Understanding the need of SO2 in wine according to grape varieties

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UNDERSTANDING THE NEED OF SO 2 IN WINE ACCORDING TO GRAPE VARIETIES

NOVA University Lisbon

CÁTIA VANESSA DE ALMEIDA SANTOS Master in Bioorganic Chemistry

DOCTORATE IN SUSTAINABLE CHEMISTRY

DEPARTAMENT OF CHEMISTRY

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UNDERSTANDING THE NEED OF SO

2

IN WINE ACCORDING TO GRAPE VARIETIES

COM UMA MUDANÇA DE LINHA FORÇADA

Adviser: Marco Diogo Richter Gomes da Silva

Associate Professor with Habilitation, NOVA University Lisbon Co-adviser: Maria João Bastos Cabrita

Associate Professor with Habilitation, Évora University

Examination Committee:

Chair: Susana Filipe Barreiros

Full Professor NOVA University Lisbon Rapporteurs: Maria Fernanda Gil Cosme Martins

Assistant Professor, Department of Biology and Environment, University of Trás-os Montes e Alto Douro

Ilda Maria Justino Caldeira

Assistant Researcher, National Institute for Agricultural and Veterinary Research Adviser: Marco Diogo Richter Gomes da Silva

Associate Professor with Habilitation, NOVA University Lisbon Members Ana Maria Costa Freitas

Retiree Full Professor, Évora University María Esperanza Valdés Sanchez

Researcher, Technological Institute of Food and Agriculture of Extremadura, Spain José de Oliveira Fernandes

Assistant Professor, Faculty of Pharmacy, University of Porto Susana Filipe Barreiros

Full Professor NOVA University Lisbon

NOVA University Lisbon September, 2022

CÁTIA VANESSA DE ALMEIDA SANTOS Master in Bioorganic Chemistry

DOCTORATE IN SUSTAINABLE CHEMISTRY

DEPARTAMENT OF CHEMISTRY

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Understanding the need of SO2 in wine according to grape varieties

The NOVA School of Science and Technology and the NOVA University Lisbon have the right, perpe- tual and without geographical boundaries, to file and publish this dissertation through printed copies reproduced on paper or on digital form, or by any other means known or that may be invented, and to disseminate through scientific repositories and admit its copying and distribution for non- commercial, educational or research purposes, as long as credit is given to the author and editor.

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To my parents for being an inspiration of strength.

“I had great mentors in my parents who always sought to understand the world around them.

And they would push me to really think things through.”

(Mae Jemison)

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Acknowledgments

I would first like to thank the doctoral program in Sustainable Chemistry for selecting me for a scholarship grand financed by National Funds through FCT - Foundation for Science and Technology under the PhD Grant [PD/BD/135081/2017]. Also, the two institutions that allowed this work to happen: Associate Laboratory for Green Chemistry (LAQV) on FCT-NOVA and the Oenology Labora- tory (MED) from Évora University.

I would like the express my gratitude and thanks to my supervisors, Professor Marco Silva and Maria João Cabrita, whose expertise was invaluable in formulating the research questions and trans- lating data into meaningful information. Your perceptive feedback pushed me to sharpen my thinking and brought my work to a higher level, we know that task was not always easy.

I am very grateful for the opportunity to work with Doctor Eduardo Mateus from CENSE. His enthusiasm, scientific curiosity and the constant challenges presented, were very motivating. Some- times leading to the loss of sense of time in the laboratory and the notion that the more we try to know the more we see what we have yet to learn.

I would thank the two teams, Resolution Lab from FCT-NOVA and Oenology Laboratory from Évora University, as growing is never an individual work. To Flávia Freitas, João Brinco, André Jorge, Paula Guedes, Joana Dionísio, Ana Rita Ferreira, Catarina Mendes, Nuno Martins and Professor Raquel Martins for their availability during the process of helping me throughout my work. I also thank all the students I contacted with in this process. In different ways, everyone taught me some- thing important in this journey.

To Rui Bicho, a special thank you, for the long hours of teamwork. Working with you was a wonderful and very enriching experience. Whether in the laboratory or in the winery, I learned plen- ty from your teachings.

To my friends Cátia Magro, Joana Almeida, Daniela Peixoto and Ana Estevão, thank you for the friendship during my PhD journey and so much more. Without this support, this journey would have been less positive and enriching.

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To Ricardo Faria, thank you for the patience, availability for scientific debate, unconditional support and understanding of the need to work in life as a team. I know it may seem like a few words but after so long I think thanks were made in the little things of everyday life and I hope I managed to do it.

To my parents, Maria José and Pedro, thank you for the encouragement in all moments of my life. Also, to my brother Rúben Santos, thank you for being there for everything and the help with all my computer software problems. I would not ask for a better brother.

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"Don't be afraid of hard work. Nothing worthwhile comes easily. Don't let others discourage you or tell you that you can't do it. In my day I was told women didn't go into chemistry.

I saw no reason why we couldn't."

(Gertrude Elion)

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Abstract

The use of sulfur dioxide (SO2) is widely accepted as a useful aid in winemaking. It is used as a preservative due to its antioxidant and antimicrobial properties in wine. Although SO2 is a by- product of fermentation, most of the SO2 present in wines is added by the winemaker at different stages of the process. There is still controversy about the use of SO2 and its real impact on consumer health. Since 2005, the EU has required an indicative warning that there are sulfites in the product because a small minority of people are highly allergic to sulfites. However, information is scarce whether the SO2 requirement depends on the wine variety and to what extent. Furthermore, the search for replacers for this enological aid, while maintaining the quality of the final product has not yet been presented, behaving with the same multiplicity of actions. For this reason, SO2 is still one of the most effective tools winemakers has to protect wine and influence flavor. Thus, in this work one studied the volatile organic composition (VOC) of several varieties of wine obtained with controlled addition of different SO2, as well as its substitution or co-addition with ascorbic acid. Headspace solid phase microextraction gas chromatography/mass spectrometry (HS-SPME-GC/MS) was used for VOC analysis. To obtain a more comprehensive characterization of these wines, amino acid (AAs) profiles were accessed through High Performance Liquid Chromatography with Diode Array Detector (HPLC-DAD).

The reduction of SO2 during the fermentation of the same must, resulted in different wines and the differences observed depend on the grape varieties used. This differentiation was observed shortly after the first 24 H of fermentation. However, these differences were reduced during storage. The same behavior was verified for ascorbic acid, which generally resulting in final products with different VOCs profiles. The results obtained indicate that any reduction in SO2 will interfere with the VOCs profile of the resulting wine, and thus may interfere with their flavor profile. The use of ascorbic acid as a substitute or co-additive did not lead to wines with the same profile as the wines treated only with SO2. Therefore, the use of these enological aids must be wisely considered as it will impact the final product.

