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Understanding Brettanomyces for improved management in the cellar

Boela Gerber

2019

Dissertation submitted to the Cape Wine Academy in partial fulfilment of the requirements for the diploma of Cape Wine Master.

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I, Boela Gerber, declare that this dissertation is my own, unaided work. It is submitted in partial fulfilment of the requirements for the diploma of Cape Wine Master to the Cape Wine Academy. It has not been submitted before for qualification of examination in this or any other educational organisation.

Boela Gerber September 2019

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Acknowledgements

I would like to thank Prof Maret du Toit and Prof Benoit Divol from the University of Stellenbosch, as well as Dr Adriaan Oelofse and Karien O’Kennedy from WineTech for their technical advice and assistance. I would also like to thank Brendan Smith for his valuable assistance. Most importantly, I would like to thank my wife Michaela, for her technical input as a scientist and academic, as well as her unconditional support throughout this journey.

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Contents

1. Introduction 1

2. History 2

3. What is Brettanomyces? 3 3.1 Confusing taxonomy 3 3.2 Genetic diversity 4 3.3 Summary 5

4. Detection and identification 6 4.1 Direct determination 6 4.2 Indirect determination 9 4.3 Summary 10

5. Sources of contamination 11 5.1 Vineyard 11 5.2 Cellar 12 5.3 Summary 13

6. Nutrient requirement 14 6.1 Carbon sources 14 6.2 Nitrogen sources 15 6.3 Summary 16

7. Wine spoilage by Brettanomyces (including sensory effects on wine) 17 7.1 Organoleptic effects 17 7.1.1 Volatile phenols 19 7.1.2 Volatile acids and volatile fatty acids 19 7.1.3 Isovaleric acid 20 7.1.4 Mousiness 20 7.2 Loss of colour 21 7.3 Haziness 22 7.4 Biogenic amines 22 7.5 Summary 24 iv

8. Impact of other microorganisms 25 8.1 Summary 26

9. Brettanomyces in white wine 28 9.1 Summary 29

10. Managing Brettanomyces in the cellar 30 10.1 Manage Brettanomyces growth 30

10.1.1 Sulphur dioxide (SO2) 30 10.1 2 Chitosan 34 10.1.3 Dimethyldicarbonate (DMDC) 35 10.1.4 Sorbic acid 37 10.1.5 High pressure processing 37 10.1.6 Fining agents 38 10.1.7 Filtration 38 10.1.8 Temperature 40 10.1.9 Cellar hygiene 41 10.1.10 Barrel maturation and sanitation 41 10.1.11 Impact of winemaking practices 43 10.2 Treatment of wine with high volatile phenols 44 10.2.1 Fining agents 44 10.2.2 Reverse osmosis 46 10.3 Summary 46

11. Latest research 48 11.1 Kaolin silver complex (KAgC) 48 11.2 Biological control 48 11.3 Disposable electrochemical biosensors 49 11.4 Summary 50

12. Conclusion 51

Addendum A: Permission for figures and tables reproduced 54

References 57 1

1. Introduction Brettanomyces is considered the most important microbiological threat to the quality of red wine. However, information on this yeast has been limited and inconsistent until recently. The purpose of this study is to clear up taxonomic confusion, to understand the genetic diversity and the adaptability of Brettanomyces. The variations between strains, from

morphology to nutrient requirements and its resistance to chemicals are discussed. Previously the detection and identification of Brettanomyces were insufficient and inaccurate. Modern identification techniques and their advantages and limitations in effectively managing Brettanomyces in the cellar will be discussed.

Brettanomyces has low carbon and nitrogen requirements which allows it to grow under extreme conditions. Brettanomyces can affect wine in various ways. Understanding the influence of other microorganisms and their impact on Brettanomyces spoilage characters is vital for the effective control of Brettanomyces. Therefore, other microbes that affect Brettanomyces or similarly affect wine will be evidenced.

Brettanomyces occurs naturally on grapes and is potentially present throughout the winemaking process. Different methods of managing Brettanomyces efficiently in the cellar include the use of sulphur. Other products and techniques will be reviewed, and new technologies will be discussed.

This review gives a broad overview of Brettanomyces, from origin and genetics to nutrient requirements and metabolites produced. This will give a better understanding of this complex yeast and how to manage it efficiently in the cellar.

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2. History Brettanomyces, meaning “British fungus”, was first isolated and identified in English stock beer in 1905 by N. Hjelte Claussen from New Carlsberg Brewery. These beers were often aged by the brewery to enhance quality and followed by a second fermentation in wooden casks. Brettanomyces, found naturally in the casks or present in the pitching yeast, gave the typical sharp tart flavour synonymous with British beers of the time. Brettanomyces was acknowledged as an official genus in the 1920’s when a similar yeast species was isolated in Belgian lambic ales (Henschke et al, 2007). Brettanomyces was isolated and identified in wine only in the middle of the 20th century by Custers (1940) followed by Agostino (1950), Barret et al. (1955) and Peynaud and Dumercq (1956). It was later isolated and described in South Africa by Van der Walt and Van Kerken (1960). Studies describing Brettanomyces bruxellensis (Froudiére and Larue, 1988; Larue et al, 1991) in wine and the associated volatile phenol production (Chatonnet et al., 1992, 1995) began to highlight the role of this yeast in wine spoilage.

Further studies revealed the presence of Brettanomyces in other products and places, such as traditional Chinese kombucha tea, apple cider and cider factories, dairy equipment and tequila (Loureiro and Malfeito-Ferreira, 2006). Brettanomyces is often found in these products due to its ability to adapt to unreceptive environments, survive for extended periods, as well as start growing in stored products. For instance, Brettanomyces is often detected in Belgian lambic beer after more than a year of ageing, at the point where no viable Saccharomyces are detected (Loureiro and Malfeito-Ferreira, 2006).

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3. What is Brettanomyces? Similar to Saccharomyces, Brettanomyces is an ethanol tolerant and Crabtree positive yeast. Crabtree positive indicates the use of fermentation in the presence of oxygen, where the yeast could utilise the respiration pathway which yields more energy. Respiration yields 18 adenosine triphosphate (ATP) per glucose molecule, as opposed to the 2 ATP generated by fermentation. (ATP is a complex organic compound that provides energy in living cells.) (Pfeiffer and Morley, 2014). While genetic events in the evolution of the Crabtree effect have been identified, the selective advantages provided by this trait remain controversial and will not be discussed further. The full classification of Brettanomyces bruxellensis is as follows:

Kingdom: Fungi Phylum: Ascomycota Class: Saccharomycetes Order: Saccharomycetales Family: Pichiaceae Genus: Brettanomyces Species: bruxellensis

3.1 Confusing taxonomy Brettanomyces has been reclassified several times since its first classification in the 1920’s. Since then, numerous species which reproduced asexually via budding were described (Custers, 1940). However, today only five species of Brettanomyces are recognised namely Brettanomyces bruxellensis, B. anomalus, B. nanus, B. custercianus and B. naardenensis of which only B. bruxellensis and B. anomalus are found in wine. Of these two species, only B. bruxellensis has consistently been isolated in tainted wine and is considered of real concern in wine (Cocolin et al., 2004, Curtin et al., 2015). A sporulating form of B. bruxellensis and B. anomalus was observed producing ascospores, which lead to the description of the genus Dekkera (Van der Walt, 1984). Dekkera pertains to the sexual reproductive stage of the two species, while the non-sporulating or asexual reproductive stage is known as Brettanomyces.

The reason for the confusing taxonomy is that fungi are mainly classified on the characteristics of their sexual reproductive structures. Many fungi reproduce only asexually and can therefore not be placed in a classification that is based on sexual characters, while others produce both sexually and asexually. In fungi that reproduce both asexually and 4

sexually, usually only one method of reproduction can be detected under specific conditions or at a specific point in time. Furthermore, fungi usually grow in mixed colonies where they sporulate between each other. For these reasons it is very challenging to link the different states of the same fungus.

The International Botanical Congress in Melbourne in July 2011 changed the International Code of Nomenclature for algae, fungi, and plants and accepted the principle of "one fungus, one name". Since 01 January 2013, one fungus can only have one name; the system allowing separate names for anamorphs then ended (Hawksworth, 2011). For the purpose of this dissertation, the author will only refer to the asexual reproductive stage Brettanomyces and discuss the species B. bruxellensis.

3.2 Genetic diversity Brettanomyces is a complex yeast, which makes research on the topic difficult. The entire Brettanomyces genome was sequenced, in a strain originating from Australian wine, for the first time in 2012 (Curtin et al., 2015). A few months later, a second strain originating from French wine was also sequenced (Piškur et al., 2012). To date, the genomes of 10 Brettanomyces strains have been sequenced (Borneman et al., 2014, Valdes et al., 2014) including the genome from a Brettanomyces strain isolated from beer (Crauwels et al., 2015).

The variations and complexities between these strains were obvious from the sequenced genomes. The chromosome numbers from the different Brettanomyces strains varied from 4 to 9 (in comparison to the chromosome number of 16 found in all strains of Saccharomyces cerevisiae sequenced). An irregularity in the ploidy (referring to the number of complete sets of chromosomes in a cell) was also detected. In addition to the varied chromosome numbers, it was found that one strain of Brettanomyces was diploid and the other strain was triploid in a study by Louw et al, (2015). The existence of strains with different levels of ploidy is not uncommon, it also occurs in S. cerevisiae. The varying number of chromosomes is unusual and therefore an important characteristic. This may give an indication of a divergence in the evolution of Brettanomyces in comparison to Saccharomyces.

A recent study by Cibrario et al. (2019), analysing 1411 wines from 21 countries, confirmed the genetic diversity of Brettanomyces. In wines produced before 1990 Brettanomyces strains were, without exception, all diploid. Some of the wines produced after 1990 contained triploid Brettanomyces strains which demonstrated increased sulphur resistance compared 5

to the diploid strains. High ethanol tolerance was also found in some of the triploid strains. The authors hypothesize that these differences may indicate diversification and subsequent adaption to modern winemaking practice. However, non-representative sampling may have biased these results. Further studies of a larger number of wineries, vintages and larger sample sizes will be needed to confirm these results. The authors hypothesize that changes in winemaking and wine styles (higher alcohol wine and increased use of SO2) could have selected stronger strains over the last three decades. This highlights the ability of this yeast to adapt to many environments.

3.3 Summary The spoilage wine yeast Brettanomyces can be viewed as a complex yeast with many variations between strains. The sequencing of certain strains has evidenced the genetic differences within this yeast. This highlights the ability of Brettanomyces to adapt to various environments and cope with the many stressors of these environments. Furthermore, with regard to management of Brettanomyces, any practices used should be carefully considered and be strain specific due to the clear variation among strains. This makes strain identification of prime importance and the management of this complex yeast extremely difficult.

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4. Detection and identification It is critically important to identify the cause of any wine spoilage before one can manage the problem. Brettanomyces can be detected either directly by culturing/microscopy or DNA- based identification, or indirectly by the analysis of metabolites.

4.1 Direct determination Direct determination of Brettanomyces utilizes culturing and DNA-based identification techniques. The traditional detection and isolation (culturing and microscopy) of Brettanomyces from a wine/winemaking environment is complicated for a number of reasons: • It is difficult to isolate Brettanomyces from a medium that is highly infected with other microorganisms (Van der Walt and Van Kerken, 1960). • The population of Brettanomyces on grapes and in the winemaking environment is often low (Fugelsang, 1997). • Low growth rate and complex nutritional demands require incubation periods of up to 14 days. Therefore, the general incubation periods for other yeasts of 3 – 6 days at 25 – 30˚C make routine screening for Brettanomyces inadequate (Rodrigues et al., 2001). • Brettanomyces can move to a dormant stage called the “viable but not culturable” (VBNC) state for long periods under unfavourable conditions. In this VBNC state the yeast cannot be cultured by regular methods but may have the ability to recover and resume growth should conditions become more favourable (Arvik and Henick-Kling, 2002). Millet and Lonvaud-Funel (2000) found that the VBNC population of dry wine can be up to 10 times higher than the culturable population. • Morphological features and other standard methods are often inadequate to confirm the presence of Brettanomyces because it has multiple morphological characteristics that make microscopic identification difficult. Some strains of Brettanomyces display normal yeast shape morphology, while others can develop pseudohyphal structures (long, branched cells), demonstrated in Figure 1. Pseudohyphal development has been reported due to nutrient deprivation (especially nitrogen) as well as oxidative stress response (Louw et al., 2016). 7

Figure 1: Light microscopy observed different cell morphologies presented by three different Brettanomyces strains at different phases (Louw et al., 2016). Reproduced with permission.

To overcome the constraints of growth media, researchers have investigated the potential of selective growth media by modifying the main components and carbon sources. This includes the addition of glycerol and trehalose (a disaccharide formed from two α-glucose units) with sucrose as carbon sources, while the addition of vitamins like thiamine and biotin can be valuable to the growth of Brettanomyces (Fugelsang, 1997).

