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    Characterisation and protein complexation of an anthocyanin-bound pectin extracted from New Zealand blackcurrant (Ribes nigrum) : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Salleh, Nurhazwani
    The main objective of this thesis was to investigate the cause of physical instability in blackcurrant juice-milk system. Poor phase stability in fruit juice-milk beverages is a major challenge for the clean-label beverage industry as milk protein can interact with fruit components, like polysaccharides and polyphenols, generating unwanted characteristics such as coagulation of milk proteins and phase separation. Hence, the principal step to understand the causes of poor phase stability was to identify and study the key interactive components of the juice, which was extracted from the New Zealand blackcurrant (Ribes nigrum), and then investigate their interactions with milk proteins. The key components of the blackcurrant juice were first isolated using mild extraction procedures, via ethanol precipitation and dialysis, and were identified as a complex fraction particularly rich in pectin and anthocyanins (Chapter 4). Proximate analysis revealed that the fraction contained carbohydrate (78% w/w), uronic acid (21% w/w), protein (4.8% w/w), anthocyanin (3.9% w/w) and calcium (2.2% w/w). The pectin-rich fraction had a net negative surface charge of -23.1 mV (at pH 4.8), a pKₐ value of 1.7 and a relatively high degree of esterification (65.2%). Constituent sugar analysis showed that the fraction was mostly made of galacturonic acid, rhamnose, arabinose and galactose, and NMR spectroscopic analysis revealed that it was rich in rhamnogalacturonans with arabinogalactan side chains. This pectic fraction was unique as it was highly pigmented, with cyanidin 3-O-rutinoside as its major anthocyanin. Liquid chromatography revealed that the anthocyanins were tightly bound to the fraction as methanol used in the technique failed to separate them. Results from size-exclusion chromatography coupled with multi-angle laser light scattering showed that the blackcurrant juice contained two major pectic fractions—≈283 kDa present at 14.6% w/w and ≈97 kDa at 85.5% w/w—with the latter producing higher UV₂₈₀ ₙₘ signal, signifying that proteins and/or polyphenols were present mainly in the second fraction. Association of anthocyanins to biopolymers like pectin and protein can occur via multiple interactive forces (electrostatic, hydrophobic and hydrogen bonding forces), and pH is known to play a significant role as it can affect the associative mechanisms of anthocyanins by changing their molecular configuration and ability to electrostatically interact. An attempt to dissociate blackcurrant anthocyanins from the blackcurrant biopolymers was carried out by disrupting electrostatic interactions and changing the planarity of anthocyanins via pH adjustments and ultra-filtration (Chapter 5). Lowering the juice pH to 2 did not result in anthocyanins dissociation, likely because anthocyanins were bound to the biopolymers by other interactive forces apart from the electrostatic bonds. Increasing the juice pH to 4.5 might have dissociated some anthocyanins from the biopolymers, but this was not reflected in the analysis of anthocyanins, probably because the freed anthocyanins had degraded before the analysis was carried out. Overall, size segregation of the juice components via ultra-filtration was relatively effective. Regardless of the pH, majority of the anthocyanins were still tightly associated with the large molecular weight biopolymers, confirming the involvement of multiple interactive forces. In order to uncover the cause of phase instability in blackcurrant juice-milk system, a complexation study between the isolated pectin-rich fraction and whey proteins was conducted (Chapter 6). The impact of bound anthocyanins on pectin-protein interactions was studied by exploring the effects of pH (pH 3.5 and pH 4.5), heating (85 °C, 15 min) and heating sequence (mixed-heated or heated-mixed). The pH was found to influence the colour, turbidity, particle size and surface charge of the mixtures, but its impact was most drastic when heating was introduced. Heating increased the amount of blackcurrant pectin within the complexes—especially at pH 3.5, where 88% w/w of the initial pectin was found in the sedimented (insoluble) fraction. Based on physical stability measurements, the mixed-heated system at pH 4.5 displayed better stability than at pH 3.5. A noteworthy finding was that heating sequence was found to be effective in preventing the destabilisation of the systems. Mixing of components before heating produced a more stable system with small complexes (<300 nm) and relatively low polydispersity. However, heating whey proteins before mixing with blackcurrant pectin prompted protein aggregation, producing large complexes (>400 nm) that worsened the destabilisation. The influence of bound anthocyanins on pectin-protein complexation was further studied by comparing two types of pectin-protein mixtures: (i) a mixture that is rich in anthocyanin (blackcurrant pectin-whey protein, BCP-WP) and (ii) a mixture that is free of anthocyanin (citrus pectin-whey protein, CP-WP) (Chapter 7). The mixtures were prepared at pH 4.5 with and without heat treatment at 85 °C. The study revealed that there was no direct relationship between anthocyanin presence and the destabilisation of mixtures. The Fourier-Transform Infrared (FTIR) spectrum of the heated and non-heated BCP-WP sedimented fractions showed the emergence of a peak at 800-1200 cm⁻¹, signifying the presence of anthocyanin-protein interactions. This peak, however, was absent in the spectrum of any of the anthocyanin-free CP-WP sedimented fractions, indicating that the bound anthocyanins of blackcurrant pectin provided the whey proteins with additional binding sites. The findings from FTIR analyses also indicated that non-electrostatic forces were most likely the governing forces of the heated BCP-WP mixture, via hydrophobic interactions and later reinforced by hydrogen bonds upon cooling. This thesis revealed that poor phase stability of the blackcurrant juice-milk system should not be attributed exclusively to the blackcurrant juice components, particularly the polyphenols. Environmental factors like pH and heat were likely the leading cause of phase instability as they could intensify the interactions that occurred in the mixed system, which eventually destabilised the mixture. This suggests that appropriate processing conditions can be applied to positively affect the blackcurrant juice-milk system.