Keywords: Wine, Volatile Organic Composition (VOC), Amino acid, Sulfur Dioxide (SO2), Ascorbic Acid, HS-SPME-GC/MS, HPLC-DAD.

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Resumo

O uso de dióxido de enxofre (SO2) é amplamente aceite como um auxiliar útil na vinificação. É usado como conservante devido às suas propriedades antioxidantes e antimicrobianas no vinho.

Embora o SO2 seja um subproduto da fermentação, a maior parte do SO2 presente nos vinhos é adi- cionado pelo enólogo em diferentes etapas do processo. Ainda há controvérsias sobre o uso do SO2

e seu real impacto na saúde do consumidor. Desde 2005, a UE exige um aviso indicativo de que há sulfitos no produto porque uma pequena minoria de pessoas é altamente alérgica a sulfitos. No entanto, a informação é escassa se a exigência de aplicação de SO2 depende da variedade do vinho e em que medida. Além disso, a produra de alternativas para o uso desse auxíliar enológico, manten- do a qualidade do produto final ainda não foi apresentada, actuando com a mesma multiplicidade de ações. Por esta razão, o SO2 ainda é uma das ferramentas mais eficazes que os vinicultores têm para proteger o vinho e influenciar o sabor. Assim, neste trabalho estudou-se a composição orgânica volátil (COV) de diversas castas de vinho obtidas com adição controlada de diferentes SO2, bem como a sua substituição ou co-adição com ácido ascórbico. Recurreu-se a cromatografia gasosa de microextração em fase sólida em espaço de cabeça/espectrometria de massa (HS-SPME-GC/MS) foi usada para análise de COV. Para obter uma caracterização mais abrangente desses vinhos, os perfis de aminoácidos (AAs) foram analisados por meio de Cromatografia Líquida de Alta Eficiência com Diodo Array Detector (HPLC-DAD).

A redução de SO2 durante a fermentação de um mesmo mosto, resultou em vinhos diferentes e as diferenças observadas dependem das castas utilizadas. Esta diferenciação foi observada logo após as primeiras 24 H de fermentação. No entanto, essas diferenças foram reduzidas durante o armazenamento. O mesmo comportamento foi verificado para o ácido ascórbico, que geralmente resulta em produtos finais com diferentes perfis de COVs. Os resultados obtidos indicam que qual- quer redução de SO2 irá interferir no perfil de COVs do vinho resultante, podendo assim interferir no seu perfil aromático. A utilização de ácido ascórbico como substituto ou co-aditivo não conduziu a vinhos com o mesmo perfil dos vinhos tratados apenas com SO2. Portanto, o uso desses auxiliares enológicos deve ser considerado com sabedoria, pois afetará o produto final.

Palavras-chave: Vinho, Composição Orgânica Volátil (COV), Aminoácido, Dióxido de Enxofre (SO2), Ácido Ascórbico, HS-SPME-GC/MS, HPLC-DAD.

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Figure index

Figure 1-1 EU data of wine production in 2020/2021, with the last update, a) by the EU member’s production and b) Portugal production by wine categories (been the principals the P.D.O wines:

Protected Designation of Origin; P.G.I wines: Protected Geographical Indication). Adaptation of European Commission in the Agri-food data portal, Agricultural markets and Wine accessed on

13-02-2022[1]. ... 3

Figure 1-2 Portuguese map division by the 14 regions consider P.G.I. An adaptation of “Vinhos e Aguardentes de Portugal Anuário” from 2018[4]. ... 4

Figure 1-3 Technics applied on wine and grape sample preparation with a few advantage and disadvantage[56][59][60]... 10

Figure 3-1 Graphical representation of three monovarietal wines studied in 2018. Study of VOC profile after fermentation of three white wine variety. ... 16

Figure 3-2 Graphical representation of three monovarietal wines studied in 2018. Study of VOC profile after short evolution of three white wine variety. ... 17

Figure 3-3 Graphical representation of Arinto and Síria wine study in 2018. Study VOC profile of antioxidant conditions on fermentation and short evolution in the presence or absence of bentonite... 18

Figure 3-4 Graphical representation of Arinto wine study in 2018. Study the amino acids content fermented in different antioxidant conditions. ... 19

Figure 3-5 Graphical representation of Arinto wine study in 2018. Study the amino acids content fermented in different antioxidant conditions in the presence or absence of bentonite... 20

Figure 3-6 Graphical representation of Antão Vaz and Blend wine study in 2018. ... 21

Figure 3-7 Graphical representation of Gouveio wine study in 2019. ... 22

Figure 3-8 Graphical representation of Arinto wine study in 2019. ... 23

Figure 3-9 Graphical representation of Aragonês wine study in 2019. ... 23

Figure 3-10 Graphical representation of Aragonês wine study in 2019, musts fermented whit different SO2 and ascorbic acid combinations additions. ... 24

Figure 4-1 Graphical representation of vinification process applied in this PhD. Project development in 2018 and 2019. a) Refers to white grape varieties and b) refers to red grape variety... 26

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Figure 4-2 Graphical representation of total area analyzed, and total number of compounds

identified for the injection mode: splitless (blue), split 1:5 (grey) and split 1:10 (black), dots are the number of compounds. ... 29 Figure 4-3 Graphical projections with the influence of NaCl concentration on the efficiency of HS-

SPME using a DVB/CAR/PDMS fibre at 30 °C, without agitation (a) n=2; b) and c) n= 3). a) and b) are two different commercial wines and c) a commercial red wine, the samples presented an ethanolic content of 12%. ... 31 Figure 6-1 Graphical representation of Arinto and Síria wine fermented with lower doses of SO2. ... 46 Figure 6-2 Graphical representation of Arinto and Síria wine fermented with higher doses of SO2. .. 46 Figure 6-3 Graphical representation of Gouveio, Arinto and Síria wine fermented with ascorbic acid.

... 46 Figure 6-4 Principal component biplot illustrating the simultaneous projection of the wine and

volatile compounds of Arinto wines and Gouveio wines fermented with higher SO2 doses range.

Black triangles – Gouveio wines; Black lozenges – Arinto wines; Dark blue dots – esters; Dark green dots – ethers; Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes;

Light grey dots – carboxylic acids; Purple dots – miscellaneous; Gray dots– unknowns. ... 59 Figure 6-5 Principal component biplot illustrating the simultaneous projection of the wine and

volatile compounds of Arinto wines and Gouveio wines fermented with higher SO2 doses range of evolution wines using wines after fermentation as reference. Black triangles – Arinto wines;

Black lozenges – Gouveio wines; Dark blue dots – esters; Dark green dots – ethers; Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes; Light grey dots – carboxylic acids;

Purple dots – miscellaneous; Gray dots – unknowns; Orange shadow – wines fermented without SO2; Grey shadow – wines fermented with 50 mg/L of SO2; Blue shadow – wines

fermented with 100 mg/L of SO2. ... 60 Figure 6-6 Principal component biplot illustrating the simultaneous projection of the wine and

volatile compounds of Arinto wines and Síria wines fermented with lower SO2 doses range.