Growth media for Brettanomyces often contain growth inhibitors like cycloheximide and/or ethanol to inhibit bacteria, Saccharomyces cerevisiae and other yeasts (Couto et al., 2005). Coumaric acid can be included in the growth media to serve as a 4-ethyl phenol precursor. The presence of Brettanomyces will result in the formation of 4-ethyl phenol with a distinctive odour which can then be confirmed by gas chromatography and olfactometry methods or a trained sensory panel. The addition of sugar to the selective culture media can limit the production of volatile acid by Brettanomyces, which can affect the sensory detection of volatile phenols (Couto et al., 2005).

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The limitations of the traditional identification techniques (culturing and microscopy) encouraged the development of faster and more reliable direct identification techniques, which lead to the development of numerous DNA-based techniques.

Polymerase chain reaction (PCR) is a technique that was developed by American biochemist Kary Mullis in 1983, and is applied in molecular biology to replicate a single copy or a very small amount of DNA segments exponentially to generate millions of copies of a certain DNA sequence. PCR principles are based on thermal cycling, exposing the DNA and reactants to 30 to 40 cycles of repeated heating and cooling. The thermal cycling employs a series of temperature-dependent reactions, primarily DNA denaturation and polymerase enzyme-driven DNA replication, to multiply the particular DNA sequence exponentially. PCR is a quick and reliable method for identifying microorganisms in wine, but it cannot provide specific information regarding the level of contamination (Delaherche et al., 2004).

A possible constraint of PCR is that the accuracy of the method relies on the level of infection or contamination, and that only a contamination of >10⁴ cfu will give a positive result (Loureiro and Malfeito-Ferreira, 2006). Brettanomyces can taint wine at infection counts below this value (Phister and Mills, 2003; Cocolin et al., 2004). Since 2006 the sample preparation protocol has been adjusted to improve the cell count limit for positive identification significantly. The direct detection and quantification of Brettanomyces cells can now successfully be done with a sample containing 10 to 10⁴ cfu/mL (Tofalo et al., 2012).

Another limitation of PCR is that it does not always accurately distinguish between living and dying cells. Several weeks can pass between the death of the cells, the loss of the integrity of the cell and the total loss of amplifiable DNA (Chatonnet, 2012). This can result in false- positive results, where cells were noted as alive while they were actually in a sublethal state, as was observed when a qPCR control test was carried out after the application of chitosan (Pillet, 2014). However, in such a study fluorescence microscopy could be effectively used in order to differentiate between live and dead cells.

Stender et al. (2001) researched a similar technique which does not require the extraction of DNA. This fluorescence in situ hybridization (FISH) method is a highly specific technique in molecular biology that utilises fluorescent probes that only bind to parts of the DNA with a high degree of sequence complementarity. The authors found this method, which uses Brettanomyces cells from a centrifuged wine, very accurate. The FISH technique can be very useful, considering the limitations of microscopic identification. When detecting the 9

presence of Brettanomyces, traditional microbiological culturing methods are frequently combined with other techniques such as fluorescence microscopy, PCR, gas chromatography mass spectrometry (GCMS) or organoleptic analysis by trained tasters (Suárez et al., 2007). Other metabolites formed by Brettanomyces are discussed in Section 7 and could possibly also contribute to the positive identification of active Brettanomyces cells.

4.2 Indirect determination by analysis of metabolites The determination of the presence of Brettanomyces by analysis of metabolites is very popular, as it is easy and results can be obtained relatively quickly (3 hours). The presence of Brettanomyces has been established by the analysis of metabolites 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) for the last 30 years. A brief schematic outlay of the metabolic pathway of the production of volatile phenols is presented in Figure 2.

Figure 2: Formation of volatile phenols via the decarboxylation of the hydroxycinnamic acid precursors (Šućur et al, 2016) Reproduced with permission.

The disadvantage of this method of indirect determination is that chemical and sensory analysis of metabolites can only establish if Brettanomyces has already affected the wine, in which case it is generally too late for preventative measures. Furthermore, 4-EP and 4-EG levels are not indicators of the presence of Brettanomyces. They only indicate that Brettanomyces was present and active at some point during the winemaking process. The accuracy of this method of analysis of metabolites has been questioned as 80% of Brettanomyces strains produce 4-EP and 4-EG, but only 50% of strains produce these volatile phenols at perceivable levels (Conterno et al., 2006). 10

4.3 Summary Traditional direct determination of Brettanomyces via culturing and microscopy has been unreliable in the past. Scientists have resorted to indirect determination of Brettanomyces, using the detection of metabolites. However, indirect determination has limitations, notably that an infection can only be detected when spoilage symptoms are already present. In addition, this method does not indicate whether the yeast is still alive. Therefore, it is advised that a combination of qPCR or fluorescence microscopy be used in conjunction with the more commonly used metabolite analysis. This would provide an accurate population density, in addition to the concentration of the spoilage metabolites in the wine.

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5. Sources of contamination Many origins of Brettanomyces have been described in earlier literature. This species is not limited to vineyards and grapes but is distributed far and wide in nature. Brettanomyces is commonly associated with fermented food products like alcoholic beverages beer, cider, Chinese kombucha tea and tequila as well as fermented milk and cheese (Loureiro and Malfeito-Ferreira, 2006). Brettanomyces has also been isolated from other sources, like carbonated drinks, olives, bees and fruit flies (Deák and Beuchat, 1995). The original entry of this yeast into these many fermented beverages is a contentious issue. Therefore, in this chapter the main niches from which Brettanomyces have been isolated will be discussed.

5.1 Vineyard The origin of Brettanomyces in wine has been a contentious topic for a long time as no study could positively point to the vineyard as the conclusive source of Brettanomyces infection (Oelofse et al., 2008). It was only as recent as 2007 that Renouf and Lonvaud-Funel successfully isolated Brettanomyces from grapes.

There are various species of bacteria and yeasts present on grape berries. Depending on its size, ripeness and sanitary state, one grape berry can host between 10⁴ - 10⁶ microbial cells (Renouf et al., 2005). In addition, the population levels between these different species vary greatly. The microflora on a grape berry is often dominated by a few major species, while the minority species (like Brettanomyces) can be limited to a few cells.

The mix of microorganisms present in must changes dramatically during fermentation, where the most adaptable organisms dominate. Because of its high ethanol tolerance, Saccharomyces usually dominates throughout the fermentation, while Brettanomyces can also be present at high levels to the end of alcoholic fermentation (Renouf et al., 2007a). The Brettanomyces population can be as high as 10⁶ cfu/mL at the end of fermentation (Renouf et al., 2006).

The low incidence of Brettanomyces in the vineyard, together with the previous difficulty in culturing and detecting Brettanomyces discussed in 4.1 Direct determination are possible reasons why the vineyard was overlooked as a source of Brettanomyces contamination in wine for many years. Progress in the knowledge of the physiology of Brettanomyces contributed to the development of selective growth media that made it possible to isolate only the Brettanomyces cells in a sample, by suppressing the development of dominating 12

species. The use of nutrient growth media tailored for Brettanomyces facilitated the identification of the vineyard as the origin of the yeast (Renouf and Lonvaud-Funel, 2007).

Brettanomyces strains identified on grapes were compared with strains found during alcoholic fermentation, maturation and after bottling. The strains isolated on grapes were found throughout the winemaking and maturation process (Miot-Sertier et al., 2006). These strains produced significant quantities of volatile phenols under laboratory conditions therefore, the production of volatile phenols in wine by Brettanomyces can be linked to strains initially present on grapes (Renouf et al., 2007b). These strains play a significant role with regards to the spoilage of wine and have been undetected, until recently.

The population of Brettanomyces on grapes changes with grape ripening, with higher levels of Brettanomyces detected at harvest than on unripe berries (Renouf and Lonvaud-Funel, 2007). The presence of Brettanomyces is related to the vineyard’s topography and its environment. Temperature and moisture contribute to a specific microclimate and are also critical parameters in microbial growth. Therefore, the microclimate of a vineyard has an influence on the probability of fungal growth and also on Brettanomyces detection (Barbin, 2006).

Brettanomyces does not appear to be associated with other organisms usually found on grapes such as Acetobacter, Aspergillus and Penicillium. A correlation has been noted between the occurrence of Brettanomyces and Botrytis cinerea on grapes, although no interaction between the two genera has been established (Barbin, 2006). Lower levels of volatile phenols associated with Brettanomyces (produced in early stages of winemaking) were found in years when conditions promoted Botrytis development and the necessary anti- fungal treatments were applied (Sturm et al., 2006). This could be explained by a possible inhibitory effect of the antifungal treatment against Botrytis and other fungal diseases on the growth of Brettanomyces on grapes.

5.2 Cellar

The isolation of Brettanomyces has been well documented in wine cellars. Brettanomyces is often associated with unhygienic cellars, infected wine and contaminated cellar equipment (Fugelsang, 1998). Numerous authors found Brettanomyces in wine pumps, transfer lines and other cellar equipment that are difficult to sterilise. Brettanomyces was also found in drains, on cellar walls and even winery air samples (Van der Walt, 1984; Alguacil et al.,1998; Fugelsang, 1998; Connel et al., 2002). Hence it is no surprise that wine cellars are regarded 13

as the principal source of Brettanomyces infection. However, as its occurrence is often inconsistent, every wine cellar poses a unique scenario that demands the determination of its own route of contamination. Therefore, the management of Brettanomyces should focus on the cellar and the potential origins of the contamination of that particular cellar.

In a recent study Cibrario et al. (2019) analysed 1411 wines from 21 different countries for Brettanomyces strains. Microsatellite markers were used to group the 340 Brettanomyces genotypes. The study found that a specific strain could repeatedly be isolated from winery samples over a period of decades, with the longest interval being from 1926 to 2012. From a practical point of view, the development of knowledge based on a history of managing a "resident strain" of Brettanomyces should improve the management of this spoilage yeast in a specific cellar.

The biggest source of infection and the highest populations of Brettanomyces found in cellars are in barrels (Malfeito-Ferreira, 2018). Contaminated wine that has been bought in or contaminated equipment could also be a source of infection (Oelofse et al, 2008). Insects and air dust are all probable modes of distributing the yeast throughout the cellar (Malfeito- Ferreira, 2018).

5.3 Summary From previous literature Brettanomyces is commonly associated with contaminated cellar equipment, where barrels show the highest population of Brettanomyces. The vineyard was long overlooked as a source of Brettanomyces infection until Miot-Sertier et al. (2006) managed to trace a specific strain of Brettanomyces from the vineyard to the cellar and eventually the bottle. This proves that all cellars are at constant risk of Brettanomyces infection, with those producers who have cooperative cellars or those who buy in grapes and wine being most at risk. In addition, it seems that each cellar or site has its own needs with regards to the management of Brettanomyces. An infection could begin in the vineyard or in the cellar, and thus each site should be treated differently with regards to the management of Brettanomyces. Before any treatment can be effective, the origin of this yeast should be determined.

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As previously evidenced in 5.2 Cellar, Brettanomyces can survive in many ecological niches. Most of these environments are varied with complex compositions and various sources of nutrients. Brettanomyces can metabolise a range of nitrogen and carbon sources, although some sources are preferred and facilitate growth better (Rozpedowska et al., 2011; Crauwels et al., 2015). There are substantial variations in nutritional requirements and the tolerance to certain growth-inhibiting factors between different Brettanomyces strains. This is due to the organism’s ability to adapt to a challenging environment, which in turn could be explained by the genetic diversity between Brettanomyces strains, as discussed under 3.2 Genetic diversity. These variations make research challenging and could possibly explain some conflicting research and controversy in literature. This chapter will focus on the elements supporting microbiological growth in wine, namely carbon and nitrogen sources.

6.1 Carbon sources Brettanomyces can, depending on the strain, utilise various carbon sources, ranging from basic C6 sugars like glucose, fructose and mannose to more complex sources such as glycerol, ethanol and acetic acid (Conterno et al., 2006, Galafassi et al., 2011). Glucose provides an efficient source of carbon and energy for Brettanomyces. A 90% conversion rate of the volatile phenol precursor coumaric acid to 4-EP was observed in a synthetic media with glucose as the only carbon source (Dias et al., 2003b). Crauwels et al. (2015) found that most of the Brettanomyces strains could grow on the C6 monosaccharides glucose, fructose, galactose and mannose. However, the growth rate and ethanol production of Brettanomyces was lower when metabolising the disaccharide maltose (Blomqvist et al., 2010).

Brettanomyces also can utilise various carbon sources via ß-glycosidase activity. This enables Brettanomyces to utilize glucose polymer dextrin (Galafassi et al, 2013). In addition, Steensels et al. (2015) reported that the ß-glucosidase enzymes can hydrolyse lignin and other wood components to produce cellobiose (Figure 3). Cellobiose is a dimer that consists of two glucose molecules and is the smallest repetitive unit of cellulose. The ß-glucosidase activity of Brettanomyces then hydrolyses the cellobiose to form glucose, a carbon source that Brettanomyces can metabolise (Steensels et al., 2015).

