Chemical and Biomolecular Engineering - Research Publications
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ItemThe effect of pH on the fat and protein within cream cheese and their influence on textural and rheological properties(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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ItemEffects of shredding on the functionality, microstructure and proteolysis of low-moisture mozzarella cheese(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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ItemStructure and functionality of almond proteins as a function of pH(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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ItemBioactives from Whey: A Sustainable Approach to Enzymatic Production of Sialyl-N-acetyllactosamine(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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ItemSynthesis of N-Acetyllactosamine and N-Acetyllactosamine-Based Bioactives(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.
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ItemImproving β-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization(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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ItemHeat induced denaturation, aggregation and gelation of almond proteins in skim and full fat almond milk(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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ItemThe role of cations in regulating reaction pathways driven by Bacillus circulans β-galactosidase(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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ItemThe application of forward osmosis to dairy processing(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.