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/lp/springer-journals/employing-oxygen-pulses-to-modulate-lachancea-thermotolerans-GOYkFQgjL5
- Publisher
- Springer Journals
- Copyright
- Copyright © 2017 by Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan
- Subject
- Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
- ISSN
- 1590-4261
- eISSN
- 1869-2044
- DOI
- 10.1007/s13213-017-1319-6
- Publisher site
- See Article on Publisher Site
Abstract
Oxygen is sometimes deliberately introduced in winemaking at various stages to enhance yeast biomass formation and prevent stuck fermentation. However, there is limited information on how such interventions affect the dynamics of yeast populations. Our previous study in synthetic grape juice showed that oxygen supply enhances the persistence of Lachancea thermotolerans, Torulaspora delbrueckii and Metschnikowia pulcherrima. The three non-Saccharomyces yeasts showed differences in growth as a function of oxygen. The present study focused on evaluating the influence of short oxygen pulses on population dynamics and the aroma profile of Chardonnay wine inoculated with L. thermotolerans and Saccharomyces cerevisiae. The results confirmed a positive effect of oxygen on the relative performance of L. thermotolerans. The mixed culture fermentation with L. thermotolerans with S. cerevisiae developed a distinct aroma profile when compared to monoculture S. cerevisiae. Specifically, a high concentration of esters, medium chain fatty acids and higher alcohols was detected in the mixed culture fermentation. The data also showed that the longer persistence of L. thermotolerans due to addition of oxygen pulses influenced the formation of major volatile compounds such as ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl caprylate, ethyl caprate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, propanol, isobutanol, butanol, isoamyl alcohol, hexanol, isobutyric acid, butyric acid, iso-valeric acid, hexanoic acid, octanoic acid, and decanoic acid. . . . . Keywords Non-Saccharomyces yeast Winemaking Oxygenation regimes Mixed cultures Yeast dynamics Introduction present in the wine ecosystem (Brandam et al. 2013;Ciani et al. 2006;Hanl etal. 2005;Hansen et al. 2001; Jolly et al. Wine fermentation is typically characterized by low pH, a 2006). Besides such broad physiological adaptations, rapid development of anaerobiosis, an increase in ethanol, S. cerevisiae also relies on some more targeted mechanisms, and, in some cases, an increase in temperature. Under these such as the production of toxic metabolites including anti- conditions, S. cerevisiae displays a better fitness than non- microbial peptides that target specific competing species. Saccharomyces yeast species, and tends to rapidly dominate For instance, antimicrobial peptides that are derived from re- the wine microflora (Albergaria and Arneborg 2016; Williams actions catalyzed by glyceraldehyde-3-phosphate dehydroge- et al. 2015). Several physiological or metabolic features con- nase has been reported as the main contributing factor in tribute to the dominance of S. cerevisiae. S. cerevisiae indeed S. cerevisiae competition against Hanseniaspora spp. shows better fermentative capacity in anaerobic conditions (Branco et al. 2014; Ciani et al. 2016). and higher ethanol tolerance than all other species that are A previous study in our laboratory showed that a continu- ous supply of oxygen at 1% and 5% dissolved oxygen allowed Lachancea thermotolerans and Torulaspora delbrueckii to Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-017-1319-6) contains supplementary dominate mixed fermentations with S. cerevisiae material, which is available to authorized users. (Shekhawat et al. 2017), thus confirming that S. cerevisiae niche construction and ecological dominance against these * Mathabatha E. Setati two yeasts was due to anaerobiosis (Brandam et al. 2013; setati@sun.ac.za Hanl et al. 2005;Hansenet al. 2001). Recently, non- Saccharomyces yeasts have become increasingly popular as Institute for Wine Biotechnology, Stellenbosch University, Stellenbosch, Western Cape, South Africa co-inoculants in mixed-starter fermentations. Indeed, in the 94 Ann Microbiol (2018) 68:93–102 past 10 years several species, including L. thermotolerans, Yeast enumeration