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Item Terroir Dynamics: Impact of Vineyard and Canopy Treatment with Chitosan on Anthocyanins, Phenolics, and Volatile and Sensory Profiles of Pinot Noir Wines from South Tyrol(MDPI (Basel, Switzerland), 2024-04-23) Tchouakeu Betnga PF; Poggesi S; Darnal A; Longo E; Rudari E; Boselli E; Rolle LThe effects of canopy treatment with chitosan and the effects of the vineyard location on the quality parameters, volatile and non-volatile profiles, and sensory profile of Pinot Noir wines from South Tyrol (Italy) were studied. Multivariate statistical analysis was applied to identify the most relevant compounds associated with the variability in phenolics and anthocyanins (analyzed by UHPLC-MS), volatile components (HS-SPME-GCxGC-ToF/MS), and basic enological parameters. A clear separation of low-altitude wines (350 m.a.s.l.), which had a high concentration of most of the identified volatile compounds, compared to high-altitude wines (800 and 1050-1150 m.a.s.l.) was pointed out. Low altitude minimized the concentration of the most significant anthocyanins in wines from a valley bottom, presumably due to reduced sun exposure. Wines obtained from chitosan-treated canopies, and, more particularly, those subjected to multiple treatments per year showed a higher amount of the main non-volatile phenolics and were sensorially described as having "unpleasant flavors" and "odors", which might suggest that grape metabolism is slightly altered compared to untreated grapevines. Thus, optimization of the treatment with chitosan should be further investigated.Item Interactive effect of pre-fermentative grape freezing and malolactic fermentation on the anthocyanins profile in red wines prone to colour instability(Springer Nature, 2023-08-01) Darnal A; Poggesi S; Ceci AT; Mimmo T; Boselli E; Longo EThe effects of pre-fermentative freezing of red grapes from Schiava variety and co-inoculation with lactic bacteria were evaluated on the profile of anthocyans of the musts and the finished wine. Peonidin-3-glucoside is the main anthocyanin in Schiava grape musts, but it was overcome by malvidin-3-glucoside at bottling. Grape freezing increased the extraction of all anthocyanins in the musts. However, the amount of all anthocyanins except peonidin-3-glucoside and malvidin-3-glucoside was lower in wines from frozen grapes than in control wines. Wines obtained with co-inoculation showed higher anthocyanin content than their respective controls. Petunidin-3-(6′′-p-coumaroyl)-glucoside, peonidin-3-(6′′-cis-p-coumaroyl)-glucoside and malvidin-3-(6′′-trans-p-coumaroyl)-glucoside were dramatically affected by the interaction of the two applied factors. Colorimetric hue (H*) was strongly correlated with peonidin-3-glucoside, and spectrophotometric tint (N) with malvidin-3-glucoside. Tint also showed a positive correlation with malolactic fermentation. Graphical abstract: [Figure not available: see fulltext.]Item Comparative Transcriptomics of Multi-Stress Responses in Pachycladon cheesemanii and Arabidopsis thaliana.(MDPI (Basel, Switzerland), 2023-07-11) Dong Y; Gupta S; Wargent JJ; Putterill J; Macknight RC; Gechev TS; Mueller-Roeber B; Dijkwel PP; You FMThe environment is seldom optimal for plant growth and changes in abiotic and biotic signals, including temperature, water availability, radiation and pests, induce plant responses to optimise survival. The New Zealand native plant species and close relative to Arabidopsis thaliana, Pachycladon cheesemanii, grows under environmental conditions that are unsustainable for many plant species. Here, we compare the responses of both species to different stressors (low temperature, salt and UV-B radiation) to help understand how P. cheesemanii can grow in such harsh environments. The stress transcriptomes were determined and comparative transcriptome and network analyses discovered similar and unique responses within species, and between the two plant species. A number of widely studied plant stress processes were highly conserved in A. thaliana and P. cheesemanii. However, in response to cold stress, Gene Ontology terms related to glycosinolate metabolism were only enriched in P. cheesemanii. Salt stress was associated with alteration of the cuticle and proline biosynthesis in A. thaliana and P. cheesemanii, respectively. Anthocyanin production may be a more important strategy to contribute to the UV-B radiation tolerance in P. cheesemanii. These results allowed us to define broad stress response pathways in A. thaliana and P. cheesemanii and suggested that regulation of glycosinolate, proline and anthocyanin metabolism are strategies that help mitigate environmental stress.Item 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, NurhazwaniThe 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.Item Molecular analysis of anthocyanin biosynthetic pathway genes in Cymbidium orchids : a thesis presentation in partial fulfilment of the requirements for the degree of Master of Science in Plant Molecular Biology at Massey University, Palmerston North, New Zealand(Massey University, 2008) Wang, LeiAnthocyanin biosynthesis was examined in cymbidium orchid (Cymbidium Swartz). Cymbidium