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    Impact of cell wall polysaccharide modifications on the performance of Pichia pastoris: novel mutants with enhanced fitness and functionality for bioproduction applications.
    (BioMed Central Ltd, 2024-02-17) Cheng B; Yu K; Weng X; Liu Z; Huang X; Jiang Y; Zhang S; Wu S; Wang X; Hu X
    BACKGROUND: Pichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities. RESULTS: Two novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in β-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage. CONCLUSIONS: Two novel P. pastoris chassis hosts with impaired β-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.
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    Characterization of the extracellular polymeric substances matrix of Pseudomonas biofilms formed at the air-liquid interface
    (Elsevier Ltd, 2025-01-27) Muthuraman S; Flint S; Palmer J
    Pseudomonas are common psychotropic food spoilage organisms that affect the quality of aerobically chilled food products. Biofilm formation of these bacteria on food contact surfaces can provide a continuous contamination source, leading to food spoilage. Pseudomonas produce proteolytic and lipolytic enzymes which lead to organoleptic degradation of stored food products. The biofilm extracellular polymeric substances matrix (EPS) protects the bacterial cells from CIP (Cleaning-In-Place) chemicals and adverse conditions. Studies on the composition of the EPS matrix and the molecules present in the EPS matrix are limited. In this study, the EPS composition of mono-species biofilms of Pseudomonas lundensis and Pseudomonas cedrina on polystyrene and stainless-steel surfaces was characterized by chemical analysis and microscopical observations. The biofilms were allowed to grow on polystyrene and stainless-steel surfaces with half-strength TSB for 2 weeks at 30 °C and cold chain temperatures of 7 °C and 4 °C. The EPS was extracted by sonication and centrifugation and chemically analysed for cellulose, total polysaccharides, total proteins, and eDNA. Pseudomonas isolates in this study formed biofilms at the air-liquid interface. The formation of ring-like structures of cells was observed on the polystyrene surface. eDNA formed as a thread-like structure on a polystyrene surface while it formed channels on a stainless-steel surface. The amount of EPS varied at different temperatures. More EPS was formed at 4 °C than 30 °C. Flagellin, Clp protease, Arginine deiminase, and ATP-Binding Cassette (ABC) transporter substrate-binding proteins were the key proteins identified in the biofilm matrix of P. lundensis.
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    Biopolymer-polyphenol conjugates: Novel multifunctional materials for active packaging
    (Elsevier B V, 2024-11) Sahraeian S; Abdollahi B; Rashidinejad A
    The development of natural active packaging materials and coatings presents a promising alternative to petroleum-based packaging solutions. These materials are engineered by incorporating functional ingredients with preservative capabilities. Concurrently, research has highlighted the diverse physicochemical, functional, and health-promoting properties of protein-polyphenol, polysaccharide-polyphenol, and protein-polysaccharide-polyphenol conjugates within various food formulations. However, a critical gap exists regarding the exploration of these biopolymers as active packaging materials. In contrast to conventional approaches for developing active packaging materials, this review presents a novel perspective by focusing on biopolymer-polyphenol conjugates. In this work, we delve into the realm of active packaging materials and coatings constructed from these conjugates, highlighting their potential as multifunctional active components in food packaging and preservation. This review comprehensively investigates the physicochemical properties, functionalities, and health-promoting activities associated with biopolymer-polyphenol conjugates. Their emulsification, antioxidant, and antimicrobial activities, coupled with enhancements in mechanical strength and permeability properties, contribute to their multifunctional nature. Furthermore, we explore the potential advantages and limitations of utilizing these conjugates in active packaging applications. Finally, the review concludes by proposing crucial research avenues for further exploration of biopolymer-polyphenol conjugates within the domain of active food packaging.