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Figure 3: Cellobiose. Reproduced with permission.

Brettanomyces displays intracellular and extracellular α-glucosidase activity (Bloem et al., 2008). This gives Brettanomyces the ability to liberate glucose from glycosidic bonds with terpene flavour compounds and even anthocyanin, giving Brettanomyces an advantage over other yeasts that do not have the ability to access these carbon sources (Mansfield et al., 2002).

Dry wine contains traces of pentose sugars like arabinose, rhamnose, ribose and xylose which cannot be metabolised by Saccharomyces, but can be utilised by Brettanomyces (Kunkee & Eschnauer, 2003). Refined cane sugar was metabolised but not preferred by Brettanomyces. The yeast showed low growth and produced low levels of ethanol in a refined sugar medium (Uscanga et al., 2007).

Even though Vigentini et al. (2008) found that Brettanomyces could not metabolise ethanol under anaerobic conditions, Dias et al. (2003a) and Conterno et al. (2006) observed that Brettanomyces could utilise ethanol as a sole carbon source. Similarly, Vigentini et al. (2008) found that most strains of Brettanomyces could not utilise glycerol as a carbon source, while Crauwels et al. (2015) observed growth with some isolates on glycerol. Dias et al. (2003a) observed the utilisation of acetic acid, even though it provided for a very low conversion of coumaric acid to 4-EP (<10%).

Brettanomyces can utilise a variety of carbon sources. Certain strains can utilise ethanol, acetic acid and even cellulose, stressing the point that Brettanomyces can grow in severely carbon depleted media.

6.2 Nitrogen sources Conclusive research on the metabolisation of nitrogen by Brettanomyces or the preferential utilisation of the various nitrogen sources is limited (Childs et al., 2015). Brettanomyces can utilise a variety of nitrogen sources according to Morneau et al. (2011), and has a low nitrogen demand under aerobic conditions, which was verified when recent studies 16

confirmed that the growth of Brettanomyces was not affected by the availability of residual nitrogen (Childs et al., 2015).

Blomqvist (2011) found that amino acids alanine, arginine, asparagine, glutamic acid, histidine and lysine stimulated growth of Brettanomyces under anaerobic conditions, but tryptophan and cysteine did not promote growth under anaerobic conditions. Certain strains of Brettanomyces were unable to grow under strict anaerobic conditions in a mineral medium without amino acids. This was confirmed by another study that found that Brettanomyces requires yeast extract or microbial growth factor casamino acids (a mixture of amino acids and peptides obtained from the hydrolysis of casein) (Rozpedowska et al., 2011). It is important to observe that Saccharomyces cerevisiae cannot metabolise nitrates and some amino acids like proline and hydroxyproline under winemaking conditions. This leaves available nitrogen for Brettanomyces after alcoholic fermentation (Ingledew et al., 1987). Valero et al. (2003) found an increase in some amino acids including proline, leucine and tryptophan, during yeast cell autolysis. Given the fact that Brettanomyces has a very low nitrogen requirement, it appears that there is sufficient nitrogen in wine under normal winemaking conditions for growth.

6.3 Summary Literature suggests that Brettanomyces can utilise a wide range of carbon and nitrogen sources. In addition, it requires sometimes lower concentrations of certain nutrients in comparison to S. cerevisiae. Brettanomyces is also able to consume certain compounds that S. cerevisiae cannot. This demonstrates how Brettanomyces is adapted to its environment and is commonly isolated in wine, well after other yeasts have subsided. There are substantial variations in nutritional requirements between Brettanomyces strains. It can utilise a range of carbon sources, from basic C6 sugars like glucose to more complex sources like ethanol, acetic acid and wood components lignin and cellobiose. Brettanomyces can metabolise various nitrogen sources and have a low nitrogen requirement under aerobic conditions. It appears that there is sufficient nitrogen available in wine to sustain Brettanomyces under normal winemaking conditions. Good winemaking practice is to analyse grape juice for yeast assimilable nitrogen (YAN) and calculate the minimum nitrogen to add to fermenting must for yeast nutrition. This should limit excess nitrogen available to spoilage organisms after alcoholic fermentation.

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7. Wine spoilage by Brettanomyces (including sensory effects on wine)

There are numerous ways that Brettanomyces can spoil wine. The most common is organoleptic, specifically volatile phenols, but Brettanomyces can also have other detrimental effects on wine quality.

7.1 Organoleptic effects The ability of Brettanomyces to produce negative aroma compounds in wine is well documented with 4-EG, 4-EP and isovaleric acid highlighted as the main contributors (Botha, 2010). In addition to the negative effect of these metabolites, Botha (2010) found that these compounds caused a suppression of the natural fruit character of wines contaminated by Brettanomyces, inducing a sickly-sweet odour. However, Brettanomyces produces a broad spectrum of aromatic compounds, some of which may be deemed positive in certain wine styles (Joseph et al., 2017, Botha, 2010).

A range of Brettanomyces strains have been observed under various nutritional and aerobic conditions, and a large number of aroma compounds have been identified. The influence of environmental factors such as oxygen availability and nutrition on Brettanomyces' ability to produce flavour compounds has been researched. All Brettanomyces strains could produce 4-EP and 4-EG under low oxygen conditions if the volatile phenol precursors coumaric acid and ferulic acid are present (Albino, 2011). However, under different conditions (i.e. aerobic conditions) and the presence of different substrates Brettanomyces can also produce a variety of other aroma compounds (Joseph et al., 2013). In a comprehensive study done by Joseph et al. (2017), 44 compounds were identified as major volatile or human detectable compounds (Table 1). Twenty-three of the compounds (52%) were reliant on the substrates available in the growth medium as well as the strain that was evaluated, while 10 compounds (23%) were only dependent on the substrate availability and another 5 (11%) were produced only by specific strains, regardless of the substrate. Six compounds (14%) did not depend on either substrate or strain and were produced by all the Brettanomyces strains under all conditions.

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Table 1: Chemical compounds produced by different strains of Brettanomyces and the aromas they produce (Joseph et al., 2017). Reproduced with permission.

Type of Substrate Strain Major Chemical compound Aroma compound dependent dependent product

2-Ethyl-1-hexanol Ester Yes No Yes Citrus, floral 2-Methyl-1-butanol Alcohol Yes No Yes Canned fruit, plastic 3-Methyl-1-butanol (isoamyl) Alcohol No No Yes Banana, whiskey, chemical 4-Ethyl guaiacol 4-EG Phenolic Yes No Yes Smoky, clove, spice, phenolic 4-Ethyl phenol 4-EP Phenolic Yes No Yes Phenolic, creosote, band-aid Ethyl 2-methyl butyrate Ester Yes Yes Yes Mint, citrus, green apple Phenethyl alcohol Alcohol, ester No No Yes Floral, rose 1-Decanol Alcohol Yes Yes Yes Waxy, floral, orange 1-Octanol Alcohol Yes Yes Yes Citrus, waxy, aldehydic, floral 2-Methyl butyric acid Fatty acid, ester Yes Yes Yes Blue cheese, rancid 2-Nonanone Ketone No No Yes Fruity, soapy, herbaceous 3-Methyl butyric acid (isovaleric) Fatty acid, ester Yes Yes Yes Sweaty feet, cheese Acetic acid Organic acid No Yes Yes Vinegar, sour β-Farnesene Terpene Yes Yes Yes Woody Butanol Alcohol Yes No Yes Alcohol Decanoic acid Fatty acid Yes No Yes Rancid, sour, fatty Ethyl acetate Ester No Yes Yes Pear, apple, nail polish remover Ethyl decanoate Ester Yes Yes Yes Fruity, apple, waxy Ethyl dodecanoate Ester Yes Yes Yes Soapy, rum, clean Ethyl isobutyrate Ester Yes No Yes Fruity, rum Ethyl octanoate Ester No No Yes Fruity, pineapple, apricot Ethyl tetradecanoate Ester Yes No Yes Waxy, violet Isobutyric acid Fatty acid Yes Yes Yes Rancid, cheese Octanoic acid Fatty acid Yes No Yes Rancid, cheesy Pentanoic acid Fatty acid No No Yes Putrid, rancid, sweat, cheese, Phenethyl acetate Ester Yes Yes Yes Floral, rose, honey Phenethyl propionate Ester No Yes Yes Musty, floral, yeasty Phenylacetaldehyde Aldehyde No Yes Yes Floral, honey 2-Methoxy-4-vinylphenol Phenolic Yes Yes No Woody, cedar, roasted nuts 4-Methoxyphenethyl methanol Alcohol, ester Yes Yes No Floral, balsamic, fruit, anise Amyl-octanoate Fatty acid Yes Yes No Wine, elderflower, orris Bisabolene Terpene Yes Yes No Woody, citrus, tropical fruit, green banana Butyric acid Fatty acid Yes Yes No Fruity, cheesy, acetic Ethyl butyrate Ester Yes Yes No Tutti-frutti, pineapple, cognac Ethyl isovalerate Ester Yes No No Fruity, esters, sharp, pineapple Ethyl valerate Ester Yes Yes No Tropical fruit, strawberry, pineapple Heptanoic acid Fatty acid Yes Yes No Fatty, animal Isoamyl alcohol Alcohol No No No Fruity, banana, whiskey Nonanal Aldehyde Yes Yes No Citrus, waxy, melon, aldehydic Ocimene Terpene Yes Yes No Fruity, floral, wet cloth Octyl butyrate Fatty acid, ester Yes Yes No Fruity, oily, fresh or green, earthy Pentyl formate Ester Yes Yes No Fruity, unripe banana, earthy Phenethyl formate Ester Yes Yes No Floral, green, watercress, hyacinth Undecanoic acid Fatty acid No Yes No Creamy, fatty coconut 19

7.1.1 Volatile phenols The most common characteristics in wine associated with Brettanomyces are volatile phenols, specifically 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) (Vigentini et al., 2008). 4-EP is reminiscent of leather and Band-Aid® characters, while 4-EG has a medicinal and spicy character (Suárez et al., 2007). Concentrations levels as low 0,14mg/L 4-EP and 0,62 mg/L 4-EG have been shown to influence the perception of wine (Chatonnet et al., 1992). The sensory perception thresholds of 4-EP and 4-EG vary considerably, and are influenced by the style of the wine, grape variety and the consumers’ wine tasting ability (Curtin et al., 2015).

Loureiro and Malfeito-Ferreira (2003), found that 20% of 474 red wines analysed from Italy, Australia and Portugal had 4-EP levels greater than 0,62 mg/L, a level above which some consumers reject the wine. Similar results were found in a study analysing 379 red Burgundy wines, where the Brettanomyces yeast was found in 50% of wines during maturation, and 25% of the wines in bottle (Gerbaux et al., 2000).

To understand wine spoilage by Brettanomyces, it is important to understand the metabolic pathways of the production of these volatile phenols. Volatile phenols are the final product of a two-step enzymatic process (Figure 2) (Chatonnet et al., 1992). The initial step is the decarboxylation of hydroxycinnamic acids to hydroxystyrenes, and the second step is the reduction of the vinyl hydroxystyrenes to the pungent-smelling ethyl derivatives.

It is important to note that the building blocks for the volatile phenols, i.e. hydroxycinnamic acids like coumaric acid, ferulic acid and caffeic acid, occur naturally in grape juice and wine. On average, 4-EP and 4-EG occur in wine at a ratio of 10:1, which correlates to the ratio of the precursors p-coumaric acid and ferulic acid in grape juice (Romano et al., 2008). The concentration and ratios of 4-EP and 4-EG depend on the grape variety and wine style. A study of Australian wine found the 4-EP to 4-EG ratio in Cabernet Sauvignon is 10:1, while it is 9:1 in Shiraz, 8:1 in Merlot and 3,5:1 in Pinot Noir (Rayne and Eggars, 2008). The relatively low levels of 4-EP in Pinot Noir is likely a function of the naturally low levels of coumaric acid in Pinot Noir grapes, providing less substrate for the production of 4-EP.

7.1.2 Volatile acids and volatile fatty acids Numerous authors found the production of acetic acid from glucose by Brettanomyces (Loureiro and Malfeito-Ferreira, 2006). Custers determined in 1940 that Brettanomyces was able to produce considerable amounts of acetic acid under aerobic conditions, but that anaerobic conditions inhibited the metabolism of glucose. Further studies showed that the 20

availability of oxygen favours the development of Brettanomyces in wine, as oxygen promotes the growth of the yeast as well as acetic acid production (Freer et al., 2003). In contrast, anaerobic conditions during winemaking may slow down the growth of Brettanomyces but will not totally prevent their development (Ciani and Ferraro, 1997). The increased concentration of acetic acid can have a negative effect on wine as it gives a vinegar or acetone aroma to the wine, and acetic acid is also associated with sluggish/stuck fermentations (Bisson, 1999).

