Interactions of Condensed Tannins with Saccharomyces cerevisiae

Article pubs.acs.org/JAFC

Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy Julie Mekoue Nguela,†,‡,#,§ Aude Vernhet,*,†,‡,# Nathalie Sieczkowski,§ and Jean-Marc Brillouet†,‡,# †

INRA, UMR 1083 SPO, F-34060 Montpellier, France Montpellier SupAgro, UMR 1083 SPO, F-34060 Montpellier, France # Université Montpellier 1, UMR 1083 SPO, F-34060 Montpellier, France § Lallemand SAS, 19 rue des Briquetiers, B.P. 59, 31702 Blagnac, France

Downloaded by FLORIDA ATLANTIC UNIV on September 8, 2015 | http://pubs.acs.org Publication Date (Web): August 24, 2015 | doi: 10.1021/acs.jafc.5b02241

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ABSTRACT: Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts’ ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components. KEYWORDS: Saccharomyces cerevisiae, cell wall mannoproteins, cytoplasmic proteins, condensed tannins, interactions, microscopy

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conditions that simulate aging on lees has been evidenced10,15 and can also contribute to changes in tannin composition and properties. All of these works thus evidence the strong impact of yeast on the phenolic composition of wines and the potential part played by their adsorption. Up to now, this adsorption has mainly been attributed to wall mannoproteins, although it has been supposed that small tannin dimers and trimers could go through the wall pores and interact with the plasma membrane.11 Inactivated yeast and yeast cell walls are now proposed in enology within the context of red winemaking to improve mouthfeel and color stability.16−18 To better identify the mechanisms involved, interactions between polyphenols and yeast cells or cell walls were studied in a previous work by adsorption isotherms in a model wine-like solution and within the framework of red wine aging.19 Polyphenols were grape seed and skin tannins with different average degrees of polymerization (up to 21) and a polyphenol fraction purified from a red wine. Yeast cells and cell walls were obtained from an enological S. cerevisiae strain. The yeast biomass was produced according to the industrial process, in aerobic conditions. Part of the biomass was frozen at −20 °C and conserved as such. Another part was inactivated as such or after autolysis and spray-dried to give inactivated and autolyzed− inactivated yeasts. Cell walls were obtained from the biomass by mechanical disruption. A very different tannin sorption was

INTRODUCTION Saccharomyces cerevisiae has been instrumental for millennia in winemaking, baking, and brewing. During red winemaking, yeast ferments sugar derived from grapes (Vitis vinifera L.) into ethanol. Concomitant in red winemaking, anthocyanins and condensed tannins (also called proanthocyanidins) from the grape berry pericarp and seed are partly solubilized. Once the alcoholic fermentation is completed, yeast may be either removed or left partially in contact with the wine during a given period (aging on lees). Yeast metabolites released during alcoholic fermentation contribute to changes in the wine phenolic composition and especially color.1,2 This is related to condensation reactions involving anthocyanins and to the formation of new pigments.3 Yeast cells have also been shown to exert a protective effect toward polyphenol oxidation during aging on lees.4 Besides, yeast cells also influence the phenolic composition of wines due to their ability to adsorb some wine phenolic compounds. Interactions between yeast cells and polyphenolic compounds have been studied during fermentation and aging5−8 or in model wine-like solutions.9−11 Anthocyanin adsorption by yeast cells during fermentation may represent up to about 6% of the whole pigments and strongly depends on the considered strain and on anthocyanin polarity.7,9 Yeast cells may also contribute to the improvement of the color of white wines by the adsorption of flavan-3-ols and of brown oxidized pigments.6,11 It is also considered that yeast cells affect the organoleptic properties of red wines related to tannins (astringency, mouthfeel) and the stability of their color during aging.12,13 This impact is mostly attributed to the release of mannoproteins by yeast cell walls during wine aging.12−14 However, significant adsorption of tannins by yeast in © 2015 American Chemical Society

Received: Revised: Accepted: Published: 7539

May 7, 2015 July 22, 2015 July 29, 2015 July 29, 2015 DOI: 10.1021/acs.jafc.5b02241 J. Agric. Food Chem. 2015, 63, 7539−7545