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    Enhanced properties of non-starch polysaccharide and protein hydrocolloids through plasma treatment: A review
    (Elsevier B V, 2023-09-30) Sahraeian S; Rashidinejad A; Niakousari M
    Hydrocolloids are important ingredients in food formulations and their modification can lead to novel ingredients with unique functionalities beyond their nutritional value. Cold plasma is a promising technology for the modification of food biopolymers due to its non-toxic and eco-friendly nature. This review discusses the recent published studies on the effects of cold plasma treatment on non-starch hydrocolloids and their derivatives. It covers the common phenomena that occur during plasma treatment, including ionization, etching effect, surface modification, and ashing effect, and how they contribute to various changes in food biopolymers. The effects of plasma treatment on important properties such as color, crystallinity, chemical structure, rheological behavior, and thermal properties of non-starch hydrocolloids and their derivatives are also discussed. In addition, this review highlights the potential of cold plasma treatment to enhance the functionality of food biopolymers and improve the quality of food products. The mechanisms underlying the effects of plasma treatment on food biopolymers, which can be useful for future research in this area, are also discussed. Overall, this review paper presents a comprehensive overview of the current knowledge in the field of cold plasma treatment of non-starch hydrocolloids and their derivatives and highlights the areas that require further investigation.
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    Impact of incorporations of various polysaccharides on rheological and microstructural characteristics of heat-induced quinoa protein isolate gels
    (Springer Science+Business Media, LLC, 2022-09) Patole S; Cheng L; Yang Z
    This study aimed to investigate the properties of heat-induced gels (85 °C for 30 min) of quinoa protein isolate (QPI) in the presence and absence of various polysaccharides including guar gum (GG), locust bean gum (LBG), and xanthan gum (XG) at pH 7. For this purpose, samples with three gum concentrations (0.05, 0.1, and 0.2 wt%) at a fixed QPI concentration (10 wt%) and a fixed ionic strength (50 mM NaCl) were studied in terms of their gelation behaviour, small and large deformation rheological properties, water holding capabilities, and microstructural characteristics. Rheological measurements revealed that all polysaccharides incorporation could improve gel strength (complex modulus, G*) and breaking stress, accelerate gel formations, and more stiffer gels were obtained at greater polysaccharide concentrations. The XG exhibited the most gel strengthening effect followed by LBG and GG. Incorporation of 0.2 wt% XG led to a 15 folds increase in G* compared to the control. Confocal laser scanning microscopy observation revealed that the polysaccharides also altered gel microstructures, with the gels containing XG showing the most compact gel structures. The findings of this study may provide useful information for the fabrication of novel QPI based food gel products with improved texture.
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    The tale of the shear-thickening mamaku polysaccharide, from forest to gut : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Food Technology), Massey University, Palmerston North, New Zealand. EMBARGOED until further notice.
    (Massey University, 2023) Bisht, Akshay
    The New Zealand black tree fern (Cyathea medullaris, ‘mamaku’ in the Māori language) is grown across the Pacific Islands and has a long history of use for therapeutic benefits or as food by Māori people. The water-soluble gum extract from mamaku fern contains a novel glucuronomannan biomacromolecule, called mamaku polysaccharide (MP), which has been shown to exhibit a unique shear-thickening (i.e., increase in viscosity on shearing) behaviour at a similar shear rate as that found in the human stomach. Herein, the objective was to gain a better technical and physiological understanding of MP for designing a novel shear-thickening ingredient for the industry with proven effects in humans. The shear-thickening behaviour of MP was sensitive to the harvesting age of mamaku fronds and industrial operations such as high temperature and shear. With the increase in harvesting age, the molecular weight of MP reduced, which consequently reduced the shear viscosity. The shear-thickening behaviour was lost in MP from old fronds. Furthermore, the temperature treatment disintegrated the backbone of MP into smaller fragments which caused a reduction in viscosity and extent of shear-thickening. Similar rheological trends were observed post-shear treatment, however, there was no evidence of depolymerisation. A combination of in vitro models revealed that mamaku gum extract could improve host gut functioning by reducing the activity of digestive enzymes (α-amylase, pepsin and lipase) and binding bile acids. Mamaku gum can act as a substrate for colonic fermentation, promote the production of short-chain fatty acids and alter the colonic microbial composition. Upon ingesting mamaku gum, the shear-thickening behaviour may develop in the oesophagus causing a possible choking hazard. Therefore, the potential of using the whole pith—natural entrapment of MP in the tissue of pith—as an alternative to gum extract was studied. Freeze-dried pith was ground to powder. The powder particles swelled upon rehydration with water and released the water-soluble MP into the continuous phase in a time-dependent manner. The presence of enough MP in the continuous phase to form polymer-polymer interactions resulted in a shear-thickening behaviour of the pith powder suspension similar to the MP extract solution. Moreover, the co-consumption of 1 h pre-hydrated mamaku pith powder with a carbohydrate-rich meal significantly reduced the postprandial glycaemic response (blood glucose peak height) in human participants. Additionally, the consumption of mamaku pith powder in rats could alter colonic microbiota. Interestingly, more than half of the MP (uronic acid) consumed by rats survived the gut transition and was obtained in faeces, suggesting that MP could potentially be used as a laxative. Thus, mamaku pith could be used as an alternative to gum extract to develop a natural shear-thickening ingredient which may potentially help to manage diabetes and improve colon health.