7.1.3. Isovaleric acid While 4-EP and 4-EG are commonly associated with Brettanomyces spoilage, the third biggest contributor to organoleptic off-odours in Brettanomyces infected wine is the volatile fatty acid isovaleric acid. Isovaleric acid has been described as “rancid”, although sensory panels often describe this compound as “sweaty” or “cheesy” (Coulter et al., 2004). Even though the concentrations of isovaleric acid are not correlated to the levels of volatile phenols, isovaleric acid can increase the perception of other Brettanomyces-associated odours (Coulter et al., 2004). It is still uncertain under which conditions isovaleric acid is produced, other than the formation of isovaleric acid via the degradation of the amino acid L- leucine that has been proven (Harwood and Canale-Parola, 1981).

7.1.4 Mousiness Mousy off-odour was first described in 1986 and is often associated with Lactobacillus species (Heresztyn, 1986). Today, three compounds are considered responsible for mousy off-flavours in wine, two of which are associated with Brettanomyces: 2- acetyltetrahydropyridine (ATHP) & 2-ethyltetrahydropyridine (ETHP) (Heresztyn, 1986). Of the two, ATHP has a lower sensory detection threshold, and is more often detected in wine. The mousy character in wine is not volatile at wine pH, but when mixed with saliva, the pH increases which releases a mousy character (Oelofse et al., 2008).

The amino acid L-lysine is an important precursor in the production of mousy flavours and so is ethanol (Snowdon et al., 2006). The mousy character occurs infrequently in wines infected with Brettanomyces, to the point where Dr Pascal Chatonnet declared that “these compounds are not of major significance when performing sensory screenings of Brettanomyces character in wine” (Chatonnet, 2012).

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7.2 Loss of colour Wines contaminated with Brettanomyces often display an undesirable colour. Several researchers referred to the glycosidic activity of many Brettanomyces strains as the source of undesirable colour in Brettanomyces affected wines (Fugelsang et al., 1993; Potgieter, 2004). A large part of the total glycoside concentration in wine involves anthocyanins, the most important red pigment in wine (Figure 4). The hydrolysis of glucose from anthocyanin will result in the conversion to a colourless pseudo base, affecting the colour of the wine negatively (Mansfield et al., 2002).

Figure 4: Generic anthocyanin molecule. The glucose molecule is indicated as "glc", R3' - R7' depict sites of possible combinations of -H, -OH or -OCH3. These combinations determine exact anthocyanin (aurantinidin, cyanidin, delphinidin, europinidin, pelargonidin, malvidin, peonidin, petunidin or rosinidin) (Valavandidis and Vlachogianni, 2013). Reproduced with permission.

Another possible explanation for the loss in colour by Brettanomyces was proposed by Oelofse et al. (2008). Wine colour is stabilised and intensified by the polymerization of anthocyanin with phenolic compounds. Some of the phenolic compounds that can polymerize with anthocyanin are vinylphenols, a compound produced by the enzymatic decarboxylation of hydroxycinnamic acids by Brettanomyces and Saccharomyces (Figure 5). However, Brettanomyces will reduce the available vinylphenol by converting the compound to ethyl derivatives (Figure 2), leaving less vinylphenol available for the formation of stable vinylphenolic pyranoanthocyanin pigments. 22

Figure 5: Enzymatic decarboxylation of hydroxycinnamic acids by Brettanomyces and Saccharomyces (Morata, Loira and Lepe, 2016). Reproduced with permission.

7.3 Haziness Other than the organoleptic influence and the loss in colour, Brettanomyces has also been responsible for turbidity or haziness in wine (Van der Walt and Van Kerken, 1958). Haziness is not a common symptom of Brettanomyces spoilage and there are few references to turbidity in Brettanomyces spoilage. The haziness in Brettanomyces affected wine is probably due to the physical presence of the spoilage yeast cells in the wine, in which case the wine is probably irreparably spoiled.

7.4 Biogenic amines Biogenic amines are nitrogen-based compounds formed by plants, animals and microorganisms. These compounds are naturally present in grapes but are principally formed in wine by the microbiological decarboxylation of free amino acids (Figure 6) (Granchi et al., 2005). Biogenic amines are associated with microbiological spoilage in food production (Silla Santos, 1996). In winemaking, biogenic amines are thought to be indicators of poor hygiene throughout the winemaking process. Putrescine and cadaverine specifically, are associated with poor sanitary conditions of grapes (Pereira et al., 2017). 23

Figure 6: Enzymatic decarboxylase of amino acids produce biogenic amines, (Suzi Smith, https://www.diagnosisdiet.com). Reproduced with permission.

When present in wine, biogenic amines may cause organoleptic faults. High levels of putrescine and cadaverine can have a negative impact on the flavour profile of a wine, often described as rancid (Pereira et al., 2017). Biogenic amines have also been linked to negative physiological effects in people sensitive to amine intolerance, mainly because of the toxicity of histamine and tyramine (Gafner, 2003). The negative physiological effects of biogenic amines in wine are aggravated due to the presence of ethanol, which reduces or inhibits the activity of the enzymes monoamine and diamine responsible for the metabolism of biogenic amines (Pereira et al., 2017).

There is no legal limit for biogenic amines in wine. Some countries have recommended maximum levels for histamine in wine, namely Germany (2 mg/L), France (8 mg/L) and Belgium (6 mg/L) (Pereira et al., 2017). A recent study by Filipe-Ribeiro et al. (2019) found that the levels of biogenic amines produced by Brettanomyces in commercial wine does not pose an increased risk of biogenic amine intake for consumers.

Most research on the production of biogenic amines in wine has been on Gram positive (lactic acid) bacteria, but Caruso et al. (2002) identified Brettanomyces as the yeast in wine with the highest production of biogenic amines. This was confirmed by a comprehensive study by Granchi et al. (2005). The biogenic amine most commonly associated with Brettanomyces is phenylethylamine. Phenylethylamine, histamine and tyramine, are the most toxic of the biogenic amines and are known to cause a wide variety of symptoms including hypotension, headaches, heart palpitations, cutaneous and gastrointestinal disorders (Granchi et al, 2005).

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7.5 Summary There are numerous ways Brettanomyces can spoil wine. Brettanomyces can produce a variety of aroma compounds, depending on the strains and conditions. The most common wine spoilage associated with Brettanomyces is organoleptic, specifically volatile phenols. All strains of Brettanomyces produce 4-ethyl phenol and 4-ethyl guaiacol under low oxygen conditions when precursors are present. 4-EP is associated with leather and Band Aid® characters while 4-EG has a medicinal, spicy character. In a study analysing wine from different countries it was found that 20% of wine had 4-EP concentrations higher than a threshold where customers reject wine.

Brettanomyces is also associated with other spoilage odours including mousiness, cheesy and rancid odours. Other than the negative organoleptic impact, Brettanomyces infection can also lead to undesirable colour and haziness in wine. Brettanomyces can also produce biogenic amines, nitrogen-based compounds that can cause a variety of symptoms, including headaches, heart palpitations, hypotension and gastrointestinal disorders.

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8. Impact of other microorganisms

Wine is not produced in a controlled, sterile environment. There are a host of other microorganisms present in wine that could possibly affect the flavour profile of a wine, directly or indirectly. Conterno et al. (2006) found that Brettanomyces is not the only microorganism that can produce 4-EP and 4-EG. The genes that allow for the decarboxylase activity involved in the production of volatile phenols (Figure 2) also exist in numerous other yeasts and bacteria, some of which are present during winemaking (Chatonnet et al., 1992). Earlier studies have found that Saccharomyces cerevisiae is not able to produce ethyl phenols and that Gram-positive bacteria, especially Lactobacillus plantarum can only produce low concentrations of ethyl phenols in wine (Chatonnet et al., 1995). However, certain strains of S. cerevisiae can produce significant amounts of vinyl phenol via the decarboxylation of cinnamic acids, which is the first step in the production of volatile phenols. Higher levels of vinyl phenol precursors can lead to undesirable concentrations of ethyl phenol produced by Brettanomyces (Nelson, 2008).

Certain strains of Meyerozyma guilliermondii (formerly known as Pichia guilliermondii) can also produce significant amounts of volatile phenols, comparable to that of Brettanomyces (Dias et al., 2003a). This could be significant, as M. guilliermondii have been found on grapes and grape juice. In contrast, Barata et al. (2006) found that these Meyerozyma spp. cannot produce significant levels of volatile phenols in wine. Joseph et al. (2017) developed an aroma wheel for Brettanomyces (Figure 7) and then analysed 30 wines from an online retailer in the US that fitted the descriptors for Brettanomyces spoilage. The 30 wines were subjected to: 1) microscopic examination, 2) plating on selective media, 3) quantitative polymerase chain reaction (qPCR) to test for Brettanomyces DNA and 4) chemical analysis to test for 4-EP and 4-EG aromas produced by Brettanomyces. When studied under a microscope and plated on selective media, 17 of the 30 wines analysed showed no sign of Brettanomyces but did show the presence of lactic acid bacteria Lactobacillus and/or Pediococcus. Joseph et al. (2017) deduced that both classes of organisms (Brettanomyces and lactic acid bacteria) can produce the same aroma compounds from amino acid precursors, as had been reported for the mousy odour derived from lysine, which was previously discussed in 7.1. Organoleptic. The study makes interesting observations, but it does not allow for the fact that, even though some of the wines analysed showed no physical evidence of Brettanomyces, these wines may previously have had Brettanomyces activity. Brettanomyces cells are on average bigger (5 μm) than 26

Lactobacillus (0,5 by 2 μm) and Pediococcus (1- 2 μm). Therefore, a 2 μm filtration at bottling may have removed Brettanomyces cells from some the wines analysed in the study, but not Pediococcus and Lactobacillus. This may give the impression that some spoilage characters were caused by malolactic bacteria, while it may have been caused by Brettanomyces.

Figure 7: Brettanomyces aroma wheel (Joseph et al., 2017). Reproduced with permission.

8.1 Summary Brettanomyces is not the only organism that can produce 4-EG and 4-EP. Many other microorganisms could be responsible for the alteration of wine aroma or organoleptic properties. Certain lactic acid bacteria, for example Lactobacillus plantarum, can produce low concentrations of ethyl phenol and some researchers found that certain strains of Meyerozyma guilliermondi can even produce significant amounts of volatile phenols. Saccharomyces cerevisiae cannot produce 4-EP and 4-EG, but can produce significant amounts of vinyl phenol, which could be converted to volatile phenols by Brettanomyces should the yeast be present in the wine in question. This higher level of precursors could 27

result in undesirable concentrations of volatile phenols. Additionally, Gram positive bacteria Lactobacillus and Pediococcus can produce the same mousy aromas from amino acid precursors as Brettanomyces. Therefore, when trying to manage a spoilt wine, a winemaker should consider other microbes when submitting samples for analysis. Various other methods for control would have to be considered for non-Brettanomyces species, for example sulphur management or even manipulating wine acidity. 28

9. Brettanomyces in white wine

The majority of research on Brettanomyces has been done on red wine. Less is known about Brettanomyces growth and spoilage in white wine.

There is a big difference in the phenolic make up of red wine and white wine. More than 85% of the phenolic content of red wine is made up from flavonoids, mainly tannins and anthocyanins (>1000 mg/L), whereas flavonoids make up less than 20% of the phenolics of white wine (<50 mg/L). The majority of the phenolics in white wine are made up primarily of non-flavonoids, that include hydroxycinnamic acids, the precursor for 4-EP and 4-EG. If the precursors are in the wine, there is potential for volatile phenols to develop, if Brettanomyces is active in the wine.

The first reported incident of Brettanomyces spoilage in white wine studied by the Australian Wine Research Institute (AWRI) was in 2000 with occasional subsequent incidents. An increase in the incidence of Brettanomyces in white wine in 2013 promoted further research (Coulter, 2014). It is interesting to note that all six white wines analysed for Brettanomyces by the AWRI in 2013 were Chardonnays, however Brettanomyces characters have also been reported in Riesling and sparkling wines. Barrels were highlighted as the biggest source of infection of Brettanomyces in 5.2 Cellars, and the highest populations of Brettanomyces found in cellars are in barrels, according to Malfeito-Ferreira (2018). As Chardonnays are often fermented and matured in barrel, this could indicate that barrel fermentation and maturation of white wine could possibly lead to Brettanomyces infection.

Volatile phenols are detectable at lower levels in white wine compared to red wine. The AWRI reported cases of Brettanomyces spoilage in white wine at 4-EP levels as low as 0,077 mg/L (Coulter, 2014). Chatonnet et al. (1992) found that 4-EP start to influence red wine at 0,14 mg/L. Coulter suggests that the solution lies in the 4-EP to 4-EG ratio, as Curtin et al. (2007) found that the perception threshold of 4-EP is decreased if the concentration of 4-EG is increased relative to 4-EP concentration. The six Chardonnays analysed by the AWRI in 2013 all had high levels of 4-EG relative to 4-EP, which intensified the organoleptic properties of the 4-EP.

