Anita Oberholster

Optimization of a Method for the Extraction and Quantification of Carotenoids and Chlorophylls during Ripening in Grape Berries (Vitis vinifera cv. Merlot)

An extraction method to identify and quantify the carotenoid and chlorophyll profile of lyophilized tissue from unripe (green) to ripe (red) Merlot grape berries was developed. The RP-HPLC method baseline separated all of the carotenoids and chlorophylls and their derivatives. Problems encountered during sample storage and extraction are discussed as well as possible alternative methods. This study confirmed that carotenoids and chlorophylls decreased on a per berry (microg/berry) and concentration (microg/g) basis from veraison to harvest over two growing seasons. The carotenoid 5,8-epoxy-beta-carotene was quantified for the first time in grapes and represents a significant amount of the total carotenoids present at harvest. All the carotenoids and chlorophylls except beta-carotene appeared to be sensitive to seasonal variation in climatic conditions. Lutein and beta-carotene were found to be the most abundant carotenoids present in Merlot grape berries together with chlorophyll a for both seasons studied.

Barrel maturation, oak alternatives and micro-oxygenation: Influence on red wine aging and quality

The impact of micro-oxygenation (MOX) in conjunction with a variety of oak alternatives on phenolic composition and red wine aging was investigated and compared with traditional barrel aging. Although several studies concluded that MOX give similar results to barrel aging, few have compared them directly and none directly compared MOX with and without wood alternatives and barrel aging. Results confirmed that MOX had a positive effect on colour density, even after 5 months of bottle aging. This is supported by an increase in polymeric phenol and pigment content not only with aging but in the MOX compared to barrel matured wine treatments. Descriptive analysis showed that MOX in combination with wood alternatives such as oak chips and staves could mimic short term (six months) barrel aging in new American and French oak barrels in regards to sensory characteristics.

Investigation of the effect of gelatine, egg albumin and cross-flow microfiltration on the phenolic composition of Pinotage wine.

The effect of fining and cross-flow microfiltration on the phenolic composition of red wine was investigated. Both gelatine (G) and egg albumin (EA) fining decreased the mean degree of polymerisation (mDP) of tannin significantly by 26.4% and 25.2%, respectively, compared to the control (C). Cross-flow microfiltration (CF) also decreased the mDP significantly by 25%. Thus, the fining agents and cross-flow microfiltration selectively removed the highly polymerised phenols. After 3.5 months of bottle ageing, differences between the different treatments and the control decreased. CF had the most significant effect on the flavan-3-ol and polymeric phenol (tannin) content of the wines compared to the control followed by G fining. CF and EA treatments significantly decreased the total pigment content compared to C. CF was also the only treatment that could be distinguished from the other treatments by sensory analysis. All treatments improved clarity of the wines with cross-flow microfiltration having the largest effect.

Effects of Cap and Overall Fermentation Temperature on Phenolic Extraction in Cabernet Sauvignon Fermentations

The phenolic content of red wine is responsible for the color, mouthfeel, and aging potential of the wine. Although many fermentation parameters and winemaking techniques affect phenolic extraction, it is generally agreed that one of the prime factors is fermentation temperature; however, temperature is not uniform during fermentation and thermal gradients form between the cap and the liquid. To determine the effects of temperature on phenolic extraction, research scale (120 L) Cabernet Sauvignon fermentations were performed in which the cap and must were either maintained at the same temperature or a constant thermal gradient was maintained between the two during the period of active fermentation. All fermentations were sampled twice daily and phenolic content was determined by reverse-phase high-performance liquid chromatography for the monomeric phenolics and UV-vis spectroscopy for the total anthocyanins and condensed tannins. These experiments showed that cap and must temperature had noticeable effects on phenolic extraction based on where the phenolics originated. For skin phenolics, temperature affected the rate of extraction but not the final concentration, and increasing temperatures favored faster extraction. For seed phenolics, increases in fermentation temperature increased both the rate of extraction and the final concentration. Results showed that must temperature was more important than cap temperature in driving extraction of phenolics.

