Browsing by Author "Nieuwoudt, Helene"

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    Analysis of volatile profiles of fermenting grape must by headspace solid-phase dynamic extraction coupled with gas chromatography-mass spectrometry (HS-SPDE GC-MS): Novel application to investigate problem fermentations
    (2009) Malherbe, S.; Watts, V.; Nieuwoudt, Helene; Bauer, Florian; Du Toit, M.
    The occurrence of stuck and sluggish wine fermentations is a persisting problem in the wine industry worldwide. This study illustrates the suitability of headspace solid-phase dynamic extraction coupled with gas chromatography-mass spectrometry (HS-SPDE GC-MS) for wine analysis and the subsequent application to discriminate between control and problem fermentations using partial least-squares discriminant analysis (PLS-DA) models. The specific analytical technique is relatively new and has not yet to the authors' knowledge been evaluated for the analysis of wine within this context of problem fermentations. HS-SPDE GC-MS was used to determine 68 volatile compounds (higher alcohols, fatty acids, esters, and carbonyl compounds) in 94 monovarietal fermenting must samples consisting of 56 red and 38 white cultivars. PLS-DA models showed the potential to discriminate between control and problem fermentations using corrected peak area headspace data for the 68 analytes. This possibility to discriminate between problem and control fermentations with only the headspace data could possibly be applied for the prediction of problem fermentations in future studies and to better understand the chemical causes of problem fermentations. ©2009 American Chemical Society.
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    Analytical techniques for wine analysis : an African perspective; a review
    (2012-02-22) De Villiers, A.; Alberts, P.; Tredoux, A. G. J.; Nieuwoudt, Helene
    Analytical chemistry is playing an ever-increasingly important role in the global wine industry. Chemical analysis of wine is essential in ensuring product safety and conformity to regulatory laws governing the international market, as well as understanding the fundamental aspects of grape and wine production to improve manufacturing processes. Within this field, advanced instrumental analysis methods have been exploited more extensively in recent years. Important advances in instrumental analytical techniques have also found application in the wine industry. This review aims to highlight the most important developments in the field of instrumental wine and grape analysis in the African context. The focus of this overview is specifically on the application of advanced instrumental techniques, including spectroscopic and chromatographic methods. Recent developments in wine and grape analysis and their application in the African context are highlighted, and future trends are discussed in terms of their potential contribution to the industry. © 2011 Elsevier B.V. All rights reserved.
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    Bioprocess monitoring of alcoholic wine fermentation using on-line NIR infrared spectroscopy.
    (2011) Nieuwoudt, Helene; Kruger, van Eck M.; Du Toit, M.; Silva Ferreira, A. C.; Tredoux, A G J.
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    Characterization of selected South African young cultivar wines using FTMIR Spectroscopy, Gas chromatography, and multivariate data analysis
    (2009) Louw, L.; Roux, K.; Tredoux, A.; Tomic, O.; Naes, T.; Nieuwoudt, Helene; Van Rensburg, P.
    The powerful combination of analytical chemistry and chemometrics and its application to wine analysis provide a way to gain knowledge and insight into the inherent chemical composition of wine and to objectively distinguish between wines. Extensive research programs are focused on the chemical characterization of wine to establish industry benchmarks and authentication systems. The aim of this study was to investigate the volatile composition and mid-infrared spectroscopic profiles of South African young cultivar wines with chemometrics to identify compositional trends and to distinguish between the different cultivars. Data were generated by gas chromatography and FTMIR spectroscopy and investigated by using analysis of variance (ANOVA), principal component analysis (PCA), and linear discriminant analysis (LDA). Significant differences were found in the volatile composition of the cultivar wines, with marked similarities in the composition of Pinotage wines and white wines, specifically for 2-phenylethanol, butyric acid, ethyl acetate, isoamyl acetate, isoamyl alcohol, and isobutyric acid. Of the 26 compounds that were analyzed, 14 had odor activity values of > 1. The volatile composition and FTMIR spectra both contributed to the differentiation between the cultivar wines. The best discrimination model between the white wines was based on FTMIR spectra (98.3% correct classification), whereas a combination of spectra and volatile compounds (86.8% correct classification) was best to discriminate between the red wine cultivars. © 2009 American Chemical Society.