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Journal of Agricultural and Food Chemistry

thermal treatment (70 °C, 15 min). The last part (A-IY) was first submitted to autolysis (pH 5.5 at 55 °C for 20 h), and cells were recovered by centrifugation and inactivated in the same conditions as IY. Both IY and A-IY were spray-dried and stored at 4 °C. Cell walls (CW) were purified at the laboratory from the biomass Y by mechanical disruption,19 according to the protocol described by Dallies et al.23 The cell wall suspension obtained was heated (72 °C, 15 min) to remove residual enzymatic activities. The suspension was then stored at 4 °C in the presence of sodium azide (0.02% w/v) until use. Cell walls represented around 20% of the whole cell dry weight, which was in accordance with literature data.24,25 Neutral sugars represented 80% of their dry weight, and Kjeldahl analyses indicated a nitrogen content of about 2% (w/w). Sorption Experiments. Suspensions were prepared as described in Mekoue Nguela et al.19 Briefly, yeast cells (Y, IY, or A-IY) (2 × 108 cells mL−1) or yeast cell walls (CW) (0.88 mg mL−1) were suspended in a model wine (12% ethanol, 2 g L−1 tartaric acid, 50 mM NaCl, pH adjusted to 3.5 with KOH, 25 mg L−1 SO2). An equal volume of grape skin tannins or wine polyphenol pool solution (8 mg mL−1 in model wine) was added to yeast cells or yeast cell wall suspensions, and the mixtures were gently stirred for 24 h. Suspensions were then centrifuged and the pellets recovered. Centrifugation conditions were 1500g for 5 min at 5 °C and 10000g for 5 min at 5 °C for yeast whole cells and cell walls, respectively. Pellets were washed four times with 1 mL of PBS buffer to remove the nonsorbed polyphenols and resuspended in 1 mL of the same buffer. Yeast cells and yeast cell walls suspended in the same conditions in a model wine without polyphenol served as controls. Light and Epifluorescence Microscopy. Samples and controls were observed under normal light and epifluorescence. Imaging was performed with a Zeiss Axiophot microscope using a DAPI filter (DAPI = 4′6-diamidino-2-phenylindole; λexc = 340−380 nm, λem = 425−800 nm). Confocal Microscopy. Confocal imaging was performed with a Zeiss Axiovert microscope 200 M 510 META fitted with a PlanApochromat ×40/1.2 W Zeiss objective. Excitations were obtained for tannins with a 405 nm blue diode, and emissions were collected for tannins with a (505−550 nm bandpass) filter.26 For images acquired in lambda scanning mode, the emission spectra were obtained on sample regions of interest (ROIs) by spectral acquisition (lambda stack, excitation at 405 nm). The detection bandwidth was set to collect emissions from 400 to 750 nm, using an array of 32 photomultiplier tube (PMT) detectors, each with a 10.7 nm bandwidth. The method of linear unmixing was applied with advanced iterative and one residual channel. Transmission Electron Microscopy (TEM). PBS was removed from the samples and controls, and the pellets were resuspended in a solution of 2% acrolein and 2% glutaraldehyde in cacodylate buffer (0.05 M, pH 7) for 12 h at 4 °C.27 They were then rinsed several times in cacodylate buffer and postfixed in a 1% osmic acid for 2 h in the dark and at room temperature. After two rinses in cacodylate buffer, the samples were dehydrated in a graded series of acetone solutions (30−100%) at room temperature. They were then embedded in EmBed 812 using automated microwave tissue processor for electronic microscopy (Leica EM AMW). Thin sections (70 nm; Leica-Reichert Ultracut E) were collected at different levels of each block. These sections were stained sequentially with uranyl acetate and lead citrate. They were then observed using a Hitachi 7100 transmission electron microscope with 75 kV accelerating voltage in the Centre de Ressources en Imagerie Cellulaire de Montpellier (France).

