Fermentation performances and aroma production of non-conventional wine yeasts are influenced by nitrogen preferences

Fermentation performances and aroma production of non-conventional wine yeasts are influenced by... Abstract Saccharomyces cerevisiae is currently the most important yeast involved in food fermentations, particularly in oenology. However, several other yeast species occur naturally in grape must that are highly promising for diversifying and improving the aromatic profile of wines. If the nitrogen requirement of S. cerevisiae has been described in detail, those of non-Saccharomyces yeasts remain poorly studied despite their increasingly widespread use in winemaking. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we explored the fermentation performances, the utilisation of nitrogen sources and the volatile compound production of 10 strains of non-conventional yeasts in pure culture. Two different conditions were tested: one mimicking the grape juice's nitrogen composition and one with all the nitrogen sources at the same level. We highlighted the diversity in terms of nitrogen preference and amount consumed among the yeast strains. Some nitrogen sources (arginine, glutamate, glycine, tryptophan and γ-aminobutyric acid) displayed the largest variations between strains throughout the fermentation. Several non-Saccharomyces strains produced important aroma compounds such as higher alcohols, acetate and ethyl esters in significantly higher quantities than S. cerevisiae. non-conventional yeasts, nitrogen consumption, fermentation performances, aroma production, alcoholic fermentation, wine INTRODUCTION Wine fermentation is a complex microbiological process in which yeasts play a fundamental role. Although Saccharomyces cerevisiae is the main microorganism involved in the alcoholic fermentation, many other species of yeasts belonging to various non-Saccharomyces genera occur in grape juice. Non-Saccharomyces yeasts have gained interest in winemaking due to their positive effects and their properties that can confer novel characteristics to the wines (Fleet 2008; Ciani et al. 2010; Jolly, Varela and Pretorius 2014; Mylona et al. 2016) meeting the consumers’ demand for new wine styles (Lleixà et al. 2016). However, most non-Saccharomyces yeasts do not reach dryness during alcoholic fermentation, thus S. cerevisiae must also be inoculated to complete the fermentation. The presence of two yeasts inoculated at high cell density automatically induces various types of interaction. For instance, previous studies have highlighted nutrient competitions between non-Saccharomyces and S. cerevisiae yeasts during the winemaking process (Medina et al. 2012; Taillandier et al. 2014; Gobert et al. 2017). Nitrogenous compounds are some of the main nutrients for yeast metabolism (growth and aroma production) during alcoholic fermentation and its deficiency is the major cause of stuck and sluggish fermentations (Blateyron and Sablayrolles 2001). In order to optimise growth and fermentation performances of specific wine yeasts, it is important to know how a given nitrogen source affects both these parameters. Over the last decades, the nitrogen requirement and uptake of nitrogen sources by S. cerevisiae have been extensively studied (Albers et al. 1996; Godard et al. 2007; Crépin et al. 2012; Gutiérrez et al. 2012), together with the impact of nitrogen on the fermentative aroma production (Carrau et al. 2008; Vilanova et al. 2012; Mouret et al. 2014a; Rollero et al. 2015). The nitrogenous compounds have been broadly classified according to their order of uptake by the yeast during the first stages of fermentation: prematurely, early and late consumed (Crépin et al. 2012). This uptake is tightly regulated. Early consumed amino acids are transported by specific permeases under Ssy1p-Ptr3p-Ssy5 (SPS)-mediated control that are expressed at the beginning of consumption (Forsberg and Ljungdahl 2001). Most nitrogenous compounds consumed late are transported by permeases under nitrogen catabolite repression (NCR; ter Schure, Riel and Verrips 2000). Therefore, the kinetic characteristics of transporters, as well as SPS and NCR, are likely to be key factors controlling the temporal sequence of uptake of nitrogen compounds and constitute a system highly conserved in S. cerevisiae strains. Compared to S. cerevisiae, the consumption of nitrogen sources by wine-related non-Saccharomyces yeasts has been poorly investigated. In 2012, Medina et al. were amongst the first authors to report on the uptake of nitrogen compounds by two non-Saccharomyces yeasts (Hanseniaspora vineae and Metschnikowia pulcherrima), inoculated as starter cultures. They showed that the amount of nitrogen assimilated by the two yeasts investigated was different and that competition for nutrients had a clear impact on fermentation kinetics, potentially leading to stuck fermentation when the initial Yeast Assimilable Nitrogen (YAN) concentration was too low. A similar result was also reported by Taillandier et al. (2014) when Torulaspora delbrueckii was inoculated together with S. cerevisiae, and Barbosa et al. (2015) confirmed that the presence of another yeast (Hanseniaspora guilliermondii in their study) had a strong influence on the transcriptome of S. cerevisiae, in particular on genes involved in the biosynthesis of vitamins as well as uptake and biosynthesis of amino acids being upregulated. Finally, Kemsawasd et al. (2015) evaluated the impact of individual and mixed nitrogen sources on biomass and fermentation kinetics of several wine-related yeast species (S. cerevisiae, Lachancea thermotolerans, M. pulcherrima, Hanseniaspora uvarum and T. delbrueckii). The latter authors showed that certain nitrogen sources were beneficial for all yeast species while others were only beneficial to specific species. Overall, the influence of nitrogen sources on yeast growth and fermentation performance differed between species, with T. delbrueckii and H. uvarum being the most similar to S. cerevisiae. They also showed that different supplementation regimes should be applied to promote growth and fermentation in different species. Furthermore, yeast species may differ in their preference for amino acids, with a potential impact on nitrogen availability for the other yeasts and on fermentative aroma production. Many aroma compounds produced by S. cerevisiae are directly related and influenced by nitrogen metabolism, such as higher alcohols and their associated fatty acids and esters (Beltran et al. 2005; Swiegers et al. 2005; Barbosa et al. 2009; Rollero et al. 2015). This could explain some of the contributions of non-Saccharomyces yeasts to secondary aroma compounds reported in literature (Rossouw and Bauer 2016; Manzanares, Vallés and Viana 2013). It is important to note that among the indigenous wine yeasts, T. delbrueckii, M. pulcherrima, Candida zemplinina, L. thermotolerans and H. uvarum have been the most largely investigated (Comitini et al. 2011; Medina et al. 2012; Sadoudi et al. 2012; Gobbi et al. 2013; Kemsawasd et al. 2015; Renault et al. 2015; Gobert et al. 2017). Nevertheless, the non-Saccharomyces population associated with grape juice is much more diverse (Setati, Jacobson and Bauer 2015) and warrants further investigation. In this study, we investigated the nitrogen preferences of 10 yeast strains not commonly occurring yet isolated from grape juice. Their fermentation performances (fermentation kinetics and aroma production) compared to those of S. cerevisiae were also assessed under winemaking conditions or when each nitrogen source (individual amino acids and ammonium) provided the same amount of assimilable nitrogen in order to overcome a possible impact of the availability of the nitrogen sources on the consumption. The results obtained in this work should extend the understanding of the nitrogen metabolism of wine-associated yeasts and pave the way to better understand the possible nutrient competition during co- or sequential inoculation with S. cerevisiae. MATERIALS AND METHODS Yeasts strains and preculture conditions Ten non-conventional wine yeasts strains isolated from grape juices in South Africa and France were used in this study (Table 1). The commercial wine strain Saccharomyces cerevisiae Lalvin EC1118® (Lallemand SA, Montreal, Canada) was used as a control yeast. The cryopreserved yeast cultures were thawed at room temperature and streaked on yeast peptone dextrose (YPD) agar (Biolab-Merck, Modderfontein, South Africa). Starter cultures of all yeast strains were prepared by inoculating a single colony into 5 ml YPD broth for each strain. The cultures were incubated at 30°C on a test tube rotating wheel for 24 h. These starter cultures were used to inoculate YPD precultures at an initial cell density of 1 × 106 cells/ml, which were incubated at 30°C on a test tube rotating wheel for 9 h. In an attempt to deplete the nitrogen present in the cells, as described by Johnston, Singer and McFarlane (1977), the yeasts were incubated in YNB containing neither amino acid nor ammonium (Difco Laboratories, Detroit, MI, USA) supplemented with 20 g/l of glucose at 30°C on a test tube rotating wheel until growth ceased. The cell population was monitored by measuring optical density at 600 nm. All the strains were inoculated from this preculture at 1 × 106 cells/ml in the synthetic grape juice-like medium. Table 1. Origins of the yeast strain studied and their H2S production. Strain Species Geographical origin Collection H2Sa IWBT Y951 Pichia burtonii South Africa IWBT 1 IWBT Y963 Hyphopichia pseudoburtonii South Africa IWBT 2 IWBT Y826 Zygoascus meyerae South Africa IWBT 1 IWBT Y1084 Zygoascus meyerae South Africa IWBT 1 IWBT Y934 Wickerhamomyces anomalus South Africa IWBT 4 IWBT Y517 Wickerhamomyces anomalus South Africa IWBT 4 MTF1103L1 Wickerhamomyces anomalus France Lallemand 4 IWBT Y885 Kluyveromyces marxianus South Africa IWBT 2 IWBT RO88 Saccharomyces paradoxus South Africa IWBT 2 Uvaferm CS2 Saccharomyces kudriavzevii × Saccharomyces cerevisiae France Lallemand 3 Lalvin EC1118 Saccharomyces cerevisiae France Lallemand 2 Strain Species Geographical origin Collection H2Sa IWBT Y951 Pichia burtonii South Africa IWBT 1 IWBT Y963 Hyphopichia pseudoburtonii South Africa IWBT 2 IWBT Y826 Zygoascus meyerae South Africa IWBT 1 IWBT Y1084 Zygoascus meyerae South Africa IWBT 1 IWBT Y934 Wickerhamomyces anomalus South Africa IWBT 4 IWBT Y517 Wickerhamomyces anomalus South Africa IWBT 4 MTF1103L1 Wickerhamomyces anomalus France Lallemand 4 IWBT Y885 Kluyveromyces marxianus South Africa IWBT 2 IWBT RO88 Saccharomyces paradoxus South Africa IWBT 2 Uvaferm CS2 Saccharomyces kudriavzevii × Saccharomyces cerevisiae France Lallemand 3 Lalvin EC1118 Saccharomyces cerevisiae France Lallemand 2 aIn a scale from 1 (no production of H2S) to 5 as described in Comitini et al. (2011). View Large Table 1. Origins of the yeast strain studied and their H2S production. Strain Species Geographical origin Collection H2Sa IWBT Y951 Pichia burtonii South Africa IWBT 1 IWBT Y963 Hyphopichia pseudoburtonii South Africa IWBT 2 IWBT Y826 Zygoascus meyerae South Africa IWBT 1 IWBT Y1084 Zygoascus meyerae South Africa IWBT 1 IWBT Y934 Wickerhamomyces anomalus South Africa IWBT 4 IWBT Y517 Wickerhamomyces anomalus South Africa IWBT 4 MTF1103L1 Wickerhamomyces anomalus France Lallemand 4 IWBT Y885 Kluyveromyces marxianus South Africa IWBT 2 IWBT RO88 Saccharomyces paradoxus South Africa IWBT 2 Uvaferm CS2 Saccharomyces kudriavzevii × Saccharomyces cerevisiae France Lallemand 3 Lalvin EC1118 Saccharomyces cerevisiae France Lallemand 2 Strain Species Geographical origin Collection H2Sa IWBT Y951 Pichia burtonii South Africa IWBT 1 IWBT Y963 Hyphopichia pseudoburtonii South Africa IWBT 2 IWBT Y826 Zygoascus meyerae South Africa IWBT 1 IWBT Y1084 Zygoascus meyerae South Africa IWBT 1 IWBT Y934 Wickerhamomyces anomalus South Africa IWBT 4 IWBT Y517 Wickerhamomyces anomalus South Africa IWBT 4 MTF1103L1 Wickerhamomyces anomalus France Lallemand 4 IWBT Y885 Kluyveromyces marxianus South Africa IWBT 2 IWBT RO88 Saccharomyces paradoxus South Africa IWBT 2 Uvaferm CS2 Saccharomyces kudriavzevii × Saccharomyces cerevisiae France Lallemand 3 Lalvin EC1118 Saccharomyces cerevisiae France Lallemand 2 aIn a scale from 1 (no production of H2S) to 5 as described in Comitini et al. (2011). View Large Fermentation condition and sampling Fermentations were carried out in synthetic medium (SM) that simulates standard grape juice as described by Bely, Sablayrolles and Barre (1990) with some modifications. The SM used in this study contained 230 g/l of sugar (115 g/l of glucose and 115 g/l of fructose); 2.5 g/l of potassium L-tartrate; 3 g/l of malic acid; 0.2 g/l of citric acid; 1.14 g/l of potassium hydrogen phosphate; 0.44 g/l of magnesium sulphate heptahydrate; 1.23 g/l of calcium chloride dihydrate; vitamins (mg/l): myo-inositol (100), calcium pantothenate (1), thiamin hydrochloride (0.5), nicotinic acid (2), pyridoxine hydrochloride (2), biotin (0.125), PABA.K (para-aminobenzoate acid K; 0.2), riboflavin (0.2), folic acid (0.2); trace elements (μg/l): manganese (II) chloride tetrahydrate (200), zinc chloride (135), iron chloride (30), copper chloride (15), boric acid (5), cobalt nitrate hexahydrate (1), sodium molybdate dihydrate (25), potassium iodate (10). Nitrogen (200 mg/l of assimilable nitrogen) was supplied as one of various mixtures of amino acids along with NH4Cl (Table 2). The reference medium, SM200, simulated the composition of a typical grape juice, and in SM200E medium, all the nitrogen compounds were included at equivalent amounts of nitrogen (9.5 mgN/l for each compound). Table 2. Initial concentrations of ammonium and amino acids in the media used in this study. Nitrogen compounds SM200 (mg/l) SM200E (mg/l) Proline 288.3 61.5 Alanine 68.3 47.8 Arginine 175.7 23.3 Asparagine 25.0 35.4 Aspartate 20.9 71.4 Cystine 6.2 64.9 GABA 100.0 73.1 Glutamine 237.7 39.1 Glutamate 56.7 78.9 Glycine 8.6 40.3 Histidine 15.4 27.7 Isoleucine 15.4 70.1 Leucine 22.8 70.1 Lysine 0.6 39.1 Methionine 14.8 79.8 Phenylalanine 17.9 88.2 Serine 36.9 56.4 Threonine 35.7 63.5 Tryptophane 84.4 54.7 Tyrosine 8.6 97.4 Valine 20.9 62.5 NH4+ 214.0 38.0 Nitrogen compounds SM200 (mg/l) SM200E (mg/l) Proline 288.3 61.5 Alanine 68.3 47.8 Arginine 175.7 23.3 Asparagine 25.0 35.4 Aspartate 20.9 71.4 Cystine 6.2 64.9 GABA 100.0 73.1 Glutamine 237.7 39.1 Glutamate 56.7 78.9 Glycine 8.6 40.3 Histidine 15.4 27.7 Isoleucine 15.4 70.1 Leucine 22.8 70.1 Lysine 0.6 39.1 Methionine 14.8 79.8 Phenylalanine 17.9 88.2 Serine 36.9 56.4 Threonine 35.7 63.5 Tryptophane 84.4 54.7 Tyrosine 8.6 97.4 Valine 20.9 62.5 NH4+ 214.0 38.0 SM200: nitrogen composition mimicking the average composition of grape juices. SM200E: all the nitrogen sources are provided in equal amount. GABA: γ-aminobutyric acid. View Large Table 2. Initial concentrations of ammonium and amino acids in the media used in this study. Nitrogen compounds SM200 (mg/l) SM200E (mg/l) Proline 288.3 61.5 Alanine 68.3 47.8 Arginine 175.7 23.3 Asparagine 25.0 35.4 Aspartate 20.9 71.4 Cystine 6.2 64.9 GABA 100.0 73.1 Glutamine 237.7 39.1 Glutamate 56.7 78.9 Glycine 8.6 40.3 Histidine 15.4 27.7 Isoleucine 15.4 70.1 Leucine 22.8 70.1 Lysine 0.6 39.1 Methionine 14.8 79.8 Phenylalanine 17.9 88.2 Serine 36.9 56.4 Threonine 35.7 63.5 Tryptophane 84.4 54.7 Tyrosine 8.6 97.4 Valine 20.9 62.5 NH4+ 214.0 38.0 Nitrogen compounds SM200 (mg/l) SM200E (mg/l) Proline 288.3 61.5 Alanine 68.3 47.8 Arginine 175.7 23.3 Asparagine 25.0 35.4 Aspartate 20.9 71.4 Cystine 6.2 64.9 GABA 100.0 73.1 Glutamine 237.7 39.1 Glutamate 56.7 78.9 Glycine 8.6 40.3 Histidine 15.4 27.7 Isoleucine 15.4 70.1 Leucine 22.8 70.1 Lysine 0.6 39.1 Methionine 14.8 79.8 Phenylalanine 17.9 88.2 Serine 36.9 56.4 Threonine 35.7 63.5 Tryptophane 84.4 54.7 Tyrosine 8.6 97.4 Valine 20.9 62.5 NH4+ 214.0 38.0 SM200: nitrogen composition mimicking the average composition of grape juices. SM200E: all the nitrogen sources are provided in equal amount. GABA: γ-aminobutyric acid. View Large Instead of adding ergosterol (yeast sterol) as in Bely, Sablayrolles and Barre (1990), SM was initially supplemented with anaerobic factors composed of phytosterols (85451, Sigma Aldrich, Saint-Louis, MO, USA), sterols naturally present in the grape juice (Le Fur et al. 1994), at a final concentration of 10 mg/l. The stock solution was composed of 5 g/l of phytosterols dissolved in Tween 80 and absolute ethanol (1:1, v/v). The pH of the SM was adjusted to 3.3 with potassium hydroxide (Saarchem, Krugersdorp, South Africa). The trace elements, vitamins, nitrogen sources and anaerobic factors were filtered through a 0.22-μm syringe filter (Starlab Scientific, Cape Town, South Africa) and added into the autoclaved SM. Each fermentation was performed in triplicate. The fermentations were carried out in cylindrical fermenters of 3.5 cm diameter and 10 cm height. The fermenters contained 70 ml of medium, so that the headspace occupied 30% of the volume of the fermenters. In order to maintain anaerobiosis, the fermenters were equipped with fermentation locks filled with water, at 25°C, with orbital agitation (125 rpm). The fermentation progress was monitored by determination of CO2 release extrapolated from the measurement of the weight loss throughout the process. At 24 h, 48 h and at the end of each fermentation, different samples were centrifuged at 4000 g for 5 min, after which the supernatants were filtered through a 0.22-μm syringe filter (Starlab Scientific, Cape Town, South Africa) and stored at –20°C for further chemical analysis. Monitoring of yeast population The yeast cell populations were monitored by plating the appropriate dilutions onto YPD agar. Plates were incubated at 30°C, generally for 2 to 3 days, until colonies were formed. Quantification of residual sugars and ammonium by enzymatic assays To quantify the residual glucose, fructose and ammonium concentrations, 400 μl of filtered sample was enzymatically analysed using the Arena 20XT (Thermo Fisher Scientific, Waltham, MA), which makes use of automated spectrophotometric readings to determine the concentrations of the various compounds. The different enzymatic assay kits are: Enzytec Fluid D-Glucose (Id-No: 5140, R-BiopharmAG, Germany) for glucose, Enzytec Fluid D-Fructose (Id-No: 5120, R-BiopharmAG, Germany) for fructose and Enzytec Fluid Ammonia (Id-No: 5390, R-BiopharmAG, Germany) for ammonium. Quantification of individual amino acids Amino acids quantification was performed by high performance liquid chromatography, Agilent 1100 (Agilent Technologies, Waldbronn, Germany) by pre-column derivatisation and fluorescence detection based upon a method previously described (Henderson and Brooks 2010) with some modifications to the derivatisation and injection. A Poroshell HPH-C18 column (4.6 mm length × 150 mm internal diameter, 2.7 μm particle size; Agilent Technologies) was used following derivatisation of the amino acids. Derivatisation was performed using three different reagents: iodoacetic acid (Sigma Aldrich) for cysteine, o-phthaldialdehyde (Sigma Aldrich) for primary amino acids and fluorenylmethyloxycarbonyl chloride (Sigma Aldrich) for secondary amino acids. Internal standards, norvaline (Sigma Aldrich) and sarcosine (Sigma Aldrich) were spiked to each sample prior to derivatisation. One millilitre of each filtered sample was analysed. Analysis of major volatile compounds The quantification of major volatiles (i.e. a selection of higher alcohols, acetate esters, fatty acids, fatty acid ethyl esters) was carried out by gas chromatography equipped with a flame ionisation detector using the Agilent GC System HP 6890 Series (Agilent, Little Falls, Wilmington, USA) as described by Louw et al. (2009) with minor modifications. Five millilitres of each of the filtered samples were used with 100 μl of 4-methyl-2-pentanol (internal standard). Diethyl ether (1 ml) was added to the mixture, which was then placed in an ultrasonic bath for 5 min to extract the volatile compounds. Thereafter, the samples were centrifuged at 4000 g for 3 min. Sodium sulphate was added to remove any water from the non-polar layer. HPChemstation software was used for data analysis. Hydrogen sulphide production Hydrogen sulphide formation was also evaluated using Difco BiGGY agar (Difco Laboratories, Detroit, MI, USA). In this medium, H2S formation is correlated with colony colour according to Comitini et al. (2011). The following arbitrary scale was used: 1, white colour (no production); 2, light brown; 3, brown; 4, dark brown; 5, black. The strain Metschnikowia pulcherrima Flavia® (Lallemand SAS, Montreal, Canada) was used as negative control. Statistical analysis Data analysis was performed with a statistical treatment and graphically (boxplots) represented using the R software version 3.3.3. (http://cran.r-project.org/). The boxplots were designed according the method described by Tukey (1977). The outliers (or atypical values) are the values located beyond adjacent values. The adjacent values are calculated using 1.5 times the interquartile space (the distance between the 1st and the 3rd quartile). The principal component analysis (PCA) was carried out with the FactoMineR package (Le, Josse and Husson 2008). A two-way analysis of variance (ANOVA) was performed to describe the diversity between the different yeasts and the two nitrogen conditions with the production of aroma compounds as a factor to detect a global effect at a P-value threshold of 0.05. For each parameter, normality of residual distributions and homogeneity of variance were studied using standard diagnostic graphics; no violation of the assumptions was detected. RESULTS Nitrogen starvation of yeasts In order to assess the nitrogen requirement and preferences of the different yeast strains during alcoholic fermentation, their intracellular reserves in nitrogen were depleted before their inoculation in the fermentation medium. The time at which growth ceased in a medium devoid of nitrogen sources (here YNB medium) was considered as the time when intracellular nitrogen reserves were depleted (Johnston, Singer and McFarlane 1977). Depending on the strains, growth stopped after a period ranging from 4 to 8 h (Fig. S1, Supporting Information). From these results, the yeasts were inoculated in the fermentation medium after a preculture in YNB lasting 4 h for Pichia burtonii and Hyphopichia pseudoburtonii, 6 h for Wickerhamomyces anomalus IWBT Y934 and MTF1103L1 and Kluyveromyces marxianus or 8 h for W. anomalus IWBT Y517, Zygoascus meyerae IWBT Y826 and Y1084, Saccharomyces paradoxus, Saccharomyces kudriavzevii × Saccharomyces cerevisiae and S. cerevisiae plus 2 h to ensure that the starvation was as effective as possible. Fermentation kinetics and growth of non-conventional wine yeast strains We first compared the fermentation kinetics and the growth of the 10 non-conventional wine yeast strains. All fermentations were performed in triplicate, in a chemically defined medium (SM200) with 200 mg/l of YAN. YAN comprises a mixture of various amino acids and ammonium ions at various concentrations (Table 2). The calculation of the YAN level excludes proline because S. cerevisiae cannot metabolise this amino acid under the anaerobic conditions occurring during alcoholic fermentation. This amino acid was nevertheless included in the experiment as it is the amino acid with the highest concentration in grape juice. As expected, only the fermentation performed with S. cerevisiae reached dryness (residual sugars <2 g/l). However, the capacity to consume sugars varied significantly among the yeasts studied (from 23 g/l to 195 g/l of residual sugars for S. kudriavzevii × S. cerevisiae UCS2 and Z. meyerae 1084, respectively). Both growth and fermentation kinetics differed considerably between strains (Fig. 1a, Table 3). Indeed, the yeasts belonging to the Saccharomyces genus displayed the highest fermentation rates without any lag phase, while the non-Saccharomyces yeasts observed a distinct lag phase (around 41 h). The maximum yeast population varied between 3.1 × 107 and 1.1 × 108 cfu/ml (for P. burtonii, and S. paradoxus and S. cerevisiae, respectively). For all the non-Saccharomyces strains studied, fermentations got stuck but stopped before any population decline could be observed (data not shown). Figure 1. View largeDownload slide Fermentation kinetics of 10 non-conventional wine yeast strains and S. cerevisiae (A) in SM200 where the nitrogen sources mimicking what was found in grape juice, (B) in SM200E where all the nitrogen sources introduced at equivalent assimilable nitrogen amount, (C) comparison between the two media for S. paradoxus, S. kudriavzevii × S. cerevisiae and S. cerevisiae, (D) comparison between the two media for the three W. anomalus strains P. burtonii (Pb) IWBT Y951, H. pseudoburtonii (Hp) IWBT Y963, Z. meyerae (Zm) IWBT Y826 and IWBT Y1084, W. anomalus (Wa) IWBT Y934, IWBT Y517 and MTF 1103L1, K. marxianus (Km) IWBT Y885, S. paradoxus (Sp) IWBT RO88, S. kudriavzevii × S. cerevisiae (Sk) Uvaferm CS2, S. cerevisiae (Sc) Lalvin EC1118®. Figure 1. View largeDownload slide Fermentation kinetics of 10 non-conventional wine yeast strains and S. cerevisiae (A) in SM200 where the nitrogen sources mimicking what was found in grape juice, (B) in SM200E where all the nitrogen sources introduced at equivalent assimilable nitrogen amount, (C) comparison between the two media for S. paradoxus, S. kudriavzevii × S. cerevisiae and S. cerevisiae, (D) comparison between the two media for the three W. anomalus strains P. burtonii (Pb) IWBT Y951, H. pseudoburtonii (Hp) IWBT Y963, Z. meyerae (Zm) IWBT Y826 and IWBT Y1084, W. anomalus (Wa) IWBT Y934, IWBT Y517 and MTF 1103L1, K. marxianus (Km) IWBT Y885, S. paradoxus (Sp) IWBT RO88, S. kudriavzevii × S. cerevisiae (Sk) Uvaferm CS2, S. cerevisiae (Sc) Lalvin EC1118®. Table 3. Maximal population reached during alcoholic fermentation (×106 cfu/ml). Strains SM200 SM200E P. burtonii IWBT Y951 31.0 ± 1.41 30.5 ± 2.67 H. pseudoburtonii IWBT Y963 34.7 ± 2.83 35.0 ± 3.21 Z. meyerae IWBT Y826 57.5 ± 3.54 61.3 ± 2.85 Z. meyerae IWBT Y1084 47.5 ± 3.51 50.4 ± 1.97 W. anomalus IWBT Y934 70.8 ± 2.84 71.0 ± 6.33 W. anomalus IWBT Y517 53.0 ± 9.93 61.8 ± 9.01 W. anomalus MTF1103L1 33.0 ± 2.12 32.0 ± 8.32 K. marxianus IWBT Y885 61.0 ± 1.41 60.0 ± 2.22 S. paradoxus IWBT RO88 110 ± 6.49 110 ± 3.43 S. kudriavzevii × S. cerevisiae Uvaferm CS2 108 ± 7.07 109 ± 6.03 S. cerevisiae Lalvin EC1118 110 ± 6.36 111 ± 7.32 Strains SM200 SM200E P. burtonii IWBT Y951 31.0 ± 1.41 30.5 ± 2.67 H. pseudoburtonii IWBT Y963 34.7 ± 2.83 35.0 ± 3.21 Z. meyerae IWBT Y826 57.5 ± 3.54 61.3 ± 2.85 Z. meyerae IWBT Y1084 47.5 ± 3.51 50.4 ± 1.97 W. anomalus IWBT Y934 70.8 ± 2.84 71.0 ± 6.33 W. anomalus IWBT Y517 53.0 ± 9.93 61.8 ± 9.01 W. anomalus MTF1103L1 33.0 ± 2.12 32.0 ± 8.32 K. marxianus IWBT Y885 61.0 ± 1.41 60.0 ± 2.22 S. paradoxus IWBT RO88 110 ± 6.49 110 ± 3.43 S. kudriavzevii × S. cerevisiae Uvaferm CS2 108 ± 7.07 109 ± 6.03 S. cerevisiae Lalvin EC1118 110 ± 6.36 111 ± 7.32 SM200: nitrogen composition mimicking the average composition of grape juices. SM200E: all the nitrogen sources are provided in equal amount. View Large Table 3. Maximal population reached during alcoholic fermentation (×106 cfu/ml). Strains SM200 SM200E P. burtonii IWBT Y951 31.0 ± 1.41 30.5 ± 2.67 H. pseudoburtonii IWBT Y963 34.7 ± 2.83 35.0 ± 3.21 Z. meyerae IWBT Y826 57.5 ± 3.54 61.3 ± 2.85 Z. meyerae IWBT Y1084 47.5 ± 3.51 50.4 ± 1.97 W. anomalus IWBT Y934 70.8 ± 2.84 71.0 ± 6.33 W. anomalus IWBT Y517 53.0 ± 9.93 61.8 ± 9.01 W. anomalus MTF1103L1 33.0 ± 2.12 32.0 ± 8.32 K. marxianus IWBT Y885 61.0 ± 1.41 60.0 ± 2.22 S. paradoxus IWBT RO88 110 ± 6.49 110 ± 3.43 S. kudriavzevii × S. cerevisiae Uvaferm CS2 108 ± 7.07 109 ± 6.03 S. cerevisiae Lalvin EC1118 110 ± 6.36 111 ± 7.32 Strains SM200 SM200E P. burtonii IWBT Y951 31.0 ± 1.41 30.5 ± 2.67 H. pseudoburtonii IWBT Y963 34.7 ± 2.83 35.0 ± 3.21 Z. meyerae IWBT Y826 57.5 ± 3.54 61.3 ± 2.85 Z. meyerae IWBT Y1084 47.5 ± 3.51 50.4 ± 1.97 W. anomalus IWBT Y934 70.8 ± 2.84 71.0 ± 6.33 W. anomalus IWBT Y517 53.0 ± 9.93 61.8 ± 9.01 W. anomalus MTF1103L1 33.0 ± 2.12 32.0 ± 8.32 K. marxianus IWBT Y885 61.0 ± 1.41 60.0 ± 2.22 S. paradoxus IWBT RO88 110 ± 6.49 110 ± 3.43 S. kudriavzevii × S. cerevisiae Uvaferm CS2 108 ± 7.07 109 ± 6.03 S. cerevisiae Lalvin EC1118 110 ± 6.36 111 ± 7.32 SM200: nitrogen composition mimicking the average composition of grape juices. SM200E: all the nitrogen sources are provided in equal amount. View Large The fermentations were repeated with a medium containing all the nitrogen sources at the same level of assimilable nitrogen (SM200E). Overall, the ranking of the strains in terms of fermentation performances remained similar in the two nitrogen conditions (Fig. 1b). However, specific differences can be highlighted. For instance, S. cerevisiae seemed to ferment more slowly with a fermentation pattern similar to that of S. paradoxus under the SM200E conditions (Fig. 1c). On the contrary, the S. cerevisiae × S. kudriavzevii UCS2 strain was able to ferment to dryness when all the nitrogen sources provided the same amount of assimilable nitrogen (Fig. 1c). Interestingly, the three W. anomalus strains displayed different behaviours (Fig. 1d). Indeed, W. anomalus IWBT Y517 fermented better (40 g CO2/l released vs 30 g CO2/l previously), while the amount of sugar consumed by W. anomalus IWBT Y934 was about 20% lower (83 g/l vs 119 g/l). The fermentation kinetics of the last strain, MTF1101L1, was not affected by the nitrogen conditions. The maximal population reached by the strains in the different nitrogen conditions remained quite similar to that achieved under the previous conditions for all the strains (Table 3). Nitrogen source consumption and preferences The nitrogen uptake of the 10 non-conventional yeasts was evaluated after 24 h, 48 h and when the fermentation was terminated. The nitrogen consumption of S. cerevisiae Lalvin EC1118® was used as a reference. For all the strains, nitrogen compounds were used sequentially throughout the growth phase. However, the order of consumption of the various sources of assimilable nitrogen was strain dependent. An overview of the nitrogen uptake of all strains under oenological conditions and for the SM200E are shown in Fig. 2a and b, respectively (concentrations for each amino acid are presented in Tables S1–S6, Supporting Information). Here, the consumption of each individual amino acid and ammonium for all the strains was considered, so the overall diversity in the nitrogen utilisation and preferences among the 11 strains is illustrated. Depending on the nitrogen conditions (SM200 or SM200E), different patterns of nitrogen uptake were observed. The consumption of the different nitrogen sources seemed to be more uniform when all the sources were provided in equal amount. By contrast, under the oenological conditions, more significant differences between the strains were observed. In both media, some amino acids were consumed later for most of the strains (glycine, alanine, tyrosine and γ-aminobutyric acid (GABA) for SM200E, and arginine, glycine and GABA for SM200), which was consistent with the few variations observed on the boxplots after 24 h (Fig. 2a and b). These amino acids appeared to be non-preferred nitrogen sources for all the strains. On the other hand, under oenological conditions, some amino acids (methionine, phenylalanine, leucine and isoleucine) displayed large variations in terms of preferences between the different yeast strains (Fig. 2a). For both nitrogen conditions, at the end of fermentation, lysine was the first consumed amino acid and it was entirely consumed by all the strains (Fig. 2a and b). The branched chain amino acids were also totally consumed by the end of fermentation but only under oenological conditions (Fig. 2a). Figure 2. View largeDownload slide Nitrogen uptake. Boxplot of nitrogen consumption during the alcoholic fermentation in (A) oenological nitrogen conditions and (B) when the nitrogen sources provide the same amount of assimilable nitrogen. Red bars represent the consumption after 24 h, green bars indicate the uptake after 48 h and blue bars at the end of fermentation. Purple stars indicate the S. cerevisiae’s consumption. Black dots represent the outliers. Figure 2. View largeDownload slide Nitrogen uptake. Boxplot of nitrogen consumption during the alcoholic fermentation in (A) oenological nitrogen conditions and (B) when the nitrogen sources provide the same amount of assimilable nitrogen. Red bars represent the consumption after 24 h, green bars indicate the uptake after 48 h and blue bars at the end of fermentation. Purple stars indicate the S. cerevisiae’s consumption. Black dots represent the outliers. Despite common characteristics in their consumption of nitrogen sources, these different yeasts displayed some specificities (identified as outliers on Fig. 2). As expected, under oenological conditions, at the end of fermentation, S. cerevisiae had consumed all the assimilable nitrogen present in the medium, except GABA (less than 50% was taken up), while Z. meyerae and S. kudriavzevii × S. cerevisiae consumed 60% of the initial concentration of GABA (Table S3, Supporting Information). However, interestingly, during the early stages of fermentation, S. cerevisiae did not always consume the highest concentration of amino acids (Tables S1 and S2, Supporting Information). In particular, the consumption of arginine and glycine at 48 h by S. cerevisiae was lower than the median (Table S2, Supporting Information). At this stage, the two Z. meyerae strains consumed more arginine (45% of initial content) and S. kudriavzevii × S. cerevisiae and W. anomalus IWBT Y517 and MTF1103L1 took more glycine up than S. cerevisiae (48%, 33% and 43%, respectively, and 20% for S. cerevisiae; Table S2, Supporting Information). Concerning K. marxianus, during the first 48 h, this yeast displayed similar requirement for nitrogen sources (around 70% of initial nitrogen was consumed after 48 h) than S. cerevisiae, except for ammonium that was sparsely consumed by K. marxianus (20% vs 60% for S. cerevisiae; Tables S1 and S2, Supporting Information). For the majority of the nitrogen sources, the variations of their uptake among the strains decreased with the fermentation progress (Tables S1–S3, Supporting Information). However, this variation increased for arginine, glutamate, glycine, tryptophan and GABA. For NH4, lysine, methionine, isoleucine, leucine and phenylalanine, no variation were observed among the strains at the end of fermentation. As observed in the previous conditions, S. cerevisiae exhausted the assimilable nitrogen (except for GABA) when all the nitrogen sources were equal (Table S6, Supporting Information). However, contrary to the previous conditions, S. cerevisiae always displayed the highest consumption for all nitrogen sources (except for GABA; Tables S4–S6, Supporting Information). The biggest variations at the end of fermentation among the 11 strains appeared for asparagine, glycine, alanine, tyrosine, methionine, tryptophan and GABA (Table S6, Supporting Information). Fermentative aroma production In order to better characterise these non-conventional strains, the fermentative aroma production was determined at the end of fermentation in the two nitrogen conditions (Fig. 3). In SM200 medium, the production of volatile compounds varied substantially depending mainly on the yeast strains (Fig. 3, in blue; Table S7, Supporting Information). Indeed, S. cerevisiae was characterised by the production of higher alcohols and isoamyl acetate, while K. marxianus displayed the highest production of phenylethyl acetate, isobutyric and isovaleric acids (Fig. 3). The three W. anomalus strains and Z. meyerae IWBT Y1084 showed small concentrations in the whole fermentative aromas, except ethyl acetate for W. anomalus strains (Fig. 3; Table S7, Supporting Information). The other strains seemed to be characterised by their production of medium-chain fatty acids and threonine-derivatives (i.e. propanol and propionic acid; Fig. 3). Interestingly, the three W. anomalus strains displayed a very similar aroma profile (as confirmed by the concentrations of aroma compounds; Table S7, Supporting Information), while the two Z. meyerae strains appeared to be distant from each other on the PCA plot (Fig. 3). Indeed, Y826 showed higher production of most of the aroma compounds than Y1084 (Table S7, Supporting Information). Figure 3. View largeDownload slide Principal component analysis of the fermentative aroma concentrations at the end of fermentation in SM200 (blue) and in SM200E (red). Dark blue: higher alcohols, light blue: acetate esters, grey blue: fusel acids, red: small and medium chain fatty acids, orange: ethyl esters. Figure 3. View largeDownload slide Principal component analysis of the fermentative aroma concentrations at the end of fermentation in SM200 (blue) and in SM200E (red). Dark blue: higher alcohols, light blue: acetate esters, grey blue: fusel acids, red: small and medium chain fatty acids, orange: ethyl esters. In SM200E medium (Fig. 3, in red), the overall aroma profile of each strain was conserved but the change of ratio between nitrogen sources compared to SM200 resulted in a significantly altered production of all the aroma compounds (ANOVA Table S7, Supporting Information). Under this condition (SM200E), the concentrations of higher alcohols and acetate esters were higher for most of the strains, with the notable exceptions of S. cerevisiae, Z. meyerae IWBT Y1084 and in a lesser extent W. anomalus MTF1103L1 for which the production of higher alcohols and their acetates decreased (Table S7, Supporting Information). Overall, the statistical analysis (2-way ANOVA) confirmed that both yeast strains and nitrogen supply impacted the production of aroma compounds (Table S7, Supporting Information). As H2S is one of the main off-flavours produced by yeast during the alcoholic fermentation, its production was assessed on BiGGY agar plates. The semi-quantitative evaluation of H2S production revealed a wide strain variability (Table 1). The three W. anomalus strains were characterised by the highest production of H2S, while P. burtonii and the two Z. meyerae yeasts displayed the lowest production. Nevertheless, these results were obtained on BiGGY agar (solid medium plate assays), which was a rich medium and should be confirmed under oenological conditions (in liquid environment and nutrient limited medium). Indeed, Jiranek, Langridge and Henschke (1995b) highlighted that the BiGGY agar plate assay provided a good indication of the maximum genetically determined sulphite reductase activity of the yeast strains but did not always correlate with the H2S production occurring during wine fermentation where this production was generally lower. DISCUSSION Over the last decades, non-Saccharomyces yeasts have been increasingly used during alcoholic fermentation and their impact on the final quality of wines was assessed in several studies (Fleet 2008; Sadoudi et al. 2012; Comitini et al. 2017; Gobert et al. 2017; Puertas et al. 2017). Nevertheless, in order to better understand their interactions with S. cerevisiae and the resulting overall impact on wine, it is essential to investigate their nutrient requirements, particularly those for nitrogen. Indeed, if the nitrogen preferences of S. cerevisiae have been studied in depth (Jiranek, Langridge and Henschke 1995a; Crépin et al. 2012), these are only partially elucidated for some non-Saccharomyces yeasts occurring in the wine environment (Andorrà et al. 2010; Kemsawasd et al. 2015; Gobert et al. 2017). In the current study, we showed that, overall, fermentation performances and biomass production remained fairly similar in the two nitrogen conditions tested for all the strains. This was previously observed by Crepin et al. (2012) for Saccharomyces cerevisiae. Nevertheless, certain yeasts (i.e. Wickerhamomyces anomalus IWBT Y517 and Y934, S. cerevisiae × Saccharomyces kudriavzevii), depending on their nitrogen preferences and requirements, were affected by the change of the nitrogen composition. An adapted nutrition could improve the fermentation performances of these yeasts and increase their impact on the final product properties. As expected, the assimilation order of nitrogen sources for S. cerevisiae confirmed previously published results (Jiranek, Langridge and Henschke 1995a; Crépin et al. 2012). The other species could be divided into three groups: (i) the strains with similar nature and quantity of nitrogen sources consumed as S. cerevisiae (i.e. S. cerevisiae × S. kudriavzevii, Saccharomyces paradoxus and Kluyveromyces marxianus), (ii) the strains with a very low nitrogen consumption (i.e. Pichia burtonii, Hyphopichia pseudoburtonii and the two Zygoascus meyerae strains) and (iii) the three W. anomalus strains displaying an intermediate pattern of assimilation. The percentage uptake of nitrogen sources correlated well with the biomass production and fermentation performances: the yeasts with the higher consumption of nitrogen produced the higher cell counts and displayed the best fermentation performances overall and vice versa. This was consistent with previous studies (Prior 2017; De Koker 2015; Kemsawasd et al. 2015) where Torulaspora delbrueckii and Lachancea thermotolerans (i.e. two fairly strong fermenters) assimilated nitrogen in a similar manner as S. cerevisiae, while P. kluyveri and Metschnikowia pulcherrima (i.e. two species fermenting poorly under winemaking conditions) displayed a low nitrogen uptake. The nature and amount of nitrogen consumed by the non-Saccharomyces yeasts could impact on S. cerevisiae’s overall fermentation performance in the case of sequential inoculation with these species, as reported previously with the stronger fermenters competing more intensely with S. cerevisiae for nitrogen sources (Medina et al. 2012; de Koker 2015). A specific nutrition, sustaining the nitrogen requirement of S. cerevisiae, could reduce the competition for nitrogen, especially when S. paradoxus and K. marxianus are to be used. On the other hand, the weaker fermenters did not consume at all some nitrogen sources during the first 24 h in a strain-dependent manner (e.g. Z. meyerae IWBT Y826 did not consume glutamine, methionine and lysine, while P. burtonii did not assimilate glutamate, phenylalanine, methionine and alanine) implying an overall limited competition for nitrogen sources with S. cerevisiae. This suggests that these species may display genomic differences in nitrogen metabolism and regulatory systems. For S. cerevisiae, the preferential assimilation of certain nitrogen sources can be explained mainly by the regulation of nitrogen source transport by permeases, including the SPS system (Ljungdahl 2009) and the NCR system (Ljungdahl and Daignan-Fornier 2012). The mechanisms have not been explored yet for non-Saccharomyces yeasts. Since the assimilation of nitrogen sources influences the volatile aroma profile of wine (Jiménez-Martí et al. 2007; Vilanova et al. 2007; Carrau et al. 2008), the production of some major volatile aroma compounds was also investigated. Differences with regard to aroma compound production were observed between different yeast species but also between the strains belonging to the same species. Indeed, it seemed that the consumption of nitrogen sources and the production of fermentative aromas were relatively conserved characteristics within the W. anomalus species, whereas they appeared to be quite dissimilar between Z. meyerae strains thereby highlighting a probable difference in the regulation of nitrogen and aroma metabolisms. However, more strains should be investigated to draw final conclusions regarding intra-species similarities and differences. Furthermore, several studies have reported a direct relation between the formation of higher alcohols and esters by S. cerevisiae and the must nitrogen concentration (Bell and Henschke 2005; Beltran et al. 2005; Hernandez-Orte et al. 2006; Mouret et al. 2014b; Fairbairn et al. 2017). The specific nitrogen requirements of certain non-Saccharomyces yeasts may also influence the final organoleptic profile of wines (Andorrà et al. 2012; de Koker 2015). For instance, W. anomalus IWBT Y517 displayed better fermentation performances associated with higher aroma production in SM200E. However, if the initial nitrogen composition did influence the overall aroma profile, the link between the concentration of amino acids entering the Ehrlich pathway and that of the corresponding volatile compound could not be clearly established in this study, suggesting that certain direct correlations observed by previous authors in S. cerevisiae (Fairbairn et al. 2017) do not apply for all yeast species. Indeed, higher consumption of nitrogen sources was not necessarily correlated with higher production of fermentative aromas. For instance, Z. meyerae IWBT Y826 was one of the best producers of higher alcohols but only consumed a few amount of the corresponding amino acid precursors (leucine, valine and phenylalanine), while K. marxianus produced the highest amount of phenylethyl acetate while consuming the same amount of phenylalanine than S. cerevisiae. Indeed, the production of higher alcohols and esters by yeasts is a highly complex process dependent on a number of parameters. Higher alcohols are produced either from their amino acid precursors or by central carbon metabolism (Styger, Prior and Bauer 2011; Mouret et al. 2014a,b,c). Recent studies (Crépin et al. 2017; Rollero et al. 2017) demonstrated that higher alcohols are mainly produced through the carbon metabolism (only 5% emanated from amino acid catabolism) in S. cerevisiae. Our combined results strongly suggest, especially for Z. meyerae, that the contribution of nitrogen metabolism to the higher alcohol synthesis was probably lower than in S. cerevisiae. Nevertheless, when all the nitrogen sources were provided in equal amounts (SM200E), higher alcohols and/or esters were usually produced in higher amounts, but in that case, the amino acid precursors (leucine, valine, phenylalanine) were provided in larger quantities (between 3 and 5.5 times more), which can explain this increase. Concerning the H2S production, the three strains of W. anomalus were identified as the highest producers. As the majority of H2S produced by yeast during wine fermentation is from the precursor of the sulphur-containing amino acids cysteine and methionine, which are required for yeast growth (Thomas and Surdin-Kerjan 1997), and grape juice usually contains very low concentrations of cysteine and methionine (<20 mg/l), we can speculate that the requirement in sulphur-containing amino acids in W. anomalus was higher than that of the other yeasts. To conclude, this study highlighted that even if non-Saccharomyces yeasts, during single culture fermentations, have lower fermentation abilities than S. cerevisiae, some non-conventional yeasts, S. paradoxus and K. marxianus, showed potential as ‘strong’ fermenters under the conditions tested. Nevertheless, their high consumption of nitrogenous compounds might comprise the success of a co-fermentation with S. cerevisiae. This work provides relevant information that could be a key to the promotion of these yeasts’ growth utilisation through the formulation of adapted nitrogen supplementation. On the other hand, the use of some of the weaker fermenters would lower the competition for nutrients and warrant fermentation success. Moreover, these strains can participate in the elaboration of a more complex wine in terms of organoleptic properties. Nevertheless, using weaker fermenter yeasts can lead to a longer lag phase, which lengthens the fermentation duration but also can lead to the development of spoilage microorganisms that could impact the quality of wines. It is therefore necessary to find a compromise between the competition for nutrients and the guarantee of a qualitative final wine. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. FUNDING This work was funding by Lallemand SAS and Stellenbosch University. Conflicts of interest. None declare. 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Google Scholar CrossRef Search ADS © FEMS 2018. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Yeast Research Oxford University Press

Fermentation performances and aroma production of non-conventional wine yeasts are influenced by nitrogen preferences

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Blackwell
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© FEMS 2018.