Black triangles – Arinto wines; Black lozenges – Síria wines; Dark blue dots – esters; Dark green dots – ethers; Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes; Light grey dots – carboxylic acids; Purple dots – miscellaneous; Gray dots – unknowns. ... 61 Figure 6-7 Principal component biplot illustrating the simultaneous projection of the wine and

volatile compounds of Arinto wines and Síria wines fermented with lower SO2 doses range of

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Purple dots – miscellaneous; Gray dots – unknowns; Orange shadow – wines fermented without SO2; Green shadow – wines fermented with 15 mg/L of SO2; Grey shadow – wines fermented with 30 mg/L of SO2; Blue shadow – wines fermented with 45 mg/L of SO2. ... 62 Figure 6-8 Principal component biplot illustrating the simultaneous projection of the wine and

volatile compounds of Arinto, Síria and Gouveio wines fermented with ascorbic acid of evolution wines using wines after fermentation as reference. Black triangles – Gouveio wines;

Black lozenges – Arinto wines; Black squares – Síria wines; Dark blue dots – esters; Dark green dots – ethers; Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes; Light grey dots – carboxylic acids; Purple dots – miscellaneous; Gray dots – unknowns; Orange shadow – wines fermented without SO2; Grey shadow – wines fermented with 50 mg/L of SO2; Blue shadow – wines fermented with 100 mg/L of SO2. ... 63 Figure 6-9 Graphical representation of total amino acid content observed on Arinto must fermented

with lower range doses of SO2 with the error bar represented. ... 126 Figure 6-10 PCA illustrating the simultaneous projection of the Arinto wines fermented with lower

range SO2 doses, in relation to the conditions of SO2 applications and the amino acid content.

Applications before fermentation: blue – 0 mg/L of SO2, black – 15 mg/L of SO2, orange – 30 mg/L of SO2 and yellow – 45 mg/L of SO2. Before fermentation: triangle – 0 mg/L of SO2, lozenge – 30 mg/L of SO2 and square – 60 mg/L of SO2. Grey dots represent the amino acid. ... 127 Figure 6-11 Graphical representation of total amino acid content observed on Arinto must

fermented with higher range doses of SO2 with the error bar represented. ... 128 Figure 6-12 PCA illustrating the simultaneous projection of the Arinto wines in relation to the

conditions of SO2 applications and the amino acid content. Applications before fermentation:

blue – 0 mg/L of SO2, black –50 mg/L of SO2 and orange – 100 mg/L of SO2. Before

fermentation: triangle – 0 mg/L of SO2 and square – 60 mg/L of SO2. Grey dots represent the amino acid. ... 128 Figure 6-13 PCA illustrating the simultaneous projection of the Arinto wines in relation to the

conditions of SO2 applications and the amino acid content fermented with and without bentonite. Applications before fermentation: blue – 0 mg/L of SO2, black – 50 mg/L of SO2 and orange – 100 mg/L of SO2. Before fermentation: triangle – 0 mg/L of SO2 and square – 60 mg/L of SO2. Grey dots represent the amino acid. Wines fermented with bentonite – inside fill with dots and wines fermented without bentonite – inside fill with lines. ... 129 Figure 6-14 Graphical representation of Gouveio wine fermented different doses of SO2 and ascorbic as as an alternative to SO2. ... 158

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Figure 6-15 Principal component biplot illustrating the simultaneous projection of musts and volatile compounds. Black triangle – musts; Dark blue dots – esters; Dark green dots – hydrocarbons;

Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes; Light green dots – carboxylic acids; Purple dots – miscellaneous; Gray dots – unknowns. ... 165 Figure 6-16 Principal component biplot illustrating the simultaneous projection of musts and volatile

compounds. Black triangle – wines; Dark blue dots – esters; Dark green dots – hydrocarbons;

Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes; Light green dots – carboxylic acids; Purple dots – miscellaneous; Gray dots – unknowns. ... 166 Figure 6-17 Principal component biplot illustrating the simultaneous projection of Arinto wines and

volatile compounds. Black triangle – wines fermented with ascorbic acid; Black dots – wines fermented without ascorbic acid; Dark blue dots – esters; Dark green dots – hydrocarbons;

Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes; Light green dots – carboxylic acids; Purple dots – miscellaneous; Gray dots – unknowns; Pink dots - ethers. ... 174 Figure 6-18 Principal component biplot illustrating the simultaneous projection of Aragones wines

and volatile compounds. Black triangle – wines fermented with ascorbic acid; Black dots – wines fermented without ascorbic acid; Dark blue dots – esters; Dark green dots – hydrocarbons;

Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes; Light green dots – carboxylic acids; Purple dots – miscellaneous; Gray dots – unknowns; Pink dots - ethers. ... 175 Figure 6-19 Principal component biplot illustrating the simultaneous projection of Aragonês wines

and volatile compounds. Black triangle – wines fermented with 25 mg/L of SO2; Black

rhombuses – wines fermented with 50 mg/L of SO2; Black dots – wines fermented with 75 mg/L of SO2; Dark blue dots – esters; Dark green dots – ethers; Yellow dots – ketones; Red dots – alcohols; Light blue dots – aldehydes; Light green dots – carboxylic acids; Gray dots –

unknowns. ... 183

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Table index

Table 4-1 Fermentation and maturation conditions applied on must of 2018 according to grape variety. ... 27 Table 4-2 Fermentation and maturation conditions applied on must of 2019 according to grape

variety. ... 28 Table 6-1 Enological parameters in all wines after fermentation (t = 0) with different doses of SO2

and ascorbic acid. ... 48 Table 6-2 Free and total SO2 content of initial wines and wines after 6 months of second antioxidant

addition. ... 50 Table 6-3 Compounds identified in wine samples (Arinto, Síria and Gouveio). ... 52 Table 6-4 Antioxidant conditions applied for the amino acid content on Arinto wine variety. ... 125 Table 6-5 Enological parameters of Gouveio wines after fermentation with different doses of SO2 and ascorbic acid. ... 159 Table 6-6 VOCs tentatively identified in all analysed samples of musts and wines of Gouveio variety

that were found in at least one samples. ... 159 Table 6-7 Enological parameters of Arinto wines after fermentation with different doses of SO2 and

ascorbic acid. ... 168 Table 6-8 Enological parameters of Aragonês wines after fermentation with different doses of SO2

and ascorbic acid. ... 168 Table 6-9 VOCs tentatively identified in all analysed samples of white and red wines (Arinto and

Aragonês respectively) found in at least one sample. ... 170 Table 6-10 Enological parameters of Aragonês wines after fermentation with combination doses of

