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A new methodology to determine cell wall mannoprotein content and release in
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wine yeasts
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Manuel Quirós1*, Pilar Morales1, Laura Pérez-Través2, José M. Barcenilla3 and Ramon
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Gonzalez1,3
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Alimentos (CSIC), Burjassot, Valencia, Spain
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Instituto de Ciencias de la Vid y del Vino (CSIC-UR-CAR), Logroño, La Rioja, Spain
Departamento de Biotecnología. Instituto de Agroquímica y Tecnología de los
Instituto de Fermentaciones Industriales (CSIC), Madrid, Spain.
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* Author to whom correspondence should be addressed:
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Instituto de Ciencias de la Vid y del Vino (CSIC-UR-CAR)
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C/ Madre de Dios, 51
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26006 Logroño, La Rioja
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Spain
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Phone: +34 941 299691
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Fax: +34 941 299608
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e-mail: mquiros@icvv.es
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Abstract
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Representing around 40% of the cell wall dry weight, mannoproteins are complex
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macromolecules structurally composed of polymers of sugar, 98% being mannose,
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covalently linked to peptides. Along the last two decades, these compounds have gained
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ground as very relevant molecules in the field of winemaking, mainly due to their
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positive contributions in the development of appreciated organoleptic features and to
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their contribution in the chemical stabilization of wine. Several methodologies have
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been recently proposed to achieve the quantification of these compounds. However,
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these methodologies are laborious, time consuming and do not allow a global
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quantification of these macromolecules. In this paper, an easy, reliable and fast forward
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methodology for the quantification of mannoproteins in model must is proposed and
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evaluated. Its application in the quantification of mannoproteins content in yeast cell
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wall is also demonstrated.
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Keywords: wine yeast, mannoproteins, cell wall, polysaccharide quantification
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1. Introduction
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Over the last two decades, yeast mannoproteins have become a hot topic in the field of
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winemaking. Numerous studies have clearly proven the multiple positive contributions
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of these molecules to the overall vinification process (Doco, Vuchot, Cheynier &
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Moutounet, 2003; Dupin et al., 2000; Dufour & Bayonove, 1999; Guadalupe &
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Ayestarán, 2008). Among their most outstanding enological features, mannoproteins
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contribute to the chemical stabilization of wine by preventing crystallization of tartrate
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salts (Feuillat, Charpentier & Nguyen van Long, 1998; Gerbaud et al., 1996) and
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protecting against protein haze (Dupin et al., 2000; Gonzalez-Ramos, Cebollero &
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Gonzalez, 2008; Gonzalez-Ramos & Gonzalez, 2006; Gonzalez-Ramos, Quirós &
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Gonzalez, 2009; Waters, Pellerin & Brillouet, 1994). These highly glycosylated proteins
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stimulate growth of lactic-acid bacteria in wine environments and thus the development
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of malolactic fermentation (Guilloux-Benatier & Chassagne, 2003) and allow a
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reduction in the concentration of some undesired compounds such as ochratoxin A
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(Ringot, Lerzy, Bonhoure, Auclair, Oriol & Larondelle, 2005). Furthermore,
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mannoproteins have a relevant impact on the sensorial properties of wine as they retain
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aroma compounds (Lubbers, Voilley, Feuillat & Charpentier, 1994), reduce astringency,
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improve the foaming properties of sparkling wines (Núñez, Carrascosa, Gonzalez, Polo
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& Martínez-Rodríguez, 2006) and increase the body, sweetness, roundness and
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mouthfeel of the final product (Guadalupe, Palacios & Ayestarán, 2007; Vidal et al.,
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2004).
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Mannoproteins are mainly located in the outermost layer of the yeast cell wall, where
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they act as structural components and are partially responsible for its permeability (Klis,
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Mol, Hellingwerf & Brul, 2002). On average, 30% of their structure corresponds to the
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protein fraction, while the remaining 70% corresponds to sugar residues, 98% of which
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are mannose. The release of yeast mannoproteins during wine making mainly takes
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place when alcoholic fermentation has come to an end. At that point, cell viability
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drastically decreases and cell death leads to the release of its constituents in a
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phenomenon known as autolysis.