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Coulter (2014) further suggests that modern winemaking trends could possibly lead to an increase in Brettanomyces in white wine. These include: Riper fruit: Increase in fruit ripeness results in higher pH and higher phenolic content. The higher pH means lowered efficiency of SO2 to control Brettanomyces, and higher phenolic content increases the substrate to produce more volatile phenols.

Natural winemaking: Limited use of SO2 will aid the growth of Brettanomyces. Also, natural alcoholic fermentation and malolactic fermentation (MLF) is often slower than inoculated alcoholic fermentation and MLF, which means more opportunity for the Brettanomyces to grow and spoil the wine. Adaptability: Brettanomyces has shown to be a very adaptive organism. It is possible that the yeast has adapted to modern winemaking techniques, including higher SO2 and alcohol levels. This is supported by Cibrario et al. (2019), as discussed in 3.2 Genetic diversity.

9.1 Summary There is a big difference in the phenolic make up of red wine and white wine. Furthermore, a lower incidence of Brettanomyces is reported in white wine, even though there are sufficient concentrations of the precursors for 4-EG and 4-EP present in white wine for Brettanomyces to convert to volatile phenols. However, the presence of this yeast in white wine is uncommon and rarely isolated in this media. As stated above, Brettanomyces spoilage in white wine has only been reported and analysed by the AWRI. Furthermore, most of the wines flagged for Brettanomyces by the AWRI were Chardonnays. Seeing that Chardonnays are often fermented and aged in barrels, which is a common area of isolation for Brettanomyces, this could possibly highlight the fact that barrel maturation of white wine could lead to the contamination of white wine by Brettanomyces. Extra care should be taken by winemakers when attempting these risk prone styles, as the resulting volatile phenols due to contamination by Brettanomyces, are detectable at lower levels in white wine compared to red wine. Barrel hygiene should also be of utmost importance when producing both white and red wine.

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10. Managing Brettanomyces in the cellar

As evidenced in 5. Sources of contamination in this dissertation, Brettanomyces occurs naturally on grapes and is potentially present throughout the winemaking process. It has proved to survive and grow under extreme conditions. Spoilage by Brettanomyces is unavoidable in an environment favouring its development. Therefore, failure to manage Brettanomyces in the cellar may lead to the production of off-odours by the yeast and ultimately result in financial loss. There are however measures that can be applied to manage the growth of this yeast.

10.1 Manage Brettanomyces growth

10.1.1 Sulphur dioxide (SO2)

Sulphur dioxide (SO2) has very strong antimicrobial properties and is widely used in the wine industry. Research on the effect of SO2 on Brettanomyces is inconsistent and inconclusive, possibly due to the great variation in SO2 tolerance between different Brettanomyces strains.

Certain authors found Brettanomyces sensitive to free SO2 levels over 30 mg/L (Chatonnet et al., 1995), while other researchers observed growth of Brettanomyces in media with a free

SO2 higher than 30 mg/L (Fraudière and Larue, 1988).

Another complicating factor is the fact that SO2 induces the VBNC state (as discussed in 4.1 Direct determination). In this VBNC state, Brettanomyces has low metabolic activity, temporarily loses the ability to produce new cells and reduce in cell size to the point that it can pass through sterile filters (Millet and Lonvaud-Funel, 2000). This may give the winemaker the impression that there is no Brettanomyces infection in the wine, but changes in the wine chemical composition and/or physical parameters (an increase in pH, a decrease in molecular SO2, and an increase in temperature) can lead to the possible revival of the yeast, and with that, the possibility of wine spoilage.

It is critical to note that the molecular portion of the free SO2 depends on many variables in the wine composition, mainly pH. For example: to achieve 0,6 mg molecular SO2/L available in a wine with a pH of 3,4, a free SO2 of 24 mg/L is required. If the pH of the wine is 3,8 then the free SO2 must be 60 mg/L to have the same amount of 0,6 mg molecular SO2/L available, as presented in Table 2. Henick-Kling et al. (2000) recommend a molecular SO2 of 0,5 – 0,8 mg/L to control Brettanomyces. A molecular SO2 of 0,5 mg/L equates to 30 31

mg/L free SO2 at pH 3,6, which correlates with Chatonnet’s recommendation of a free SO2 of 30 mg/L.

Table 2: Free SO2 required for 0,6 and 0,8 mg/L molecular SO2 at different pH levels (VinLab Technical Guide) Reproduced with permission.

Vigentini et al. (2013) reported a diverse and highly strain dependent SO2 tolerance among

Brettanomyces. SO2 tolerance ranged from 0,03 mg/L to 0,60 mg/L molecular SO2. This is echoed by an earlier study of 41 Brettanomyces strains by the AWRI that found SO2 tolerance from 0,22 to 0,55 mg/L molecular SO2 (Curtin et al., 2012). In a more recent study, Dimopoulou et al. (2019) analysed 46 Greek wines and found 22 Brettanomyces strains that could be divided into three major groups based on genetic similarities using microsatellite analysis. The study found that the three genetic groups correlated with three 32

geographic regions of origin of the wines analysed. Furthermore, the three groups displayed different SO2 tolerance. Strains from the one group were sensitive to SO2 as low as 0,2 mg/L molecular SO2, strains from the second group were sensitive to SO2 at 0,4 mg/L while strains from the third group displayed SO2 tolerance and could grow in a synthetic medium at 0,6 mg/L molecular SO2. This reiterates the need for the winemaker to identify

Brettanomyces at strain level to determine SO2 tolerance in order to manage the yeast effectively.

Longin et al. (2016) did a comprehensive study investigating the efficiency of different levels of molecular SO2 on various strains of Brettanomyces as well as different population sizes of Brettanomyces. The result echoes that of other studies, that different strains of

Brettanomyces displayed different SO2 resistance. An important observation from Longin et al. (2016) was the correlation between Brettanomyces population size and SO2 resistance where a higher yeast concentration resulted in lower SO2 efficiency over time. This is clearly displayed in the three graphs shown in Figure 8, where the addition of 0,5 mg/L molecular

SO2 led to a decrease in the yeast population size to the point that no viable or culturable cells could be detected for the remainder of the experiment at starting population levels of a) 10³ and b) 10⁴ cells/mL. However, at a population of c) 10⁵ cells/mL, the addition of 0,9 mg/L molecular SO2 led to the decline of Brettanomyces, but a viable population was identified after 14 days, and this strain could be cultured again after 30 days.

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Figure 8: Viable and culturable population of Brettanomyces bruxellensis strain L02E2 over time without SO2 and with 0.5, 0.9 and 1.1mg/L molecular SO2. Initial populations were a) 10³, b) 10⁴ and c) 10⁵ cells per mL. (Longin et al., 2016). Reproduced with permission.

A survey undertaken by the Australian Wine Research Institute discovered a trend among winemakers in Australia to use frequent but small SO2 additions, rather than the application of large SO2 additions less frequently. This practice is not recommended, as it can lead to the unintended selection of more SO2 resistant Brettanomyces species (Coulter et al., 2004). In a recent seminar at Stellenbosch University (Brettanomyces Workshop, 19 July 2018),

Prof Benoit Divol summarised that SO2 is a very important tool in the management of Brettanomyces but stressed that the management of Brettanomyces is complex and that the winemaker cannot rely on SO2 alone. 34

10.1.2 Chitosan Chitosan is a linear polysaccharide consisting of β-(1→4)-linked D-glucosamine and N- acetyl-D-glucosamine. Chitosan has wide applications in the cosmetic, agricultural, pharmaceutical, food, textile and water treatment industries due to its unique properties (Kong et al., 2010). The antimicrobial properties of chitosan in the food industry have been investigated for years (Ouattara et al., 2000) and the application of chitosan in the wine industry has been accepted by the International Organisation of Vine and Wine (OIV) since 2009 (Organisation Internationale de la Vigne et du Vin, 2009).

The principal component of chitosan is chitin, the second most common sugar in nature after cellulose (Aider, 2010). Chitosan is manufactured on a commercial scale by the deacetylation of chitin (Figure 9), which is the structural component in the exoskeleton of shellfish and insects, and cell walls of fungi. Deacetylation implies the removal of an acetyl group and this is commonly done by treating the chitin with an alkaline substance, like sodium hydroxide.

Figure 9: Chitosan formation via chitin deacetylation (Younes and Rinaudo, 2015). Reproduced with permission.

It is important to note that only chitosan and chitin-glucan derived from Aspergillus niger specifically is allowed for oenological use in South Africa according to the latest SAWIS Regulations Annotated 2014 (SAWIS, 2019).

Determining the effective dosage of chitosan Bornet and Teisseidre (2008), found that doses of 2 – 6 g/hL of chitosan were adequate to eliminate the Brettanomyces population, determined by qPCR in a contaminated wine. Further trials, done by Pillet (2014) at the Institut Oenologique de Champagne (IOC) laboratory in Burgundy, verified and refined this observation. In all cases, Pillet found that 4 35

g/hL chitosan was sufficient to eradicate the Brettanomyces population of contaminated wines, as counted on selective growth gel media.

Petrova et al. (2016) is not convinced that chitosan eliminates Brettanomyces. It was found that chitosan is efficient in reducing the population of Brettanomyces, but that the population of Brettanomyces can recover to >10³ cfu/mL after 10 weeks. Petrova et al. (2016) recommend that chitosan could be applied to reduce the population but not necessarily eradicate Brettanomyces from wine. These conflicting results suggests it is possible that certain strains of Brettanomyces may be resistant to chitosan. This effect has been noted when evaluating the effect of SO2 and could be similar for chitosan. Further studies should focus on the effect of chitosan on various strains of Brettanomyces.

The impact of chitosan on other wine-related microorganisms: It should be noted that chitosan is particularly effective against other wine microorganisms (Table 3). Each microorganism will be discussed below. Saccharomyces cerevisiae Numerous studies found chitosan to have an extended lag phase effect on S. cerevisiae, even though Bağder Elmacı et al. (2015) observed that the use of chitosan did not have a significant impact on the viability or fermentative performance of S. cerevisiae. Non-Saccharomyces yeast: The addition of chitosan had a stronger inhibitory effect on non-Saccharomyces yeast compared to S. cerevisiae. The addition of 20 g/hL chitosan had no significant reduction in the viable population of Hanseniaspora uvarum and Zygosaccharomyces bailii, but a higher dose of chitosan at 40 g/hL completely inactivated H. uvarum and Z. bailii (Bağder Elmacı et al, 2015). Lactic acid bacteria The addition of 20 g/hL chitosan completely inactivated lactic acid bacteria Lactobacillus hilgardi and Oenococcus oeni. In contrast, Lactobacillus plantarum showed more resistance and required chitosan additions of up to 100 g/hL to reduce the viable cfu count to below 10¹.

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Table 3: Growth of wine-related microorganisms in yeast extract-peptone-glycerol medium in the presence of various concentrations of chitosan (Bağder Elmaci et al., 2015). Reproduced with permission.

Incubation Microorganism time (days) Viable count (log cfu/ml) Chitosan concentration (g/hL) 0 20 40 60 80 100 120 140 160 180 200 H. uvarum 0 7,40 7,40 7,40 7,40 7,40 7,40 7,40 7,40 7,40 7,40 7,40 3 8,07 7,81 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 6 7,86 7,9 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 Z. bailii 0 7,23 7,23 7,23 7,23 7,23 7,23 7,23 7,23 7,23 7,23 7,23 3 7,94 7,62 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 6 7,6 6,68 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 L. plantarum 0 8,74 8,74 8,74 8,74 8,74 8,74 ND ND ND ND 8,74 3 9,30 6,52 3,54 3,4 <1,00 <1,00 ND ND ND ND <1,00 6 9,41 9,31 7,94 7,34 5,64 6,06 ND ND ND ND <1,00 L. hilgardii 0 7,47 7,47 7,47 7,47 7,47 7,47 7,47 7,47 7,47 7,47 7,47 3 6,74 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 6 6,29 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 O. oeni 0 8,98 8,98 8,98 8,98 8,98 8,98 8,98 8,98 8,98 8,98 8,98 3 8,01 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 6 7,95 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 B. bruxellensis 0 5,24 5,24 5,24 5,24 5,24 5,24 5,24 5,24 5,24 5,24 5,24 3 4,83 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 6 6,5 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00 <1,00

It is important for winemakers to understand the effect of chitosan on other wine-related microorganisms, as this will determine the timing of the use of this additive. Considering chitosan’s bactericidal properties against lactic acid bacteria, it is advisable to use chitosan after completion of malolactic fermentation.