SO2 and ascorbic acid. ... 178 Table 6-11 VOCs tentatively identified in all analysed samples of Aragonês wines found in at least

one samples. ... 180

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Scheme index

Scheme 1-1 Representation of SO2 acid-base equilibrium in wine pH (3 to 4) adapted from Guerrero et al. in 2015[19]. Represented in blue are the bound forms, black the free forms and in bold the form with higher concentration. ... 6 Scheme 1-2 Scheme adapted from the proposed mechanism of wine oxidation under the specific

antioxidant combination of SO2 and ascorbic acid[33][34]. The ascorbic acid action is

represented in black and the action of SO2 is represented in blue. ... 7 Scheme 1-3 Ascorbic acid oxidation cycle without SO2, in blue are the main reactive species formed,

adapted from Manzocco et al. in 1998 and Bradshaw et al. in 2011[25][36]. ... 8 Scheme 1-4 Adaptation for the protein precipitation mechanism in the presence of SO2 on white

wines[43]. ... 8 Scheme 2-1 Research questions divided by stages of winemaking were this PhD project focuses on.

... 13

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Acronym index

ANOVA: Analysis of variance

CARMIM: Cooperativa Agrícola de Reguengos de Monsaraz CG: Gas Chromatography

DNA: Deoxyribonucleic acid DOI: Digital Object Identifier

DVB/CAR/PDMS: Divinylbenzene/Carboxen/Polydimethylsiloxane FAO: Food and Agriculture Organization

GC × GC/TOF-MS: Two-dimensional Gas Chromatography/Time of flight Mass Spectrometry GC-FID: Gas Chromatography-Mass Spectrometry-Flame ionization detection

GC-O: Gas Chromatography-Olfactometry

HPLC-DAD: High Performance Liquid Chromatograph with Diode Array Detector HS-SPME: Headspace Solid-Phase Microextraction

HS-SPME-GC/MS: Headspace Solid Microextraction Gas Chromatography/Mass Spectrometry LCA: Life Cycle Assessment

LCT: Life Cycle Thinking

LRIcal: Linear retention indices calculated

LRIlit: Linear retention indices reported in the literature MLF: Malolactic Fermentation

OIV: International Organization of Vine and Wine P.D.O: Protected Designation of Origin

P.G.I: Protected Geographical Indication PCA: Principal Components Analysis RNA: Ribonucleic acid

ROS: Reactive Oxygen Species RPA: Relative Peak Area

RSD: Relative Standard Deviation VOC: Volatile Organic Compound WHO: World Health Organization

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Ciência Téc. Vitiv. 37(1) 39-59. 2022

Part I

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1 Introduction

European Union (EU) is one of the top market leaders in the wine industry, responsible for two-thirds of global wine exportation. According to data for 2020/2021 in EU, Portugal is among the 5 largest wine producers representing 2.4% of production (Figure 1-1a) being, its production, mainly distributed in two sectors: Protected Designation of Origin (P.D.O) and Protected Geo- graphical Indication (P.G.I) wines (Figure 1-1b). Portugal showed in recent years a competitive advantage in the world market against the so-called new producers (for example: Australia, South Africa and Argentina) therefore, this industry has a significant economic weight[1][2].

Figure 1-1 EU data of wine production in 2020/2021, with the last update, a) by the EU member’s production and b) Portugal production by wine categories (been the principals the P.D.O wines: Protected Designation of Origin; P.G.I wines: Protected Geographical Indication). Adaptation of European Commission in the Agri-food data portal, Agricultural markets and Wine accessed on 13-02-2022[1].

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Portugal is divided into 14 wine regions consider P.G.I (Figure 1-2) being Alentejo one of them., Alentejo integrates the top three main regions in terms of production area, economic rele- vance and is also consider has P.D.O for wine production[3][4].

Figure 1-2 Portuguese map division by the 14 regions consider P.G.I. An adaptation of “Vinhos e Aguardentes de Portugal Anuário” from 2018[4].

Today, there is a change in consumer trends and what they look for in wines. The demand for wines designated as “Natural wine”, a designation that is not yet consensual in the literature in terms of definition, has grown rapidly[5]. Indeed, consumers attribute value in the relation to the environmental and sustainability aspects of a product, being wine an example. If wine producers want to remain competitive and even grow in the sector, some adaptations need to be taken into account, in order to attract this new market[5][6][7].

When a proper evaluation of the wine process or product sustainability is made, a Life Cycle Thinking (LCT) needs to be adopted to improve the sector. In the case of wine, two main perspec- tives can be applied. The perspective of the wine industry, including agriculture, marketing, waste

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sustainable wine. Actions on a set of indicators and certifications were proposed to promote con- tinuous improvement[10]. Assessing the Life Cycle Assessment (LCA) of Portuguese wines, a list of indicators responsible for the carbon emission footprint were reported: sulphur dioxide (SO2), yeast applied, and bentonite are some of them in the winemaking stage. However, this stage is not reported to have the main carbon emission footprint, but it can present an important point on the subject of wastewater and energy consumption[8][9][11].

Additionally, to environmental concerns, an increasing interest in health related to food quality led winemakers to reduce the use of oenological additives. One of these products is SO2, a multifunctional food additive. The joint World Health Organization (WHO) & Food and Agriculture Organization (FAO) of the United Nations expert committee on food additives, concluded that the dietary intake of sulphites in populations exceed the acceptable daily intake[12]. This excessive exposure may increase allergic response episodes with a range of symptoms, including, dermatitis, urticaria, angioedema, abdominal pain, diarrhoea, bronchoconstriction, and anaphylaxis[13][14].

Aiming to address this issue, sulphites were introduced in the list of substances/products triggering allergies or intolerance[15]. Consequently, the EU has required a warning in the label indicating the presence of sulphites when present above 10 mg/kg or 10 mg/L[16][17]. Also, in 2015 the International Organization of Vine and Wine (OIV) determined the new maximum value of total SO2 according to sugar content in wines: 150 - 300 mg/L for red wines, 200 - 400 mg/L for white wines, and 200 - 300 mg/L for rosé wines[18]. As a consequence of these limitations the search for replacers of SO2 or co-additives that allow a reduction of SO2 concentration increased in the last few years[19].

Since the 19^th century SO2 has been studied and applied as a well-accepted preserving agent in the food industry. In the wine industry, it is used at different stages of the process due to its different functions, namely, the ability to react as an antioxidant and antiseptic as the two most relevants[20][21][22]. This additive can be used on grapes usually in the form of powder, on must and wines in the form of gas, salt (powder or tables) or solution. The doses to apply depend on a combination of factors: grape condition, must pH, level of hygiene in the cellar and applied techniques. For example, for musts from healthy grapes with lower pH the recommended dose is be-

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tween 30 - 50 mg/L of SO2, but with higher pH the recommended dose is between 50 - 90 mg/L of SO2[23]. Regardless, lower doses are being used in wineries and even no SO2 addition at all.