orchids lack true red and purple/blue flowers, because they accumulate cyanidin and peonidin (pink) anthocyanins, but not pelargonidin (red) or delphinidin (blue). Transient gene expression studies showed that the use of a heterologous flavonoid biosynthetic gene, dihydroflavonol reductase (DFR) from Anthurium, enhanced production of pelargonidin in cymbidium floral tissues. Similarly, delphinidin was produced when a pansy flavonoid 3', 5'-hydroxylase (F3'5'H) was introduced. The maize Lc/C1 transcription factors in combination with DFR or F3'5'H was necessary in order to observe upregulation of the anthocyanin pathway and changes in anthocyanin pigment accumulation. The presence of the new anthocyanins was confirmed by TLC and HPLC. cDNA clones of the flavonoid biosynthetic genes chalcone synthase (CHS), DFR and flavonoid 3' hydroxylase (F3'H) were isolated using PCR. The full length cymbidium CHS (1173 bp) was ~85% identical at DNA level with CHS from Oncidium 'Gower Ramsey', a Phalaenopsis hybrid cultivar, as well as bibenzyl synthase from Phalaenopsis sp. The 792 bp partial-length F3'H sequence was ~66% identical with F3'H from Pelargonidiumxhortorum, Verbena hybrida and Sorghum bicolor, while the DFR sequence was highly homologous with the published cymbidium DFR. The deduced protein sequences contained domains or conserved residues typical of CHS and F3'H. Southern analysis showed both cymbidium CHS and F3'H are represented by small gene families, with CHS consisting of at least three members and F3'H up to three genes. By contrast, DFR is likely to be presented as a single gene. Using different coloured cymbidium cultivars, it was shown that DFR expression correlated with cyanidin production in the flower. The CHS clone was most highly expressed in leaf tissues and in late developmental stages in floral tissues of Vanguard Mas Beauty (a green cultivar). This expression pattern did not correlate with pigment production, and hence this gene is unlikely to be involved in anthocyanin production in flowers. F3'H expression was not detected in leaf and floral tissues at any developmental stages examined. DFR and CHS promoters were isolated by genome walking, in an attempt to identify organ specific promoters suitable for use in cymbidium. A 1544 bp DFR promoter and a 1561 bp CHS promoter were cloned upstream of a GFP reporter gene and transient gene expression studies showed that CHS promoter had the ability to drive GFP production in white and pink petal tissues and in leaf tissues. However, the DFR promoter activated GFP expression only in the white petal tissues. These transient gene expression studies also demonstrated that maize Lc/C1 transcription factors greatly enhanced the activities of both CHS and DFR promoters. The success of this transient expression system indicates that MYB and bHLH transcription factor are likely to be involved in anthocyanin production in cymbidium. Together, the results of this study confirm that a suite of molecular strategies to modify flower colour in cymbidium are feasible, as well as providing essential information on flavonoid and anthocyanin genes that expand our knowledge and understanding of this complex flowering plant.Item Natural variation in the serially duplicated Production of Anthocyanin Pigment loci and anthocyanin accumulation in Arabidopsis thaliana (Brassicaceae) : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Plant Biology at Massey University, Palmerston North, New Zealand(Massey University, 2013) Butcher, MatthewThe TTG1-regulatory gene network regulates the development of all epidermal cell fates in Arabidopsis thaliana. Four members of the TTG1 complex, the serially duplicated R2R3-MYB PRODUCTION OF ANTHOCYANIN PIGMENT (PAP) genes, have previously been implicated in regulating the late stages of anthocyanin bionsynthesis in Arabidopsis thaliana. To study the effects of gene duplication, we sought to determine the extent of variation in each PAP gene compared to a single copy gene of the TTG1 network, WEREWOLF, using 48 naturally occurring A. thaliana accessions. It appears that the predominantly expressed PAP1 gene demonstrates a biallelic pattern, consistent with other A. thaliana genes. All four genes fall below the average nucleotide diversity levels observed across A. thaliana; however, WEREWOLF demonstrates almost complete sequence conservation across the 48 accessions used in this study. We attempted to determine the relative ages of the four PAP genes, though this does not appear to correlate with accumulation of genetic variation. To investigate the genetic architecture of anthocyanin accumulation in A. thaliana, we performed an heritability and quantitative trait loci mapping analysis using a recombinant inbred line population derived from 19 natural A. thaliana accessions. While QTL were mapped for anthocyanin accumulation near several of the PAP genes, we observed a number of loci with no obvious candidate genes, providing novel insights into the genetic architecture of anthocyanin accumulation in A. thaliana. This work contributes to a greater understanding of the roles of regulatory genes in biosynthesis and the molecular basis of regulation as