10.1.3 Dimethyldicarbonate (DMDC) Dimethyldicarbonate (DMDC), known by the trade name Velcorin®, has been assessed for the control of Brettanomyces in wine. Delfini et al. (2002) established that 250 mg/L could inhibit the growth of Brettanomyces. Subsequent studies found variable results with different strains of Brettanomyces (Renouf et al., 2007c, Malfeito-Ferreira et al., 2004, Malfeito- Ferreira 2018). Malfeito-Ferreira et al. (2004) also observed that the efficiency of DMDC was dependent on the ethanol content of the wine, while Threlfall and Morris (2002) found that the efficiency of DMDC is not influenced by the pH of wine. Malfeito-Ferreira (2018), found DMDC to be efficient against the growth of Brettanomyces when present at a relatively low initial cell count (<500 cfu/mL) however, Brettanomyces could not be inhibited with the maximum legal dose of 200 mg/L of DMDC at cell counts higher than 10⁴ cfu/mL. The hydrolysis of DMDC releases methanol that should be considered if a high concentration of methanol is suspected. Furthermore, the low solubility of DMDC requires the use of an approved dosing machine.

From a legal perspective, it must be mentioned that in the European Union the use of DMDC is only allowed as an additive to wine with more than 5 g/L of residual sugar and additions 37

may only be added just prior to bottling. The maximum DMDC dosage allowed by the Organisation Internationale de la Vigne et du Vin (OIV), is 200 mg/L (Organisation Internationale de la Vigne et du Vin, 2009). DMDC should therefore only be considered if the dose required would be lower than 200 mg/L and if the resulting amount of methanol produced is within legal limits.

10.1.4 Sorbic acid Sorbic acid was also investigated as it has well-documented anti-fungal properties, but Brettanomyces was found as one of the yeast species that is the most tolerant against sorbic acid (Benito et al., 2009). This was confirmed by Malfeito-Ferreira (2018) that Brettanomyces is resistant to the maximum legal dose of 200 mg/L in wines. Therefore, sorbic acid should not be considered when managing a possible Brettanomyces contamination.

10.1.5 High pressure processing Commercial technology that applies uniform pressure of 100 – 600 MPa (1000 – 6000 bar pressure) to inactivate harmful microbes in food is commonly known as high pressure processing (HPP) (Milani et al., 2016). The usage of HPP technology worldwide has grown exponentially since 2000, and is commercially implemented in Japan, the USA and Europe. HPP processed products include fruit and vegetables, fruit juice and other beverages, pre- cooked meals and meat- and fish-based products. Industrial machines with a capacity of 3000 L/h are available, and the annual global production capacity currently exceeds 250 000 tons. HPP requires high capital investment and is therefore limited to high value products. HPP has a minor or negligible impact on the sensory properties of beer (Milani et al., 2016). Puig et al. (2003) observed > log 6 reduction of Brettanomyces population when HPP of 400 MPa was applied for five minutes. HPP of 300 MPa for only one-minute lead to a log 7 reduction in Brettanomyces population, while 100 MPa for seven minutes resulted in no significant reduction in the Brettanomyces population (Gonzalez-Arenzana et al., 2016). These results were confirmed by Van Wyk and Silva (2017), who found that no significant inactivation was achieved at 100 MPa, but treatments of 400 MPa and 600 MPa for five seconds led to total elimination of Brettanomyces (> log 6 reduction of population). The study also demonstrated that the HPP inactivation of Brettanomyces is a function of pressure and treatment time.

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10.1.6 Fining agents A fining agent reacts with wine components either chemically or physically, to form a new coagulant that can be separated from the wine. The purpose of using a fining agent in wine is to soften or reduce bitterness; remove proteins or reduce colour by the adsorption and precipitation of polymeric phenols and tannins.

These fining agents could also be used to manage Brettanomyces in the cellar. Brettanomyces populations can be reduced by 40 – 2000-fold by treatment with fining proteins. A liquid gelatine fining can reduce the Brettanomyces population in red wine from

10⁴ cfu/mL to 170 cfu/mL. Fining agents should also increase the efficiency of SO2, mainly by reducing general microbial population. This would make fining agents a good option to manage Brettanomyces when used in conjunction with SO2 or other methods.

10.1.7 Filtration Filtration is a valuable tool for reducing spoilage microorganisms. Even though a filter acts as a mechanical barrier, it is more than just a simple strainer. Some filters act as surface filters, while depth filter and pleated depth filters increase the filtration surface even further. There is some resistance against filtering of quality wine because of the belief that filtration with small pore membranes may remove valuable compounds from the wine. Studies from Canas et al. (2011) have shown that adequate filtration does not substantially affect red wine composition or sensory properties.

Another question over the timing and efficiency of filtration arose after the observation of

Curtin et al. (2015) that the addition of SO2 and the subsequent VBNC state of Brettanomyces can influence the morphology of the Brettanomyces and lead to a 22% decrease in cell size. As discussed in 10.1.1 SO2, Millet and Lonvaud-Funel (2000) found that Brettanomyces in the VBNC state could go through a sterile 0,45 μm filter. However,

Umiker et al. (2012) found that exposure to SO2 did not affect cell removal by nylon membrane filtration. In a series of experiments, some strains were retained by filtration through 1.2 µm membranes, while other strains required 0.8 µm membranes. The results showed that even though the morphology of Brettanomyces was affected by SO2 additions, it did not influence the removal of Brettanomyces by membrane filtration (Umiker et al., 2012).

Duarte et al. (2017) did a comprehensive study comparing several commercially available filters as pleated cartridges for the wine industry regarding their ability to remove Brettanomyces from wine. The filters differed in composition and micron rating, namely 39

depth filters made of polypropylene (PP) (0.6 and 1.0 μm), borosilicate glass microfiber (GF) (X and V rating where X was equivalent to 0.5 μm and V was equivalent to 0.8 μm), and polyether sulfone membrane filters (PES, 0.45, 0.65, and 1.0 μm). The red wine used in the experiment was sterile filtered and inoculated with Brettanomyces to achieve a cell count of 1,6 x 10⁶ cells/mL.

The polypropylene filters had a low retention of Brettanomyces. For the PP 1.0 μm filter, it was not possible to count the colonies in any of the filtrated volume analysed because the colony numbers largely exceeded 400 cfu. Higher retention of Brettanomyces cells was observed for the PP 0.6 μm filters, however it still exceeded 400 cfu (Table 4). In general, the PP filters showed poor efficiency, as a high number of cells could pass through these filters.

The borosilicate glass microfibre GF V filters presented differences in the duplicate results, but for the GF X filters, no culturable Brettanomyces cells were detected (Table 4). The results, summarised in Table 4, obtained with the three polyether sulfone filters indicated no detection of culturable Brettanomyces cells in any of the duplicates. It is interesting to note that the PES 1,0 μm filters were more efficient than the PP 0,6 μm filters. This highlights the relevance and importance of filtration as a removal mechanism. However, the type of filter and filter material should be considered. Furthermore, the timing of the filtration action and the addition of SO2 should be considered, due to the alteration of Brettanomyces morphology in certain cases.

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Table 4: Results obtained by plate counting of the filtrated wine for duplicate tests performed with each filter (Duarte et al., 2017). Reproduced with permission.

Filter code Yeast population PP 1.0 >4.0 × 102 cfu/mL >4.0 × 102 cfu/mL PP 0.6 >4.0 × 102 cfu/mL >4.0 × 102 cfu/mL GF V 2.7 × 101 cfu/mL >4.0 × 102 cfu/mL GF X <1.0 cfu/50 mL <1.0 cfu/50 mL PES 1.0 <1.0 cfu/50 mL <1.0 cfu/50 mL PES 0.65-V <1.0 cfu/50 mL <1.0 cfu/50 mL PES 0.65-N <1.0 cfu/50 mL <1.0 cfu/50 mL PES 0.45-V <1.0 cfu/50 mL <1.0 cfu/50 mL PES 0.45-N <1.0 cfu/50 mL <1.0 cfu/50 mL PP: polypropylene with 0.6 and 1.0 μm micron ratings GF: borosilicate glass microfiber with X and V grades PES: polyethersulfone membrane with 0.45, 0.65, and 1.0 μm micron ratings

10.1.8 Temperature The heating of wine to inactivate Brettanomyces has been investigated. Couto et al. (2005) established that a Brettanomyces population of 10⁶ cfu/ml could be eliminated with heat treatment of 37,5°C for 6 minutes and 41°C for 0,6 minutes. The main disadvantage of heat treatment is the effect on the organoleptic quality of the treated wine. It is worth noting that the heating of grapes is not uncommon practice in red wine production. The process of thermovinification entails the heating of grapes to 50 – 60˚C for 24 - 48 hours to extract colour. This technique produces a particular style of wine and is associated with Beaujolais Nouveau wines.

The use of low temperatures during storage to limit spoilage by Brettanomyces has been documented (Edwards and Oswald, 2018). A recent study investigating the interactive effects of alcohol, temperature and SO2 on Brettanomyces growth in prepared wine found significantly lower production of volatile phenols when a wine with a total SO2 of 60 mg/L was stored at 15˚C for 100 days (0,862 mg/L 4-EP and 0,137 mg/L 4-EG) as opposed to the same wine stored at 18˚C for 100 days (2,363 mg/L 4-EP and 0,342 mg/L 4-EG) (Edwards and Oswald, 2018). These results support the use of temperature control as a useful tool 41

within a multiple control approach in the management of Brettanomyces. Future studies could investigate the cooling of grape must and its effect on the Brettanomyces population.

10.1.9 Cellar hygiene As mentioned in 5.2 Cellar (as source of Brettanomyces infection), Brettanomyces has been commonly associated with contaminated cellar equipment, to the extent that wineries are often regarded as the principal source of Brettanomyces infection. Efficient hygiene along with SO2 management, is extremely important in the effective management of Brettanomyces in the cellar. This includes proper sterilisation of second-hand equipment, especially barrels, as well as full analysis of bought in wine to prevent contamination.

Ozone gas is very efficient to sterilise winery equipment. Some literature found a decrease in the Brettanomyces population with a combination of hot water and ozone gas treatment, while other literature found a reduction of up to 99% with ozonated water (Cantacuzene et al., 2003, Coggan, 2003).

10.1.10 Barrel maturation and sanitation Wines are very susceptible to Brettanomyces spoilage during barrel maturation for numerous reasons. The porous structure of the oak permits small amounts of oxygen into the barrel, and the presence of cellobiose that can serve as a carbon source, are important properties of new and old oak barrels that benefit Brettanomyces growth (Chatonnet et al., 1992).

Barata et al. (2013) found that Brettanomyces can penetrate the wooden staves of a barrel up to 8 mm, which correlates to the maximum depth that wine will penetrate the barrel. This is important to note, as it complicates efficient barrel sterilisation and complete removal of Brettanomyces cells. Furthermore, the elimination of established populations of Brettanomyces in wine barrels becomes increasingly difficult in older barrels, as microbiological cells, colour pigments and other material block the pores of the wood (Oelofse et al., 2008). Apart from the staves, Brettanomyces can also survive around the bung holes and in the lees in the barrel (Laureano et al., 2005).

Barrel sanitation is critical in the efficient management of Brettanomyces, but literature on which method is most effective is inconsistent. Chatonnet et al. (1992) recommend > 7 g

SO2 gas per barrel to sanitise the wood. Laureano et al. (2005) investigated a time consuming method of rinsing the barrel with cold water, followed by three cycles of hot water rinse at 70˚C and air drying followed by soaking the barrel with a SO2-water solution with 42

200 mg/L SO2 at pH3 for one month, or another cycle of cold water rinse followed by low pressure steam (0,5 kg/cm) for 10 minutes. The steam sterilization appeared to be very effective, although Brettanomyces cells were still recovered from the barrels after all the successive treatments from Laureano et al. (2005).

Laboratory tests showed that ultrasound can effectively kill Brettanomyces in a chemically defined medium. More than 97% of a 4,4 x 10⁶ cfu/mL population of Brettanomyces was eradicated with ultrasound at a power rating of 50 watts for 90 – 100 seconds (Yap et al., 2007). The practical and commercial viability of this practice has not been fully investigated (Yap et al., 2007).

Another technique of shaving and re-toasting barrels has been studied by Pollnitz et al. (2000) who found a reduction of up to 85% in volatile phenols in wine matured in the treated barrels compared to the untreated barrels. The quality of the wood component extracted has not been evaluated.

Fabrizio et al. (2015) found hot water treatment for the purpose of eliminating Brettanomyces from contaminated barrels to be very efficient. Three different 3-year old barrels were microbiologically analysed before and after hot water treatment, where the barrels were exposed to hot water at 60˚C for 19 minutes. This hot water treatment resulted in the elimination of Brettanomyces population to below the detection limit as shown in Table 5.

Table 5: Yeast cell concentrations found in different samples of 3-year-old barrels collected before and after heat treatment in water (60°C for 19minutes) (Fabrizio et al., 2015). Reproduced with permission.

Plate counts before Plate counts after Barrel heat treatment heat treatment (CFU/100 ml) (CFU/100 ml) 1 2.5 x 10⁴ <1 2 2.1 x 10⁶ <1 3 2.3 x 10⁸ <1

A combination of steam, ozone and sulphur could be considered as an integrated approach to barrel sanitation for the management of Brettanomyces populations. These methods are efficient and low cost, and when used together, have the potential to vastly decrease high Brettanomyces populations.

