circulans (Biolacta FN5) can catalyse transgalactosylation reactions with lactose as a donor. In addition to their function as cofactors and structural stabilisers in biocatalytic reactions, cations can play a role in salt-bridge interactions and electrostatic charge screening of proteins. In this work, we investigated the impact of calcium, magnesium, sodium and potassium, commonly found in dairy whey systems, on the transgalactosylation kinetics of the β-galactosidase from Bacillus circulans. Both molecular modeling and quantitative experimental methods were used to assess enzyme aggregation and resulting loss in enzyme activity that is initiated by high concentrations of these cations. The effect of this loss in activity with time was studied during the transgalactosylation of N-acetylglucosamine (GlcNAc) to N-acetyllactosamine (LacNAc) using lactose as the donor. No significant change in hydrolysis or transgalactosylation reaction kinetics was observed at low concentrations of divalent cations (Ca2+ or Mg2+) or up to 100 mM of monovalent cations (Na+ or K+). The enzymatic yield and selectivity, however, were significantly affected at concentrations of 100 mM of Ca2+ or Mg2+. These changes were the result of both the loss in enzyme activity and a reduction in the reaction rate constant for hydrolysis and formation of the undesired isomer, Allo-LacNAc. In particular, addition of magnesium enhanced the selectivity for LacNAc over Allo-LacNAc, with no significant reduction in the LacNAc yield. These findings suggest that cations can be employed to regulate the action of β-galactosidase during transgalactosylation through the formation of protein aggregates.
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    The application of forward osmosis to dairy processing
    Chen, GQ ; Gras, SL ; Kentish, SE (Elsevier, 2020-09-01)
    This work assesses the feasibility for concentrating process streams within dairy processing facilities using commercial forward osmosis membranes; to increase their total solids concentrations before entering energy intensive unit operations including thermal evaporators and spray dryers. These streams include demineralised whey, lactose, whey protein concentrate, sweet whey and skim milk. FTSH2O cellulose acetate (CTA) and Aquaporin flat sheet membranes are used with magnesium chloride concentrations of 1.66 ± 0.12 M as the draw solution. The experimental data are fitted to conventional mathematical models for forward osmosis, further modified by considering the nonlinear relationship between osmotic pressure and solute concentration. The diffusion coefficients of magnesium chloride in 1.6 M solutions at 10 °C, 20 °C and 50 °C are obtained and reported for the first time. Minimal fouling and a significantly smaller degree of concentration polarisation was observed on the membrane surface during lactose concentration compared to the concentration of other dairy solutions, due to the absence of proteins and calcium phosphate salts. The transfer of magnesium into the concentrated products was monitored and shown to be below 100 mg per 100 g dry powder. Acid cleaning alone was not effective in recovering pure water flux, and enzyme cleaners at neutral pH were needed given the limited pH tolerance (3–8) of the CTA membranes. Total solids concentrations of the concentrated dairy streams by forward osmosis (up to 40%) exceed those which can be achieved by nanofiltration and reverse osmosis (i.e., 15–20%). This study shows that forward osmosis is an effective approach to concentrate relevant dairy streams to achieve high concentration factors (e.g. >4 for sweet whey samples) without jeopardising product quality.
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    A proteomic characterization shows differences in the milk fat globule membrane of buffalo and bovine milk
    Nguyen, HTH ; Ong, L ; Hoque, A ; Kentish, SE ; Williamson, N ; Ang, C-S ; Gras, SL (Elsevier, 2017-09-01)
    The proteins of the milk fat globule membrane (MFGM) have a number of functions, such as the regulation of milk fat secretion and metabolism, the uptake and transportation of fatty acids in the intestine, and potential protection from bacterial or viral infection. While the proteome of the MFGM in bovine milk has been extensively characterized, knowledge of these proteins in buffalo milk is limited. In this study, a proteomic approach was used to characterize the proteome of the buffalo MFGM. Multiple extraction techniques were used to increase the number of proteins identified, while label free relative quantitative liquid chromatography tandem mass spectrometry was used for comparison between the buffalo and bovine MFGM proteomes. A total of 220 buffalo MFGM proteins and 234 bovine MFGM proteins were identified after being filtered from the initial dataset of 757 and 680 proteins, respectively. A sixfold higher concentration of xanthine oxidoreductase was identified per mass of buffalo MFGM protein extracted, together with significantly greater concentrations of platelet glycoprotein 4, heat shock cognate and calcineurin B homologous protein. The expression of xanthine oxidoreductase in the MFGM of buffalo milk, which can affect milk shelf-life and flavor, was confirmed by Western blot analysis and a heterogeneous distribution of this protein observed in situ on the surface of the MFGM. The high concentration of fat in buffalo milk, together with the differences in the MFGM proteome provide insights into the differences in nutritional profile, biological function and properties of these two milk products.
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    The Effect of Salt on the Structure of Individual Fat Globules and the Microstructure of Dry Salted Cheddar Cheese
    Ong, L ; D’Incecco, P ; Pellegrino, L ; Nguyen, HTH ; Kentish, SE ; Gras, SL (Springer, 2020-03)
    Salting is an essential step in the production of Cheddar and other cheese varieties and is a well-studied process but the effect of salt addition on the microstructure of the milk ingredients and resulting cheese is not well known. This study provides insights into how the primary components in milk and the cheese matrix respond to salting. High concentrations of salt (15–25% (w/w) NaCl) disrupted fat globules due to the increased osmotic pressure. This led to fat coalescence, resulting in large fat globules >10 μm in diameter, together with submicron sized fat globules ~ 120–500 nm in diameter. Salt addition also prevented the visualization of the milk fat globule membrane when added at high concentrations (25% (w/w) NaCl) and induced asymmetry in liquid ordered domains at lower concentrations (10% (w/w) NaCl). The microstructure of the surface of the milled curd was compacted by salt, appearing coarse with 5% (w/w) NaCl or more hydrated with a denser protein structure with 2.5% (w/w) NaCl. After pressing, the curd junctions were fine and thin within the unsalted sample but coarse and thick where 5% (w/w) NaCl was added. Such coarse junctions appear to reduce binding between curd particles leading to a less cohesive cheese. Our results show that NaCl can significantly impact on the structure of fat and protein matrix of the curd surface if salt is not evenly distributed during dry salting. High concentrations of salt can also change the microstructure and texture of the cheese, resulting in a more heterogeneous product.
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    The addition of calcium chloride in combination with a lower draining pH to change the microstructure and improve fat retention in Cheddar cheese
    Ong, L ; Soodam, K ; Kentish, SE ; Powell, IB ; Gras, SL (Elsevier, 2015-07-01)
    Calcium chloride addition and the whey draining pH are known to impact on cheese making. The effect of 100 or 300 mg kg−1 calcium chloride (CaCl2) and the whey draining pH (6.2 or 6.0) on the microstructure of Cheddar cheese was assessed using confocal and cryo scanning electron microscopy. The gel made with 300 mg kg−1 CaCl2 was found to have a denser protein network and smaller pores than the gel with lower or no CaCl2 addition. CaCl2 addition reduced fat lost to the sweet whey. The texture of the cheeses with a lower draining pH was harder and moisture content lower. Our results show that the combination of calcium addition and lower draining pH could be used to increase network formation at the early stages of cheese making to improve fat retention while maintaining a similar level of total calcium in the final cheese.