evidenced between whole cells and cell walls. Seed and skin tannins exhibited a high affinity for whole yeast cells, associated with high adsorption levels (between 300 and 700 mg g−1 for average degree of polymerization (aDP) 21 skin tannins) and low reversibility (about 20%). By contrast, adsorption remained very low and mostly reversible with cell walls (on the order of 50 mg g−1 for aDP 21 skin tannins, with 60% reversibility). Experiments were repeated with red wine polyphenols. Adsorption primarily concerned oligomeric and polymeric tannins and pigments. That of monomers was very small. Once again, wine tannin and pigment sorption strongly differed between whole cells and cell walls, with maximum adsorbed amounts around 350 and 20−30 mg g−1, respectively. This last point was quite unexpected and led us to wonder about the exact part played by cell walls in the adsorption of wine polyphenols. We suggested that grape and wine tannins, even those with high molecular weight, were able to diffuse quite easily through the wall network of dead cells and that the differences observed were related to their accumulation in the periplasmic space and/or their interactions with cell membrane. The aim of the present work was to identify the location of polyphenol compounds in yeast cells. This was achieved through scanning electron microscopy, light epifluorescence, and confocal microscopy.

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MATERIALS AND METHODS

Purification of Grape Skin Tannins and Red Wine Polyphenols. The grape skin tannin fraction was extracted from the skin of Muscat berries according to the procedure proposed by Mane.20 It was further purified by extractions with hexane, followed by adsorption on a styrene/divinylbenzene Diaion resin and chromatography on a Toyopearl TSK HW-50 (F) gel (Tosoh Corp., Tokyo, Japan), as described in detail previously.19 This procedure allowed the recovery of a high molecular weight tannin fraction, which was freezedried and stored at −80 °C before use. The composition of the fraction was determined by UPLC after acid-catalyzed cleavage in the presence of thioglycolic acid.21 Its mean degree of polymerization was 20.1 ± 0.7, and its percentage of epigallocatechin units was 16.8 ± 1.2%. The yield of the depolymerization reaction was 73.9%. This fraction was named skin21. The red wine polyphenol fraction (WP) was purified from a Merlot wine by chromatography on a styrene/divinylbenzene Diaion resin and analyzed as detailed in our previous work.19 This fraction contained residual polysaccharides and nitrogenous compounds, which represented about 15% of the dry weight. Its polyphenol composition was as follows: 29.3 mg g−1 anthocyanins, 7.7 mg g−1 flavan-3-ol monomers, 32.7 mg g−1 phenolic acids, 24.2 mg g−1 stilbenes, 5.9 mg g−1 flavonols, and 95.0 mg g−1 tannins. The average degree of polymerization and composition of the tannins, determined by phloroglucinolysis, were aDP, 7.3 ± 1.1; epigallocatechin units (EgC), 24.2 ± 2.3%; and epicatechin-gallate (Ec-G), 3.3 ± 0.3%. The yield of the depolymerization reaction was very low (around 13%). This was attributed to the presence in this fraction of a large amount of derived pigments and tannins. The latter are formed by chemical reactions during winemaking and aging and are resistant to usual depolymerization methods.1,21,22 Yeast Cells and Preparation of Yeast Cell Walls. The yeast strain used was a commercial S. cerevisiae strain (Lallemand, Montreal, Canada), commonly used in enology. The yeast biomass (Y), the inactivated yeast (IY), and the yeast inactivated after autolysis (A-IY) were produced by the manufacturer according to the protocols described previously.19 The yeast biomass was produced in aerobic conditions. Part of this biomass was frozen at −20 °C and conserved as such until further use. The viability of the cells was determined before interaction experiments. Results obtained by flow cytometry (1%) and growth on Petri plates (2.4%) indicated that most cells in Y were dead. Another part of the biomass (IY) was inactivated by

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RESULTS AND DISCUSSION Epifluorescence and Confocal Microscopy. Untreated yeast cells (Y) were observed in light microscopy (Figure 1A) as regular spheres (average diameter = 3 μm). Yeast cells after contact with grape skin tannins (treated cells) showed the same morphology as untreated cells and formed stacks (Figure 1B). Untreated yeast cells exhibited no fluorescence (Figure 1C), whereas treated ones fluoresced strongly in blue (Figure 1D). 7540

DOI: 10.1021/acs.jafc.5b02241 J. Agric. Food Chem. 2015, 63, 7539−7545

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Figure 1. Views of untreated (A, C) and treated (B, D) yeast cells with grape skin tannins under light and epifluorescence microscopy (λexc = 340−380 nm, λem = 425−800 nm).