and isolation T. delbrueckii, Metschnikowia pulcherrima and Pichia kluyveri have been commercialized and are available as mono- For initial yeast identification of Chardonnay grape juice, se- culture active dry yeasts or as blends (Jolly et al. 2014). Our rial dilutions were prepared in 0.9% (w/v) NaCl solution and current study employed L. theromotolerans as it was found to spread on Wallerstein laboratory nutrient (WLN) agar, Sigma- require the least amount of oxygen to dominate S. cerevisiae Aldrich). Yeast enumeration and isolation were performed compared to T. delbrueckii and M. pulcherrima.This yeast is from plates that contained between 30 and 300 yeast colonies. known to enhance the concentration of higher alcohols (par- Colonies with clearly identifiable features (color, texture, size, ticularly 2-phenylethanol), L-lactic acid, glycerol, and esters in shape, margin) were further purified. At least three represen- wine (Benito et al. 2016; Gobbi et al. 2013). Moreover, using tative colonies per colony morphology were streaked out from L. thermotolerans in sequential fermentation with each plate. The isolates were further stored in glycerol 20% (v/ S. cerevisiae at low temperatures was reported to bring down v) at −80 °C (Bagheri et al. 2015). For further yeast enumer- the levels of ethanol in wine (Gobbi et al. 2013). ation of mixed and single culture inoculated fermentations, Oxygen is typically introduced in winemaking especially samples were taken every 2nd day; both species were distin- in the production of red wine through punch downs, pump guished based on colony morphology on YPD plates (Fig. −1 over, and transfers. Such methods can add up to 6 mg L S1). The plates were incubated for 4 days at 30 °C for the oxygen (du Toit et al. 2006;Moenne et al. 2014). These oxy- colonies to be clearly distinguishable. gen additions are common practice in most wineries as they promote yeast biomass synthesis, contribute to sound wine Yeast identification in chardonnay grape juice fermentation, and enhance the aroma profile of wine. However, there is little to no information on how this influ- For the initial yeast identification, the genomic DNA was ex- ences the growth and development non-Saccharomyces yeast tracted from 1 mL of the sample using the rapid yeast DNA inoculants. extraction method (Hoffman 2003). The ITS1-5.8S rDNA- In the current study, we employed L. thermotolerans to ITS2 region amplification was performed by PCR using the evaluate the effect of low oxygen input on its persistence primer set ITS1 (5′-TCCGTAGGTGAACCTCGCG-3′) and and contribution to the aroma of Chardonnay. The aim of ITS4 (5′-TCCTCCGCTTTATTGATATGC-3′)(Esteve- the study was to assess how commercially realistic oxygen Zarzoso et al. 1999). PCR amplification was done in a final input levels would influence the growth and persistence of volume of 25 μL containing 0.4 mM dNTP mix, 0.25 μMof L. thermotolerans in mixed culture fermentation with each primer, 1 U Ex-Taq polymerase (TaKara, Kyoto, Japan), S. cerevisiae, and gain insight on the organoleptic properties 1× buffer, 1 mM MgCl and 100 ng template DNA. Further, of the Chardonnay wines produced through these the PCR products were purified using the ZymocleanTM Gel interventions. DNA recovery kit (Zymo, Irvine, CA) following the manu- facture’s instruction. Restriction fragment length polymor- phism (RFLP) was performed by digesting the ITS-5.8S rDNA PCR amplicons with HaeIII, HinfI, and CfoIin sepa- Materials and methods rate reactions as described by Esteve-Zarzoso et al. (1999). For further identification, the yeast isolates were grouped ac- Microorganisms and media cording to distinct restriction patterns, and previously se- quenced species were digested with the same enzymes and Astrain of L. thermotolerans (IWBT Y-1240) was obtained used as references to identify the current isolates (Bagheri from the yeast culture collection of Institute for Wine et al. 2015). Biotechnology (Stellenbosch University), while S. cerevisiae (Cross evolution 285) was obtained from Lallemand SAS Fermentations (Blagnac, France). The cryogenically (−80 °C) maintained yeast strains were streaked out on YPD agar plates containing Fermentations were performed in Chardonnay grape juice. (per liter) 20 g glucose, 20 g peptone, 10 g yeast