well as the effects of gene duplication on nucleotide variation in the resulting genes.Item Phytochemical optimisation of blueberry juice: a research report presented in partial fulfilment of the requirements for the degree of Master of Engineering in Food Technology, Massey University, Turitea campus, Palmerston North, New Zealand(Massey University, 2011) Birt, NatashaBlueberries contain a high concentration and diversity of anthocyanins which are responsible for the blue/purple pigment of their skin. Like many other fruits and vegetables they also contain a large amount of chlorogenic acid (CGA) within the fruit flesh and seeds. Together these phytochemicals appear to account for most of the high antioxidant activity of the fruit, although within the scientific community a consensus has not been reached as to their effects on human health. Fresh blueberries have a limited season and are perishable unless stored frozen. Processing of blueberries into juice allows year round sale, and importantly less market-driven pricing. Therefore the aim of this research project was to investigate and optimise factors which may be significant in producing a high phytochemical blueberry juice. The pigment of blueberries is concentrated in the skin, and therefore smaller blueberries have the highest anthocyanin concentrations on a weight basis. Consequently the three highest concentrations were found in cultivars ‘Elliot’, ‘Burlington’ and ‘Duke’. Conversely chlorogenic acid is not restricted to the blueberry skin; ‘Elliot’, ‘Bluecrop’ and ‘Burlington’ had the three highest concentrations, while ‘Duke’ had the lowest. The profile of individual anthocyanins was also found to be diverse amongst cultivars. Therefore if individual anthocyanin(s) are shown to be important for specific health conditions further consideration should be given to cultivar selection. Upon review of the current blueberry juice manufacturing process, large anthocyanin losses were seen at three key steps: after thawing of the fruit, removal of the press cake and holding of the juice between pressing and pasteurisation. Two alternative processes were compared where a slightly higher anthocyanin concentration was achieved for ‘hot press juice’ but a lower chlorogenic acid concentration than the alternative ‘cold press’ juice. Modifications to the manufacturing process were investigated using the pilot plant at Massey University. It was found that blanching of frozen blueberries before pressing was successful in significantly increasing the anthocyanin and chlorogenic acid concentration of the juice (about a 5 fold increase of anthocyanins and a 4 fold increase in chlorogenic acid from previous levels). However, this did alter the sensory properties of the juice significantly, with a more cooked flavour and thicker texture. Other variations that were trialled, such as milling, ii variation in holding time and temperature between pressing and pasteurising, had comparatively little effect on the anthocyanin and chlorogenic acid concentration. Storage tests on blueberry juice showed a clear relationship between the storage temperature and anthocyanin retention, where warmer temperatures resulted in larger anthocyanin degradation. At the end of the six month storage period, juice storage at 5°C gave 63% anthocyanin retention while juice storage at 25°C gave only 8% retention. Some protection was also afforded to juice packed in glass bottles rather than plastic and stored in the dark rather than the light; but this difference was far smaller than the effect of temperature. Chlorogenic acid levels appeared to be comparatively less affected; only relatively small amounts of degradation were observed. It is important to note that when the antioxidant capacity was measured for stored juice at six months under the various treatments, there was only a small degree of degradation for all samples as compared with at time zero. Previously researchers had encountered a similar phenomenon and suggested that unknown anthocyanin degradation products may still be able to contribute to the juice’s antioxidant capacity. However, here it is also suggested that chlorogenic acid may have had a more significant contribution to the antioxidant capacity that it is usually credited with, due to the large amount present with relatively little degradation throughout the storage period. This information may be used to produce and market a juice with high anthocyanin, chlorogenic acid and/or antioxidant properties. Of the health effects evaluated here, currently, research in cardiovascular disease and neuroprotection effects are looking the most promising with regard to dietary blueberry supplementation in humans, although there is still a lack of double blind randomised placebo controlled studies to come to any consensus within the scientific community. Additionally, at the present time, the use of health claims on food products in New Zealand is being revised (Food Standards Authority proposal P293). As the current state of nutrition research surrounding plant polyphenols is inconclusive it may be important to use generic statements such as ‘high in antioxidants’ rather than statements about specific compounds.