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    Polysaccharide-DNA strings for single molecular polysaccharide studies : a thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Biophysics and Soft Matter, School of Natural Sciences, Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Mohandas, Nimisha
    For several decades DNA has been the workhorse of single molecule experiments, owing to its large controllable size and simplicity of end group attachment. The use of DNA handles to study DNA-protein conjugates has also previously been employed to understand the behaviour of proteins at the single molecular level. In contrast, single molecule studies of polysaccharides are not widely known. This project attempts to develop a methodology in order to facilitate single molecule polysaccharide studies with optical tweezers (OT). Homogalacturonan (HG), a polysaccharide component extracted from pectin, a key component in plant cell walls, was chosen to be the subject of this study. The proposed strategy was to utilise DNA strands as "handles" with one end attached onto HG, and the other coupled to beads, to allow for stretching of HG, and other single molecule studies. In order to attach HG between different DNA handles, the chemistry present at the reducing and non-reducing ends of the polysaccharide which can be used to form bonds with end functionalised DNA strands was the point of focus. Ultimately the DNA-polysaccharide connection was mediated by streptavidin moieties linking biotin-functionalised ends. Streptavidin is a tetrameric protein, renowned for its strong binding to biotin that has to led to multitudinous applications. By separating streptavidin species that have differing numbers of binding sites plugged, "linking hubs" with trivalent, divalent and monovalent functionality were obtained. Species identity, and the plugging process were studied with capillary electrophoresis, which in this case provides several advantages over traditional gels. Subsequently, divalent linkers were used to concatenate two biotin-terminated 5 kb pieces of double stranded DNA, and the resulting string stretched in an optical tweezers experiment, demonstrating the "plug-and-play" potential of the methodology for coupling and extending molecules for use in single molecule biophysical experiments.
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    Structure-rheology relationships of protein-polysaccharide complexes at oil/water interfaces : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics at Biophysics and Soft Matter Group, School of Fundamental Science, Massey University
    (Massey University, 2021) Ramamirtham, Sashikumar
    The complexation of proteins with polysaccharides to form bio-complexes is being utilized in a variety of applications including food formulations, microencapsulation, protein separation and bioactive deliveries. Understanding the impact of these biomolecules on each other with discernment will not only improve our existing usages but also aid in devising newer applications. The duo of beta -lactoglobulin (beta -lg), a surface active globular whey protein, and pectin, a plant-derived polysaccharide, is the model protein-polysaccharide system of this study. Beta -lg and pectin have been reported to undergo complexation driven by electrostatic attraction leading to contrasting interfacial rheological properties depending on the fine structures of the polysaccharide. The aim of this thesis is to understand the role of fine structures of the polysaccharide in protein adsorption and the interfacial film formation. Given that beta -lg is the interfacially active molecule in this study, assemblies of beta -lg at dodecane/water