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Although little is known about the genetic determinants involved in the secretion of
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mannoproteins during wine fermentations, several recent papers have cast light onto this
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issue (Gonzalez-Ramos et al., 2006; 2008; 2009). The results presented in these works
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have led to the development of a new methodology to obtain wine yeast strains
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overproducing mannoproteins (Quirós, Gonzalez-Ramos, Tabera & Gonzalez, 2010).
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While working on this topic, the need to establish a standardized and reliable
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methodology to perform the quantification of these molecules became obvious. Some
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currently available methodologies are either unspecific (Segarra, Lao, López-Tamames
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& de la Torre-Boronat, 1995) or complicated and laborious and do not allow a clear
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overall quantification of the release of mannoproteins (Palomero, Benito, Morata,
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Tesfaye, González & Suárez-Lepe, 2009)
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In the present paper, a simple and reliable methodology to quantify the release of
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mannoproteins by yeast strains in a synthetic medium and in model must and to
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determine their concentration in the cell wall is proposed and evaluated. Correlation
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studies to predict the potential behaviour of yeast strains in mannoproteins release in
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wine fermentations have also been investigated.
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2. Materials and Methods
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2.1. Strains and culture conditions
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Eight different S. cerevisiae strains and one hybrid S. cerevisiae x S. kudriavzevii were
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used in this work. Within the first set of strains, BM45, D254, EC1118, T73 and VRB
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are wine yeast strains commercialised by Lallemand Inc. (Ontario, Canada). IFI87 and
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IFI475 are non-commercial winemaking strains obtained from the Instituto de
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Fermentaciones Industriales culture collection (CSIC)while BY4743 is a reference
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laboratory strain obtained from the Euroscarf consortium. Finally, W27, the only natural
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S. cerevisiae x S. kudriavzevii hybrid used (Gonzalez, Barrio, Gafner & Querol, 2006)
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was originally isolated in Wädenswil, Switzerland, and is also commercialised by
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Lallemand Inc. Strains were routinely maintained on YPD plates, containing 2%
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glucose, 2% peptone, 1% yeast extract and 2% agar.
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The release of mannoproteins and polysaccharides and the mannoprotein content in the
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cell wall of the selected strains was studied in two different media. Firstly,
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fermentations were performed in 50 mL of GCY, a synthetic medium containing 2%
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glucose (Sigma-Aldrich, St. Loius, MO), 2% BactoTM Casamino Acids (BD, Sparks,
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USA) and 0.67% DifcoTM Yeast Nitrogen Base (BD) during 24 hours at 30 ºC and 150
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rpm shaking. In a second step, industrial strains were used in order to perform
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fermentations in a synthetic must (pH 3.5) containing 10% glucose, 10% fructose, 0.6%
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citric acid, 0.6% malic acid (all of them from Sigma-Aldrich), 0.17% YNB without
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aminoacids and ammonium sulphate (Difco), 306 mg/L NH4Cl (Panreac, Barcelona,
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Spain) and 2/3 of the amount of aminoacids used in the control synthetic grape must
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(CNC) described by Beltran, Novo, Rozès, Mas and Guillamon (2004), corresponding
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to 200 mg/L of yeast assimilable nitrogen. In this case, fermentations were performed in
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100 mL borosilicate glass bottles (Schott AG, Mainz, Germany) containing 50 mL of
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medium. Bottles were capped with Müller valves filled with vaseline oil and incubated
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at 25 ºC without shaking. Fermentation time courses were monitored by determining the
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production of CO2 expressed as weight loss until weight was constant.
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Inocula were grown in YPD broth o/n, washed twice in sterile distilled water and
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inoculated at a final concentration of 106 cells/mL (approximately 0.1 units O.D.600nm).