This fluorescence was within the range of that of tannins in the applied experimental conditions. Confocal microscopy was performed on yeast cells (Y), inactivated yeasts (IY, A-IY), and yeast cell walls (CW) (Figure 2). Untreated yeast cells and inactivated yeasts showed an extremely faint and almost invisible blue fluorescence; the same was observed for purified cell walls (Figure 2). After contact with skin21 (grape skin tannins), yeast cells and inactivated yeasts showed an intense blue fluorescence in their inner core (Figure 2); some plasmolyzed cells were observed with Y, and they showed, in addition to their fluorescent core, a corona fluorescing in blue. Cell walls exhibited a blue fluorescence and were devoid of internal content. With Y and CW, some scarce residual fluorescent cytoplasmic elements were observed (Figure 2). An examination of treated cells (Y) by confocal spectral analysis revealed particles (diameter = 3 μm) (Figure 3A) exhibiting an autofluorescence emission spectrum (Figure 3B) with several maxima in the 500−600 nm range. Spectral analysis of this fluorescence in comparison with purified grape tannins26 indicated this was due to tannins. Thus, after contact with skin21, the yeast biomass (Y) and the inactivated yeasts (IY and A-IY) showed a strong blue fluorescence in their cytoplasm. This indicated the presence of tannins inside the cells. Another site of fluorescence, the cell wall, was identified on plasmolyzed cells and purified cell walls. Transmission Electron Microscopy. TEM was performed on yeast cell biomass (Y) and yeast cell wall (CW) without and after contact with grape skin tannins (Figure 4) or with wine polyphenol pool (Figure 5). Untreated yeast cells (Y) appeared typical, in comparison with published TEM images28−30 being spherical and with the electron-dense fibrillary network of the cell wall (thickness = 200 nm) evident (Figures 4A−C and 5A−C). At higher magnification (Figures 4B,C and 5B,C), the distinction of regions within the cell wall became evident. Yeast cell wall is described as a two-layered structure made of β-(1→ 3) and β-(1→6)-glucans, mannoproteins, and chitin, which can be covalently linked to form higher order complexes.25,31 TEM views showed the more electron-dense outer brush-like layer enriched in mannoprotein, over the more electron transparent microfibrillar glucan-rich layer.30,32 In particular, one must note the regular design of mannoproteins bound to the outer face of the cell wall; it appears as fibers extending from the wall. Within

Figure 2. Confocal images of untreated and treated yeast cells (Y, IY, A-IY) and untreated and treated yeast cell walls (CW) with grape skin tannins. Asterisk focuses on fluorescent cytoplasm; white arrows, cell walls; blue arrow, remnants of cytoplasm out of the cells; Y, whole yeast cells; IY, inactivated yeast cells; A-IY, yeast inactivated after autolysis. λexc = 405 nm, λem = 505−550 nm.

yeast cells, plasma membrane and ultrastructure granular elements were visible. Following exposure to grape tannins (Figure 4) or wine polyphenols (Figure 5), cells and cell walls, compared with untreated cells, showed obvious differences at their cell wall and cytoplasm levels. The outer brush-like layer of cell walls appeared as electron-dense aggregated elements (Figures 4D− F and 5D−F). Another major difference was observed in the cytoplasm of polyphenol-treated cells: indeed, and contrary to untreated cells, the entire cytoplasm appeared as highly electron-dense. Finally, at the junction between the cytoplasm and the cell wall, an electron-dense thin layer was visible (Figure 5D−F). The enhanced electron-dense character observed in the outer mannoprotein layer and within the cytoplasm can be attributed to the presence of tannins. Indeed, postfixation of samples for TEM observations was performed with osmic acid, and the tendency of osmium to strongly stain tannins has been evidenced.26 Whereas the grape skin tannin fraction consists only of flavan-3-ol polymers with an aDP of 21, red wine polyphenols consist of a complex mixture of native 7541


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Figure 3. (A) Confocal image of yeast cells treated with grape skin tannins and (B) spectral analysis showing their autofluorescence emission spectrum (λem = 430−800 nm range), similar to that of grape tannins.26 ROI, region of interest. (C) Untreated yeast cells (control) and (D) spectral analysis showing their autofluorescence emission spectrum.