interfaces at pHs 3 and 4, and at different conditions of ionic strength, salt type and temperature were studied. These parameters were tuned to vary the relative amounts of two native species, namely, monomer and its smallest aggregate, the dimer, while the interface was monitored using rheology and tensiometry. Unfolding of beta -lg dimers at the interface triggers the formation of disulfide linkages between the free thiol groups located at cys121 of the monomers. In this way, it is demonstrated here for the first time that beta -lg dimers are the smallest elastic network building unit of the protein. A higher concentration of dimers increases the final interfacial elastic strength of the network. The lack of the elastic film forming ability of beta -lg monomer is attributed to the absence of multiple free thiol groups. Moreover, beta -lg monomer exhibited minimal reduction in interfacial tension akin to a pure buffer solution. This fundamental relation between the quaternary structure of beta -lg and its subsequent interfacial network suggests a possible interfacial role in its biological function. Besides, these results will also be used as control for assessing the behaviors of beta -lg/polysaccharide complexes. In the next phase of this study, transient interfacial rheology of pre-mixed solutions of beta -lg and polysaccharides with different lengths and charge densities at pHs 3 and 4 are presented. It was found that, while the interfacial activity of beta -lg/pectin complexes is dictated by the amount of charge on the polysaccharide, the kinetics of the complexed beta -lg’s adsorption and its subsequent interfacial film formation is largely controlled by the contiguity of the charges on the polysaccharide molecule. Using subphase injection techniques, it is further shown that the structure of the beta -lg in the protein/polysaccharide complex prior to adsorption is the major contributor to the lag time duration before the onset of an elastic film formation. This is exemplified by the contrasting behaviors of beta -lg/pectin complexes with high polysaccharide charge density as compared to beta -lg/pectin complexes with low polysaccharide charge density, where the latter can be used as a one shot delivery system to obtain reinforced oil/water interfaces. It is further proposed that the mechanism by which a polysaccharide molecule reinforces beta -lg interfacial film is by concatenating multiple protein units and establishing cross-links in the aqueous subphase. The final phase of this study presents microrheology measurements of oil/water interfaces laden with beta -lg and beta -lg/polysaccharide complexes. Microrheology further ascertains the viscous nature of beta -lg monomer laden interfaces and the elastic nature of the interfaces with beta -lg dimers. In addition, the presence of heterogeneity in the entangled films made of beta -lg dimers in the form of confinements was also observed. A sharp transition was exhibited from an inelastic to elastic interface occurring around a surface dimer concentration of 56 ng/m2 at pH 3, 15 mM NaCl. Further, a slightly denser interface was observed for almost all the beta -lg/polysaccharide complexes at pH 4. The heterogeneity that was observed at dimeric interfaces was not seen for interfaces with beta -lg/polysaccharide complexes indicating the presence of the polysaccharide molecules beneath the interfacial film. On the whole, this thesis demonstrates the advantages of using of interfacial rheological techniques to tease out the structure-rheology relationships of biomolecules such as proteins and protein/polysaccharide complexes and thereby provide valuable insights about molecular manipulations.