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10.1.11 Impact of winemaking practices Apart from the obvious winemaking practices like cellar hygiene and barrel sanitation, there are other winemaking practices that can be implemented to limit the growth of Brettanomyces in wine.

- The efficiency of SO2 is pH-dependent (10.1.1 SO2). Low acidity and the corresponding high pH can be adjusted by acidification. - Prevent sluggish/stuck fermentation as it creates an environment favourable for a range of microbiological spoilage organisms. - Oxygen has a positive influence on the development of Brettanomyces, therefore exposure to oxygen should be limited as far as possible. Antioxidants like ellagitannins can be used to reduce available oxygen, thus limiting the growth of Brettanomyces. Regular topping of barrels reduces the contact between wine and oxygen, and also the solution of oxygen in wine. The practice of micro- oxygenation, the addition of small, measured quantities of oxygen to wine, should be applied with caution. - The concentration of hydroxycinnamic acids, the precursors of volatile phenols 4- EG and 4-EP, depends on grape variety and grape quality, but also on maceration and extraction methods employed during the winemaking process. High maceration temperature, along with the use of pectolytic enzymes with cinnamoyl esterase activity for colour extraction can promote the extraction of volatile phenol precursors resulting in higher concentrations of volatile phenols produced by Brettanomyces (Oelofse et al., 2008). - The most critical stages for Brettanomyces contamination in the winemaking process are malolactic fermentation and the ageing of wine in barrels that have

been used before (Chatonnet et al., 1995; Suárez et al., 2007). Low free SO2 and available nutrients released by yeast cell autolysis, paired with low microbiological competition, create favourable conditions for Brettanomyces to develop during malolactic fermentation. Oxygen allowed into the barrel by the porous structure of oak and the availability of cellobiose that can be utilised as carbon source as mentioned in 10.1.10 Barrel maturation and sanitation add to the favourable conditions for Brettanomyces. Furthermore, the difficulty and complexity of used barrel sterilisation increase the probability of Brettanomyces contamination in wine. When malolactic fermentation is done in previously used barrels, the characteristics mentioned can promote the development of Brettanomyces in wine (Chatonnet et al., 1992)

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10.1 Treatment of wine with high volatile phenols

10.2.1 Fining agents

Guilloux-Benatier et al. (2001) observed a reduction in in 4-EG and 4-EP levels in red wine with its lees when compared to the same wine without its lees. Chassagne et al. (2005) then researched the influence of active dried wine yeast and yeast lees on the concentration of volatile phenols and discovered that active dried yeast of Saccharomyces cerevisiae eliminated 33% of 4-EP and 26% of 4-EG.

Milheiro et al. (2016) did a comprehensive study of the influence of various fining agents on the volatile phenol levels of infected wine, as well the impact of the fining agents on the phenolic compounds in the wine. The trial was carried out on red wine samples with the 4- EP and 4-EG levels adjusted to the highest levels found in scientific journals (300 μg/L for 4- EG and 1500 μg/L for 4-EP). The 8 fining agents analysed in the study include isinglass, egg albumin, gelatine, potassium caseinate, carboxymethylcellulose, chitosan, bentonite and activated carbon (Table 6). All the fining agents were tested at the maximum dosage suggested by its supplier. Activated carbon was the most effective, reducing the 4-EP concentration by 58% and the 4-EG concentration by 56%. Egg albumin was also effective, reducing the 4-EP concentration by 20% and the 4-EG concentration by 17%. The other products in the trial did not have a significant decrease in the volatile phenol concentration of the wine.

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Table 6: The effect of fining agents on the reduction of total concentration of 4-ethylphenol and 4-ethylguiacol (Milheiro et al., 2016) Reproduced with permission.

Fining agents Percentage of total concentration reduction (%)

Protein 4-Ethylphenol 4-Ethylguiacol Isinglass 0 0 Egg albumin 20,4 17,4 Gelatine 4,9 8,3 Potassium caseinate 0 4

Polysaccharide Carboxymethylcellulose 0 2,8 Chitosan 1,3 0

Mineral Bentonite 0 0,6

Other Activated carbon 58,3 56,1

Studying the impact of these fining agents on the phenolic composition of the wine, Milheiro et al. (2016) found only carboxymethylcellulose (CMC) caused a significant decrease in colour intensity. Even though bentonite, activated carbon, isinglass, egg albumin and potassium caseinate caused a decrease in total anthocyanins, the wine colour intensity did not decrease significantly (Table 7). In most cases, there was no significant decrease in phenolic acids and flavonoids observed after treatment.

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Table 7: The effect of fining agents on the colour intensity, hue, total anthocyanins, total phenols, non-flavonoid phenols, flavonoid phenols, polymerized pigments, coloured anthocyanins and total pigments of both unfined and fined red wine (Milheiro et al., 2016) Reproduced with permission.

Non- Total Flavenoid Polymerised Coloured Total Colour Total phenols flavenoid Hue Anthocyanins phenols pigments anthocyanins pigments Intensity AU (mg/L) phenols mg/L (mg/L) AU AU AU Fining agents (mg/L) Unfined wine 14,98 0,619 231,66 1999 365 1634 4,61 2,60 13,89

Protein Isinglass 14,46 0,624 215,91 2000 372 1628 3,39 1,19 13,46 Egg albumin 14,19 0,619 217,00 2016 347 1669 4,86 3,46 14,27 Gelatine 13,84 0,638 247,62 1912 404 1507 5,01 3,36 14,87 Potassium caseinate 14,81 0,619 207,81 1962 315 1647 5,05 3,45 13,53

Polysaccharide Carboxymethylcellulose 12,91 0,628 176,09 2025 366 1659 4,80 2,79 11,99 Chitosan 15,14 0,621 223,12 2020 344 1677 4,58 2,49 14,90

Mineral Bentonite 14,59 0,640 202,34 1954 341 1612 5,01 2,87 13,51

Other Activated carbon 15,05 0,645 191,19 2025 329 1697 5,22 2,89 12,57

10.2.2 Reverse osmosis Reverse osmosis and selective absorption are very efficient solutions to reduce ethyl phenols in wine. The process involves the use of tangential flow filtration over a selective permeable membrane and treatment with hydrophobic absorbent resin. Ugarte et al. (2005) observed a significant reduction in volatile phenols when performing reverse osmosis and selective absorption on a naturally contaminated wine. The 4-EP levels were reduced by 88% (from 5700 μg/L to 690 μg/L) and the 4-EG levels were reduced by 74% (from 204 μg/L to 52 μg/L). Unfortunately, a decrease in other aromatic compounds was also observed.

10.3 Summary The need for an integrated approach to Brettanomyces management is evident, as no single

method is infallible. Even though research on the effect of SO2 on Brettanomyces is inconsistent, it is still considered as a valuable tool in the management of Brettanomyces.

Therefore, the use of SO2 with other methods could be beneficial. Although research results agree that the use of chitosan is an important practice in the management of Brettanomyces, there are inconsistencies in the optimal dosage of chitosan to eliminate Brettanomyces. The use of fining agents like gelatine can also reduce the Brettanomyces population but will not eradicate it. Adequate filtration with appropriate filters (1,0 μm polyether sulfone or 0,5 μm borosilicate glass microfiber) will remove all viable Brettanomyces cells without compromising wine quality. Heat treatment of infected wine is efficient to eradicate Brettanomyces cells but the impact of this treatment on wine quality remains a concern. 47

New technology like high pressure processing is expensive at present but may have future potential.

Cellar hygiene is extremely important in the effective management of Brettanomyces. Barrels are cited as the biggest source of infection in wine cellars. Hot water treatment is very efficient in eliminating Brettanomyces from contaminated barrels, and more so when used in conjunction with SO2 and ozone treatments.

Numerous other winemaking practices can be implemented to limit the growth of Brettanomyces in wine. These practices include the use of acid to lower wine pH to improve the efficiency of SO2, prevention of sluggish fermentation, minimizing dissolved oxygen in wine and management of malolactic fermentation.

Regarding the volatile phenols post contamination, activated carbon is considered an efficient fining agent to remove volatile phenols from wine that is already tainted. However, the overall impact on wine quality remains a concern. Reverse osmosis and selective absorption can reduce other volatile compounds and is a very efficient technique to reduce the volatile phenol concentration in wine.

It would be beneficial to establish which strain(s) of Brettanomyces are present in a cellar to facilitate specific action. This is theoretically possible with methods like PCR, however this service is not commercially available yet. When a Brettanomyces infection is detected in a winery, the safest option is to assume the worst-case scenario and treat as if the strain present is the most resistant to whatever treatment(s) is applied.

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11. Latest research

11.1 Kaolin silver complex (KAgC) Kaolin silver is manufactured under patent. It is a grey powder with a particle size of approximately 30 nm and is insoluble in ethanol and water. It consists of kaolin, a silica rich clay, and silver nanoparticles (<10 nm).

Recent studies revealed that silver nanomaterials have antimicrobial properties towards a wide range of bacteria, and also have some antifungal as well as antiviral properties (García -Ruiz et al, 2015). Regardless of the potential in the application of silver nanomaterials in the wine industry, there have been very few studies done on the practice of silver as an antimicrobial treatment in wine production. In recent trials, Izquierdo-Canas et al. (2018) demonstrated that 1 g/L KAgC was effective against Brettanomyces and acetic acid bacteria (Table 8). Table 8: Populations of Brettanomyces and acetic acid bacteria (genomic units/mL) in wine naturally contaminated before and after 10 days of treatment with KAgC and chitosan (Izquierdo-Canas et al., 2018) Reproduced with permission

B. bruxellensis Acetic acid bacteria 10 days after 10 days after Initial wine Initial wine treatment treatment Control 1.0 × 10⁴ 3.7 × 10⁴ 1.1 × 10⁵ 8.3 × 10⁵ KAgC 1.0 × 10⁴ 1.2 × 10² 1.1 × 10⁵ 1.7 × 10³ Chitosan 1.0 × 10⁴ 3.0 × 10² 1.1 × 10⁵ 3.5 × 10³

Further trials found 1 g/L KAgC was not as efficient against a higher population (1,0 x 10⁶ cfu/mL) of Brettanomyces in an infected wine (Izquierdo-Canas et al., 2018). Further trials are therefore warranted to determine the efficacy of this product against Brettanomyces.

11.2 Biological control

Managing Brettanomyces with non-Saccharomyces yeasts Various strains of non-Saccharomyces yeast have shown the capacity to produce killer toxins with a broad spectrum of activity, acting on Saccharomyces species as well as non- Saccharomyces genera (Petruzzi et al., 2017). Killer yeast strains have the capability of producing protein-like toxins that are lethal to sensitive yeasts. Several studies (Berbegal et al., 2017, Table 9), focussing on yeast killer toxins specifically aimed at Brettanomyces have been conducted recently, and a host of non-Saccharomyces species that are able to produce toxins with specific killer activity against several Brettanomyces strains have been identified. 49

Table 9: Non-Saccharomyces yeast capable of producing killer toxins against Brettanomyces (Adapted from Berbegal et al., 2017). Reproduced with permission.

Yeast/fungus species Killer toxin Mode of action Kluyveromyces wickerhamii Kwkt - Pichia anomala Pikt - Pichia membranifaciens PMTK2 Cell cycle arrest Candida pyralidae CpKT1 Cell Wall and membrane disruption Candida pyralidae CpKT2 - Ustilago maydis KP6 K+ depletion Torulospora delbrueckii TdKT Cell wall disruption

All the toxins listed above were stable at acidic wine pH and at temperatures between 20 - 25˚C, which are comparable to winemaking conditions. The killer toxins were applied in trial fermentations without affecting the population of Saccharomyces cerevisiae (Santos et al., 2011).

Inhibiting of Brettanomyces with Saccharomyces yeasts Branco et al. (2019) found that Saccharomyces cerevisiae produces a biocide called saccharomycin, which is composed of antimicrobial peptides derived from a glycolytic enzyme. This saccharomycin is efficient against several wine-related yeast species, including Brettanomyces. However, the levels of naturally produced saccharomycin is not sufficient to ensure the complete elimination of Brettanomyces. Branco et al. (2019) constructed genetically modified Saccharomyces cerevisiae that over-produced saccharomycin to levels of 1000 – 2000 μg/mL, which is about 10 times the level naturally produced by S. cerevisiae. In this experiment, the modified S. cerevisiae strains were able to eliminate Brettanomyces population larger than 10⁶ cells/mL (Branco et al., 2019). Unfortunately, genetically modified yeasts are not permitted in the wine industry.