extract and Clarified juice (4 L) was inoculated in 5 L bottles sealed with 20 g bacteriological agar. For further use, cultures were fermentation caps. Oxygen was added to the bottles with the maintained at 4 °C for a short period. The chemical help of Norprene tubing using an oxygen cylinder. The oxy- analysis of Chardonnay grape juice was obtained from gen concentration was monitored by using oxygen sensor Fourier transform infrared (FT-IR) spectroscopy using spots (Pst-3; PreSens, Regensburg, Germany) fitted inside the Grape Scan 2000 instrument (FOSS Electric, each bottle. The fermentation kinetics were monitored Hillerød, Denmark). The analysis revealed a total sugar by weighing the bottles every 2nd day until the weight −1 concentration of 218 g L and pH 3.7. was stable. Ann Microbiol (2018) 68:93–102 95 Inoculation strategies The split ratio was 15:1 and the split flow rate −1 49.5 mL min . The column flow rate was 3.3 mL −1 The yeast strains were first inoculated in 5 mL YPD broth min using hydrogen as carrier gas. The detector tem- overnight followed by a transfer of 1 mL to 100 mL YPD perature was 250 °C (Louw et al. 2010). Duplicate in- broth, which was allowed to grow overnight (±16 h) at jections were carried out per sample. 30 °C with agitation at 100 rpm. To obtain a higher cell con- centration; the 100 mL pre-culture was re-cultured into 1 L Statistical analysis YPD broth and incubated until mid-exponential growth phase. Cells were harvested by centrifugation at 5000 g for 5 min, The chemical analysis of all compounds was performed on and re-suspended into 0.9% (w/v) NaCl solution. For single three independent biological repeats of fermentations in bot- culture fermentations, S. cerevisiae CE 285 and tles, and all the values are stated as means ± SD. The signif- L. thermotolerans Y1240 were inoculated into separate ves- icant differences between measurements within different treat- 6 7 −1 sels at 10 and 10 cells mL , respectively, while for mixed ments were determined using analysis of variance [a least- culture fermentations, S. cerevisiae CE 285 and significant-difference (LSD) test] with the statistical software L. thermotolerans were co-inoculated simultaneously in the Statistica version 13.0 (Stat Soft, Palo Alto, CA) and differ- 6 7 −1 same vessel with cell density of 10 and 10 cells mL , ences were considered significant when P values were ≤0.05. respectively. To analyze the significant differences in major volatiles due to aeration and mixing, the two-way ANOVA was performed Sample analysis using XLSTAT 2017 software (Addinsoft, New York, NY). For multivariate data analysis, principle component analysis Glucose, fructose, glycerol and acetic acid were mea- (PCA) was created using SIMCA-P software version 14.0 sured using specific enzymatic kits, Enytec™ Fluid D- (Umetrics, Umea, Sweden). glucose, fructose, acetic acid (Thermo Fisher Scientific, Vantaa, Finland), Boehringer Mannheim / R-Biopharm- acetaldehyde (R-Biopharm, Darmstadt, Germany) and Results analyzed using Arena 20XT photometric analyzer (Thermo Electron, Helsinki, Finland) (Schnierda et al. Grape juice analysis: initial yeast identification 2014). Ethanol was analyzed by high-performance liquid chromatography (HPLC) on an AMINEX HPX-87H ion The analysis of the initial yeast diversity of the Chardonnay exchange column using 5 mM H SO as the mobile grape juice revealed the presence of nine different yeast spe- 2 4 phase. Agilent RID and UV detectors were used in tan- cies (Fig. 1). Hanseniaspora uvarum was the most abundant 4 −1 dem for peak detection and quantification. The final species (74%, 5.8 × 10 cfu mL ), followed by Candida 4 −1 analysis was done using the HPChemstation software apicola (7%, 1.2 × 10 cfu mL ), Candida oleophila (5%, 3 −1 (Rossouw et al. 2012). The liquid–liquid extraction 4× 10 cfu mL ), Starmerella bacillaris (5%, 3. 