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2.2. Quantification of total mannoproteins and polysaccharides released during
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fermentation
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When all fermentations were finished, i.e. sugar was depleted, cultures were centrifuged
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at 10,000 rpm for 5 min and 3 mL of each supernatant gel filtered through 30 x 10 mm
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Econo-Pac® 10 DG disposable chromatography columns (Bio-Rad Laboratories,
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Hercules, CA) and eluted with 4 mL distilled water in order to isolate the non-retained
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macromolecular fraction. For samples corresponding to fermentations in synthetic must,
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3 mL of the eluted fraction were filtered again using the same type of columns and
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eluted with 4 mL of distilled water. For samples corresponding to fermentations in GCY
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medium, this second filtration step was not necessary. Once the macromolecular
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fraction was obtained, it was concentrated and subjected to a double analysis. On one
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side, 3 aliquots of 200 μL were subjected to the phenol-sulphuric acid method described
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by Segarra et al. (1995) in quadruplicates. Absorbance at 490 nm was determined using
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a UV-1800 Shimadzu spectrophotometer (Shimadzu Corp., Kyoto, Japan). In parallel,
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two aliquots of 2 mL were concentrated in 2 mL screw-capped microtubes (Sarstedt,
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Nümbrecht, Germany) using a miVac DNA centrifugal concentrator (Genevac Ltd,
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Suffolk, UK) at 60 ºC until complete evaporation. Resulting pellets were carefully
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resuspended in 100 µL of 1 M H2SO4. Tubes were tightly capped and placed in an oven
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at 100 ºC for 4 hours to undergo acid hydrolysis. After this treatment, tubes were briefly
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spun down, 10 fold diluted using 900 µL of miliQ water, filtered through 0.45 µm pore
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size Nylon filters (Scharlab, Sentmenat, Spain) and subjected to HPLC analysis for
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quantification of the glucose and mannose released during hydrolysis. For the
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preparation of a standard curve, serial aqueous dilutions of commercial mannan from S.
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cerevisiae (Sigma-Aldrich) containing 10 different concentrations, ranging from 250 to
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25 mg/L, were prepared and subjected to the double hydrolysis described above.
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2.3. Determination of the relative mannoprotein content of the yeast cell wall
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In order to obtain an indicative value of the mannoprotein content in the yeast cell wall,
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two samples of 3 mL of each of the fermentations performed in GCY and synthetic
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must were centrifuged, supernatant discarded and cells washed twice with 5 mL sterile
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distilled water. Pellets were then subjected to acid hydrolysis following the protocol
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described above (subsection 2.2). The glucose and mannose content was determined by
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HPLC and the mannose:glucose ratio of the cell wall calculated.
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2.4. Stability of the hydrolysed samples
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In order to check the stability of the hydrolysed samples, a cell wall hydrolysis
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corresponding to strain EC1118 was prepared as mentioned above, aliquoted in
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eppendorf tubes and stored at -20, 4 and 28 ºC for 5 and 15 days. After that time,
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samples were analyzed by HPLC and the mannose:glucose ratio obtained compared to
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the one observed for the same sample analyzed just after the hydrolysis had been
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performed.
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2.5. Method reproducibility and repeatability
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In order to characterize the reproducibility and repeatability of the methodology
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proposed and compare it to those obtained for the phenol-sulphuric method used as
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reference, 15 mL of a macromolecular fraction from two independent cultures of the
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strains EC1118 and T73 in synthetic must were obtained by column filtration as
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described above. Three independent manipulators performed a double hydrolysis of the
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concentrated macromolecular fraction followed by HPLC analysis and a triple
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determination of the total polysaccharide content using the phenol-sulphuric method in
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two different types of commercial 2 mL tubes in order to analyze the possible effect of
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the shape and/or the plastic material in the determination.
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2.6. HPLC analysis of hydrolysates, musts and wines
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To determine the concentration of glucose and mannose resulting from the acid
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hydrolysis of both the macromolecular fractions and yeast cell walls samples were
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filtered through 0.22 pore size nylon filters (Symta, Madrid, Spain) and injected in
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duplicate in a Surveyor Plus chromatograph (Thermo Fisher Scientific, Waltham, MA)
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equipped with a refraction index detector (Surveyor RI Plus Detector). The column
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employed was a HyperREZTM XP Carbohydrate H+ 8μm (Thermo Fisher Scientific)
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assembled to its correspondent guard column. 1.5 mM H2SO4 was used as the mobile
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phase with a flux of 0.6 mL/min and a column temperature of 50 ºC. Each sample was
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run for 20 minutes. Standard solutions of mannose and glucose used for the construction
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of the calibration curves were prepared using a 100 mM H2SO4 aqueous solution instead
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of miliQ water to mimic the conditions found after hydrolysis.