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    Comparative metabolic profiling to investigate the contribution of O. oeni MLF starter cultures to red wine composition
    (2011) Malherbe, S.; Tredoux, A. G. J.; Nieuwoudt, Helene; Du Toit, M.
    In this research work we investigated changes in volatile aroma composition associated with four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures in South African Shiraz and Pinotage red wines. A control wine in which MLF was suppressed was included. The MLF progress was monitored by use of infrared spectroscopy. Gas chromatographic analysis and capillary electrophoresis were used to evaluate the volatile aroma composition and organic acid profiles, respectively. Significant strain-specific variations were observed in the degradation of citric acid and production of lactic acid during MLF. Subsequently, compounds directly and indirectly resulting from citric acid metabolism, namely diacetyl, acetic acid, acetoin, and ethyl lactate, were also affected depending on the bacterial strain used for MLF. Bacterial metabolic activity increased concentrations of the higher alcohols, fatty acids, and total esters, with a larger increase in ethyl esters than in acetate esters. Ethyl lactate, diethyl succinate, ethyl octanoate, ethyl 2-methylpropanoate, and ethyl propionate concentrations were increased by MLF. In contrast, levels of hexyl acetate, isoamyl acetate, 2-phenylethyl acetate, and ethyl acetate were reduced or remained unchanged, depending on the strain and cultivar evaluated. Formation of ethyl butyrate, ethyl propionate, ethyl 2methylbutryate, and ethyl isovalerate was related to specific bacterial strains used, indicating possible differences in esterase activity. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, although further investigation is needed in this regard. This study provided insight into metabolism in O. oeni starter cultures during MLF in red wine. © 2011 Society for Industrial Microbiology and Biotechnology.
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    Comprehensive survey of the distribution of colour and phenolics of different red grape wine vineyard blocks from the Robertson area in South Africa
    (South African Society for Enology and Viticulture, 2012) Van der Merwe, H.; Nieuwoudt, Helene; De Beer, D.; Du Toit, W. J.
    Colour and phenolic content of red grapes are two of the most important constituents required to produce a quality red wine. In the Robertson grape growing area, difficulty is sometimes experienced with colour development of grapes. This is especially linked to location and most probably greatly influenced by season. Forty four vineyard blocks of the cultivars Pinotage, Merlot, Cabernet Sauvignon and Shiraz, were studied over 3 seasons primarily to focus on colour and phenolic content, but secondly on total soluble solids, titratable acidity and pH of the grapes. High performance liquid chromatography and spectrophotometric methods were used to determine various colour and phenolic parameters present at harvest. This data was used to indicate how colour and phenolic constitution of a part of the Robertson grape growing area was distributed in relation to various factors, such as cultivar and season. GPS points were used to map data for the blocks visually. Results showed variable colour and phenolic content for these grapes based on blocks and phenolic compounds investigated. Shiraz displayed a wider distribution of certain phenolic compounds over the three seasons than the other 3 cultivars. Seasonality had a great influence on these results, with outlying blocks being identified.
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    Effect of different extraction methods on the quality and biochemical attributes of pomegranate juice and the application of Fourier transformed infrared spectroscopy in discriminating between different extraction methods
    (Frontiers Media, 2021-08-23) Arendse, Ebrahiema; Nieuwoudt, Helene; Fawole, Olaniyi Amos; Linus Opara, Umezuruike
    This study investigated the effects of extraction methods on the physicochemical, phytochemical, and antioxidant properties of pomegranate juice (cv. Wonderful). In addition, the application of attenuated total reflectance Fourier transformed mid-infrared (ATR-FT-MIR) spectroscopy and chemometrics were explored in order to discriminate between different extraction methods. Juice variants evaluated included juice extracted without crushing the seeds (arils only) using a juice extractor (JE), juice extracted by crushing the seeds using a blender (arils plus seed) (JB), and juice extracted from half fruit using a commercial hand press juicer (CH). Juice extracted from CH had higher total soluble solid (TSS) content (18.20%), TSS/TA ratio (15.83), and color properties (a* = 32.67, b* = 11.80, C* = 34.77) compared with extraction methods JE and JB. The juice extracted from JB showed the highest titratable acidity (2.17%), cloudiness (0.43), and lowest pH value (2.69). The total phenolics and anthocyanin content in the investigated juice ranged from 1.87 to 3.04 g gallic acid equivalent (GAE)/L and 37.74–43.67 mg cyanidin 3-glucoside equivalent/L of crude juice, respectively. Juice extracted from JB and CH was significantly higher in phenolic and anthocyanin compared with JE. Orthogonal partial least squares discriminant analysis (OPLS-DA) and principal component analysis (PCA) were used for classification. Classification accuracy of 100% was achieved between the three methods. The S-line plot revealed that the corresponding wavelength bands within the following regions 1,090, 1,250, 1,750, and 3,200 cm−1 were responsible for discrimination between the different extraction methods. Our results suggest that the main contributor to the discrimination between extraction methods were TSS, TSS/TA, color attributes, and anthocyanin content. Overall, this study has demonstrated that ATR-FT-MIR spectroscopy provides a powerful way to discriminate between juice extraction methods.