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1567-1356
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1567-1364
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10.1093/femsyr/foy055
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Abstract

Abstract Saccharomyces cerevisiae is currently the most important yeast involved in food fermentations, particularly in oenology. However, several other yeast species occur naturally in grape must that are highly promising for diversifying and improving the aromatic profile of wines. If the nitrogen requirement of S. cerevisiae has been described in detail, those of non-Saccharomyces yeasts remain poorly studied despite their increasingly widespread use in winemaking. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we explored the fermentation performances, the utilisation of nitrogen sources and the volatile compound production of 10 strains of non-conventional yeasts in pure culture. Two different conditions were tested: one mimicking the grape juice's nitrogen composition and one with all the nitrogen sources at the same level. We highlighted the diversity in terms of nitrogen preference and amount consumed among the yeast strains. Some nitrogen sources (arginine, glutamate, glycine, tryptophan and γ-aminobutyric acid) displayed the largest variations between strains throughout the fermentation. Several non-Saccharomyces strains produced important aroma compounds such as higher alcohols, acetate and ethyl esters in significantly higher quantities than S. cerevisiae. non-conventional yeasts, nitrogen consumption, fermentation performances, aroma production, alcoholic fermentation, wine INTRODUCTION Wine fermentation is a complex microbiological process in which yeasts play a fundamental role. Although Saccharomyces cerevisiae is the main microorganism involved in the alcoholic fermentation, many other species of yeasts belonging to various non-Saccharomyces genera occur in grape juice. Non-Saccharomyces yeasts have gained interest in winemaking due to their positive effects and their properties that can confer novel characteristics to the wines (Fleet 2008; Ciani et al. 2010; Jolly, Varela and Pretorius 2014; Mylona et al. 2016) meeting the consumers’ demand for new wine styles (Lleixà et al. 2016). However, most non-Saccharomyces yeasts do not reach dryness during alcoholic fermentation, thus S. cerevisiae must also be inoculated to complete the fermentation. The presence of two yeasts inoculated at high cell density automatically induces various types of interaction. For instance, previous studies have highlighted nutrient competitions between non-Saccharomyces and S. cerevisiae yeasts during the winemaking process (Medina et al. 2012; Taillandier et al. 2014; Gobert et al. 2017). Nitrogenous compounds are some of the main nutrients for yeast metabolism (growth and aroma production) during alcoholic fermentation and its deficiency is the major cause of stuck and sluggish fermentations (Blateyron and Sablayrolles 2001). In order to optimise growth and fermentation performances of specific wine yeasts, it is important to know how a given nitrogen source affects both these parameters. Over the last decades, the nitrogen requirement and uptake of nitrogen sources by S. cerevisiae have been extensively studied (Albers et al. 1996; Godard et al. 2007; Crépin et al. 2012; Gutiérrez et al. 2012), together with the impact of nitrogen on the fermentative aroma production (Carrau et al. 2008; Vilanova et al. 2012; Mouret et al. 2014a; Rollero et al. 2015). The nitrogenous compounds have been broadly classified according to their order of uptake by the yeast during the first stages of fermentation: prematurely, early and late consumed (Crépin et al. 2012). This uptake is tightly regulated. Early consumed amino acids are transported by specific permeases under Ssy1p-Ptr3p-Ssy5 (SPS)-mediated control that are expressed at the beginning of consumption (Forsberg and Ljungdahl 2001). Most nitrogenous compounds consumed late are transported by permeases under nitrogen catabolite repression (NCR; ter Schure, Riel and Verrips 2000). Therefore, the kinetic characteristics of transporters, as well as SPS and NCR, are likely to be key factors controlling the temporal sequence of uptake of nitrogen compounds and constitute a system highly conserved in S. cerevisiae strains. Compared to S. cerevisiae, the consumption of nitrogen sources by wine-related non-Saccharomyces yeasts has been poorly investigated. In 2012, Medina et al. were amongst the first authors to report on the uptake of nitrogen compounds by two non-Saccharomyces yeasts (Hanseniaspora vineae and Metschnikowia pulcherrima), inoculated as starter cultures. They showed that the amount of nitrogen assimilated by the two yeasts investigated was different and that competition for nutrients had a clear impact on fermentation kinetics, potentially leading to stuck fermentation when the initial Yeast Assimilable Nitrogen (YAN) concentration was too low. A similar result was also reported by Taillandier et al. (2014) when Torulaspora delbrueckii was inoculated together with S. cerevisiae, and Barbosa et al. (2015) confirmed that the presence of another yeast (Hanseniaspora guilliermondii in their study) had a strong influence on the transcriptome of S. cerevisiae, in particular on genes involved in the biosynthesis of vitamins as well as uptake and biosynthesis of amino acids being upregulated. Finally, Kemsawasd et al. (2015) evaluated the impact of individual and mixed nitrogen sources on biomass and fermentation kinetics of several wine-related yeast species (S. cerevisiae, Lachancea thermotolerans, M. pulcherrima, Hanseniaspora uvarum and T. delbrueckii). The latter authors showed that certain nitrogen sources were beneficial for all yeast species while others were only beneficial to specific species. Overall, the influence of nitrogen sources on yeast growth and fermentation performance differed between species, with T. delbrueckii and H. uvarum being the most similar to S. cerevisiae. They also showed that different supplementation regimes should be applied to promote growth and fermentation in different species. Furthermore, yeast species may differ in their preference for amino acids, with a potential impact on nitrogen availability for the other yeasts and on fermentative aroma production. Many aroma compounds produced by S. cerevisiae are directly related and influenced by nitrogen metabolism, such as higher alcohols and their associated fatty acids and esters (Beltran et al. 2005; Swiegers et al. 2005; Barbosa et al. 2009; Rollero et al. 2015). This could explain some of the contributions of non-Saccharomyces yeasts to secondary aroma compounds reported in literature (Rossouw and Bauer 2016; Manzanares, Vallés and Viana 2013). It is important to note that among the indigenous wine yeasts, T. delbrueckii, M. pulcherrima, Candida zemplinina, L. thermotolerans and H. uvarum have been the most largely investigated (Comitini et al. 2011; Medina et al. 2012; Sadoudi et al. 2012; Gobbi et al. 2013; Kemsawasd et al. 2015; Renault et al. 2015; Gobert et al. 2017). Nevertheless, the non-Saccharomyces population associated with grape juice is much more diverse (Setati, Jacobson and Bauer 2015) and warrants further investigation. In this study, we investigated the nitrogen preferences of 10 yeast strains not commonly occurring yet isolated from grape juice. Their fermentation performances (fermentation kinetics and aroma production) compared to those of S. cerevisiae were also assessed under winemaking conditions or when each nitrogen source (individual amino acids and ammonium) provided the same amount of assimilable nitrogen in order to overcome a possible impact of the availability of the nitrogen sources on the consumption. The results obtained in this work should extend the understanding of the nitrogen metabolism of wine-associated yeasts and pave the way to better understand the possible nutrient competition during co- or sequential inoculation with S. cerevisiae. MATERIALS AND METHODS Yeasts strains and preculture conditions Ten non-conventional wine yeasts strains isolated from grape juices in South Africa and France were used in this study (Table 1). The commercial wine strain Saccharomyces cerevisiae Lalvin EC1118® (Lallemand SA, Montreal, Canada) was used as a control yeast. The cryopreserved yeast cultures were thawed at room temperature and streaked on yeast peptone dextrose (YPD) agar (Biolab-Merck, Modderfontein, South Africa). Starter cultures of all yeast strains were prepared by inoculating a single colony into 5 ml YPD broth for each strain. The cultures were incubated at 30°C on a test tube rotating wheel for 24 h. These starter cultures were used to inoculate YPD precultures at an initial cell density of 1 × 106 cells/ml, which were incubated at 30°C on a test tube rotating wheel for 9 h. In an attempt to deplete the nitrogen present in the cells, as described by Johnston, Singer and McFarlane (1977), the yeasts were incubated in YNB containing neither amino acid nor ammonium (Difco Laboratories, Detroit, MI, USA) supplemented with 20 g/l of glucose at 30°C on a test tube rotating wheel until growth ceased. The cell population was monitored by measuring optical density at 600 nm. All the strains were inoculated from this preculture at 1 × 106 cells/ml in the synthetic grape juice-like medium. Table 1. Origins of the yeast strain studied and their H2S production. Strain Species Geographical origin Collection H2Sa IWBT Y951 Pichia burtonii South Africa IWBT 1 IWBT Y963 Hyphopichia pseudoburtonii South Africa IWBT 2 IWBT Y826 Zygoascus meyerae South Africa IWBT 1 IWBT Y1084 Zygoascus meyerae South Africa IWBT 1 IWBT Y934 Wickerhamomyces anomalus South Africa IWBT 4 IWBT Y517 Wickerhamomyces anomalus South Africa IWBT 4 MTF1103L1 Wickerhamomyces anomalus France Lallemand 4 IWBT Y885 Kluyveromyces marxianus South Africa IWBT 2 IWBT RO88 Saccharomyces paradoxus South Africa IWBT 2 Uvaferm CS2 Saccharomyces kudriavzevii × Saccharomyces cerevisiae France Lallemand 3 Lalvin EC1118 Saccharomyces cerevisiae France Lallemand 2 Strain Species Geographical origin Collection H2Sa IWBT Y951 Pichia burtonii South Africa IWBT 1 IWBT Y963 Hyphopichia pseudoburtonii South Africa IWBT 2 IWBT Y826 Zygoascus meyerae South Africa IWBT 1 IWBT Y1084 Zygoascus meyerae South Africa IWBT 1 IWBT Y934 Wickerhamomyces anomalus South Africa IWBT 4 IWBT Y517 Wickerhamomyces anomalus South Africa IWBT 4 MTF1103L1 Wickerhamomyces anomalus France Lallemand 4 IWBT Y885 Kluyveromyces marxianus South Africa IWBT 2 IWBT RO88 Saccharomyces paradoxus South Africa IWBT 2 Uvaferm CS2 Saccharomyces kudriavzevii × Saccharomyces cerevisiae France Lallemand 3 Lalvin EC1118 Saccharomyces cerevisiae France Lallemand 2 aIn a scale from 1 (no production of H2S) to 5 as described in Comitini et al. (2011). View Large Table 1. Origins of the yeast strain studied and their H2S production. Strain Species Geographical origin Collection H2Sa IWBT Y951 Pichia burtonii South Africa IWBT 1 IWBT Y963 Hyphopichia pseudoburtonii South Africa IWBT 2 IWBT Y826 Zygoascus meyerae South Africa IWBT 1 IWBT Y1084 Zygoascus meyerae South Africa IWBT 1 IWBT Y934 Wickerhamomyces anomalus South Africa IWBT 4 IWBT Y517 Wickerhamomyces anomalus South Africa IWBT 4 MTF1103L1 Wickerhamomyces anomalus France Lallemand 4 IWBT Y885 Kluyveromyces marxianus South Africa IWBT 2 IWBT RO88 Saccharomyces paradoxus South Africa IWBT 2 Uvaferm CS2 Saccharomyces kudriavzevii × Saccharomyces cerevisiae France Lallemand 3 Lalvin EC1118 Saccharomyces cerevisiae France Lallemand 2 Strain Species Geographical origin Collection H2Sa IWBT Y951 Pichia burtonii South Africa IWBT 1 IWBT Y963 Hyphopichia pseudoburtonii South Africa IWBT 2 IWBT Y826 Zygoascus meyerae South Africa IWBT 1 IWBT Y1084 Zygoascus meyerae South Africa IWBT 1 IWBT Y934 Wickerhamomyces anomalus South Africa IWBT 4 IWBT Y517 Wickerhamomyces anomalus South Africa IWBT 4 MTF1103L1 Wickerhamomyces anomalus France Lallemand 4 IWBT Y885 Kluyveromyces marxianus South Africa IWBT 