67 × 3 −1 3 method was used for volatile compound analysis using 10 cfu mL ), Candida intermedia (4%, 3.3 × 10 cfu −1 3 −1 GC-FID, where 5 mL sample of synthetic grape juice mL ), and Candida californica (2%, 1.66 × 10 cfu mL ), was added with internal standard 4-methyl-2-pentanol while Metschnikowia pulcherrima, Zygoascus meyerae, −1 (final concentration 5 mg L ). To perform liquid–liquid Bandoniozyma visegradensis each accounted for approxi- 3 −1 extraction 1 mL diethyl ether was added to each sample mately 1% (0.67 × 10 cfu mL ) of the population. and sonicated for 5 min. The wine/ether mixture was then centrifuged at 4000 g for 5 min, and the ether Fermentation kinetics and yeast dynamics layer (supernatant) was transferred to new vials and dried on Na SO to remove the excess of water. For In the current study, small-scale wine fermentations were per- 2 4 gas chromatography (GC) a DB-FFAP capillary column formed in 5 L fermentation bottles without agitation, and ox- (Agilent, Little Falls, DE) with dimensions 60 m length ygen was pulsed once a day for monoculture fermentations, ×0.32mmi.d.× 0.5 μm film thickness and a Hewlett and three times a day for mixed culture fermentations com- Packard 6890 Plus GC instrument (Hewlett-Packard, pared to no oxygen provision. The monoculture and mixed Little Falls, DE) equipped with a split/splitless injector culture fermentations of L. thermotolerans and S. cerevisiae −1 and a flame ionization detector (FID) was used. The with both types of oxygen pulses reached dryness (< 5 g L initial oven temperature was 33 °C, held for 17 min, sugar) in 10 days while fermentation without oxygen addition after which the temperature was increased by 12 °C took 12 days to achieve dryness (Fig. 2). −1 min to 240 °C, and held for 5 min. Diethyl-ether The data show that, in the monoculture fermentation, extract (3 μL) was injected at 200 °C in split mode. L. thermotolerans could be detected only for the first 4 days 96 Ann Microbiol (2018) 68:93–102 Fig. 1 Percentage distribution of 1% 1% yeast species retrieved from 5% Chardonnay grape juice through 4% 7% 2% culture-dependent methods 5% 1% 74% Metschnikowia pulcherrima Zygoascus meyerae Candida apicola Hanseniaspora uvarum Bandoniozyma visegradensis Starmerella bacillaris Candida californica Candida intermedia Candida oleophila under anaerobic conditions, before the indigenous yeast be- oxygen pulses three times a day, improved the came dominant. However, when oxygen was pulsed three L. thermotolerans cell concentrations from the initial inocu- 7 −1 9 −1 times a day, the viability of L. thermotolerans was sustained lum level of 10 cfu mL to 10 cfu mL within the first for 6 days before the indigenous population surpassed it (Fig. 2 days. S. cerevisiae displayed a steady increase in growth 6 −1 8 −1 2). In mixed culture fermentation under anaerobic conditions, from 10 cfu mL to a maximum of 6.3 × 10 cfu mL and 8 −1 L. thermotolerans maintained viability at the initial inoculum 5.2 × 10 cfu mL in 4 days under anaerobic conditions and level for 4 days and then declined below detection (Fig. 3). with oxygen pulsed once a day, respectively (Fig. 3). In con- Oxygen pulses once a day resulted in a slight increase in cell trast, when oxygen was pulsed three times a day, S. cerevisiae 8 −1 concentrations in the first 2 days of fermentation as well as increased to a maximum of 7.0 × 10 cfu mL within 2 days sustained viability up to 6 days of fermentation. Conversely, and remained stable for 4 days before starting to decline. 1.00E+10 450 Fig. 2 Growth and fermentation kinetics of Lachancea thermotolerans and Saccharomyces cerevisiae 1.00E+09 monoculture fermentations under anaerobic (AN) oxygenation once per day (1xPulse) and three times 1.00E+08 per day (3xPulse) 1.00E+07 1.00E+06 1.00E+05 0 02468 10 12 Fermentation Period (Days) Lt-3D Sc-3D Sc-AN Lt-AN Sc-AN-Ferm Sc-3xPulse Lt-AN-Ferm Lt-3xPulse -1 Yeast Growth (CFU mL ) CO Release (g) 2 Ann Microbiol (2018) 68:93–102 97 1.00E+10 450 1.00E+09 1.00E+08 1.00E+07 1.00E+06 1.00E+05 0 02468 10 12 14 Fermentation Period (Days) Lt-AN Sc-AN Lt-1xPulse Sc-1xPulse Lt-3xPulse Sc-3xPulse AN-Ferm 1xPulse 3xPulse Fig. 3 Growth and fermentation kinetics of L. thermotolerans and S. cerevisiae in mixed culture fermentation under AN conditions, and with oxygenation 1xPulse and 3xPulse Impact of oxygen pulses on dry biomass and ethanol differences observed in the concentration of acetic acid and content glycerol in the different fermentations. The dry biomass was determined only at the end of fermenta- Impact of oxygen pulses on major volatile tion. Higher biomass was obtained under aerobic conditions compounds of Chardonnay grape juice compared to anaerobic conditions (Fig. 4). In mixed fermen- −1 tations, the dry biomass increased from 3.23 g L under an- The data revealed a significant increase in the concentration of −1 −1 aerobic conditions to 3.93 g L and 4.15 g L ,whenoxygen higher alcohols, esters, and fatty acids in mixed fermentations was pulsed once a day and three times a day, respectively (Fig. in comparison to monoculture fermentation of S. cerevisiae 4). Oxygen pulsing three times a day in mixed fermentations under anaerobic conditions. The further influence of aeration, resulted in significant reduction in ethanol levels compared to mixing or both was analyzed using two-way ANOVA for single S. cerevisiae anaerobic fermentation with a difference mixed and single fermentations. The two-way ANOVA re- −1 of 2.2gL ethanol (Table 1). There were no significant vealed that the addition of oxygen and co-inoculation of L. thermotolerans/ S. cerevisiae influenced the volatile profile of aerated single and mixed fermentations in comparison to their anaerobic mono-culture fermentation. For instance, in comparison to single S. cerevisiae anaerobic fermentation, the concentration of ethyl acetate was significantly decreased in anaerobic mixed fermentation due to presence of L. thermotolerans, this concentration of ethyl acetate further gradually decreased in aerated mixed fermentation because of extended survival of L. thermotolerans. Similar results were also noted for ethyl butyrate, ethyl hexanoate, ethyl caprylate, ethyl caprate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, Sc+Lt-AN Sc+Lt-1D Sc+Lt-3D Sc-AN Sc-3D Lt-AN Lt-3D propanol, isobutanol, butanol, isoamyl alcohol, hexanol, Fermentations isobutyric acid, butyric acid, iso-valeric acid, hexanoic acid, Fig. 4 Dry mass produced by S. cerevisiae/ L. thermotolerans mixed and octanoic acid, and decanoic acid, where the concentration of single culture fermentations in bottles with AN conditions, at 1xPulse and 3xPulse these compounds was influenced in anaerobic mixed -1 Biomass in g L -1 Yeast Growth (CFU mL ) CO Release (g) 2 98 Ann Microbiol (2018) 68:93–102 Table 1 Fermentation parameters and products of candidate Lachancea thermotolerans and Saccharomyces cerevisiae in mixed and pure culture fermentations in bottles (values are the mean of triplicates). Sc S. cerevisiae, Lt L. thermotolerans, AN Anaerobic, 1D oxygenation once per day, 3D oxygenation three times per day −1 −1 Fermentation Ethanol yield Ethanol (%) Residual sugar Ethanol (g L ) Acetic acid Glycerol (g L ) −1 −1 (g/g sugar) (g L ) (g L ) Sc + Lt-AN 0.509 ± 0.002 10.90 2.0 ± 0.04 109.8 ± 1.57 0.12 ± 0.02 5.56 ± 0.24 Sc + Lt-1D 0.503 ± 0.01 10.80 4.0 ± 0.02 108 ± 0.48 0.19 ± 0.005 5.69 ± 0.11 Sc + Lt-3D 0.49 ± 0.008 10.70 4.2 ± 0.003 107 ± 1.92 0.22 ± 0.11 5.75 ± 0.08 S. cerevisiae-AN 0.51 ± 0.007 11.00 3.0 ± 0.002 110 ± 0.99 0.19 ± 0.02 5.54 ± 0.22 S. cerevisiae-3D 0.49 ± 0.01 10.90 0.4 ± 0.009 109.2 ± 0.79 0.14 ± 0.02 5.59 ± 0.24 L. thermotolerans-AN 0.50 ± 0.04 10.90 0.3 ± 0.07 109.3 ± 0.45 0.20 ± 0.005 5.80 ± 0.13 L. thermotolerans-3D 0.49 ± 0.06 10.80 0.5 ± 0.05 108 ± 0.76 0.25 ± 0.08 5.89 ± 0.33 fermentation by co-inoculation, and by both in aerated mixed demonstrated the influence of oxygen on yeast dynamics and fermentation, while by aeration in single aerated fermentations volatile compounds. The addition of oxygen showed the nu- (indicated in red in Table S1). In contrast, the concentration of merical dominance of L. thermotolerans, and increased the isoamyl acetate, 3-methyl-1-pentanol and valeric acid were concentration of higher alcohols. However, excessive contin- influenced mainly by different oxygenation regimes in mixed uous levels of oxygen were applied, which is not a realistic as well as single aerated fermentations in comparison to their strategy in a commercial cellar (Shekhawat et al. 2017). single anaerobic fermentations; while the formation of ethyl Therefore, the current study applied oxygen levels like those lactate, diethyl succinate, 3-ethoxy-1-propanol and propionic that may be achieved through common winemaking practices acid was influenced only by co-inoculation (Table S1). such as punch downs and pump-overs. Although in white The score plot showed that the first two principal compo- wine, the punch down does not take place, the current study nents explain 