Chemical and Biomolecular Engineering - Research Publications

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    Application of mechanistic modelling in membrane and fiber chromatography for purification of biotherapeutics - A review
    Qu, Y ; Baker, I ; Black, J ; Fabri, L ; Gras, SL ; Lenhoff, AM ; Kentish, SE (ELSEVIER, 2024-02-08)
    Mechanistic modelling is a simulation tool which has been effectively applied in downstream bioprocessing to model resin chromatography. Membrane and fiber chromatography are newer approaches that offer higher rates of mass transfer and consequently higher flow rates and reduced processing times. This review describes the key considerations in the development of mechanistic models for these unit operations. Mass transfer is less complex than in resin columns, but internal housing volumes can make modelling difficult, particularly for laboratory-scale devices. Flow paths are often non-linear and the dead volume is often a larger fraction of the overall volume, which may require more complex hydrodynamic models to capture residence time distributions accurately. In this respect, the combination of computational fluid dynamics with appropriate protein binding models is emerging as an ideal approach.
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    The Transition from Resin Chromatography to Membrane Adsorbers for Protein Separations at Industrial Scale
    Qu, Y ; Bekard, I ; Hunt, B ; Black, J ; Fabri, L ; Gras, SLL ; Kentish, SEE (TAYLOR & FRANCIS INC, 2023-01-01)
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    Economic optimization of antibody capture through Protein A affinity nanofiber chromatography
    Qu, Y ; Bekard, I ; Hunt, B ; Black, J ; Fabri, L ; Gras, SL ; Kentish, SE (ELSEVIER, 2024-01)
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    The effect of pH on the fat and protein within cream cheese and their influence on textural and rheological properties
    Ong, L ; Pax, AP ; Ong, A ; Vongsvivut, J ; Tobin, MJ ; Kentish, SE ; Gras, SL (Elsevier BV, 2020-12-01)
    The effect of variation in acid gel pH during cream cheese production was investigated. The gel microstructure was denser and cheese texture firmer, as the pH decreased from pH 5.0 to pH 4.3, despite the viscoelasticity of these gels remaining similar during heating. Protein hydration and secondary structure appeared to be key factors affecting both cheese microstructure and properties. Proteins within the matrix appeared to swell at pH 5.0, leading to a larger corpuscular structure; greater β-turn structure was also observed by synchrotron-Fourier transform infrared (S-FTIR) microspectroscopy and the cheese was softer. A decrease in pH led to a denser microstructure with increased aggregated β-sheet structure and a firmer cheese. The higher whey protein loss at low pH likely contributed to increased cheese hardness. In summary, controlling the pH of acid gel is important, as this parameter affects proteins in the cheese, their secondary structure and the resulting cream cheese.
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    Effects of shredding on the functionality, microstructure and proteolysis of low-moisture mozzarella cheese
    Pax, AP ; Ong, L ; Kentish, SE ; Gras, SL (ELSEVIER SCI LTD, 2021-06)
    Low-moisture mozzarella cheese (LMMC) is commonly shredded before packaging, however, the effects of shredding are not fully understood. Industrially-produced block and shredded LMMC were studied during 8 weeks of storage at 4 °C. Cheese shredded on 15 d and at 8 weeks of age, coated with microcrystalline cellulose and stored in a modified atmosphere (70% N₂ and 30% CO₂), had an altered microstructure after 8 weeks compared with vacuum-packed block cheese. In the latter case the fat formed a more dispersed phase. Proteolysis was higher in shredded samples and a higher level of two bacterial proteases was detected. Despite these differences, the meltability and stretchability of the block and shredded LMMC were similar. The microstructure and functionality of cheese shredded at 15 d and stored for a further 6 weeks was similar to cheese shredded at 8 weeks, suggesting there is a flexible period for performing cheese shredding processes.