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To determine the initial and final concentration of the main metabolites present in the
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fermentations performed in synthetic must, i.e. glucose, fructose, ethanol and glycerol,
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the same system was used. Prior to injection, samples were centrifuged for cell removal,
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filtered through 0.45 nylon filters and diluted 5 or 10-fold in miliQ water. In this case,
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samples were run for 30 minutes.
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2.7. Statistical analysis of data
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Determination of all parameters was performed in duplicate, except for the
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determination of the polysaccharides released following the phenol-sulphuric method,
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performed, if not differently stated, in quadruplicate. Reproducibility was analysed by
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one-way ANOVA and the Dunnett test for comparison of means. Bivariate correlation
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between the variables analysed was determined by Pearson’s correlation coefficient. In
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both cases the SPSS 15.0 software (SPSS Inc., Chicago, IL) was used. Regression
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analysis of data was performed using Microsoft® Excel 2000 software.
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3. Results and discussion
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3.1. Method robustness
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In order to test if the proposed methodology was suitable for the quantification of the
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total mannoproteins content of an aqueous solution, a serial dilution of commercial
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mannan from S. cerevisiae was subjected to a double analysis: acid hydrolysis with
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H2SO4 followed by HPLC determination of mannose and glucose, and determination of
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total polysaccharides performed by the phenol-sulphuric method. Sulphuric acid was
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chosen to perform the hydrolysis because it has been proven as the most efficient and
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reliable acid for this purpose (Garleb, Bourquin & Fahey, 1989).
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The relationship found between the concentration of mannan in the solution and the
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concentration of mannose released
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quantification could be perfectly fitted to a regression line (y = 0.772x + 1.663) with a
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R2 = 0.999. The limits of detection and quantification of this method were calculated as
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3 and 10 times the signal/noise ratio, respectively. The limit of detection for glucose and
after acid hydrolysis determined by HPLC
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mannose was 0.40 and 0.39 mg/L while the limit of quantification was 1.34 and 1.30
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mg/L, respectively.
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Similar results were obtained using the phenol-sulphuric methodology used for
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comparison. In this case, data from the concentration of mannan and absorbance at 490
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nm could also be fitted to a regression line presenting a very reliable regression
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coefficient (y = 0.010x + 0.047, R2 = 0.999). When the correlation between all the
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variables analysed in this two sets of experiments, i.e. mannan concentration, mannose
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concentration after hydrolysis and absorbance at 490 nm, was studied, a Pearson’s
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correlation coefficient of 1.00 was obtained for all of them. However, our experience
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using the phenol-sulphuric method during several years made us realize that it was quite
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difficult to obtain an optimal repeatability using such technique and the reproducibility
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obtained when different manipulators were involved in the analysis was deficient. This
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fact will be tackled in depth in a subsequent section.
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3.2. Mannoprotein content of GCY after yeast fermentation
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Once proven that the concentration of mannose correlated significantly with the
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concentration of a complex polymer such as mannan with the proposed methodology
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(p<0.01), we decided to check if this method could also be applied to a macromolecular
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fraction isolated from a synthetic media after yeast fermentation. As a result of the
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release of mannoproteins and other polysaccharides during fermentation, this medium
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would be rich in complex macromolecules presenting a similar chemical structure.