polyphenols monomers (mainly phenolic acids, flavanol monomers, and anthocyanins) and tannins, along with socalled derived pigments and tannins formed during winemaking.1,3,21,22 We evidenced previously that interactions between red wine polyphenols and yeast cells or cell walls mainly concerned oligomeric and polymeric forms.19 Thus, TEM observations confirmed that interactions between yeast cells and native or derived condensed tannins/pigments occur not only in the cell wall but also with their cytoplasmic components. Plasmolysis of some yeast cells was helpful for description of interactions between tannins and mannoproteins. On intact cells, mannoproteins bound to the outer face of the plasma membrane were hardly visible, the plasma membrane being stuck to the wall (Figure 5A,B); after contact with tannins, only a fine osmophilic layer appressed between the wall and plasmalemma was visible (Figure 5D−F). After plasmolysis, fine structures were unveiled in the space generated: on treated cells, mannoproteins were seen attached by GP1 anchor (glycosyl-phosphatidylinositol)25 to the outer face of the plasmalemma (Figure 6A−C). The inner mannoproteins formed large aggregated electron-dense structures still attached through their GPI anchor to the plasmalemma (Figure 6B,D). In this area, we noted that, under the influence of plasmolysis tearing force, the mannoproteins were cleaved, a large proportion remaining attached to the inner face of the wall.

Interactions of the plasmalemma proteins with tannins were also well visible on plasmolyzed yeast cells (Figure 6C). The weakly electron-opaque cell wall [mainly β-(1→3)-glucan and chitin] did not show any typical structure. Tannin and Red Wine Polyphenol Interactions with Dead/Inactivated Yeast Cells and Cell Walls. These results evidenced that interactions between yeast cells and grape or wine tannins are not necessarily limited to cell walls when dealing with dead and/or inactivated cells. Grape tannins and wine tannins/pigments are liable to enter the periplasmic space through the cell wall and to interact with the cell membrane and its cytoplasmic content. According to their average degree of polymerization, a 3 nm average hydrodynamic volume could be estimated for aDP 21:33 thus, the polysaccharide wall in our study was permeable to molecules of this size. These observations explained the very large differences observed in our previous work19 between whole cells and cell walls regarding their ability to bind grape and red wine tannins. Indeed, adsorption isotherms evidenced that the amount of polyphenols adsorbed at concentrations corresponding to those found in red wines was about 10-fold higher with whole cells (nonviable yeast biomass and inactivated cells) than with cell walls. In addition, the reversibility of the interactions was also much more important with cell walls. The property of tannins to form complexes with proteins is well-known.34,35 Proteins relevant to the yeast wall include 7542


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Figure 6. TEM images of (A) treated yeast cell wall with wine polyphenol pool, showing aggregated outer and inner mannoproteins, (B, C) a treated cell showing the plasmalemma, and (D) drawing of plasmolyzed outer region of untreated and treated yeast cells. cw, polysaccharidic cell wall; mp, mannoproteins; pe, periplasm; pl, plasmalemma; red circles, GPI anchor; blue arrows, contrasted knots in the inner layer of mannoproteins; green arrows, contrasted knots at the outer layer of mannoproteins; red arrows, clumps of clotted plasmalemma proteins; on scale.

Figure 4. TEM images at diverse magnifications of (A−C) untreated and (D−F) treated yeast cells (Y) and cell walls (CW) with grape skin tannins. Red arrows, mannoproteins; blue arrows, cell wall thickness.