2 IWBT RO88 Saccharomyces paradoxus South Africa IWBT 2 Uvaferm CS2 Saccharomyces kudriavzevii × Saccharomyces cerevisiae France Lallemand 3 Lalvin EC1118 Saccharomyces cerevisiae France Lallemand 2 aIn a scale from 1 (no production of H2S) to 5 as described in Comitini et al. (2011). View Large Fermentation condition and sampling Fermentations were carried out in synthetic medium (SM) that simulates standard grape juice as described by Bely, Sablayrolles and Barre (1990) with some modifications. The SM used in this study contained 230 g/l of sugar (115 g/l of glucose and 115 g/l of fructose); 2.5 g/l of potassium L-tartrate; 3 g/l of malic acid; 0.2 g/l of citric acid; 1.14 g/l of potassium hydrogen phosphate; 0.44 g/l of magnesium sulphate heptahydrate; 1.23 g/l of calcium chloride dihydrate; vitamins (mg/l): myo-inositol (100), calcium pantothenate (1), thiamin hydrochloride (0.5), nicotinic acid (2), pyridoxine hydrochloride (2), biotin (0.125), PABA.K (para-aminobenzoate acid K; 0.2), riboflavin (0.2), folic acid (0.2); trace elements (μg/l): manganese (II) chloride tetrahydrate (200), zinc chloride (135), iron chloride (30), copper chloride (15), boric acid (5), cobalt nitrate hexahydrate (1), sodium molybdate dihydrate (25), potassium iodate (10). Nitrogen (200 mg/l of assimilable nitrogen) was supplied as one of various mixtures of amino acids along with NH4Cl (Table 2). The reference medium, SM200, simulated the composition of a typical grape juice, and in SM200E medium, all the nitrogen compounds were included at equivalent amounts of nitrogen (9.5 mgN/l for each compound). Table 2. Initial concentrations of ammonium and amino acids in the media used in this study. Nitrogen compounds SM200 (mg/l) SM200E (mg/l) Proline 288.3 61.5 Alanine 68.3 47.8 Arginine 175.7 23.3 Asparagine 25.0 35.4 Aspartate 20.9 71.4 Cystine 6.2 64.9 GABA 100.0 73.1 Glutamine 237.7 39.1 Glutamate 56.7 78.9 Glycine 8.6 40.3 Histidine 15.4 27.7 Isoleucine 15.4 70.1 Leucine 22.8 70.1 Lysine 0.6 39.1 Methionine 14.8 79.8 Phenylalanine 17.9 88.2 Serine 36.9 56.4 Threonine 35.7 63.5 Tryptophane 84.4 54.7 Tyrosine 8.6 97.4 Valine 20.9 62.5 NH4+ 214.0 38.0 Nitrogen compounds SM200 (mg/l) SM200E (mg/l) Proline 288.3 61.5 Alanine 68.3 47.8 Arginine 175.7 23.3 Asparagine 25.0 35.4 Aspartate 20.9 71.4 Cystine 6.2 64.9 GABA 100.0 73.1 Glutamine 237.7 39.1 Glutamate 56.7 78.9 Glycine 8.6 40.3 Histidine 15.4 27.7 Isoleucine 15.4 70.1 Leucine 22.8 70.1 Lysine 0.6 39.1 Methionine 14.8 79.8 Phenylalanine 17.9 88.2 Serine 36.9 56.4 Threonine 35.7 63.5 Tryptophane 84.4 54.7 Tyrosine 8.6 97.4 Valine 20.9 62.5 NH4+ 214.0 38.0 SM200: nitrogen composition mimicking the average composition of grape juices. SM200E: all the nitrogen sources are provided in equal amount. GABA: γ-aminobutyric acid. View Large Table 2. Initial concentrations of ammonium and amino acids in the media used in this study. Nitrogen compounds SM200 (mg/l) SM200E (mg/l) Proline 288.3 61.5 Alanine 68.3 47.8 Arginine 175.7 23.3 Asparagine 25.0 35.4 Aspartate 20.9 71.4 Cystine 6.2 64.9 GABA 100.0 73.1 Glutamine 237.7 39.1 Glutamate 56.7 78.9 Glycine 8.6 40.3 Histidine 15.4 27.7 Isoleucine 15.4 70.1 Leucine 22.8 70.1 Lysine 0.6 39.1 Methionine 14.8 79.8 Phenylalanine 17.9 88.2 Serine 36.9 56.4 Threonine 35.7 63.5 Tryptophane 84.4 54.7 Tyrosine 8.6 97.4 Valine 20.9 62.5 NH4+ 214.0 38.0 Nitrogen compounds SM200 (mg/l) SM200E (mg/l) Proline 288.3 61.5 Alanine 68.3 47.8 Arginine 175.7 23.3 Asparagine 25.0 35.4 Aspartate 20.9 71.4 Cystine 6.2 64.9 GABA 100.0 73.1 Glutamine 237.7 39.1 Glutamate 56.7 78.9 Glycine 8.6 40.3 Histidine 15.4 27.7 Isoleucine 15.4 70.1 Leucine 22.8 70.1 Lysine 0.6 39.1 Methionine 14.8 79.8 Phenylalanine 17.9 88.2 Serine 36.9 56.4 Threonine 35.7 63.5 Tryptophane 84.4 54.7 Tyrosine 8.6 97.4 Valine 20.9 62.5 NH4+ 214.0 38.0 SM200: nitrogen composition mimicking the average composition of grape juices. SM200E: all the nitrogen sources are provided in equal amount. GABA: γ-aminobutyric acid. View Large Instead of adding ergosterol (yeast sterol) as in Bely, Sablayrolles and Barre (1990), SM was initially supplemented with anaerobic factors composed of phytosterols (85451, Sigma Aldrich, Saint-Louis, MO, USA), sterols naturally present in the grape juice (Le Fur et al. 1994), at a final concentration of 10 mg/l. The stock solution was composed of 5 g/l of phytosterols dissolved in Tween 80 and absolute ethanol (1:1, v/v). The pH of the SM was adjusted to 3.3 with potassium hydroxide (Saarchem, Krugersdorp, South Africa). The trace elements, vitamins, nitrogen sources and anaerobic factors were filtered through a 0.22-μm syringe filter (Starlab Scientific, Cape Town, South Africa) and added into the autoclaved SM. Each fermentation was performed in triplicate. The fermentations were carried out in cylindrical fermenters of 3.5 cm diameter and 10 cm height. The fermenters contained 70 ml of medium, so that the headspace occupied 30% of the volume of the fermenters. In order to maintain anaerobiosis, the fermenters were equipped with fermentation locks filled with water, at 25°C, with orbital agitation (125 rpm). The fermentation progress was monitored by determination of CO2 release extrapolated from the measurement of the weight loss throughout the process. At 24 h, 48 h and at the end of each fermentation, different samples were centrifuged at 4000 g for 5 min, after which the supernatants were filtered through a 0.22-μm syringe filter (Starlab Scientific, Cape Town, South Africa) and stored at –20°C for further chemical analysis. Monitoring of yeast population The yeast cell populations were monitored by plating the appropriate dilutions onto YPD agar. Plates were incubated at 30°C, generally for 2 to 3 days, until colonies were formed. Quantification of residual sugars and ammonium by enzymatic assays To quantify the residual glucose, fructose and ammonium concentrations, 400 μl of filtered sample was enzymatically analysed using the Arena 20XT (Thermo Fisher Scientific, Waltham, MA), which makes use of automated spectrophotometric readings to determine the concentrations of the various compounds. The different enzymatic assay kits are: Enzytec Fluid D-Glucose (Id-No: 5140, R-BiopharmAG, Germany) for glucose, Enzytec Fluid D-Fructose (Id-No: 5120, R-BiopharmAG, Germany) for fructose and Enzytec Fluid Ammonia (Id-No: 5390, R-BiopharmAG, Germany) for ammonium. Quantification of individual amino acids Amino acids quantification was performed by high performance liquid chromatography, Agilent 1100 (Agilent Technologies, Waldbronn, Germany) by pre-column derivatisation and fluorescence detection based upon a method previously described (Henderson and Brooks 2010) with some modifications to the derivatisation and injection. A Poroshell HPH-C18 column (4.6 mm length × 150 mm internal diameter, 2.7 μm particle size; Agilent Technologies) was used following derivatisation of the amino acids. Derivatisation was performed using three different reagents: iodoacetic acid (Sigma Aldrich) for cysteine, o-phthaldialdehyde (Sigma Aldrich) for primary amino acids and fluorenylmethyloxycarbonyl chloride (Sigma Aldrich) for secondary amino acids. Internal standards, norvaline (Sigma Aldrich) and sarcosine (Sigma Aldrich) were spiked to each sample prior to derivatisation. One millilitre of each filtered sample was analysed. Analysis of major volatile compounds The quantification of major volatiles (i.e. a selection of higher alcohols, acetate esters, fatty acids, fatty acid ethyl esters) was carried out by gas chromatography equipped with a flame ionisation detector using the Agilent GC System HP 6890 Series (Agilent, Little Falls, Wilmington, USA) as described by Louw et al. (2009) with minor modifications. Five millilitres of each of the filtered samples were used with 100 μl of 4-methyl-2-pentanol (internal standard). Diethyl ether (1 ml) was added to the mixture, which was then placed in an ultrasonic bath for 5 min to extract the volatile compounds. Thereafter, the samples were centrifuged at 4000 g for 3 min. Sodium sulphate was added to remove any water from the non-polar layer. HPChemstation software was used for data analysis. Hydrogen sulphide production Hydrogen sulphide formation was also evaluated using Difco BiGGY agar (Difco Laboratories, Detroit, MI, USA). In this medium, H2S formation is correlated with colony colour according to Comitini et al. (2011). The following arbitrary scale was used: 1, white colour (no production); 2, light brown; 3, brown; 4, dark brown; 5, black. The strain Metschnikowia pulcherrima Flavia® (Lallemand SAS, Montreal, Canada) was used as negative control. Statistical analysis Data analysis was performed with a statistical treatment and graphically (boxplots) represented using the R software version 3.3.3. (http://cran.r-project.org/). The boxplots were designed according the method described by Tukey (1977). The outliers (or atypical values) are the values located beyond adjacent values. The adjacent values are calculated using 1.5 times the interquartile space (the distance between the 1st and the 3rd quartile). The principal component analysis (PCA) was carried out with the FactoMineR package (Le, Josse and Husson 2008). A two-way analysis of variance (ANOVA) was performed to describe the diversity between the different yeasts and the two nitrogen conditions with the production of aroma compounds as a factor to detect a global effect at a P-value threshold of 0.05. For each parameter, normality of residual distributions and homogeneity of variance were studied using standard diagnostic graphics; no violation of the assumptions was detected. RESULTS Nitrogen starvation of yeasts In order to assess the nitrogen requirement and preferences of the different yeast strains during alcoholic fermentation, their intracellular reserves in nitrogen were depleted before their inoculation in the fermentation medium. The time at which growth ceased in a medium devoid of nitrogen sources (here YNB medium) was considered as the time when intracellular nitrogen reserves were depleted (Johnston, Singer and McFarlane 1977). Depending on the strains, growth stopped after a period ranging from 4 to 8 h (Fig. S1, Supporting Information). From these results, the yeasts were inoculated in the fermentation medium after a preculture in YNB lasting 4 h for Pichia burtonii and Hyphopichia pseudoburtonii, 6 h for Wickerhamomyces anomalus IWBT Y934 and MTF1103L1 and Kluyveromyces marxianus or 8 h for W. anomalus IWBT Y517, Zygoascus meyerae IWBT Y826 and Y1084, Saccharomyces paradoxus, Saccharomyces kudriavzevii × Saccharomyces cerevisiae and S. cerevisiae plus 2 h to ensure that the starvation was as effective as possible. Fermentation kinetics and growth of non-conventional wine yeast strains We first compared the fermentation kinetics and the growth of the 10 non-conventional wine yeast strains. All fermentations were performed in triplicate, in a chemically defined medium (SM200) with 200 mg/l of YAN. YAN comprises a mixture of various amino acids and ammonium ions at various concentrations (Table 2). The calculation of the YAN level excludes proline because S. cerevisiae cannot metabolise this amino acid under the anaerobic conditions occurring during alcoholic fermentation. This amino acid was nevertheless included in the experiment as it is the amino acid with the highest concentration in grape juice. As expected, only the fermentation performed with S. cerevisiae reached dryness (residual sugars <2 g/l). However, the capacity to consume sugars varied significantly among the yeasts studied (from 23 g/l to 195 g/l of residual sugars for S. kudriavzevii × S. cerevisiae UCS2 and Z. meyerae 1084, respectively). Both growth and fermentation kinetics differed considerably between strains (Fig. 1a, Table 3). Indeed, the yeasts belonging to the Saccharomyces genus displayed the highest fermentation rates without any lag phase, while the non-Saccharomyces yeasts observed a distinct lag phase (around 41 h). The maximum yeast population varied between 3.1 × 107 and 1.1 × 108 cfu/ml (for P. burtonii, and S. paradoxus and