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Fermentations with seven industrial S. cerevisiae strains and one hybrid S. cerevisiae x
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S. kudriavzevii were performed in duplicate on the complex medium GCY. The
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polysaccharide content of the macromolecular fractions isolated from the supernatants
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of these cultures was analysed both by the phenol-sulphuric method and the proposed
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methodology. Fig. 1 depicts the relationship found between the concentration of total
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polysaccharides and the concentration of mannose (Fig. 1A) or glucose (Fig. 1B)
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determined by HPLC after acid hydrolysis. Again, and as observed in the standard curve
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performed with commercial mannan, the relationship between concentration of mannose
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and the concentration of total polysaccharides can be fitted to a regression line that
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presents a R2 of 0.994.
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This relationship does not exist in the case of the concentration of glucose measured
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after hydrolysis and the concentration of total polysaccharides in the supernatants (Fig.
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2B). Indeed, when correlation studies were performed, mannose content and total
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polysaccharides significantly correlated (p<0.01) in the two sets of fermentations while
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glucose and total polysaccharides did not (even setting down the significance level,
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p<0.05).
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Another interesting fact is that the mannose content after acid hydrolysis also correlated
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with the mannose:glucose ratio of the macromolecular fraction of the supernatants in
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both sets of fermentations. This indicates that the differences in mannoproteins release
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found within the strains studied does not necessarily imply an increased release in some
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other cell wall polysaccharides as, for example, β-glucans (measured as glucose after
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acid hydrolysis). The differences observed in the release of total polysaccharides
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between the strains can be mainly explained by a difference in the secretion of
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mannoproteins (measured as the total amount of mannose).
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3.3. Mannoprotein content of synthetic must after yeast fermentation
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Fig. 2A shows the relationship found between the mannose obtained after acid
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hydrolysis of the macromolecular fraction of the supernatants (see a representative
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chromatogram in Fig. 3A) and the total polysaccharides measured by the phenol-
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sulphuric method. In this case, these two variables could be fitted to a regression line
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with a R2=0.810. Correlation between these variables was highly significant (Pearson’s
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correlation coefficient = 0.900, p<0.01). When the sum of the concentrations of
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mannose and glucose obtained after hydrolysis was plotted against the concentration of
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total polysaccharides measured using the phenol-sulphuric method (Fig. 2B) data could
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be fitted to a significantly improved regression line (R2=0.977). Correlation analysis
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between these variables rendered a Pearson’s coefficient of 0.988, p<0.01.
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These data indicate that for one of the wine yeast strains used in this study, W27, the
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difference in the release of total polysaccharides to the medium cannot only be
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explained by an increase in mannoprotein release but also by an over secretion of
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another polysaccharide that does not present mannose as the main sugar. Fig. 4 shows
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the values of glucose and mannose after acid hydrolysis of the macromolecular fractions
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of two fermentations in synthetic must (A and B). Excluding W27, values of glucose for
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all the strains tested ranged between 1.98 and 4.33 mg/L in both sets of fermentations.
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Glucose values obtained for W27 were clearly and significantly different from the rest
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of the strains (8.45±0.27 for fermentation A and 10.26±0.003 for fermentation B).
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This data support, once again, that acid hydrolysis of the macromolecular fractions
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isolated after fermentation in a synthetic must is a suitable strategy for the quantification
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of the mannoproteins released. In this case, it was necessary to filter the
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macromolecular fractions through the 10 DG chromatography columns twice, as some
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malic acid was eluted in the first filtration and interfered in the HPLC quantification of
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glucose and mannose.
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3.4. Mannoproteins content in the cell wall
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As the methodology had proven its usefulness for determining the amount of
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mannoproteins released during yeast fermentation, we decided to apply it in order to
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determine the mannose:glucose ratio in the yeast cell wall since this feature could be
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indicative for the potential of the release of mannoproteins in wine fermentations.
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The application of the proposed methodology to the quantification of mannoproteins of
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cell wall was inspired by the method published by François (2006). It should be
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mentioned that our methodology does not aim to determine the polysaccharide
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composition of the yeast cell wall quantitatively but to quantify the mannoproteins in it,
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measured as the amount of mannose released after hydrolysis.