Tracing flavonoid degradation in grapes by MS filtering with stable isotopes

Anthocyanin degradation has been proposed as one of the primary causes for reduced colour and quality in red wine grapes grown in a warm climate. To study anthocyanin degradation we infused berries with L-phenyl-(13)C₆-alanine and then tracked the fate of the anthocyanins comparing normal (25 °C) and warm (45 °C) temperature conditions. An untargeted metabolomics approach was aided by filtering the MS data using software algorithms to extract all M and M+6 isotopic peak pairs, allowing the analysis to focus solely on the metabolites of phenylalanine. A paired-comparison t-test was performed over the 8 biological replicates revealing 13 metabolites that were statistically different between 25 °C and 45 °C treatments. Most of these features had lower abundances in 45 °C samples, confirming that 45 °C treatment caused anthocyanin degradation. In addition, resveratrol was significantly reduced following heat treatment. However, 5 metabolites increased following the 45 °C treatment. These unidentified metabolites are therefore suspects for anthocyanin degradation products.

Investigating the Effect of Cold Soak Duration on Phenolic Extraction during Cabernet Sauvignon Fermentation.

The impact of increasing cold soak (CS) duration (0, 1, 4, 7, and 10 days at 10 °C) on the extraction of phenolic compounds during the CS period and primary fermentation as well as the final composition of Cabernet Sauvignon wine was investigated. The results showed that CS duration had no effect on hydroxycinnamate and flavonol extractions. Greater amounts of gallic acid, (+)-catechin, (−)-epicatechin, and total tannins were extracted with increasing CS duration, with differences maintained during bottle aging. Anthocyanin extraction and color density increased with longer periods of CS; however, by the end of primary fermentation, as well as three months’ bottle aging, there were no significant differences due to CS duration. The wines made with seven and 10 days of CS had higher seed tannin contributions and total tannin compared to the non-CS wine, which could potentially result in increased astringency.

Monitoring the Effect of Micro-oxygenation before Malolactic Fermentation on South African Pinotage Red Wine with Different Colour and Phenolic Analyses

The use of micro-oxygenation and its effect on the quality of the red wine varietal, Pinotage, is largely unknown. The influence of adding different oxygen dosages before malolactic fermentation on the phenolic composition and colour stabilisation of wine made from Pinotage was studied, and the suitability of certain spectrophotometric and RP-HPLC analyses to determine these changes were assessed in tanks of commercial lengths. Total oxygen dosages of 0 mg/L, 16 mg/L and 32 mg/L were applied, after which the wines underwent malolactic fermentation and maturation in the same tanks for two additional months. Decreases in anthocyanin concentration showed a strong inversely proportional correlation with increasing polymeric pigment concentration. This suggests that the addition of oxygen contributed to the early stabilisation of wine colour in Pinotage red wine. Overall, tannin concentrations were not significantly influenced by the oxygen treatments, although lower levels were observed in wines treated with oxygen. An important finding of the study was that there appeared to be little difference in the colour and phenolic composition of the wines between the 16 mg/L and 32 mg/L oxygen treatments. Good correlations were found between certain spectrophotometric techniques and the RP-HPLC analysis used to study changes induced by micro-oxygenation (total anthocyanins, total phenols). The colour and phenolic composition of Pinotage wine can be influenced before malolactic fermentation by micro-oxygenation. Some spectrophotometric phenolic analyses showed the same tendencies as observed with RP-HPLC (anthocyanins, monomeric flavan-3-ols, tannins), indicating their suitability to follow phenolic and colour changes induced by micro-oxygenation in Pinotage red wine.

Assessing Spatial Variability of Grape Skin Flavonoids at the Vineyard Scale Based on Plant Water Status Mapping