11.3 Disposable electrochemical biosensors

Villalonga et al. (2018) created disposable amperometric biosensors for quick detection and quantification of Brettanomyces. The biosensors include the use of core-shell Fe3O4@SiO2

Nanocaptors, a core of Fe3O4 nanoparticles coated by a stable and inert SiO2 shell to protect the nanoparticles from oxidation and accumulation. The NanoCaptors are enabled with different bioreceptors, a carbohydrate-binding protein from jack beans called Concanavalin A (Con A) for total yeast analysis, and Brettanomyces polyclonal antibodies 50

for Brettanomyces detection. The biosensor equipped with the Con A bioreceptor could accurately determine the total yeast count in red wine from 10 to 10⁶ cfu/mL and the biosensor functionalized with Brettanomyces antibodies could accurately detect Brettanomyces in red wine samples from 10 to 10⁶ cfu/mL. Future commercial availability of this kind of technology could enable winemakers to detect Brettanomyces growth accurately and early in the winemaking process and enable them to act accordingly to prevent spoilage.

11.4 Summary

Silver nanomaterials like kaolin silver complex are efficient in reducing the population of Brettanomyces. It is not yet legal to use kaolin silver complexes in the wine industry and further research should be conducted to determine when it would be most effective against Brettanomyces. Various strains of non-Saccharomyces have shown the ability to produce killer toxins with specific killer activity against Brettanomyces. If one considers sulphur to be an issue for consumers, then microbiological alternatives such as killer toxins should be considered. Killer toxins produced by non-Saccharomyces yeast such as Kluyveromyces wickerhamii and Pichia anomala appear to have promising applications in winemaking for the control of Brettanomyces. If these killer toxins could be produced on a larger scale, they could be used for commercial purposes. However, killer toxins should first be accepted by the OIV as a legal additive to wine.

Saccharomyces cerevisiae produces low levels of the biocide saccharomycin that is effective against Brettanomyces. Genetically modified S. cerevisiae is able to produce sufficient quantities of saccharomycin to eliminate large populations of Brettanomyces efficiently, but genetically modified yeasts are currently not permitted in the wine industry. Directed evolution could be carried out to attain new strains of yeasts that would be able to produce higher levels of the biocide saccharomycin. Further studies of the effect of silver nanomaterials and biocides like saccharomycin on wine quality and health implications have to be conducted before these possible solutions can be considered for commercial use.

New technology like electrochemical biosensors may facilitate the detection of Brettanomyces. This can provide a very useful tool to winemakers, as early detection is key to prevent Brettanomyces spoilage.

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12. Conclusion Brettanomyces is a complex yeast with many variations, including genetic variations, between different strains. The variations described are an indication of the ability of Brettanomyces to adapt under stress conditions, making identification and management extremely difficult.

Traditional direct determination of Brettanomyces via culturing and microscopy has been unreliable until recently. Scientists resorted to indirect determination of Brettanomyces, including the analysis of metabolites. Indirect determination has limitations, notably that an infection is only detected when spoilage symptoms are already present, and indirect determination does not indicate whether the yeast is still alive or not. Better understanding of Brettanomyces has led to improved culturing methods, while new technology like qPCR gives an accurate indication of the presence of the yeast.

For the effective management of Brettanomyces, the use of multiple detection methods should be considered, i.e. direct and indirect. It would be beneficial to establish which strain(s) are present in a cellar to facilitate specific action, which is theoretically possible with methods like PCR. This is however not possible at the moment. When a Brettanomyces infection is detected in a winery, it is safest to treat as if the strain present is the most resistant to the treatment is applied.

Brettanomyces is commonly associated with contaminated cellars and equipment. The vineyard was previously overlooked as a source of Brettanomyces, but it has since been isolated from many niches from vineyard to bottle. Because Brettanomyces can utilise many carbon and nitrogen sources, winemakers should make sure that the alcoholic fermentation runs efficiently to completion with the absolute minimum residual sugar in the wine after alcoholic fermentation. Basic analysis of yeast available nitrogen on grape juice before fermentation can be used to calculate the minimum nitrogen required to limit available nitrogen in the wine after alcoholic fermentation. Acid management should be considered on high pH wines in order to reduce the pH to allow for added SO2 to be more effective.

In order to combat the presence of Brettanomyces in wine, an integrated approach should be considered. It is important to accept that all cellars are under risk of Brettanomyces infection. Therefore, prevention is of prime importance, and this includes good cellar hygiene, especially barrel hygiene. Most of the studies showed that various treatments are effective against low populations of Brettanomyces, so it is important to ensure that the population never gets high. SO2 is an efficient way to manage Brettanomyces. Maintaining a free SO2 of 30 mg/L during barrel maturation is considered good practice, to protect the wine from oxidation as well as protecting the wine from a broad spectrum of microbiological 52

spoilage, including Brettanomyces. The use of SO2 in conjunction with chitosan shows a lot of promise. Fining should be considered during the maturation of red wine to help reduce the population levels of Brettanomyces. Adequate filtration with carefully selected filter material is also a very effective way of reducing Brettanomyces populations.

The style of wine produced could also be considered when managing Brettanomyces in the cellar. This includes riper fruit with higher pH and higher phenolic content, which means lower SO2 efficiency and more phenolic substrate. Natural winemaking includes limited use of SO2, as well as natural alcoholic and malolactic fermentation, which could lead to a higher incidence of infection. The use of oak, new oak as well as used barrels, brings potential risk to Brettanomyces infection and growth in wine.

If 4-EG and 4-EP is detected in wine, it should be noted that other organisms can contribute to these aromas. Therefore, it would be beneficial to first identify the microbe in question before effective management of the microbe can be carried out. Saccharomyces cerevisiae can produce significant amounts of vinyl phenol therefore yeast starter cultures that produce less of these precursors could be selected to limit possible volatile phenol production. Additionally, Lactobacillus and Pediococcus can produce the same mousy aromas as Brettanomyces and that may increase the perception of Brettanomyces spoilage. There are ways to treat wine with 4-EG and 4-EP levels above the perception threshold, such as fining agents and reverse osmosis, but the best way is to prevent Brettanomyces spoilage in the first place. New technologies such as high-pressure processing, silver nanomaterials and killer toxins shows potential, but these practices still need to be refined for the wine industry and be accepted for commercial use.

As a winemaker, the management of Brettanomyces is a balancing act in the cellar. Brettanomyces is an important microbiological threat to the wine industry, but it is only one component of wine. Winemakers should focus on understanding the risk of Brettanomyces infection and best management practices, rather than adjusting wine style to avoid infection. Based on the available literature, winemakers should aim for dry fermentation and quick malolactic fermentation. A pH of 3,6 after malolactic fermentation is ideal. To limit oxygen exposure barrels should be topped up regularly. A free SO2 of 30mg/L during maturation is recommended, and barrel sanitation is of utmost importance. Wines in barrel should be screen tested every six months for volatile phenols as an indicator for possible Brettanomyces infection. Brettanomyces free wine cannot be guaranteed, even when following recommended practices. Treatment of infected wine depends on the concentration 53

of Brettanomyces population and the matrix of the wine. Generally, adequate filtration followed by the recommended dose of chitosan should prevent further growth. Follow up analysis of volatile phenols and Brettanomyces cells via PCR testing will indicate if the treatment was effective.

54

Addendum A: Permission for figures and tables reproduced

Figure 1: Light microscopy observed different cell morphologies presented by three different Brettanomyces strains at different phases (Louw et al., 2016).

Reprinted from International Journal of Food Microbiology, 238, Louw, M., Du Toit, M., Alexandre, H. and Divol, B. 2016. Comparative morphological characteristics of three Brettanomyces bruxellensis wine strains in the presence/absence of sulfur dioxide. pp79–88. 2016, with permission from Elsevier.

Figure 2: Formation of volatile phenols via the decarboxylation of the hydroxycinnamic acid precursors (Šućur et al., 2016).

Reprinted from Acta Agriculturae Slovenica, 107(2), Šućur, S., Čadež, N. and Košmerl, T., Volatile phenols in wine: Control measures of Brettanomyces/Dekkera yeasts. pp.453-472, 2016. Open access article

Figure 3: Cellobiose.

Reproduced with permission of Dr. J. David Rawn.

Table 1: Chemical compounds produced by different strains of Brettanomyces and the aromas they produce (Joseph et al., 2017).

© 2017 American Society for Enology and Viticulture. Catalyst 1:12-20.

Figure 4: Generic anthocyanin molecule. The glucose molecule is indicated as "glc", R3' - R7' depict sites of possible combinations of -H, -OH or -OCH3. These combinations determine exact anthocyanin (aurantinidin, cyanidin, delphinidin, europinidin, pelargonidin, malvidin, peonidin, petunidin or rosinidin) (Valavandidis and Vlachogianni, 2013).

Reprinted from Studies in Natural Products Chemistry, Valavandidis, A. and Vlachogianni, T., Chapter 8: Plant polyphenols: Recent Advances in Epidemiological Research and Other Studies on Cancer Prevention. pp 269-295. 2013, with permission from Elsevier.

Figure 5: Enzymatic decarboxylation of hydroxycinnamic acids by Brettanomyces and Saccharomyces (Morata, Loira and Lepe, 2016).

Open access article 55

Figure 6: Enzymatic decarboxylase of amino acids phenyalanine to produce biogenic amines

Image credit: Suzi Smith, https://www.diagnosisdiet.com, used with permission.

Figure 7: Brettanomyces aroma wheel (Joseph et al., 2017).

© 2017 American Society for Enology and Viticulture. Catalyst 1:12-20.

Table 2: Free SO2 required for 0,6 and 0,8 mg/L molecular SO2 at different pH levels (VinLab Technical Guide).

Reproduced with permission from VinLab.

Figure 8: Viable and culturable population of Brettanomyces bruxellensis strain L02E2 over time without SO2 and with 0.5, 0.9 and 1.1mg/L molecular SO2. Initial populations were a) 10³, b) 10⁴ and c) 10⁵ cells per mLl. (Longin et al., 2016).

Reprinted from Food Research International, 89, Longin, C., Degueurce, C., Julliat, F., Guilloux-Benatier, M., Rousseaux, S. and Alexandre, H., Efficiency of population-dependent sulfite against Brettanomyces bruxellensis in red wine., 620–630, 2016, with permission from Elsevier.

Figure 9: Chitosan formation via chitin deacetylation (Younes and Rinaudo, 2015).

Open access article.

Table 3: Growth of wine-related microorganisms in yeast extract-peptone-glycerol medium in the presence of various concentrations of chitosan (Bagder Elmaci et al., 2015).

Reprinted from Antonie Van Leeuwenhoek 107(3), Bağder Elmacı, S., Gülgör, G., Tokatlı, M., Erten, H., İşci, A., and Özçelik, F., Effectiveness of chitosan against wine-related microorganisms, 2016, with permission of Springer Nature.

Table 4: Results obtained by plate counting of the filtrated wine for duplicate tests performed with each filter (Duarte et al., 2017).

Reproduced with permission from American Journal of Enology and Viticulture, © 2017 American Society for Enology and Viticulture. AJEV 68:504-508. 56

Table 5: Yeast cell concentrations found in different samples of 3-year-old barrels collected before and after heat treatment in water (60°C for 19 minutes) (Fabrizio et al., 2015).

Reprinted from Letters in Applied Microbiology, 61(2), Fabrizio, V., Vigentini, I., Parisi, N., Picozzi, C., Compagno, C. and Foschino, R., Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment., pp.186-191, 2015, with permission from John Wiley and Sons.

Table 6: The effect of fining agents on the reduction of total concentration of 4-ethylphenol and 4-ethylguiacol (Milheiro et al., 2016).

Reprinted from Journal of Chromatography B, 1041-1042, Milheiro, J., Filipe-Ribeiroa, L., Cosmea, F. and Nunes, F., A simple, cheap and reliable method for control of 4-ethylphenol and 4-ethylguaiacol in red wines., 183–190, 2016, with permission from Elsevier.

Table 7: The effect of fining agents on the colour intensity, hue, total anthocyanins, total phenols, non-flavonoid phenols, flavonoid phenols, polymerized pigments, coloured anthocyanins and total pigments of both unfined and fined red wine (Milheiro et al., 2016).

Reprinted from Journal of Chromatography B, 1041-1042, Milheiro, J., Filipe-Ribeiroa, L., Cosmea, F. and Nunes, F., A simple, cheap and reliable method for control of 4-ethylphenol and 4-ethylguaiacol in red wines., 183–190, 2016, with permission from Elsevier.

Table 8: Populations of Brettanomyces and acetic acid bacteria (genomic units/mL) in wine naturally contaminated before and after 10 days of treatment with KAgC and chitosan (Izquierdo-Canas et al., 2018).

Reprinted from Journal of Food Science and Technology, 55(5), Izquierdo-Canas, P., Lopez- Martin, R., Garcia-Romero, E., Gonzales-Arenzana, L., Minguez-Sanz, S., Chatonnet, P., Palacios-Garcia, A. and Puig-Pujol, A., 1823-1831, 2018, with permission from Springer Nature.

Table 9: Non-Saccharomyces yeast capable of producing killer toxins against Brettanomyces (Adapted from Berbegal et al., 2017).

Open access article which permits unrestricted use

57

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