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    Effect of saccharomyces, non-saccharomyces yeasts and malolactic fermentation strategies on fermentation kinetics and flavor of Shiraz Wines
    (MDPI, 2017) Du Plessis, Heinrich; Du Toit, Maret; Nieuwoudt, Helene; Van der Rijst, Marieta; Kidd, Martin; Jolly, Neil
    The use of non-Saccharomyces yeasts to improve complexity and diversify wine style is increasing; however, the interactions between non-Saccharomyces yeasts and lactic acid bacteria (LAB) have not received much attention. This study investigated the interactions of seven non-Saccharomyces yeast strains of the genera Candida, Hanseniaspora, Lachancea, Metschnikowia and Torulaspora in combination with S. cerevisiae and three malolactic fermentation (MLF) strategies in a Shiraz winemaking trial. Standard oenological parameters, volatile composition and sensory profiles of wines were investigated. Wines produced with non-Saccharomyces yeasts had lower alcohol and glycerol levels than wines produced with S. cerevisiae only. Malolactic fermentation also completed faster in these wines. Wines produced with non-Saccharomyces yeasts differed chemically and sensorially from wines produced with S. cerevisiae only. The Candida zemplinina and the one L. thermotolerans isolate slightly inhibited LAB growth in wines that underwent simultaneous MLF. Malolactic fermentation strategy had a greater impact on sensory profiles than yeast treatment. Both yeast selection and MLF strategy had a significant effect on berry aroma, but MLF strategy also had a significant effect on acid balance and astringency of wines. Winemakers should apply the optimal yeast combination and MLF strategy to ensure fast completion of MLF and improve wine complexit
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    Effect of skin contact before and during alcoholic fermentation on the chemical and sensory profile of South African Chenin Blanc white wines
    (South African Society for Enology and Viticulture, 2015) Aleixandre-Tudo, Jose Luis; Weightman, C.; Panzeri, V.; Nieuwoudt, Helene; Du Toit, W. J.
    The volatile and phenolic composition of Chenin blanc wines made with different skin contact treatments was studied. One batch of grapes was used to make a dry white wine according to two different treatments, namely pre-fermentative skin contact and complete fermentation on the skins. A white wine fermented without any skin contact was used as control. Fermentation on the skins and skin contact before fermentation led to significantly lower levels of terpenes, esters, acids and thiols, and the highest significant levels of alcohols and phenolic compounds. However, this effect was less pronounced in wines with skin contact before fermentation. Sensory analysis of all the experimental wines was also performed. The results showed a significant shift from the sensory attributes of fresh and tropical fruits of the control Chenin blanc wines towards riper fruit notes in the skin contact treatments. This observation was correlated with the length of the skin contact period. Possible reasons to explain the results observed in this study are discussed.