Plant water stress affects grape (Vitis vinifera L. cv. Cabernet Sauvignon) berry composition and is variable in space due to variations in the physical environment at the growing site. We monitored the natural variability of grapevine water stress by stem water potential (Ψstem) and leaf gas exchange in an equi-distant grid in a commercial vineyard. Spatial differences were measured and related to topographical variation by modeling. Geospatial analysis and clustering allowed researchers to differentiate the vineyard block into two distinct zones having severe and moderate water stress where it varied by 0.2 MPa. Differences in stem water potential affected stomatal conductance, net carbon assimilation, and intrinsic water use efficiency that were different in all measurement dates. The two zones were selectively sampled at harvest for measurements of berry chemistry. The water status zones did not affect berry mass or yield per vine. Significant difference in total soluble solids was observed (3.56 Brix), and in titratable acidity, thus indicating a direct effect of water stress on ripening acceleration. Berry skin flavonol and anthocyanin composition and concentration were measured by C18 reversed-phased high-performance liquid chromatography (HPLC). The anthocyanins were most affected by the two water stress zones. The dihydroxylated anthocyanins were more affected than trihydroxylated; therefore, the ratio of the two forms increased. Flavonols were different in total amounts, but hydroxylation patterns were not affected. Proanthocyanidin isolates were characterized by acid catalysis in the presence of excess phloroglucinol followed by reversed-phase HPLC. Proanthocyanidins showed the least significant difference, although (+)-catechin terminal subunits were important predictors in a partial least square model used to summarize the multivariate relationships, predicting Ψstem or the management zone. The results provide fundamental information on vineyard water status to discriminate harvest or direction to vineyard operators to modify irrigation management to equilibrate berry composition at harvest.

Chemical Gradients in Pilot-Scale Cabernet Sauvignon Fermentations and Their Effect on Phenolic Extraction

The phenolic content of red wine is responsible for the color and mouthfeel of the wine. Phenols are extracted from the skins and seeds of the crushed grapes through contact with the juice. Formation of a skin cap at the top of the fermentation decreases this contact, necessitating cap management techniques such as punch-downs or pump-overs. While it is clear that these techniques are necessary for extraction, the kinetics of phenolic release, adsorption, and reaction in this heterogeneous system are not well understood. To better understand these kinetics, pilot-scale (2000 L) Cabernet Sauvignon fermentations were performed in two consecutive harvests (2011 and 2012) using a tank modified with a custom sampling array that allowed sampling of the fermentations at four depths (two in the cap and two in the liquid), with three replicate sampling points at each depth. Chemical gradients were observed for several phenolics, including gallic acid, (+)-catechin, (−)-epicatechin, caftaric acid, malvidin-3-O-glucoside, total anthocyanins, and condensed tannins. Chemical gradients for skin phenolics, such as the anthocyanins, were observed to develop early during fermentation and to diminish as fermentation progressed. Phenolics found primarily within the seed, such as (+)-catechin, exhibited chemical gradients throughout the fermentation. Seed extraction trends were confirmed with phloroglucinolysis. Interestingly, the phenolic gradients were eliminated following a pump-over event, but were re-established during the subsequent few hours and approached a saturation point ~8 hrs after the pump-over for skin phenolics, while saturation behavior was not as pronounced for the seed phenolics.

Impact of Mechanical Harvesting and Optical Berry Sorting on Grape and Wine Composition

The impact of mechanical harvesting, optical berry sorting, and their possible synergistic effect on grape and wine composition was investigated. Pinot noir grapes from the Russian River Valley American Viticultural Area were harvested by hand, by a standard bow-rod mechanical harvester, or by a mechanical harvester with a Selectiv’ Process on-board. For each harvest method, half of the grapes were unsorted and half were optically sorted at the winery. The grapes, wines at bottling, and wines after three months of bottle aging were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC), ultraviolet-visible spectroscopy (UV-vis), and headspace solid-phase microextraction (HS-SPME) gas chromatography mass spectrometry (GC-MS) for color expression and phenolic and aroma profiling. The machine-harvested grapes had higher levels of β-damascenone, linalool, β-myrcene, and α-terpinene, potentially caused by glycosidic hydrolysis triggered by berry damage during harvest or from induced synthesis as a wounding response. In general, differences in wine composition attributable to harvest method were diminished or eliminated by optical sorting. The machine harvester with the Selectiv’ Process on-board led to wines with the most phenolics, although these differences may have been decreased or eliminated had the grapes been crushed before fermentation as the wines were produced by whole-berry fermentation. Descriptive sensory analysis conducted on wines three months after bottling determined that the wines made from hand-harvested fruit had significantly greater tropical fruit aroma, while wines made from optically sorted treatments had less hue saturation. With only two significant differences among the 18 aroma, taste, and mouthfeel attributes tested, it was concluded that all treatments led to wines of similar character.