Although wine contains natural compounds with antioxidant characteristics due to the presence of phenolic compounds, in order to prevent oxidative degradation, it is necessary an exogenous addition of preservative agents such as SO2[24][25]. The total SO2 present in wine (by natural or exogenous addition) are commonly divided into two fractions. The “free SO2”: bisulphite ion (HSO3-), sulphate ion (SO24-), and molecular SO2 and the “bound SO2”: SO2 mainly bound to unsatu- rated compounds (Scheme 1-1). At typical wine pH (3 to 4) an acid-base equilibrium occur with HSO3- form (the more representative at this pH) and a small portion in the SO2 form[19].

Scheme 1-1 Representation of SO2 acid-base equilibrium in wine pH (3 to 4) adapted from Guerrero et al. in 2015[19].

Represented in blue are the bound forms, black the free forms and in bold the form with higher concentration.

The protective function of SO2 appears not to be by direct reaction with O2 but by scaveng- ing hydrogen peroxide (H2O2) with HSO3- form. O2 in must and wine appears to be reduced by iron to the hydroperoxyl radical ([HO2]^.) among other reactive oxygen species (ROS). ROS reacts with ketones, aldehydes, or phenolic compounds from must and wine producing quinones, which results in loss of characteristic aromas. However, with SO2 addition a preventing mechanism occurs, and, additionally, quinones formed during oxidation are reduced back to their phenol forms[26][27][28].

SO2 can be used also as an antiseptic agent inhibiting the development of all types of micro-

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tions on microorganism, by deamination and transamination reactions damaging DNA and/or RNA thus impacting the protein metabolisms[29][30].

However, due to the health concerns by the report of allergic reaction in some groups of the population the search for replacers or co-additives emerges[13][31]. Ascorbic acid (vitamin C) has been indicated to be used in white musts and wines mainly as an antioxidant, a scavenger of molecular oxygen before the oxidation of phenolic compounds via a copper and/or iron redox cycling.

However, it is not indicated to be used alone due to the limited inhibitory functions when compared to SO2, with a maximum dose of 250 mg/L used in must (Scheme 1-2)[32].

Scheme 1-2 Scheme adapted from the proposed mechanism of wine oxidation under the specific antioxidant combination of SO2 and ascorbic acid[33][34]. The ascorbic acid action is represented in black and the action of SO2 is represented in blue.

The use of ascorbic acid as a complementary antioxidant product allows to decrease the amounts of SO2 needed and is becoming an increasingly popular procedure[35]. However, the combined addition needs to be well-balanced. Indeed, ascorbic acid at higher concentration acts as an antioxidant and, when in lower concentrations, presents a pro-oxidative influence by the generation of radical species. Scheme 1-3 illustrates the ascorbic acid oxidation cycle without SO2. When present in lower concentrations all ascorbic acid is oxidized to the dehydroascorbic acid and an excess of H2O2 is produced, leading to the oxidation of phenolic compounds. Despite this risk,

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some authors describe that the risk/benefit pays off in color and flavor protection, especially in the bottling step[34][36].

Scheme 1-3 Ascorbic acid oxidation cycle without SO2, in blue are the main reactive species formed, adapted from Manzocco et al. in 1998 and Bradshaw et al. in 2011[25][36].

Another factor to be considered by winemakers is the formation of turbidity during storage or transportation, caused by the presence of undissolved matter in the wines. These sediments may have different sources like minerals, organic molecular aggregates, or microorgan- isms[37][38]. One of the main reasons for this turbidity relies on the aggregation and insolubility of proteins[39]. The formation of protein aggregates is described to be multi-factorial, in which protein content, ionic strength, organic acids content, exposure to high temperatures, pH and the presence of sulphate are some of them. When wines are subjected to an increase of temperature it leads to a conformation loss of proteins which are temperature sensitive. The hydrophobic pockets are exposed and SO2 reduces the disulphide bonds. After cooling, these proteins have their configuration modified and mechanism of aggregation occurs (Scheme 1-4)[40][41][42].

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The loss of wine clarity, an important oenological feature, prevent the sale of the product and represents a producer´s concern. To mitigate this problem bentonite has been used in different winemaking steps[44][45]. Bentonite, a clay absorbent, is efficient to remove proteins associated with this problem and may modulate the presence of aroma compounds. However, its use represents a high financial effort and the need for additional filtration steps for its removal, presenting an economic drawback with environmental impact[39][46].

All these factors contribute to a continued need for adaptation and transformation in wine industry maintaining wine quality. The quality of wine is associated with the appreciation of the final product, in terms of color, translucency, bouquet, taste, and mouthfeel. All those factors depend on the chemical composition, mainly the volatile organic composition (VOC) of wine and their interactions related to grape quality, microflora present on grape must, pre- and post- fermentative treatments, maturation, and ageing conditions. Primarily, the type of grape is responsible for the varietal aroma, which reflects the contribution of, e.g., isoprenoid monoter- penes. Despite this differentiation, the fermentative activity generates additional transformations, leading to direct or indirect fermentative aromas. As direct fermentative VOC contribution is possible to find ethanol, glycerol and acetic acid and as indirect metabolism, higher alcohols and re- spective esters and volatile carboxylic acids are observed. VOC profile of wine is a dynamic system showing changes during the fermentative steps and ageing, giving wines a more mature aroma.

Esters, namely, ethyl esters and acetates, linear or branched-chained, long-chain alcohols and volatile fatty acids are some of the chemical groups that present evolution in the whole vinification process[47][48][49]. Taking advantage of the dynamic transformation capacity of the wine, some authors proposed a short controlled maturation over lees. With this methodology, it is possible to increase the amino acids content improving the flavor characteristics during maturation and ageing[50][51][52].

All of these factors contribute to VOCs combinations leading to one of the most important factor on consumers acceptance, wine aroma[6]. Studying the complex combination of VOCs present in the wine can help to better understand this complex matrix. Indeed, more than 800 aroma compounds were already reported in the volatile fraction of wine[53]. Although studies indicate that only a minority of VOCs found in wine play an important role in the flavor characteristics of

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the matrix, there is no unanimity on their role in terms of concentration, complementary role or even an enhance in a specific odor profile[48][53]. In this perspective, it is important to develop analytical techniques that allow measuring the effects of changes in viticulture and winemaking techniques in order to identify key compounds. The application of headspace solid-phase microextraction (HS-SPME) techniques has proved to be an effective tool for this analysis[53][54][55].

Nevertheless, other techniques have been applied on wine and grape sample preparation, Figure 1-3 presents a summary with advantage and disadvantage of their application[56][57]. The application of this technique (HS-SPME) has increased in the past few years on wine analyses, being more common the use of the following conditions: DVB/CAR/PDMS fiber, moderate extraction temperature (between 35 °C and 45 °C), and volume ratio of 1:2 for sample: vial. However, prelim- inary tests should be run in order to determine the conditions more suitable for HS-SPME for each sample wine to be analysed [57][58].