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In first place, hydrolysis of cells harvested from 1, 3 and 5 mL of cultures were
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performed for the EC1118 strains in order to analyze if significant differences in the
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mannose:glucose ratio were found depending on the amount of biological material
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analyzed. No statistically significant differences were found between the three amounts
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used (data not shown). From that moment onwards, all cell wall hydrolysis were
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performed on cells harvested from 3 mL of culture (Fig. 3B). It is important to point out
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that cells were not dried prior to hydrolysis and, therefore, the concentration of
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mannoproteins per g of dry weight was not calculated. This data can easily be obtained
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if an equivalent volume of culture is used for such determination.
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It should be remarked that the reduction of, among others, this step allows an important
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reduction of time and reagents compared to the methodology described by François
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(2006). While preparation of 1 to 30 samples prior to quantification by HPLC will not
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take more than 6 hours, it would take around two and a half days in the protocol
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proposed by François (2006). Furthermore, based on our experience, steps performed
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for drying the biomass, wetting the dry cell wall mass before hydrolysis and
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neutralizing after hydrolysis are not reproducible and, furthermore, contribute to sample
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instability (data not shown). For these reasons these steps have been skipped in the
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proposed methodology. This was possible because the HPLC column employed in the
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quantification of the samples can perfectly tolerate high concentrations of H2SO4 and
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the mobile phase used is an aqueous solution with 1.5 mM sulphuric acid.
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3.5. Correlation between cell wall parameters and mannoproteins released
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The correlation between the different variables measured in the two culture media used
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in the present work was studied in order to analyze if any of the data that could be
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obtained from a quick fermentation performed in GCY (24 h) could give us some
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information on the amount of total polysaccharides and mannoproteins that would be
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released when these strains would be used in industrial grape must fermentations.
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In the two sets of fermentations performed in GCY medium, the mannose:glucose ratio
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in the cell wall positively correlates with three different variables: the total amount of
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polysaccharides released to the medium measured using the phenol-sulphuric method
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(r=0.813), the concentration of mannose in the macromolecular fraction of the
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supernatant (r=0.853) and the mannose:glucose ratio found in the macromolecular
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fraction of the supernatant (r=0.847) (p<0.05).
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However, in the case of synthetic must, no correlation was found between the
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mannose:glucose ratio of the cell wall and any of the other variables analyzed and
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measured in the macromolecular fraction of the supernatants.
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No correlation was found either between the mannose:glucose ratio of the cell wall and
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the mannose content in GCY and the same two variables measured in synthetic must.
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This fact indicates that the mannoprotein content of the cell wall cannot be used to
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predict the potential of the release of mannoproteins by wine yeast during fermentation.
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3.6. Stability of the hydrolysed samples
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Our main goal in this section was to prove if samples were stable after hydrolysis. This
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fact would allow an accurate quantification of the concentration of mannose and glucose
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even if the samples have to be stored for several days prior HPLC analysis. The
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concentration of these two sugars after 5 days of storage of the samples at the three
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temperatures tested, i.e. -20, 4 and 28 ºC, did not significantly differ from that obtained
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when the samples were analyzed just after hydrolysis (data not shown). This proves that
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sample neutralization is not needed in order to achieve its stabilization. Indeed, when
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the resulting acid pH of the samples after hydrolysis was neutralized using Ba(OH)2, as
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indicated by François (2006), this stability was not achieved. It is also noteworthy to
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mention that the temperature at which the sample is stored prior to injection does not
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seem to affect the stability either.
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3.7. Method reproducibility and repeatability
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During several years, our group has focused on the study of the genetic determinants of
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mannoproteins release in wine yeast (Gonzalez-Ramos et al., 2006; 2008; 2009). In all
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these studies, the phenol-sulphuric method has always been the chosen methodology to
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evaluate the release of polysaccharides in must fermentation. When performing these
365
measurements, it became apparent that factors such as the type of eppendorf tube used
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in the determinations, the time elapsed since the phenol solution had been prepared or
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the speed of the pipette flush in the addition of the sulphuric acid had a relevant impact
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on the results, which clearly hamper both the reproducibility and repeatability of the
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experiments. This is the reason why, in all the experiments, only one manipulator
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performed five replicates for the measurement of every sample following exactly the
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same methodology and material. All the sources of variation mentioned above are based
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on our experience but were also originally reported by Dubois, Gilles, Hamilton, Rebers
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and Smith (1956).