68% of the variability between different fermen- used white grape juice in order to repeat the data of synthetic tations (Fig. 5). PC1 explained 52% variability, and separated grape juice, which is closer to the white grape juice matrix. the fermentations according to different oxygen regimes. The fermentation profiles of anaerobic fermentations were separat- Impact of oxygen pulses on persistence ed from aerobic fermentations, and the separation was driven of L. thermotolerans mainly by higher alcohols (2-phenylethanol, isobutanol, isoamyl-alcohol, hexanol), ethyl phenyl acetate, valeric acid Our data revealed that oxygen inputs as low as pulses once a and isobutyric acid (Fig. 5). The fermentations are further day can enhance the persistence of L. thermotolerans. separated along PC2 with 16% of the variance. The anaerobic However, when inoculated in monoculture such an improve- fermentation of L. thermotolerans clearly formed a separate ment in viability could only be sustained for a short period, group from the rest of the fermentations due to higher concen- before an indigenous population, presumably of S. cerevisiae, trations of ethyl lactate, ethyl acetate and ethyl-3- outcompetes the L. thermotolerans inoculant. In contrast, in hydroxybutanoate. The anaerobic fermentation of mixed culture fermentation, oxygen pulses had a clear and S. cerevisiae and mixed anaerobic fermentations clearly considerable influence not just on the persistence of formed separate groups from fermentation with oxygenation L. thermotolerans but also on its growth. Indeed, pulsing ox- either once or three times per day. The metabolic profile of the ygen once and three times a day, resulted in a 10-fold and 100- anaerobic monoculture of S. cerevisiae and the anaerobic fold increase in maximum cell concentration after 48 h com- mixed-fermentation grouped together due to a higher concen- pared to no oxygen input where a decline in population was tration of medium chain fatty acids and esters. evident during the same time period. In particular, pulsing three times a day resulted in higher cell concentrations of L. thermotolerans compared to S. cerevisiae within the first Discussion 48 h. These results are in accordance with previous studies, and clearly show a positive effect of oxygenation and stirring The incorporation of oxygen at various stages of the wine on growth of L. thermotolerans (Hansen et al. 2001;Contreras making process has an impact on fermentative rate and wine et al. 2014; Quirós et al. 2014, Shekhawat et al. 2017). Nissen quality, as well as on yeast physiology (Aceituno et al. 2012; et al. (2004) performed population dynamics with Ingledew et al. 1987; Rosenfeld et al. 2003; Valero et al. L. thermotolerans and S. cerevisiae, and the study showed 2001). Our previous data generated on synthetic grape juice the need for higher oxygen in L. thermotolerans than Ann Microbiol (2018) 68:93–102 99 Fig. 5 a Score and b loading plots of the first principle components showing major volatiles produced by L. thermotolerans and S. cerevisiae single species and mixed fermentations with and without oxygen in bottles 100 Ann Microbiol (2018) 68:93–102 S. cerevisiae. Quirós et al. (2014) showed higher oxygen re- (branched chain amino acid permease) is reported as a result quirement of Lachancea species (Kluyveromyces lactis/ of oxygen addition (Verbelen et al. 2008). Therefore, the marxianus) with respiratory quotient (RQ) of 0.8-1.25. This higher levels of these alcohols could be due to the higher most certainly explains why L. thermotolerans seemed to be persistence of L. thermotolerans as well more uptake of these affected by the changes in oxygen availability. Besides the amino acids by the yeasts under oxygenation conditions. role of oxygen and stirring on growth of L. thermotolerans, Similarly, a higher concentration of ethyl phenylacetate, ethyl considering the previous studies our data of the yeast growth lactate, diethyl succinate and isobutyric can be linked to the in bottle fermentation also suggests that L. thermotolerans longer persistence of L. thermotolerans due to oxygenation. died off earlier not only because of less oxygen and stirring, The levels of most esters, except ethyl phenylacetate, ethyl but also possibly because of the presence of S. cerevisiae