S. cerevisiae, respectively). For all the non-Saccharomyces strains studied, fermentations got stuck but stopped before any population decline could be observed (data not shown). Figure 1. View largeDownload slide Fermentation kinetics of 10 non-conventional wine yeast strains and S. cerevisiae (A) in SM200 where the nitrogen sources mimicking what was found in grape juice, (B) in SM200E where all the nitrogen sources introduced at equivalent assimilable nitrogen amount, (C) comparison between the two media for S. paradoxus, S. kudriavzevii × S. cerevisiae and S. cerevisiae, (D) comparison between the two media for the three W. anomalus strains P. burtonii (Pb) IWBT Y951, H. pseudoburtonii (Hp) IWBT Y963, Z. meyerae (Zm) IWBT Y826 and IWBT Y1084, W. anomalus (Wa) IWBT Y934, IWBT Y517 and MTF 1103L1, K. marxianus (Km) IWBT Y885, S. paradoxus (Sp) IWBT RO88, S. kudriavzevii × S. cerevisiae (Sk) Uvaferm CS2, S. cerevisiae (Sc) Lalvin EC1118®. Figure 1. View largeDownload slide Fermentation kinetics of 10 non-conventional wine yeast strains and S. cerevisiae (A) in SM200 where the nitrogen sources mimicking what was found in grape juice, (B) in SM200E where all the nitrogen sources introduced at equivalent assimilable nitrogen amount, (C) comparison between the two media for S. paradoxus, S. kudriavzevii × S. cerevisiae and S. cerevisiae, (D) comparison between the two media for the three W. anomalus strains P. burtonii (Pb) IWBT Y951, H. pseudoburtonii (Hp) IWBT Y963, Z. meyerae (Zm) IWBT Y826 and IWBT Y1084, W. anomalus (Wa) IWBT Y934, IWBT Y517 and MTF 1103L1, K. marxianus (Km) IWBT Y885, S. paradoxus (Sp) IWBT RO88, S. kudriavzevii × S. cerevisiae (Sk) Uvaferm CS2, S. cerevisiae (Sc) Lalvin EC1118®. Table 3. Maximal population reached during alcoholic fermentation (×106 cfu/ml). Strains SM200 SM200E P. burtonii IWBT Y951 31.0 ± 1.41 30.5 ± 2.67 H. pseudoburtonii IWBT Y963 34.7 ± 2.83 35.0 ± 3.21 Z. meyerae IWBT Y826 57.5 ± 3.54 61.3 ± 2.85 Z. meyerae IWBT Y1084 47.5 ± 3.51 50.4 ± 1.97 W. anomalus IWBT Y934 70.8 ± 2.84 71.0 ± 6.33 W. anomalus IWBT Y517 53.0 ± 9.93 61.8 ± 9.01 W. anomalus MTF1103L1 33.0 ± 2.12 32.0 ± 8.32 K. marxianus IWBT Y885 61.0 ± 1.41 60.0 ± 2.22 S. paradoxus IWBT RO88 110 ± 6.49 110 ± 3.43 S. kudriavzevii × S. cerevisiae Uvaferm CS2 108 ± 7.07 109 ± 6.03 S. cerevisiae Lalvin EC1118 110 ± 6.36 111 ± 7.32 Strains SM200 SM200E P. burtonii IWBT Y951 31.0 ± 1.41 30.5 ± 2.67 H. pseudoburtonii IWBT Y963 34.7 ± 2.83 35.0 ± 3.21 Z. meyerae IWBT Y826 57.5 ± 3.54 61.3 ± 2.85 Z. meyerae IWBT Y1084 47.5 ± 3.51 50.4 ± 1.97 W. anomalus IWBT Y934 70.8 ± 2.84 71.0 ± 6.33 W. anomalus IWBT Y517 53.0 ± 9.93 61.8 ± 9.01 W. anomalus MTF1103L1 33.0 ± 2.12 32.0 ± 8.32 K. marxianus IWBT Y885 61.0 ± 1.41 60.0 ± 2.22 S. paradoxus IWBT RO88 110 ± 6.49 110 ± 3.43 S. kudriavzevii × S. cerevisiae Uvaferm CS2 108 ± 7.07 109 ± 6.03 S. cerevisiae Lalvin EC1118 110 ± 6.36 111 ± 7.32 SM200: nitrogen composition mimicking the average composition of grape juices. SM200E: all the nitrogen sources are provided in equal amount. View Large Table 3. Maximal population reached during alcoholic fermentation (×106 cfu/ml). Strains SM200 SM200E P. burtonii IWBT Y951 31.0 ± 1.41 30.5 ± 2.67 H. pseudoburtonii IWBT Y963 34.7 ± 2.83 35.0 ± 3.21 Z. meyerae IWBT Y826 57.5 ± 3.54 61.3 ± 2.85 Z. meyerae IWBT Y1084 47.5 ± 3.51 50.4 ± 1.97 W. anomalus IWBT Y934 70.8 ± 2.84 71.0 ± 6.33 W. anomalus IWBT Y517 53.0 ± 9.93 61.8 ± 9.01 W. anomalus MTF1103L1 33.0 ± 2.12 32.0 ± 8.32 K. marxianus IWBT Y885 61.0 ± 1.41 60.0 ± 2.22 S. paradoxus IWBT RO88 110 ± 6.49 110 ± 3.43 S. kudriavzevii × S. cerevisiae Uvaferm CS2 108 ± 7.07 109 ± 6.03 S. cerevisiae Lalvin EC1118 110 ± 6.36 111 ± 7.32 Strains SM200 SM200E P. burtonii IWBT Y951 31.0 ± 1.41 30.5 ± 2.67 H. pseudoburtonii IWBT Y963 34.7 ± 2.83 35.0 ± 3.21 Z. meyerae IWBT Y826 57.5 ± 3.54 61.3 ± 2.85 Z. meyerae IWBT Y1084 47.5 ± 3.51 50.4 ± 1.97 W. anomalus IWBT Y934 70.8 ± 2.84 71.0 ± 6.33 W. anomalus IWBT Y517 53.0 ± 9.93 61.8 ± 9.01 W. anomalus MTF1103L1 33.0 ± 2.12 32.0 ± 8.32 K. marxianus IWBT Y885 61.0 ± 1.41 60.0 ± 2.22 S. paradoxus IWBT RO88 110 ± 6.49 110 ± 3.43 S. kudriavzevii × S. cerevisiae Uvaferm CS2 108 ± 7.07 109 ± 6.03 S. cerevisiae Lalvin EC1118 110 ± 6.36 111 ± 7.32 SM200: nitrogen composition mimicking the average composition of grape juices. SM200E: all the nitrogen sources are provided in equal amount. View Large The fermentations were repeated with a medium containing all the nitrogen sources at the same level of assimilable nitrogen (SM200E). Overall, the ranking of the strains in terms of fermentation performances remained similar in the two nitrogen conditions (Fig. 1b). However, specific differences can be highlighted. For instance, S. cerevisiae seemed to ferment more slowly with a fermentation pattern similar to that of S. paradoxus under the SM200E conditions (Fig. 1c). On the contrary, the S. cerevisiae × S. kudriavzevii UCS2 strain was able to ferment to dryness when all the nitrogen sources provided the same amount of assimilable nitrogen (Fig. 1c). Interestingly, the three W. anomalus strains displayed different behaviours (Fig. 1d). Indeed, W. anomalus IWBT Y517 fermented better (40 g CO2/l released vs 30 g CO2/l previously), while the amount of sugar consumed by W. anomalus IWBT Y934 was about 20% lower (83 g/l vs 119 g/l). The fermentation kinetics of the last strain, MTF1101L1, was not affected by the nitrogen conditions. The maximal population reached by the strains in the different nitrogen conditions remained quite similar to that achieved under the previous conditions for all the strains (Table 3). Nitrogen source consumption and preferences The nitrogen uptake of the 10 non-conventional yeasts was evaluated after 24 h, 48 h and when the fermentation was terminated. The nitrogen consumption of S. cerevisiae Lalvin EC1118® was used as a reference. For all the strains, nitrogen compounds were used sequentially throughout the growth phase. However, the order of consumption of the various sources of assimilable nitrogen was strain dependent. An overview of the nitrogen uptake of all strains under oenological conditions and for the SM200E are shown in Fig. 2a and b, respectively (concentrations for each amino acid are presented in Tables S1–S6, Supporting Information). Here, the consumption of each individual amino acid and ammonium for all the strains was considered, so the overall diversity in the nitrogen utilisation and preferences among the 11 strains is illustrated. Depending on the nitrogen conditions (SM200 or SM200E), different patterns of nitrogen uptake were observed. The consumption of the different nitrogen sources seemed to be more uniform when all the sources were provided in equal amount. By contrast, under the oenological conditions, more significant differences between the strains were observed. In both media, some amino acids were consumed later for most of the strains (glycine, alanine, tyrosine and γ-aminobutyric acid (GABA) for SM200E, and arginine, glycine and GABA for SM200), which was consistent with the few variations observed on the boxplots after 24 h (Fig. 2a and b). These amino acids appeared to be non-preferred nitrogen sources for all the strains. On the other hand, under oenological conditions, some amino acids (methionine, phenylalanine, leucine and isoleucine) displayed large variations in terms of preferences between the different yeast strains (Fig. 2a). For both nitrogen conditions, at the end of fermentation, lysine was the first consumed amino acid and it was entirely consumed by all the strains (Fig. 2a and b). The branched chain amino acids were also totally consumed by the end of fermentation but only under oenological conditions (Fig. 2a). Figure 2. View largeDownload slide Nitrogen uptake. Boxplot of nitrogen consumption during the alcoholic fermentation in (A) oenological nitrogen conditions and (B) when the nitrogen sources provide the same amount of assimilable nitrogen. Red bars represent the consumption after 24 h, green bars indicate the uptake after 48 h and blue bars at the end of fermentation. Purple stars indicate the S. cerevisiae’s consumption. Black dots represent the outliers. Figure 2. View largeDownload slide Nitrogen uptake. Boxplot of nitrogen consumption during the alcoholic fermentation in (A) oenological nitrogen conditions and (B) when the nitrogen sources provide the same amount of assimilable nitrogen. Red bars represent the consumption after 24 h, green bars indicate the uptake after 48 h and blue bars at the end of fermentation. Purple stars indicate the S. cerevisiae’s consumption. Black dots represent the outliers. Despite common characteristics in their consumption of nitrogen sources, these different yeasts displayed some specificities (identified as outliers on Fig. 2). As expected, under oenological conditions, at the end of fermentation, S. cerevisiae had consumed all the assimilable nitrogen present in the medium, except GABA (less than 50% was taken up), while Z. meyerae and S. kudriavzevii × S. cerevisiae consumed 60% of the initial concentration of GABA (Table S3, Supporting Information). However, interestingly, during the early stages of fermentation, S. cerevisiae did not always consume the highest concentration of amino acids (Tables S1 and S2, Supporting Information). In particular, the consumption of arginine and glycine at 48 h by S. cerevisiae was lower than the median (Table S2, Supporting Information). At this stage, the two Z. meyerae strains consumed more arginine (45% of initial content) and S. kudriavzevii × S. cerevisiae and W. anomalus IWBT Y517 and MTF1103L1 took more glycine up than S. cerevisiae (48%, 33% and 43%, respectively, and 20% for S. cerevisiae; Table S2, Supporting Information). Concerning K. marxianus, during the first 48 h, this yeast displayed similar requirement for nitrogen sources (around 70% of initial nitrogen was consumed after 48 h) than S. cerevisiae, except for ammonium that was sparsely consumed by K. marxianus (20% vs 60% for S. cerevisiae; Tables S1 and S2, Supporting Information). For the majority of the nitrogen sources, the variations of their uptake among the strains decreased with the fermentation progress (Tables S1–S3, Supporting Information). However, this variation increased for arginine, glutamate, glycine, tryptophan and GABA. For NH4, lysine, methionine, isoleucine, leucine and phenylalanine, no variation were observed among the strains at the end of fermentation. As observed in the previous conditions, S. cerevisiae exhausted the assimilable nitrogen (except for GABA) when all the nitrogen sources were equal (Table S6, Supporting Information). However, contrary to the previous conditions, S. cerevisiae always displayed the highest consumption for all nitrogen sources (except for GABA; Tables S4–S6, Supporting Information). The biggest variations at the end of fermentation among the 11 strains appeared for asparagine, glycine, alanine, tyrosine, methionine, tryptophan and GABA (Table S6, Supporting Information). Fermentative aroma production In order to better characterise these non-conventional strains, the fermentative aroma production was determined at the end of fermentation in the two nitrogen conditions (Fig. 3). In SM200 medium, the production of volatile compounds varied substantially depending mainly on the yeast strains (Fig. 3, in blue; Table S7, Supporting Information). Indeed, S. cerevisiae was characterised by the production of higher alcohols and isoamyl acetate, while K. marxianus displayed the highest production of phenylethyl acetate, isobutyric and isovaleric acids (Fig. 3). The three W. anomalus strains and Z. meyerae IWBT Y1084 showed small concentrations in the whole fermentative aromas, except ethyl acetate for W. anomalus strains (Fig. 3; Table S7, Supporting Information). The other strains seemed to be characterised by their production of medium-chain fatty acids and threonine-derivatives (i.e. propanol and propionic acid; Fig. 3). Interestingly, the three W. anomalus strains displayed a very similar aroma profile (as confirmed by the concentrations of aroma compounds; Table