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HS-SPME is a sample preparation technique easily coupled with gas chromatography (CG), it is solvent-free, relatively fast and easy to operate manually and also through automatic sampling, economical and suitable for direct analysis of complex matrices, and provides lower detection limits than other headspace techniques[61]. CG methodologies usually applied in wine VOCs analysis coupled to HS-SPME are two-dimensional gas chromatography-time-of-flight-mass spectrometry (GC × GC-TOF-MS), gas chromatography-mass spectrometry (GC/MS), gas chromatography- mass spectrometry-flame ionization detection (GC-FID) and gas chromatography-olfactometry (GC-O) [53][56][62][63][64][65].

This Ph.D. project aimed to contribute with knowledge for a better “big picture” of the need of SO2 for some relevant grape varieties, mainly on wine VOC, if total or partial replacement of SO2

is performed, either during wine fermentation, maturation, or ageing. To study these impacts a set of fermentation, maturation, and ageing trials under a controlled SO2 environment will be made, with Alentejo grape varieties: Arinto, Síria, Gouveio, Antão Vaz and Aragonês. These varieties are of great importance in the production of the region, both in terms of vineyard area and the wine produced[3][66]. VOC will be analysed by headspace-solid phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS). Also, the amino acid content will be evaluated after maturation over lees by high-performance liquid chromatography with a diode-array detector (HPLC-DAD), since several VOCs are precisely originated from amino acid metabolisms[47][67].

Their presence also contributes to increasing the gustatory intensity of wines, including sweetness[48][67].

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2 Objectives

The main objective of this PhD project was to acquire a better knowledge of VOCs profile due to SO2 content on wine considering grape varieties. To achieve this objective the work was divided in different subobjectives that are presented on Scheme 2-1.

Scheme 2-1 Research questions divided by stages of winemaking were this PhD project focuses on.

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3 Work plan

The specific objectives and its achievements through this project are summarized as follows, having Alentejo vineyards as a source of grapes:

a. To study the VOC profile of monovarietal wines after fermentation under different SO2 environments, musts of three different white varieties were used and fermented with different doses of SO2 and ascorbic acid as SO2 (Figure 3-1). In this study, VOCs were analysed after fermentation by HS-SPME-GC/MS. Results obtained in this study are presented on section 6.1.

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Figure 3-1 Graphical representation of three monovarietal wines studied in 2018. Study of VOC profile after fermentation of three white wine variety.

b. VOC profile of monovarietal wines resulting from the previous topic (a) were kept for 3 months on fine lees and 3 months in bottle in different antioxidant conditions (Figure 3-2).

This analysis was carried out by HS-SPME-GC/MS, and the wines of the previous point (a) were used as a reference, studying the effect of a short evolution on VOC profile. Results obtained in this study are presented on section 6.1.

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Figure 3-2 Graphical representation of three monovarietal wines studied in 2018. Study of VOC profile after short evolution of three white wine variety.

c. To study the influence of the combination of SO2 and bentonite on fermentative aroma and the evolution of VOCs profile after maturation over fine lees in different SO2 environments.

Two musts were fermented under different antioxidant conditions in the presence and absence of SO2. They were analysed after maturation for three months over fine lees and then three months on bottle, Figure 3-3. In this study, VOCs were analyzed in the initial wine, after six months (three months in contact with lees and after three months stored in bottle) by HS-SPME-GC/MS. Results obtained in this study are presented on section 6.2.

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Figure 3-3 Graphical representation of Arinto and Síria wine study in 2018. Study VOC profile of antioxidant conditions on fermentation and short evolution in the presence or absence of bentonite.

d. To study the amino acid content of wines after fermentation in the presence of different doses of SO2 and after three months over fine lees, Figure 3-4. In this study, amino acid content was analyzed after fermentation as reference by HPLC-DAD and after three months over fine lees. Also, the amino acid content was analysed regarding different antioxidant

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Figure 3-4 Graphical representation of Arinto wine study in 2018. Study the amino acids content fermented in different antioxidant conditions.

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Figure 3-5 Graphical representation of Arinto wine study in 2018. Study the amino acids content fermented in different antioxidant conditions in the presence or absence of bentonite.

e. Evolution comparation after sort ageing on amino acid content and VOCs profile were also studied by splitting the same wine after fermentation, Figure 3-6. In this study, VOCs were analysed in the initial wine, after three months in contact with fine lees and after three and nine months stored in bottle by HS-SPME-GC/MS. The amino acid content was also analysed after three months in contact with fine the lees by HLPC-DAD. Results obtained in this study are presented on section 6.4.

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Figure 3-6 Graphical representation of Antão Vaz and Blend wine study in 2018.

f. VOC profile of a monovarietal must were analysed 24 H after antioxidation addition by HS- SPME-GC/MS and used as reference. The same analysis was performed after fermentation comparing de different profile evolution during fermentation, Figure 3-7. Results obtained in this study are presented on section 6.5.

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Figure 3-7 Graphical representation of Gouveio wine study in 2019.

g. VOC profile analysis after fermentation of two different wines, one white and one red varieties by HS-SPME-GC/MS. Two monovarietal musts were fermented in different antioxidant condition, Figure 3-8 for white wine and Figure 3-9 for red wine. In this study, VOCs were analysed in the musts 24 H after antioxidation addition and after fermentation. Results obtained in this study are presented on section 6.6.

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Figure 3-8 Graphical representation of Arinto wine study in 2019.

Figure 3-9 Graphical representation of Aragonês wine study in 2019.

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h. Compare the VOCs profile upon SO2 and ascorbic acid coaddition as antioxidant agent on fermentation, Figure 3-10. In this study, VOCs were analysed after fermentation by HS- SPME-GC/MS. Results obtained in this study are presented on section 6.7.

Figure 3-10 Graphical representation of Aragonês wine study in 2019, musts fermented whit different SO2 and ascorbic acid combinations additions.

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4 Materials and methodologies

4.1 Vinification

The grapes used were harvested in September 2018 and 2019, and immediately processed.

Figure 4-1 is a representation of the general vinification process applied. White grapes (Figure 4- 1a) were destemmed, crushed, pressed and after a 24 H cold static sedimentation, the must was divided into duplicate vessels and inoculated with commercial yeast, fermentations took place at 16 °C. At the end of alcoholic fermentation samples were collected and frozen (-32ºC) until analysis and wines were kept over fine lees before bottling. After bottling samples were kept vertically at 16 °C. Red grapes (Figure 4-1b) were destemmed and crushed, and the alcoholic fermentation occurs at room temperature. The fermenting musts were punched down twice a day to promote pomace contact and when the amount of reducing sugar were less than 3 g/L, free run wine was separated from pomace without pressing. Malolactic fermentation (MLF) took place at room temperature without lactic bacteria addition, and after MLF was complete, wines were separated from lees and samples were collected and frozen (-32 ºC) until analysis. Wines were bottled and storage horizontally at 20 °C. Frozen samples were thawed in a controlled protocol, having been placed at 4 °C for 20 H and then 1 H at 25 °C before being prepared for analysis.