lactate and diethyl succinate, were reduced in response to (Luyt 2015;Nissen et al. 2004). Previous studies have pre- oxygen exposure. Volatile esters are enormously important sented results where metabolic and cell–cell interaction be- for the flavor profile of the wine. Numerous different enzymes tween L. thermotolerans, T. delbrueckii with S. cerevisiae take part in the formation of esters, and the best characterized seems to be responsible for early decline of these non- are the alcohol acetyl transferases I and II, which are encoded Saccharomyces yeasts (Nissen et al. 2003; Renault et al. by the genes ATF1 and ATF2, respectively (Malcorps and 2013). Dufour 1992). The expression of these transferases has been shown to be down-regulated in response to oxygen exposure Major volatile compound profile of wine (Mason and Dufour 2000). Therefore, the reduction in these esters could be due to the result of repression of these genes. The mixed fermentation of S. cerevisiae/ L. thermotolerans Unsaturated fatty acids (UFAs) are essential for maintain- resulted in less acetic acid production than pure S. cerevisiae ing membrane integrity, function, as well as for adapting to cultures, confirming our previous results in synthetic grape fermentation stresses, such as high sugar and ethanol toxicity. juice. In mixed culture fermentation, L. thermotolerans influ- These unsaturated fatty acids are derived from desaturation of enced the final levels of higher alcohols, esters and medium small chain and MCFAs in presence of oxygen (Duan et al. chain fatty acids (MCFAs) in wines, in comparison to 2015). Therefore, in fermentations with oxygenation, the de- S. cerevisiae pure culture fermentation. The current dataset crease in MCFAs can be explained by the phenomenon of of S. cerevisiae/ L. thermotolerans mixed fermentation is in unsaturated fatty acid (UFA), sterols formation. This has been accordance with our previous study and other literature reports reported to occur in the presence of oxygen, through the action (Howell et al. 2006; Gobbi et al. 2013; Milanovic et al. 2012). of ERG1, which has also been reported previously as a reason The increase in concentration of secondary metabolites in for the partial removal of toxic MCFAs (C8-C12) and accel- mixed culture fermentation has been attributed to metabolic erated the synthesis of long-chain (C16- C18) fatty acids and interaction between S. cerevisiae and non-Saccharomyces sterols. These factors can contribute to an enhanced sugar yeasts (Barbosa et al. 2015; Luyt 2015). Therefore, the current uptake through the cell membrane and accelerated yeast sur- data set also suggest that perhaps the higher concentration of vival (Ingledew and Kunkee 1985; Ribereau-Gayon 1985; higher alcohols, esters and medium chain fatty acids in anaer- Schneider 1998; Varela et al. 2012). obic mixed culture fermentation could be due to metabolic To understand the fermentative behavior of yeast while interaction between L. thermotolerans and S. cerevisiae. fermenting real grape juice, the use of synthetic grape juice Our statistical analysis showed a combined influence of co- has been considered the best approach at laboratory scale. Yet, inoculation as well as aeration. For instance, the formation of it is often difficult to extrapolate the behavior of yeast strains propanol, isobutanol, butanol, isoamyl alcohol, 2-phenyl eth- in synthetic grape juice under laboratory conditions to the anol, hexanol and isobutyric acid was influenced by co- behavior of the same yeast strain in real grape juice conditions. inoculation as well as aeration. We think that an increase in It is therefore necessary to validate the behavior of the same these compounds could be due to increased persistence of yeast strains in both juice compositions. The comparison of L. thermotolerans in aerobic fermentation as mixed fermenta- the major volatile compounds between the data set obtained tion even in anaerobic condition led to increase in concentra- from synthetic grape juice in our previous work (Shekhawat tion of these higher alcohols. et al. 2017) and Chardonnay grape must in the current study The formation of these higher alcohols takes places via the for S. cerevisiae/ L. thermotolerans show similar trend for Ehrlich pathway, which involves uptake of