not all, of the proteins in these two groups are glycosylated. The third group consists of single-pass plasma membrane proteins, mechanosensors that detect cell wall stress.25 TEM observations evidenced that among cell wall constituents (mannoproteins, proteins, β-glucans, and chitin), mannoproteins are likely primarily involved in the interaction with tannins. This observation is in accordance with previous works evidencing interactions in solution between mannoproteins and tannins or tannin aggregates.36−38 Considering whole cells and cytoplasmic constituents, it can be supposed that interactions occur with the proteins of the plasmalemma or of other cell membranes or with cytoplasmic proteins. Indeed, proteins represent between 40 and 50% of the yeast cell dry weight.39 The much higher reversibility of the interactions between cell walls and tannins compared to that observed with whole yeast cells and tannins can be related to the high glycosylation level of mannoproteins: it has been demonstrated that mannosylated yeast invertase presents a 10-fold lower ability to bind red wine tannins than bovine serum albumin.36 Potential Implications in Winemaking. The present results have several potential implications in winemaking and aging. The latter concerns first the role of yeast-derived products, especially inactivated yeasts and yeast cell walls, and their use in red winemaking for their potential impact on wine mouthfeel and color stability. Although the exact mechanisms are not solved yet, this impact has mainly been attributed to interactions between mannoproteins and wine tannins. Interactions may occur in solution, due to the release of mannoproteins from cell walls,15,40,41 or at the interface, due to tannin interactions with mannoproteins located in the outer layer of walls.10,15 Interactions between condensed grape skin tannins or red wine tannins/derived pigments and mannoproteins in the cell walls have been evidenced here by TEM for the first time. They induce aggregate formation in the initially brush-like structure of the outer mannoprotein layer. However,

Figure 5. TEM images at diverse magnifications of (A−C) untreated and (D−F) treated yeast cells (Y) and cell walls (CW) with wine polyphenol pool. Red arrows, mannoproteins; blue arrows, antennary oligo(manno)-saccharides; pl, plasma membrane.

hydrolases or transglycosidases, nonenzymatic mating agglutinins, flocculins, and β-(1→3)-glucan cross-connectors. Most, if 7543


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grape skin tannins with an average degree of polymerization of 21; TEM, transmission electron microscopy; WP, wine polyphenols; Y, yeast biomass

when dealing with whole dead or inactivated cells, most of the interactions (about 90%) occur with the yeast cellular constituents. It can then be expected that a quite different impact on red wine polyphenol composition will be obtained depending on the exact structure and composition of the yeastderived product used:17 inactivated yeast, yeast inactivated after autolysis treatment and autolysis conditions, yeast walls or hulls. Indeed, autolysis may strongly affect the yeast cell wall composition and cytoplasmic content42−45 and thus their interactions with wine polyphenols. The autolyzed and inactivated yeast (A-IY) used in our study did not present changes in their ultrastructure detectable by TEM, and only small differences were observed between IY and A-IY in their interactions with red wine polyphenols.19 Besides the degree of autolysis, also the yeast strain and culture conditions have a potential impact on the composition of the yeast-derived products that deserves to be further explored.12,17 Inactivated cells and cell walls are proposed as wine quality enhancers to replace aging on lees. Cell growth conditions used to produce yeast biomass and yeast-derived products strongly differ from those found in winemaking. This may induce different behavior of dead cells toward their interactions with wine polyphenols by comparison to the yeast biomass Y used in our work. However, for a close initial tannin concentration, the amount of polyphenols adsorbed by the yeast biomass Y in our study was similar to that adsorbed by yeast lees in the previous work of Mazauric et al.10,15 In that case, lees were obtained after alcoholic fermentation in a model must and the strain was not the one used in the present study. This tends to indicate that results obtained in the present work are not related only to the strain and its growth conditions (aerobiosis versus anaerobiosis). This will need to be further explored. Likewise, we focused in the present work on condensed tannins and red wine polyphenols. Although their phenolic compositions are very different, white and rosé wines also contain flavan-3-ol oligomers and derived pigments that can develop physicochemical interactions with yeast cells. For instance, it has been hypothesized that direct interactions between small flavan-3-ols and yeast cytoplasmic membrane lipids could explain the protective effect of yeast lees toward polyphenol oxidation in white wines. Thus, it will be of importance to identify the exact sites of interaction between white and rosé polyphenols and yeast cells to better understand the mechanisms involved in the impact of yeasts and yeast-derived products on wine quality.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*(A.V.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Chantal Cazevieille [Centre de Ressources en Imagerie Cellulaire (CRIC), Université Montpellier I, Montpellier, France] and Elodie Jublanc (INRA, Montpellier, France) for their technical assistance in transmission electron and confocal microscopy, respectively. We thank Stephanie Carrillo for the purification of skin tannins.

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ABBREVIATIONS aDP, average degree of polymerization; A-IY, autolyzed and inactivated yeast; CW, cell wall; IY, inactivated yeast; skin 21, 7544


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Interactions of Condensed Tannins with Saccharomyces cerevisiae

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