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In order to prove that the proposed methodology allows an improved repeatability and
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reproducibility compared to the phenol-sulphuric method, two macromolecular fractions
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isolated from two different fermentations in synthetic must were independently
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analyzed in duplicate by three manipulators using both methodologies. Results obtained
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after the analysis are depicted in Fig. 5. Panels A and B show the results for the
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polysaccharide concentration of two must fermentations determined by the phenol-
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sulphuric method in two different types of eppendorf tubes (Tube. 1 and 2). It is
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noteworthy to mention the differences observed both between manipulators
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(reproducibility) and between the measurements performed by the same manipulator in
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the same and different tubes.
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The values obtained individually by each manipulator in the three replicates performed
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following the phenol-sulphuric method were first analyzed. The relative standard
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deviation (RSD) obtained for the concentration of polysaccharides ranged from 3.2%
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(T73, manipulator no. 3, tube no. 1, Fig. 5B) to 21.9% (EC1118, manipulator no. 1, tube
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no. 1, Fig 6A). When the results obtained by the different manipulators for the same
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macromolecular fraction were compared and statistically analyzed, the RSD ranged
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from 5.8% (T73, tube no. 1, Fig. 5B) to 13.9% (T73, tube no. 2, Fig. 5B). Furthermore,
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a clear effect of the type of eppendorf tube used during sample analysis was observed.
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When the results obtained by the same manipulator for the same macromolecular
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fraction in the different tubes were compared, the RSD ranged from 0.6% (EC1118,
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manipulator no. 2, Fig. 5A) to 15,8% (EC1118, manipulator no. 3). The influence of the
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tube used in the analysis was also pointed out by Dubois et al. (1956), stating that an
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specific diameter would allow a good mixing without dissipating the heat generated in
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the reaction too rapidly. This represents a key factor, as a high maximum temperature is
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desired to increase the sensitivity of the reagent. All these data clearly prove the poor
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repeatability and reproducibility of the phenol-sulphuric method.
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Fig. 5C and 5D represent the values of glucose and mannose obtained after acid
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hydrolysis and HPLC quantification of the same macromolecular fractions analyzed in
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Figs. 5A and 5B. The RSD obtained individually by each independent manipulator for
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the mannose concentration after the double hydrolysis of the macromolecular fraction
404
ranged from 0.23% (T73, manipulator no. 3, Fig. 5D) to 3.1% (EC1118, manipulator
405
no. 2, Fig. 5C). When the results obtained by the three manipulators for the same
406
macromolecular fraction were compared, the RSD for the mannose concentration was
407
2.3% and 0.9% for EC1118 and T73 respectively, while the RSD for the glucose
408
determination in both fermentations was 1.5 and 1.6%. All these statistical data prove
409
how the proposed methodology allows a significant reduction of the relative standard
410
deviation of all the determinations performed individually by each manipulator
411
(repeatability) and a significant improvement of the reproducibility when data from the
412
three independent manipulators were compared.
413
414
4. Conclusions
415
It can be concluded that the proposed method represents a suitable strategy for the
416
overall quantification of yeast mannoproteins in fermentations performed in synthetic
417
media. This methodology presents a high reproducibility and repeatability when
418
compared with other currently available methodologies usually applied in the field and
419
can be performed with inexpensive and stable reagents and a HPLC equipment. Another
420
relevant advantage is that it allows the quantification of mannoproteins out of the total
17
421
amount of polysaccharides released and it can also be applied to the quantification of
422
the mannoproteins content in the yeast cell wall.
423
424
Acknowledgements
425
This work was supported by the Spanish Ministerio de Ciencia e Innovación (project
426
AGL2006-02558). The authors are grateful to Rubén Martínez Moreno and Vanessa
427
Penacho Martín for participating in the reproducibility assays included in this work and
428
to Laura López Berges and Cristina Juez Ojeda for excellent technical assistance.
