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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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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ItemCytochrome P450-mediated N-demethylation of noscapine by whole-cell biotransformation: process limitations and strategies for optimisation(Springer Verlag, 2020-07-01)Cytochrome P450 enzymes catalyse reactions of significant industrial interest but are underutilised in large-scale bioprocesses due to enzyme stability, cofactor requirements and the poor aqueous solubility and microbial toxicity of typical substrates and products. In this work, we investigate the potential for preparative-scale N-demethylation of the opium poppy alkaloid noscapine by a P450BM3 (CYP102A1) mutant enzyme in a whole-cell biotransformation system. We identify and address several common limitations of whole-cell P450 biotransformations using this model N-demethylation process. Mass transfer into Escherichia coli cells was found to be a major limitation of biotransformation rate and an alternative Gram-positive expression host Bacillus megaterium provided a 25-fold improvement in specific initial rate. Two methods were investigated to address poor substrate solubility. First, a biphasic biotransformation system was developed by systematic selection of potentially biocompatible solvents and in silico solubility modelling using Hansen solubility parameters. The best-performing biphasic system gave a 2.3-fold improvement in final product titre compared to a single-phase system but had slower initial rates of biotransformation due to low substrate concentration in the aqueous phase. The second strategy aimed to improve aqueous substrate solubility using cyclodextrin and hydrophilic polymers. This approach provided a fivefold improvement in initial biotransformation rate and allowed a sixfold increase in final product concentration. Enzyme stability and cell viability were identified as the next parameters requiring optimisation to improve productivity. The approaches used are also applicable to the development of other pharmaceutical P450-mediated biotransformations.
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ItemHigh-Efficiency Biocatalytic Conversion of Thebaine to Codeine(American Chemical Society, 2020-04-28)An enzymatic biosynthesis approach is described for codeine, the most widely used medicinal opiate, providing a more environmentally sustainable alternative to current chemical conversion, with yields and productivity compatible with industrial production. Escherichia coli strains were engineered to express key enzymes from poppy, including the recently discovered neopinone isomerase, producing codeine from thebaine. We show that compartmentalization of these enzymes in different cells is an effective strategy that allows active spatial and temporal control of reactions, increasing yield and volumetric productivity and reducing byproduct generation. Codeine is produced at a yield of 64% and a volumetric productivity of 0.19 g/(L·h), providing the basis for an industrially applicable aqueous whole-cell biotransformation process. This approach could be used to redirect thebaine-rich feedstocks arising from the U.S. reduction of opioid manufacturing quotas or applied to enable total biosynthesis and may have broader applicability to other medicinal plant compounds.
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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.
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ItemThe Effect of Salt on the Structure of Individual Fat Globules and the Microstructure of Dry Salted Cheddar Cheese(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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ItemTailoring the structure of casein micelles through a multifactorial approach to manipulate rennet coagulation properties(Elsevier Inc., 2020-04-01)The properties of casein micelles are known to be affected by modifications to the environment, such as variations in pH or the addition of salts, yet the scientific literature typically considers the effects of one factor at a time, while in industrial processes, several modifications are performed simultaneously. The aim of this study was to assess the impact of multifactorial environmental modifications on the colloidal, structural and rennet coagulation properties of casein micelles in a simplified model system. A key finding was that dense regions (~20 nm in size) could be released from the casein micelle. The addition of NaCl and CaCl2 had opposing effects, i.e. enhancing or limiting this micellar disruption, respectively. A decrease in pH had the strongest impact on the mineral balance, causing the colloidal CaP to solubilize and the micelle to swell. The rennet clotting time was impacted by variations in pH and NaCl content. Interestingly, a consideration of all three levels of casein micelle structure and their interactions was needed to explain variations in the firmness of rennet gels. This study illustrates the complex interplay of factors affecting micellar structure and improves our understanding of how micelles can be manipulated to control their properties.
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ItemSimulation and economic assessment of large-scale enzymatic N-acetyllactosamine manufacture(Elsevier BV, 2020-02-15)N-acetyllactosamine (LacNAc) is an important lactose-derived molecule which can act as an effective prebiotic. In this study a process for the enzymatic synthesis and downstream purification of LacNAc was designed based on the use of thermostable β-galactosidases from Bacillus circulans (BgaD-D), Thermus thermophilus HB27 or Pyrococcus furiosus (CelB) respectively. Four configurations for the purification stage were simulated; anion-exchange chromatography, an activated charcoal-Celite column, N-acetylglucosamine (GlcNAc) crystallization and an activated charcoal-Celite column, as well as selective crystallization. While the enzyme CelB has greater stability at higher temperatures, this enzyme gives a lower LacNAc yield, leading to significant capital investment. For the design based on the BgaD-D biocatalyst and anion exchange chromatography, recovery of GlcNAc improved the project profitability when the GlcNAc price was greater than $10 per kg. GlcNAc was the main contributor to the raw material costs for most processes, although methanol contributed 72% of these costs for the process based on an activated charcoal column. The use of a crystallizer for GlcNAc separation before this column, reduced this methanol consumption by 73%. The use of selective crystallization proved the best approach, reducing the minimum LacNAc sales price to $2 per gram. The plant was more economic when the acceptor to donor ratio was reduced from 10 to 4 and the lactose concentration increased from 50 mM to 550 mM.