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    Fermentation-derived aroma compounds in varietal young wines from South Africa
    (SASEV, 2010-09) Louw, L.; Tredoux, A. G. J.; Van Rensburg, P.; Kidd, M.; Naes, T.; Nieuwoudt, Helene
    The volatile composition of 925 single cultivar young Sauvignon blanc, Chardonnay, Pinotage, Merlot, Shiraz and Cabernet Sauvignon wines of vintages 2005 to 2007, was determined using gas chromatography-flame ionisation detection. Compositional data were compared to published data on young wines from South Africa and other countries. South African young wines analysed in this study had a largely similar volatile composition to that reported in the literature. Significant between-vintage and between-cultivar differences were observed in the volatile composition of the wines investigated in this study. The concentration ranges of four compounds in red wines, hexanol, propanol, diethyl succinate and ethyl lactate, and four compounds in white wines, 2-phenylethanol, hexanoic acid, isoamyl acetate and propanol, were not influenced by vintage effects. This finding was interpreted as the first indication that typical concentration ranges for some aroma compounds can be established for South African young cultivar wines. A trend was observed in the white wines that the alcohols and their respective acetate esters, as well as fatty acids and their ethyl esters, were responsible for the vintage-related effects. Differences in volatile composition between Chardonnay and Sauvignon blanc wines could also largely be explained on the same basis. Classification models were established to discriminate between individual red wine cultivars and between the two white wine cultivars and correct classification rates of respectively, 79 % and 85 % were achieved.
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    Glycerol and wine
    (Stellenbosch : Stellenbosch University, 2004-03) Nieuwoudt, Helene; Prior, B. A.; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of AgriScience. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Key research areas in modern enology are related to the production of wine of consistent quality, as well as to the improvement of existing wine quality through the enhancement of the sensory properties of wine. The formation of glycerol during alcoholic fermentation is highly relevant to both these issues. Since the early years of the zo" century, glycerol has been positively associated with the quality of wine and it is thought to impart important mouth-feel sensations such as "viscosity", "smoothness" and "body". In general, it is considered that glycerol concentrations higher than those normally found in wine, can contribute towards the improvement of wine quality. It has also been suggested that increased concentrations of glycerol can enhance the aroma of wine. On the basis of these perceptions, several strategies have been developed to favour the production of glycerol during the fermentation process and over a period of years, a large volume of data has been collected that relates to various aspects regarding glycerol production during alcoholic fermentation. To date, however, several aspects regarding the relationship between glycerol and wine quality remain unclear. The reasons for this situation can mainly be ascribed to the lack of reliable analytical data to serve as a basis for investigating the relationship between glycerol and wine quality, as well as the preponderance of empirical and anecdotal evidence. Despite numerous opinions regarding optimal glycerol concentrations in wine, glycerol is indeed seldom assayed on a routine basis and targets with respect to specific wine grape cultivars and glycerol concentrations have largely remained unspecified. To date, very little information regarding glycerol concentrations in South African wines has been published. The analytical techniques that are most frequently used for the quantification of glycerol in grape juice, fermenting must and wine are not easily automated and this aspect placed severe limitations on the generation of large volumes of analytical data on glycerol concentrations in these matrices. This project was undertaken with the aim to holistically address some of the unresolved issues relating to the relationship between glycerol and wine quality. This also implied the development and optimisation of analytical techniques suitable for the rapid and accurate determination of glycerol in fermentation media, as well as in finished wine. In the first stage of this project a quantitative database was established that contained the analytical data on the glycerol concentrations of a statistically significant number of wines of adjudged quality, as well as additional information for each wine regarding the geographic origin, vintage, routine chemical analyses and the yeast strain(s) used for the production of the wine. The relevance of glycerol in wine for the modern South African winemaker was evaluated through the establishment of a quantitative database that contained the opinions of an expert panel of 15 South African winemakers, enologists and wine chemists on topics relating to glycerol in wine. In the second stage of the project the data captured in the databases were used to investigate aspects regarding the relationship between glycerol and wine quality. From the data captured in the qualitative database, it was clear that the topic regarding glycerol in