429
430
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499
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500
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501
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502
winemaking products. American Journal of Enology and Viticulture, 46(4), 564-570.
503
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504
Waters, E., (2004). The mouth-feel properties of polysaccharides and anthocyaneins in a
505
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506
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507
that protects wine from protein haze. Carbohydrate Polymers 23(3), 185-191.
508
509
510
511
512
513
514
515
516
517
518
21
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Figure captions
520
521
Fig. 1. Relationship between the concentration of mannose (A) or glucose (B) released
522
after acid hydrolysis of the concentrated macromolecular fraction of supernatants
523
obtained after yeast fermentation in GCY medium, determined by HPLC, and the
524
concentration of total polysaccharides determined using the phenol-sulphuric method.
525
526
Fig. 2. Relationship between the concentration of mannose (A) or the sum of glucose
527
and mannose (B) released after acid hydrolysis of the concentrated macromolecular
528
fraction of supernatants obtained after fermentation of a synthetic must with 8 different
529
industrial yeast strains, determined by HPLC, and the concentration of total
530
polysaccharides determined using the phenol-sulphuric method.
531
532
Fig. 3. HPLC chromatograms depicting the peaks of glucose and mannose obtained
533
after acid hydrolysis of a concentrated macromolecular fraction isolated from a
534
synthetic must fermented by the yeast strain EC1118 (A) and after applying the same
535
methodology to EC1118 yeast cells (B).
536
537
Fig. 4. Concentration of glucose and mannose determined by HPLC after acid
538
hydrolysis of the concentrated macromolecular fraction derived from two sets of
539
fermentations in synthetic must with the eight industrial yeast strains included in this
540
study.
541
542
Fig. 5. Comparison of the repeatability and reproducibility of the proposed
543
methodology with the phenol-sulphuric method.
22
544
Panels A) and B): concentration of total polysaccharides measured by three different
545
manipulators in a macromolecular fraction isolated from the fermentation of a synthetic
546
must with two different yeast strains (EC1118, panel A; T73, panel B) using two
547
different types of eppendorf tubes (Tube 1, white columns; Tube 2, black columns).
548
Panels C) and D): concentration of mannose and glucose determined by HPLC after
549
acid hydrolysis of a macromolecular fraction isolated from the fermentation of a
550
synthetic must with two different yeast strains (EC1118, panel A; T73, panel B)
551
performed by three different manipulators (Man. 1, white columns; Man. 2, black
552
columns; Man. 3, grey columns).
553
23
A
Total polysaccharides (mg/L)
555
Figure 1
60
y = 1,755x - 0,385
R² = 0,993
50
40
30
20
10
0
0
10
20
30
40
Mannose (mg/L)
B
Total polysaccharides (mg/L)
554
60
y = -2,600x + 37,69
R² = 0,005
50
40
30
20
10
0
0,0
0,5
1,0
1,5
2,0
2,5
Glucose (mg/L)
24
556
Figure 2
557
B
y = 1,743x - 6,683
R² = 0,837
Total polysaccharides (mg/L)
Total polysaccharides (mg/L)
A 80
60
40
20
80
y = 1,263x + 3,864
R² = 0,982
60
40
20
0
0
0
20
40
Mannose (mg/L)
60
0
20
40
60
Glucose + Mannose (mg/L)
25
558
Figure 3
559
A
B
26
560
Figure 4
561
60
Glucose A
Mannose A
Glucose B
Mannose B
50
(mg/L)
40
30
20
10
0
27
Figure 5
Phenol-sulphuric method
A
B
EC1118
Tube 1
Tube 2
60
Total polysaccharides (mg/L)
Total polysaccharides (mg/L)
60
40
20
0
T73
Tube 1
Tube 2
40
20
0
Man. 1
Man. 2
Man. 3
Man. 1
Man. 2
Man. 3
Acid hydrolysis + HPLC
C
D
EC1118
60
60
Man. 1
Man. 2
Man. 3
(mg/L)
40
(mg/L)
562
20
T73
Man. 1
Man. 2
Man. 3
40
20
0
0
Glucose
Mannose
Glucose
Mannose
28