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    Structure and functionality of almond proteins as a function of pH
    Devnani, B ; Ong, L ; Kentish, S ; Gras, SL (ELSEVIER, 2021-10)
    Almond proteins have potential utility in a range of food and beverages but it is not clear how pH affects protein structure and function. The behaviour of almond protein isolate was examined under conditions of neutral and acidic pH (pH 7 and 4). The isolate was highly soluble (70–80%) at either pH. An increase in acidity lead to protein unfolding, an increase in random coil structure and the appearance of lower molecular weight proteins due to acidic hydrolysis. These structural changes at pH 4 increased the capacity for foam formation and foam stability, increased viscosity and led to concentration and age dependent thickening. Gels, similar in strength but with distinct microstructures and properties were obtained following heating. At pH 7, a particulate type gel with an interconnected protein network was formed, while the gel at pH 4 had a dense continuous protein matrix. The gels differed in their susceptibility to chemical disruption, suggesting different underlying molecular interactions. The ability to alter protein structure and properties as a function of pH and heating could be used to broaden the application of almond proteins and develop a variety of food products, such as protein supplements and vegan alternatives to traditional products.
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    Bioactives from Whey: A Sustainable Approach to Enzymatic Production of Sialyl-N-acetyllactosamine
    Alavijeh, MK ; Zeuner, B ; Meyer, AS ; Gras, SL ; Kentish, SE (AMER CHEMICAL SOC, 2022-05-16)
    The use of dairy whey to manufacture pharmaceutical products fosters sustainable environmental and economic development. This study represents a new strategy for upgrading of whey to 3′-sialyl-N-acetyllactosamine (3′-SLN) as an important structural component of glycoproteins and a receptor analog capable of forming complexes with hemagglutinins on influenza viruses. N-Acetyllactosamine (LacNAc) was enzymatically produced and purified directly from whey with no pretreatment required. An engineered and recombinantly produced sialidase with trans-sialylation ability from the non-pathogenic Trypanosoma rangeli was then used to transfer sialic acid from whey-derived, sialylated casein glycomacropeptide (CGMP) to this LacNAc. A maximum of 0.92 mM 3′-SLN was obtained at an equimolar ratio of LacNAc to bound sialic acid in CGMP; on the other hand, a molar ratio of 10 gave a fourfold greater 3′-SLN concentration. The variations in the concentration of 3′-SLN and free sialic acid during the hydrolysis reaction were modeled under different reaction conditions using machine learning and mechanistic approaches. The mechanistic analysis of the reaction indicated that the relative initial trans-sialylation rate to hydrolysis rate is directly proportional to the initial LacNAc concentration, with the ratio of trans-sialylation to hydrolysis rate constants equal to 111 M-1. The maximum 3′-SLN yield obtained was 75% based on α-2,3-sialic acid bound to CGMP. Separation of CGMP and reuse of enzyme were also investigated in an enzymatic membrane reactor.
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    Physicochemical and rheological properties of commercial almond-based yoghurt alternatives to dairy and soy yoghurts
    Devnani, B ; Ong, L ; Kentish, SE ; Scales, PJ ; Gras, SL (Elsevier, 2022-12-01)
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    Industrial freezing and tempering for optimal functional properties in thawed Mozzarella cheese
    Pax, AP ; Ong, L ; Pax, RA ; Vongsvivut, J ; Tobin, MJ ; Kentish, SE ; Gras, SL (ELSEVIER SCI LTD, 2023-03-30)
    Mozzarella cheese was industrially frozen (-18 °C), stored for up to six months, tempered at 4 °C for one or three weeks and the structure and functionality compared to cheese stored at 4 °C and cheese aged at 4 °C for four weeks prior to freezing. When combined with ageing or tempering, the slow industrial freezing minimised changes to the protein network as detected by confocal microscopy and arrested proteolysis. Cheese functionality improved with three weeks of tempering, with properties similar to cheese refrigerated for one month, potentially due to increased proteolysis and protein rehydration. Frozen storage induced β-sheet and β-turn structures, as detected by S-FTIR microspectroscopy, with longer tempering leading to structural recovery in the cheese. This study indicates the proteolysis and functionality of frozen cheese can be optimised with tempering time. It also provides new insights into heat transfer during the industrial freezing and tempering of cheese.
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    Synthesis of N-Acetyllactosamine and N-Acetyllactosamine-Based Bioactives
    Alavijeh, MK ; Meyer, AS ; Gras, SL ; Kentish, SE (AMER CHEMICAL SOC, 2021-07-14)
    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.