wine was important to the South African winemakers and it was also evident that there was a need for the development and optimisation of methods suitable for the routine analysis of the glycerol concentrations in grape juice, fermenting must and wine. The opinions of the panel members also highlighted the issue that the mouth-feel property of wine was considered to be an area where the quality of some wines could be further improved. The quantitative database contained the information on the glycerol concentrations of 450 commercial South African table wines of adjudged quality. The premium cultivars Chardonnay, Chenin blanc, Sauvignon blanc, Cabernet Sauvignon, Merlot, Shiraz and Pinotage were used for the purpose of investigating the relationship between glycerol concentration and wine quality. The wines represented a wide variety of wine styles, including dry white, off-dry white, dry red and late harvest wines. The average glycerol concentration was significantly associated with the wine style. In white wines the average glycerol concentration was much lower than in the red wines (6.82 gIL versus 10.49 gIL, respectively). No significant relationship between the final glycerol concentration and the geographic origin, vintage and the yeast strain used for the fermentation was found. Wine quality could not be significantly associated with glycerol concentration in the red wines. In the white wines, the relationship between glycerol concentration and wine quality was significant, but due to the very small differences in the average glycerol concentrations of the wines of different quality ratings, the statistical significance is probably of little practical value. The effect of glycerol on the volatility of a selection of esters and higher alcohols was also investigated. Solid-phase microextraction, followed by gas chromatography, was used to analyse the composition of the headspace at equilibrium between the liquid phase and the gas phase of a model wine, and a dry white wine that contained a basal concentration of 5.4 gIL glycerol. Results showed that incremental increases in the glycerol concentrations over a range of 1 -10 gIL in the model wine and in a Chenin blanc wine, were not accompanied by a proportional increase or decrease in the abundance of the aroma components in the headspace. The volatile components tested were isoamyl acetate, ethyl butyrate, ethyl valerate, ethyl lactate, ethyl hexanoate, hexyl acetate, isoamyl alcohol and isobutanol. For all the volatile components tested, the difference between the headspace composition of samples containing the lowest glycerol concentration, and those containing the highest glycerol concentration, was not significant. However, sufficient experimental evidence was obtained to indicate that increasing glycerol concentrations had an effect on the volatility of aroma components, and that the effect is of a complex and non-linear nature. In the third phase of the work Fourier transform infrared spectroscopy (FT-IR) was used to establish and optimise methods for the accurate and rapid quantification of glycerol in wine. For this purpose calibrations were developed for the quantification of glycerol in dry wine and late harvest wines. The accuracy of prediction was evaluated by means of the standard error of prediction that was 0.38 gIL for the dry wines and 0.65 gIL for the sweet wines. Large variations are introduced in the FT-IR spectra of wine by factors such as process technology, cultivar and geographic origin, and this variation can have an effect on the accuracy of the analytical data generated when employing FT-IR spectroscopy. Using glycerol prediction in wine as a model system, principal component analysis of the FT-IR spectra was done in order to establish quality control measures for the detection of poorly predicted, or outlier samples. A classification model, based on principal component analysis, was established that enabled the interpretation and classification of the outlier samples in the data set in 100% of the cases tested. This work forms the basis for expanding the quality control measures for the detection of wines of which the FT-IR spectra are highly unnatural, as well as for establishing quality control measures to ensure that accurate analytical data are generated when FT-IR is used. FT-IR spectroscopy was also used to develop a rapid screen for the evaluation of the fermentation profiles of wine yeasts. For this purpose, a selection of wine yeasts, which included commercial wine yeasts frequently used in winemaking in South Africa, as well as a selection of hybrid Saccharomyces cerevisiae yeasts, that were obtained through a selective breeding strategy aimed at increasing glycerol concentrations were used. Calibrations necessary for the accurate quantification of glycerol, volatile acidity, ethanol, reducing sugar and glucose, in Chenin blanc must and a synthetic must were developed and optimised. This work forms the basis upon which the scope of the analysis, both in terms of the number of components that can be measured, as well as the medium in which the yeasts are being evaluated, can be enlarged. This would be valuable for future applications in both the research as well as the industrial environment. The method that was developed serves to illustrate how this application can play a supportive role in yeast development programmes, through the speeding up of the initial stages of yeast strain evaluation.