branched-chain oxygen treatments (Fig. S2). For instance, the total concentra- amino acids (leucine, isoleucine, and valine) and synthesizes tion of higher alcohols in Chardonnay grape juice mixed cul- higher alcohols (isoamyl alcohol, active amyl alcohols and ture fermentation was increased from anaerobic conditions to −1 isobutanol respectively). Studies have shown accelerated tran- oxygenation once a day and three times a day: 248 g L , −1 −1 scripts of permeases that are responsible for uptake of these 325gL and336gL , respectively. This trend for higher amino acids. For instance, higher expression of BAP2 alcohols was similar to those obtained in synthetic grape juice Ann Microbiol (2018) 68:93–102 101 nitrogen-limited enological conditions. Appl Environ Microbiol 78: under anaerobic, 1%, 5% and 21% levels of oxygen: 191 g 8340–8352 −1 −1 −1 −1 L , 426 g L , 466 g L and 633 g L , respectively. In Barbosa C, Mendes Faia A, Lage P, Mira NP, Mendes Ferreira A (2015) contrast in both matrices the total concentration of esters and Genomic expression program of Saccharomyces cerevisiae along a MCFA decreased as a result of oxygen addition as showed in mixed-culture wine fermentation with Hanseniaspora guilliermondii. Microb Cell Factories 14:124 Fig. S2. Branco P, Francisco D, Chambon C, Hébraud M, Arneborg N, Gabriela In conclusion, our data set from real grape juice confirms MG, Jorge CMA, Albergaria H (2014) Identification of novel the result of synthetic grape juice. A reduced oxygen avail- GAPDH-derived antimicrobial peptides secreted by ability greatly affected the growth of L. thermotolerans and Saccharomyces cerevisiae and involved in wine microbial interac- tions. Appl Microbiol Biotechnol 9:843–853 volatile compounds. However, presence of S. cerevisiae and Brandam C, Lai QP, Julien-Ortiz A, Taillandier P (2013) Influence of stirring also influenced the growth of L. thermotolerans. oxygen on alcoholic fermentation by a wine strain of Toluraspora Based on literature, the current data also suggest a possible delbrueckii: kinetics and carbon mass balance. Biosci Biotechnol metabolic interaction between the two yeasts which influences Biochem 77:1848–1853 the growth of L. thermotolerans and formation of secondary Ciani M, Beco L, Comitini F (2006) Fermentation behaviour and meta- bolic interactions of multistarter wine yeast fermentations. Int J Food metabolites. Furthermore, the use of oxygen pulses resulted in Microbiol 108:239–245 no major difference in acetic acid concentration. Therefore, Ciani M, Capece A, Comitini F, Canonico L, Siesto G, Romano P (2016) use of oxygen pulses in the form of pump-over or punch- Yeast interactions in inoculated wine fermentation. Front Microbiol down seems more feasible to sustain L. thermotolerans for a 7:555. https://doi.org/10.3389/fmicb.2016.00555 longer period to attain the higher concentration of volatile Contreras A, Hidalgo C, Henschke PA, Chambers PJ, Curtin C, Varela C (2014) Evaluation of non-Saccharomyces yeast for the reduction of compounds, while avoiding the risk of formation of undesir- alcohol content in wine. Appl Environ Microbiol 80:1670–1678 able compounds such as acetic acid. Du Toit WJ, Marais J, Pretorius IS, du Toit M (2006) Oxygen in must and wine: a review. S Afr J Enol Vitic 27:76–94 Acknowledgments We would like to thank Prof. Wessel du Toit Duan LL, Shi Y, Jiang R, Yang Q, Wang YQ, Liu PT, Duan CQ, Yan GL (Department of Viticulture and Oenology) for his guidance with oxygen (2015) Effects of adding unsaturated fatty acids on fatty acid com- measurements in the cellar and providing all necessary information re- position of saccharomyces cerevisiae and major volatile compounds garding pst-3 spot. This research for the current study was funded by the in wine. S Afr J Enol Vitic 36:285–294 National Research Foundation (NRF)Grant Unique Number 83471 and Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A (1999) Identification the National Research Foundation-Technology and HumanResources for of yeasts by RFLP analysis of the 5.8S rRNA gene and the two Industry Programme (grant TP2011060600013). The opinions expressed ribosomal internal transcribed spacers. 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Journal
Annals of Microbiology – Springer Journals
Published: Dec 22, 2017