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    Fruit modulation of the effects of fatigue on cognitive performance : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Psychology at Massey University, Albany, New Zealand
    (Massey University, 2018) Peart, Natalie
    Cognitive fatigue hinders performance in social, academic and physical environments and has a profound effect on the ability of a person to function and make decisions. Research into reducing or eliminating cognitive fatigue and its effects have been largely inconclusive but an emerging area of research is focussing on phytochemicals effects on optimising cognition. The present study examined the effects of blackcurrant supplementation on cognitive fatigue and physical markers of performance. This required 11mg of freeze-dried blackcurrant powder and water mixture to be consumed by participants before completing a psychometric and exercise test. Blackcurrants are a high source of phytochemicals but are under-researched compared to other berry fruit, such as blueberries. Sixty participants completed two sessions which composed of a familiarisation session and an intervention session. Participants were randomly assigned to the blackcurrant supplementation or the control group. Each session consisted of six blocks of the Stroop test, a Standard VO2max test and followed by a post-exercise Stroop test. The purpose of the Standard VO2max test to exhaustion was to induce physical and cognitive fatigue. At the intervention session, participants ingested either a blackcurrant or sugar-controlled juice one hour before testing. Analyses demonstrated that the blackcurrant supplement had no effect on cognitive performance or physical markers. However, it was questionable as to whether the study had enough statically power to test for the small effect sizes due to participants being unable to complete testing and some data sets unable to be used. Future research should focus on larger sample sizes and high doses of anthocyanin to observe if blackcurrant can have cognitive and physiological effects.
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    An analysis of polyphenolic blackcurrant (Ribes nigrum) extracts for the potential to modulate allergic airway inflammation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutritional Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2009) Taylor, Janet Lynley
    The allergic disease of asthma is characterized by an infiltration of inflammatory cells to the lung, a process co-ordinated by T-helper (TH) cells. The TH2 cytokine Interleukin (IL)-4 promotes infiltration of eosinophils to sites of inflammation. Eosinophil-selective chemoattractant cytokines (eg. eotaxins) are synthesized by lung epithelial cells. Eotaxin-3 is expressed at high levels in the asthmatic lung, predominantly after IL-4 stimulation. Eotaxin-3 is therefore a marker of inappropriate airway inflammation. Polyphenolic (PP) compounds found in high concentrations in berries may have beneficial effects in inflammatory conditions. Plant and Food Research produced high-PP extracts of blackcurrant (BC) cultivars that were tested for inflammation modulating effects. Since high doses of PPs have been shown to cause cell death, we tested two BC cultivars at a range of concentrations in a cell viability (WST-1) assay. While no toxic effects were attributable to the BC extracts (1-50µg/ml), a dose-related trend in cell death was observed and therefore 10µg/ml was chosen for further experiments Ten BC cultivars were compared for efficacy by measuring eotaxin-3 production in IL-4 stimulated human lung epithelial (A549) cells in vitro. Cells were incubated with BC extracts (10µg/ml) and IL-4 (10ng/ml) for 24 hours. The supernatants were then quantified for eotaxin-3 levels by an enzyme-linked immunosorbent assay (ELISA). All ten BC extracts reduced eotaxin-3 levels after stimulation with IL-4, and six BC extracts were effective by statistically significant levels (P<0.05), (BC cultivars -01, -02, -03, -05, -09 & -10). Of those, BC extracts of four cultivars demonstrated a reduction of more than 65% from the IL-4 stimulated control. In addition, a positive trend in inflammation modulation vs. one anthocyanin (ACN) in the BC extracts was shown. This study has demonstrated the beneficial inflammation modulatory effects of polyphenolic BC extracts, which could be related to cyanidin 3-O-rutinoside content. These results may have therapeutic potential for asthma.