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    Glycerol in South African table wines : an assessment of its relationship to wine quality
    (South African Society for Enology and Viticulture, 2002) Nieuwoudt, Helene; Prior, B. A.; Pretorius, I. S.; Bauer, Florian
    Glycerol is an important by-product of glycolysis and is quantitatively one of the major components of wine. While the physicochemical and sensory characteristics of pure glycerol are well established, the impact of varying levels of glycerol on general wine quality remains a topic of debate. Previous reports have relied on limited numbers of either commercial or experimental wines to assess the role of glycerol, leading to contradictory conclusions. Here we report on a large-scale assessment of the relationship between glycerol concentration and wine quality, based on the analysis of a significant number of commercial South African table wines of adjudged quality. The mean glycerol concentrations of 237 dry red (10.49 g/L), 158 dry white (6.82 g/L), 22 off-dry white (6.55 g/L), 16 special late harvest (8.26 g/L) and 14 noble late harvest wines (15.55 g/L) were found to be associated with considerable variation within each respective style. The final glycerol concentrations were significantly associated with the wine style (P<0.05). Shiraz wines had a mean glycerol concentration (10.22 g/L) which was significantly lower than that of Cabernet Sauvignon (10.81 g/L), Pinotage (10.46 g/L) and Merlot (10.62 g/L) wines (P<0.05). In both the dry white and off-dry white styles, the mean glycerol concentrations of Sauvignon blanc wines (6.31and5.42 g/L, respectively) were significantly lower (P<0.05) than those of the Chardonnay wines (7.08 and 7.03 g/L, respectively) and the Chenin blanc wines (6.81 and 6.85 g/L, respectively). No significant association between the final glycerol concentrations in commercial wines and the vintage, geographic origin or yeast strain used in inoculated fermentations could be established (P>0.05). The mean glycerol concentrations for South African dry red wines were significantly higher than those of dry white and off-dry white wines. Wine quality could not be significantly associated with glycerol concentrations in the dry red wines (P>0.05). For the dry white, off-dry white and late harvest wines this association was significant (P<0.05), although the exact nature of the association was somewhat different for the respective styles. Despite this positive statistical association, the observed differences between the mean glycerol concentrations of dry white and off-dry white wines of different quality ratings were too small to be of major practical value. The relationship between glycerol concentration and wine quality is reassessed on the basis of results obtained in this study as well as on recent reports in the literature.
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    Identification of wine yeast strains with FT-NIRS and effect of spectral variation due to sample preparation and repeated analyses
    (2007) Muller, C. A.; Manley, M.; Lambrechts, M.; Pretorius, I. S.; Nieuwoudt, Helene
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    Modelling the sensory space of varietal wines : mining of large, unstructured text data and visualisation of style patterns
    (Nature Publishing Group, 2018) Valente, Carlo C.; Bauer, Florian; Venter, Fritz; Watson, Bruce; Nieuwoudt, Helene
    The increasingly large volumes of publicly available sensory descriptions of wine raises the question whether this source of data can be mined to extract meaningful domain-specific information about the sensory properties of wine. We introduce a novel application of formal concept lattices, in combination with traditional statistical tests, to visualise the sensory attributes of a big data set of some 7,000 Chenin blanc and Sauvignon blanc wines. Complexity was identified as an important driver of style in hereto uncharacterised Chenin blanc, and the sensory cues for specific styles were identified. This is the first study to apply these methods for the purpose of identifying styles within varietal wines. More generally, our interactive data visualisation and mining driven approach opens up new investigations towards better understanding of the complex field of sensory science.
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    Modulation of wine flavor using hanseniaspora uvarum in combination with different saccharomyces cerevisiae, lactic acid bacteria strains and malolactic fermentation strategies
    (MDPI, 2019-07-2019) Du Plessis, Heinrich; Du Toit, Maret; Nieuwoudt, Helene; Van Der Rijst, Marieta; Hoff, Justin; Jolly, Neil
    Hanseniaspora uvarum is one of the predominant non-Saccharomyces yeast species found on grapes and in juice, but its effect on lactic acid bacteria (LAB) growth and wine flavor has not been extensively studied. Therefore, the interaction between H. uvarum, two Saccharomyces cerevisiae yeast strains, two LAB species (Lactobacillus plantarum and Oenococcus oeni) in combination with two malolactic fermentation (MLF) strategies was investigated in Shiraz wine production trials. The evolution of the different microorganisms was monitored, non-volatile and volatile compounds were measured, and the wines were subjected to sensory evaluation. Wines produced with H. uvarum in combination with S. cerevisiae completed MLF in a shorter period than wines produced with only S. cerevisiae. Sequential MLF wines scored higher for fresh vegetative and spicy aroma than wines where MLF was induced as a simultaneous inoculation. Wines produced with H. uvarum had more body than wines produced with only S. cerevisiae. The induction of MLF using L. plantarum also resulted in wines with higher scores for body. H. uvarum can be used to reduce the duration of MLF, enhance fresh vegetative aroma and improve the body of a wine.
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