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    Charged polysaccharides as model polyelectrolytes : computational studies of transport and conformational properties : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physics at Massey University, Palmerston North, New Zealand
    (Massey University, 2018) Irani, Amir Hossein
    Homogalacturonans (HGs) are polysaccharide co-polymers of galacturonic acid and its methylesterified counterpart, that play a crucial role in the mechanobiology of the cell walls of all land plants. When extracted, in solution, at pH values above the pKa, the carboxyl groups carried by the unmethylesterified residues endow the polymer chains with charge, making these systems interesting polyelectrolytes. The inter- and intra-molecular distributions of the non-charged methylesterified residues and their charged methylesterified counterparts are vital behaviour-determining characteristics of a sample's structure. Previous work has led to the development of techniques for the control of the amount and distribution of charges, and with these tools and samples available in different degrees of polymerisation, including small oligomers, the system offers a flexible test-bed for studying the behaviour of biological polyelectrolytes. This thesis is rooted in exploring the use of computational approaches, in particular molecular dynamics, to calculate the conformation of such polyelectrolytes in solution and to describe their transport properties in electric fields. The results of simulations are, in all cases, compared with the results of experimental work in order to ground the simulations. First, in chapter 2, these simulations are applied to calculate the free solution electrophoretic mobilities of galacturonides, charged oligosaccharides derived from digests of partially methylesterified HGs. The simulations are compared with experiment and were found to correctly predict the loss of resolution of electrophoretic mobilities for fully-charged species above a critical degree of polymerisation (DP), and the ionic strength dependence of the electrophoretic mobilities of different partially charged oligosaccharides. Next, in chapter 3, molecular dynamics (MD) simulations are used to calculate the electrophoretic mobilities of HGs that have different amounts and distributions of charges placed along the backbone. The simulations are shown to capture experimental results well even for samples that possess high charge densities. In addition they illuminate the role that local counterion condensation can play in the determination of the electrophoretic mobility of heterogeneous blocky polyelectrolytes that cannot be adequately described by a single chain-averaged charge spacing. Finally, in chapter 4, the last part of the research focusses on the configurations of these polyelelectrolytes in dilute solution, and on how the interactions between several chains can lead to the spatially heterogenous nature of polyelectrolyte solutions. Such questions are of long standing interest in the polyelectrolyte field and the results are compared with results from Small Angle X-Ray Scattering(SAXS). Overall the work demonstrates how state of the art MD approaches can provide insights into experimental results obtained from fundamentally interesting and biologically relevant polyelectrolytes.
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    Extraction, encapsulation and in-vitro stomach digestion of mamaku extract : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2018) Tresidder, Rebecca Emily
    Process development aimed to scale up the extraction of Mamaku, with a resulting yield of 56%wt/wt (mass of liquid extract/100kg of fronds) and 2.8%wt/wt (mass of freeze-dried material/100kg of fronds); this being higher than the yield previously obtained at a small scale (~1% freeze-dried Mamaku). The concentration of the liquid extract followed by freezing, as opposed to freeze-drying it, improved the shear-thickening properties of the Mamaku solutions. The critical temperature for processing was identified to be 63oC, whereby mamaku can be treated for 60 minutes without degradation. Higher temperatures were detrimental for the rheological properties of this polysaccharide, exhibiting a complete loss of shear-thickening when heated to 110oC. The shear- thickening properties of Mamaku solutions were also reduced when exposed to body temperature (37oC) and at the acidic pH found in the human stomach (pH 2-4). Encapsulation experiments aimed to allow mamaku to be swallowed safely, targeting release in the stomach in a hydrated form, and without incorporating calories if used as an ingredient in functional foods aiming to target weight loss. Gelatin was chosen as the encapsulating agent and 7.5%wt/v concentration was selected, being this quantity able to successfully trap concentrated mamaku (4.5%wt/wt), but also exhibiting a melting point high enough (~31oC) to avoid melting in the mouth when consumed. A number of different encapsulation techniques were trialled. The most promising technique was the fluid gel system. The nozzle techniques proved not be suitable for the properties of mamaku. The emulsion templating system showed challenges around removing the oil. The micro-injector based system produced beads likely to be too large for practical use in a food product. Limitations in the encapsulation techniques included: (i) a low mamaku concentration was used (4.5%wt/v—obtained after concentration) and (ii) the gelatin gel appeared to be unstable when transferred to an aqueous environment. The in-vitro stomach digestion results highlighted that encapsulated mamaku by gelatin will be released in the stomach, allowing the shear-thickening properties to re-form. Above 2.2%wt/v Mamaku, some shear thickening was observed (at high shear rates) after digestion of the Mamaku + gelatin mixtures. At least a 4.0%wt/v Mamaku concentration was needed in these mixtures, to obtain similar shear-thickening viscosity values after digestion, as those found by placing the mixture just under the acidic stomach conditions. Overall, a minimum of 10%wt/v of rehydrated mamaku should be delivered to the stomach to gain optimal shear-thickening properties at biological shear rates (1-10s-1) similar to those found at native pH (~pH 5.3). Overall, more research is needed to conclude about the viability of encapsulating Mamaku using gelatin as well as optimizing the encapsulation process based on fluid gel formation.