White musts used on this project were inoculated with commercial yeast. Samples from 2018 a mixture of Saccharomyces cerevisiae (mixture 1:1 of LEVULINE FB from Oeno France and IOC 18-2007 from Lallemand OEnology) was used and for samples from 2019 Saccharomyces cere- visiae (Fermol Arôme Plus from AEB) was used for white wines. SO2 was added to samples (must and wines) into two forms, a commercial 6% aqueous solution of sodium bissulfite (SAI, SOLFOX 6

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Nº CE: 231-870-1) for samples from 2018 and by sodium metabisulfite salt (AnalaR NORMAPUR CAS: 7681-57-4) for samples from 2019. To samples fermented with ascorbic acid (MERCK CAS: 50- 81-7), the commercial compound was added. The application of bentonite (MICROCOL AL-PHA, LAFFORT), was performed in an aqueous solution of 10% (w/v) to a final concentration of 0.1 g/L.

Figure 4-1 Graphical representation of vinification process applied in this PhD. Project development in 2018 and 2019. a) Refers to white grape varieties and b) refers to red grape variety.

During this PhD project two years of vinification was monitored, 2018 and 2019. In 2018, four white wines with Alentejo grape varieties: one Síria and two Arinto from Évora and one Gouveio from Portel were harvest and fermented. Table 4-1 presents a summary of conditions used in 2018 fermentations all samples were performed in duplicate. In these trials the effect of different doses of SO2 on monovarietal musts, presence of bentonite and ascorbic acid were studied on fermentation. Also, a comparison of wines during maturation under different SO2 conditions was studied.

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Table 4-1 Fermentation and maturation conditions applied on must of 2018 according to grape variety.

ARINTO SÍRIA GOUVEIO

SO2 APPLIED BEFORE FERMENTATION (mg/L)

0 50 100

0 15 30 45

0 50 100 ASCORBIC ACID APPLIED BEFORE FERMENTATION (100 mg/L) Yes* No Yes* Yes*

BENTONITE APPLIED BEFORE FERMENTATION (100 mg/L) Yes/No No Yes/No No

SO2 ADDITION AFTER FERMENTATION (mg/L) 0 60

0 30 60

0 60

MATURATION/AGEING TIME (MONTHS) 6 6 6 6

* Without addition of SO2 before fermentation.

In the same year two white wines, one varietal of Antão Vaz and one blend (Síria (32%), Rabo de Ovelha (17%), Antão Vaz (10%), Viosinho (6%), Fernão Pires (6%), Arinto (5%), Verdelho (5%), Manteúdo (4%), Roupeiro (4%), Gouveio (4%), Semillon (3%), Perrum (3%) and Diagalves (1%)) were also used. The two wines were produced by CARMIM - Cooperativa Agrícola de Re- guengos de Monsaraz, Portugal. Both were fermented, in large volume (500000 L) tanks, following CARMIM vinification protocol. At the end of alcoholic fermentation, when reducing sugars were less than 2 g/L, a portion of wine was separated, distributed among several glass carboyls and SO2

at different doses (0, 30, 60, 90 and 120 mg/L) was applied using a commercial 6% solution (SAI, SOLFOX 6 Nº CE: 231-870-1). Wines were kept in contact with lees for 3 months. After this period, were bottled and stored for 3 and 9 months in vertical and at 16 °C.

In 2019 four musts were harvest and fermented, three white wines (Arinto and Síria from Évora and Gouveio from Portel) and one red wine (Aragonês from Évora). Table 4-2 presents a summary of 2019 used conditions, in which all samples were performed in duplicate. In these trials, the effect of different SO2 concentrations, ascorbic acid, and a combination SO2/ascorbic acid on monovarietal musts were studied during fermentation step. In the Aragonês, red must fermentation was performed using 25, 50 and 75 mg/L of SO2 with 50, 100 and 150 mg/L of ascorbic acid.

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Table 4-2 Fermentation and maturation conditions applied on must of 2019 according to grape variety.

ARINTO GOUVEIO ARAGONÊS

SO2 APPLIED BEFORE FERMENTATION (mg/L)

0 20 40 60 80

0 40 60

0 20 40 60 80

25 50 75

ASCORBIC ACID APPLIED

BEFORE FERMENTATION (mg/L) Yes* Yes* Yes*

50 100 150

* Only for musts without addition and addition of 60 mg/L of SO2 before fermentation with a concentration of 100 mg/L of ascorbic acid.

4.2 Oenological parameters

The analysis of oenological parameters as alcohol content (distillation, Method OIV-MA- AS312-01B) , pH (potenciometry, Method OIV-MA-AS313-15), total (potentiometric titration, Method OIV-MA-AS313-01) and volatile acidity (destillation and titration, Method OIV-MA-AS313- 02), sulphur dioxide (iodometric titration, Method OIV-MA-AS323-04B) and reducing substances (distillation and titration, Method OIV-MA-AS311-01A) were measured according to OIV methods[68].

4.3 HS-SPME sampling of wine volatiles

Wine is a chemical complex matrix with more than 800 aroma compounds reported in the volatile fraction[62]. To be able to understand this complex matrix, is important to apply analytical methodologies with the capacity to measure the many volatiles has possible[53]. However, some components of the matrix can interfere with the analysis, lead to the necessity of extract, and con- centrate the volatile compounds. Classic methodologies require sample manipulation, with application of solvents that can be a source of sample contamination and relatively low

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depend on many factors like, fiber coating, ionic strength, extraction and desorption conditions[70]. In 2007 Syekova et al. and in 2012 Barros et al., reported that Divinylben- zene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) 50/30 µm fiber presented the best performance for the whole range of compounds of different volatile or/and polarities on wine volatile fraction[55][71].

In this work the HS-SPME method used was an adaptation of the method reported by Barros et al., however no stirring was applied due to technical limitations and the extraction temperature was reduced to 30 °C to prevent VOCs degradation during extraction[71]. Nevertheless, desorption in split or splitless was tested (Figure 4-2). A commercial white wine (5 mL) was extracted and then injected in splitless and split modes of 1:5 and 1:10, on the sample extraction no NaCl was added.

It was possible to observe that for a total volatile analysis a splitless injection was the best option, presenting the highest number of compounds analysed and the highest areas, regarding the analysed total area and chemical classes.

Figure 4-2 Graphical representation of total area analyzed, and total number of compounds identified for the injection mode: splitless (blue), split 1:5 (grey) and split 1:10 (black), dots are the number of compounds.