S7, Supporting Information), while the two Z. meyerae strains appeared to be distant from each other on the PCA plot (Fig. 3). Indeed, Y826 showed higher production of most of the aroma compounds than Y1084 (Table S7, Supporting Information). Figure 3. View largeDownload slide Principal component analysis of the fermentative aroma concentrations at the end of fermentation in SM200 (blue) and in SM200E (red). Dark blue: higher alcohols, light blue: acetate esters, grey blue: fusel acids, red: small and medium chain fatty acids, orange: ethyl esters. Figure 3. View largeDownload slide Principal component analysis of the fermentative aroma concentrations at the end of fermentation in SM200 (blue) and in SM200E (red). Dark blue: higher alcohols, light blue: acetate esters, grey blue: fusel acids, red: small and medium chain fatty acids, orange: ethyl esters. In SM200E medium (Fig. 3, in red), the overall aroma profile of each strain was conserved but the change of ratio between nitrogen sources compared to SM200 resulted in a significantly altered production of all the aroma compounds (ANOVA Table S7, Supporting Information). Under this condition (SM200E), the concentrations of higher alcohols and acetate esters were higher for most of the strains, with the notable exceptions of S. cerevisiae, Z. meyerae IWBT Y1084 and in a lesser extent W. anomalus MTF1103L1 for which the production of higher alcohols and their acetates decreased (Table S7, Supporting Information). Overall, the statistical analysis (2-way ANOVA) confirmed that both yeast strains and nitrogen supply impacted the production of aroma compounds (Table S7, Supporting Information). As H2S is one of the main off-flavours produced by yeast during the alcoholic fermentation, its production was assessed on BiGGY agar plates. The semi-quantitative evaluation of H2S production revealed a wide strain variability (Table 1). The three W. anomalus strains were characterised by the highest production of H2S, while P. burtonii and the two Z. meyerae yeasts displayed the lowest production. Nevertheless, these results were obtained on BiGGY agar (solid medium plate assays), which was a rich medium and should be confirmed under oenological conditions (in liquid environment and nutrient limited medium). Indeed, Jiranek, Langridge and Henschke (1995b) highlighted that the BiGGY agar plate assay provided a good indication of the maximum genetically determined sulphite reductase activity of the yeast strains but did not always correlate with the H2S production occurring during wine fermentation where this production was generally lower. DISCUSSION Over the last decades, non-Saccharomyces yeasts have been increasingly used during alcoholic fermentation and their impact on the final quality of wines was assessed in several studies (Fleet 2008; Sadoudi et al. 2012; Comitini et al. 2017; Gobert et al. 2017; Puertas et al. 2017). Nevertheless, in order to better understand their interactions with S. cerevisiae and the resulting overall impact on wine, it is essential to investigate their nutrient requirements, particularly those for nitrogen. Indeed, if the nitrogen preferences of S. cerevisiae have been studied in depth (Jiranek, Langridge and Henschke 1995a; Crépin et al. 2012), these are only partially elucidated for some non-Saccharomyces yeasts occurring in the wine environment (Andorrà et al. 2010; Kemsawasd et al. 2015; Gobert et al. 2017). In the current study, we showed that, overall, fermentation performances and biomass production remained fairly similar in the two nitrogen conditions tested for all the strains. This was previously observed by Crepin et al. (2012) for Saccharomyces cerevisiae. Nevertheless, certain yeasts (i.e. Wickerhamomyces anomalus IWBT Y517 and Y934, S. cerevisiae × Saccharomyces kudriavzevii), depending on their nitrogen preferences and requirements, were affected by the change of the nitrogen composition. An adapted nutrition could improve the fermentation performances of these yeasts and increase their impact on the final product properties. As expected, the assimilation order of nitrogen sources for S. cerevisiae confirmed previously published results (Jiranek, Langridge and Henschke 1995a; Crépin et al. 2012). The other species could be divided into three groups: (i) the strains with similar nature and quantity of nitrogen sources consumed as S. cerevisiae (i.e. S. cerevisiae × S. kudriavzevii, Saccharomyces paradoxus and Kluyveromyces marxianus), (ii) the strains with a very low nitrogen consumption (i.e. Pichia burtonii, Hyphopichia pseudoburtonii and the two Zygoascus meyerae strains) and (iii) the three W. anomalus strains displaying an intermediate pattern of assimilation. The percentage uptake of nitrogen sources correlated well with the biomass production and fermentation performances: the yeasts with the higher consumption of nitrogen produced the higher cell counts and displayed the best fermentation performances overall and vice versa. This was consistent with previous studies (Prior 2017; De Koker 2015; Kemsawasd et al. 2015) where Torulaspora delbrueckii and Lachancea thermotolerans (i.e. two fairly strong fermenters) assimilated nitrogen in a similar manner as S. cerevisiae, while P. kluyveri and Metschnikowia pulcherrima (i.e. two species fermenting poorly under winemaking conditions) displayed a low nitrogen uptake. The nature and amount of nitrogen consumed by the non-Saccharomyces yeasts could impact on S. cerevisiae’s overall fermentation performance in the case of sequential inoculation with these species, as reported previously with the stronger fermenters competing more intensely with S. cerevisiae for nitrogen sources (Medina et al. 2012; de Koker 2015). A specific nutrition, sustaining the nitrogen requirement of S. cerevisiae, could reduce the competition for nitrogen, especially when S. paradoxus and K. marxianus are to be used. On the other hand, the weaker fermenters did not consume at all some nitrogen sources during the first 24 h in a strain-dependent manner (e.g. Z. meyerae IWBT Y826 did not consume glutamine, methionine and lysine, while P. burtonii did not assimilate glutamate, phenylalanine, methionine and alanine) implying an overall limited competition for nitrogen sources with S. cerevisiae. This suggests that these species may display genomic differences in nitrogen metabolism and regulatory systems. For S. cerevisiae, the preferential assimilation of certain nitrogen sources can be explained mainly by the regulation of nitrogen source transport by permeases, including the SPS system (Ljungdahl 2009) and the NCR system (Ljungdahl and Daignan-Fornier 2012). The mechanisms have not been explored yet for non-Saccharomyces yeasts. Since the assimilation of nitrogen sources influences the volatile aroma profile of wine (Jiménez-Martí et al. 2007; Vilanova et al. 2007; Carrau et al. 2008), the production of some major volatile aroma compounds was also investigated. Differences with regard to aroma compound production were observed between different yeast species but also between the strains belonging to the same species. Indeed, it seemed that the consumption of nitrogen sources and the production of fermentative aromas were relatively conserved characteristics within the W. anomalus species, whereas they appeared to be quite dissimilar between Z. meyerae strains thereby highlighting a probable difference in the regulation of nitrogen and aroma metabolisms. However, more strains should be investigated to draw final conclusions regarding intra-species similarities and differences. Furthermore, several studies have reported a direct relation between the formation of higher alcohols and esters by S. cerevisiae and the must nitrogen concentration (Bell and Henschke 2005; Beltran et al. 2005; Hernandez-Orte et al. 2006; Mouret et al. 2014b; Fairbairn et al. 2017). The specific nitrogen requirements of certain non-Saccharomyces yeasts may also influence the final organoleptic profile of wines (Andorrà et al. 2012; de Koker 2015). For instance, W. anomalus IWBT Y517 displayed better fermentation performances associated with higher aroma production in SM200E. However, if the initial nitrogen composition did influence the overall aroma profile, the link between the concentration of amino acids entering the Ehrlich pathway and that of the corresponding volatile compound could not be clearly established in this study, suggesting that certain direct correlations observed by previous authors in S. cerevisiae (Fairbairn et al. 2017) do not apply for all yeast species. Indeed, higher consumption of nitrogen sources was not necessarily correlated with higher production of fermentative aromas. For instance, Z. meyerae IWBT Y826 was one of the best producers of higher alcohols but only consumed a few amount of the corresponding amino acid precursors (leucine, valine and phenylalanine), while K. marxianus produced the highest amount of phenylethyl acetate while consuming the same amount of phenylalanine than S. cerevisiae. Indeed, the production of higher alcohols and esters by yeasts is a highly complex process dependent on a number of parameters. Higher alcohols are produced either from their amino acid precursors or by central carbon metabolism (Styger, Prior and Bauer 2011; Mouret et al. 2014a,b,c). Recent studies (Crépin et al. 2017; Rollero et al. 2017) demonstrated that higher alcohols are mainly produced through the carbon metabolism (only 5% emanated from amino acid catabolism) in S. cerevisiae. Our combined results strongly suggest, especially for Z. meyerae, that the contribution of nitrogen metabolism to the higher alcohol synthesis was probably lower than in S. cerevisiae. Nevertheless, when all the nitrogen sources were provided in equal amounts (SM200E), higher alcohols and/or esters were usually produced in higher amounts, but in that case, the amino acid precursors (leucine, valine, phenylalanine) were provided in larger quantities (between 3 and 5.5 times more), which can explain this increase. Concerning the H2S production, the three strains of W. anomalus were identified as the highest producers. As the majority of H2S produced by yeast during wine fermentation is from the precursor of the sulphur-containing amino acids cysteine and methionine, which are required for yeast growth (Thomas and Surdin-Kerjan 1997), and grape juice usually contains very low concentrations of cysteine and methionine (<20 mg/l), we can speculate that the requirement in sulphur-containing amino acids in W. anomalus was higher than that of the other yeasts. To conclude, this study highlighted that even if non-Saccharomyces yeasts, during single culture fermentations, have lower fermentation abilities than S. cerevisiae, some non-conventional yeasts, S. paradoxus and K. marxianus, showed potential as ‘strong’ fermenters under the conditions tested. Nevertheless, their high consumption of nitrogenous compounds might comprise the success of a co-fermentation with S. cerevisiae. This work provides relevant information that could be a key to the promotion of these yeasts’ growth utilisation through the formulation of adapted nitrogen supplementation. On the other hand, the use of some of the weaker fermenters would lower the competition for nutrients and warrant fermentation success. Moreover, these strains can participate in the elaboration of a more complex wine in terms of organoleptic properties. Nevertheless, using weaker fermenter yeasts can lead to a longer lag phase, which lengthens the fermentation duration but also can lead to the development of spoilage microorganisms that could impact the quality of wines. It is therefore necessary to find a compromise between the competition for nutrients and the guarantee of a qualitative final wine. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. FUNDING This work was funding by Lallemand SAS and Stellenbosch University. Conflicts of interest. None declare. 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FEMS Yeast ResearchOxford University Press

Published: May 7, 2018

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