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Then the salting-out condition was analyzed with splitless injection. Two commercial white wines and one commercial red wine were analyzed. On Figure 4-3 are present the graphical projections comparing: salting-out conditions, NaCl (w/v) at 0, 10, 20, 30, 40 and 50%, total area analyzed and the average of relative standard deviation (RSD) for each condition. The ideal conditions would present the largest area analyzed with a lower average of RSD. In Figure 4-3 the conditions shown in purple will be those tending towards the optimal system. In Figure 4-3 the conditions shown in purple will be those tending towards the optimal system. In the case of the wine presented in a), the application of 40% w/v of NaCl, despite not being the condition with the highest concentration analyzed, has the lowest RDS in the analysis. Referring to Figure 4-3 b) their analysis indicated two conditions of lower RDS, however only the condition of 40% w/v NaCl was found near the highest area. The same was observed in the case of red wine in Figure 4-3c).

Through the observation of the obtained projections, it was possible to conclude that for the system used, the most favorable extraction condition was the extraction with 40% of NaCl (w/v). Indeed, the application of salting-out condition to wine analyses is not consensual on literature. Some authors point out that no advantages result in what sensitivity increasing concerns, within the range of VOCs studied[58].

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Figure 4-3 Graphical projections with the influence of NaCl concentration on the efficiency of HS-SPME using a DVB/CAR/PDMS fibre at 30 °C, without agitation (a) n=2; b) and c) n= 3). a) and b) are two different commercial wines and c) a commercial red wine, the samples presented an ethanolic content of 12%.

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The application of this methodology on this work aim to observed and compared VOCs profile of wines subjected to difference fermentation and ageing conditions with the small sample manipulation as possible. With this previous experiment, final condition was established as follow.

HS-SPME sampling experiments of wines were carried out by exposing 1 cm of a DVB/Carb/PDMS fiber, 50/30 μm film thickness (df), supplied from Supelco, (Bellefonte, PA, USA).

Prior to use, the fiber was conditioned following the manufacturer's recommendations. Fiber blanks were run periodically to ensure the absence of contaminants and/or carryover. The samples were prepared by addition of 5 mL of wine to 2 g of sodium chloride in a 20 mL vial, in an adaptation of Barros et al,[71] and sealed with a Teflon-lined rubber septum/magnetic screw cap. The vial was equilibrated for 5 min at 30 °C and then extracted for 30 min at the same temperature. Ther- mal desorption of the analytes was carried out by exposing the fiber in the GC injection port at 260

°C for 3 min in splitless mode, for the same time period. All samples were analysed in duplicate and with less than 24 H between preparation and analysis.

4.4 GC/MS analysis of wine volatiles

The analyses of wine samples were performed on a GC/MS system consisting on a Bruker GC 456 with a Bruker mass selective detector Scion TQ. An automatic sampler injector was used: CTC Analysis autosampler CombiPAL. Data were acquired via MSWS 8.2 Bruker and analysed with Bruker MS Data Review 8.0. Chromatographic separation was achieved on a SupelcoWaxTM 10 PLUS capillary column (60 m × 0.25 mm i.d., 1.0 μm df), supplied by Supelco Analytical (Supelco, Bellefonte, PA). The oven temperature program began at 40 °C hold for 5 min, raised at 4 °C/min up to 240 °C holding for 5 min. Helium was used as carrier gas at constant flow of 1.7 mL/min at the Electronic flow control (EFC 21). The MS transfer line and source temperatures were set at 260

°C. Spectra were matched with NIST MS Search Program Version 2.3. To determine the retention times and characteristic mass fragments, electron ionization (EI) at 70 eV mass spectra of the ana-

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chromatographic conditions. The relative amounts of individual components are expressed as per- cent peak areas relative to the total peak area of the chromatogram (Relative Peak Area - RPA).

4.5 Amino acid analysis by HPLC-DAD

After a derivatization step of 1 mL of wine to obtain the aminoenone derivatives, a HPLC- DAD from Waters Alliance System 2695 series Separation Module equipped with Alliance Series Column Heater, a Photodiode Array Detector (2998 PDA Detector) (Waters, USA) with an ACE HPLC C18 column (4.6 × 250 mm, 5 µm particle size) column was used to separate, detect, and quantify AAs. Sample preparation and analytical methodology were adapted from Gómez-Alonso et al., 2007[71] and are fully described in Pereira et al. 2021[65], in which the validation of the chromatographic method is also described. Calibration curves, presenting determination coeffi- cients (R²) from 0.994 to 0.998 were used for amino acid quantification. All the standards, alanine (Ala), asparagine (Asn), arginine (Arg), aspartic acid (Asp), cysteine (Cys), glycine (Gly), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), δ-aminobutyric acid (GABA), internal standard (IS) (L-2- aminoadipic acid) and derivatizing agent diethyl ethoxymethylenemalonate (DEEMM) were analytical grade purchased from Sigma Aldrich (USA). Sodium hydroxide and acetonitrile HPLC grade were purchased from VWR International (USA). Sodium azide and boric acid were purchased from Sigma Aldrich (USA). Glacial acetic acid (analytical grade) and methanol (HPLC grade) were purchased from Fisher Scientific, and Hydrochloric acid was purchase from Honeywell, Fluka. The wa- ter used in all experiments was distilled and purified by a Milli-Q system (Millipore, Bedford, MA, USA).

4.6 Statistical Analysis

In the study of two white wines (one monovarietal and one blend) one-way and two-way analysis of variance (ANOVA) using multiple comparison Bonferroni test was performed to compare the means at the level of significance of p < 0.05, using GraphPad Prism version 9.0.0 by

Understanding the need of SO2 in wine according to grape varieties

UNDERSTANDING THE NEED OF SO 2 IN WINE ACCORDING TO GRAPE VARIETIES

NOVA University Lisbon

CÁTIA VANESSA DE ALMEIDA SANTOS Master in Bioorganic Chemistry

DOCTORATE IN SUSTAINABLE CHEMISTRY

DEPARTAMENT OF CHEMISTRY

UNDERSTANDING THE NEED OF SO

IN WINE ACCORDING TO GRAPE VARIETIES

COM UMA MUDANÇA DE LINHA FORÇADA

CÁTIA VANESSA DE ALMEIDA SANTOS Master in Bioorganic Chemistry

Acknowledgments

Abstract

Resumo

Contents

Figure index

Table index

Scheme index

Acronym index

Part I

1 Introduction

2 Objectives

3 Work plan

4 Materials and methodologies

4.1 Vinification

4.2 Oenological parameters

4.3 HS-SPME sampling of wine volatiles

4.4 GC/MS analysis of wine volatiles

4.5 Amino acid analysis by HPLC-DAD

4.6 Statistical Analysis