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Antonie van Leeuwenhoek (2007) 91:151157 DOI 10.



Effect of Agave tequilana juice on cell wall polysaccharides of three Saccharomyces cerevisiae strains from different origins
Blanca Aguilar-Uscanga Javier Arrizon Jesu Solis-Pacheco s Ramirez Josue

Received: 15 February 2006 / Accepted: 20 July 2006 / Published online: 21 November 2006 Springer Science+Business Media B.V. 2006

Abstract In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the b(1,3)-glucanase lytic activity and the b-glucan/ mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake asks with 30 g l1 sugar concentration of A. tequilana juice and with the control YPD using 30 g l1 of glucose. The three yeasts strains showed different levels of b-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast. Keywords Agave tequilana Cell wall b-Glucan Mannan Yeasts

Introduction Tequila is a Mexican alcoholic beverage obtained by distilling and rectifying the fermented agave juice and is protected by a guarantee of origin. The process is divided into four main phases: cooking, milling, fermenting, and distilling o 1995). First the agaves are harvested (Ceden (68 years old), then the plant leafs are pilled out to obtain the pines. The agave pines are cooked in autoclaves or brick ovens. During this process, most of the assimilable nitrogen is degraded by Maillard reactions due to heating (MancillaMargalli and Lopez 2002), which in turn produces compounds toxic to yeasts like furfural (Palmqvist et al. 1999) and vanillin (Fitzgerald et al. 2004), and make the juice poor in nitrogen content (Arrizon and Gschaedler 2002). Once the agave pines are cooked, they are milled to obtain the agave juice, which is then diluted to reach 1214 Brix degrees (80100 g/of agave sugar). In some cases, an inorganic nitrogen source is added before the beginning of fermentation. The fermentation can occur with or without inoculation of a starter culture, with yeasts isolated from agave must or other sources like wine or bakerys. It has been found that when tequila is produced at high Agave tequilana sugar fermentation (HASF), the strains isolated from different agave musts produced more alcohol and volatile components than wine strains

B. Aguilar-Uscanga (&) J. Arrizon J. Ramirez J. Solis-Pacheco n y Asistencia en Tecnolog a y Centro de Investigacio o del Estado de Jalisco A.C., Normalistas 800 Disen Col. Colinas de la Normal, Guadalajara, Jalisco C.P. xico 44270, Me e-mail:



Antonie van Leeuwenhoek (2007) 91:151157

(Arrizon et al. 2006). Furthermore, these yeasts, specically non-Saccharomyces strains, are tolerant to stress conditions such as sulte and ethanol resistant, which could be derived from the cell wall structure (Fiore et al. 2005). During the fermentation of different alcoholic beverages, the ethanol produced is one of the most important growth inhibitors, as a result of the inhibition of sugar and amino acids uptake (Boulton et al. 1996; Leao and Van Uden 1983). In addition the cytoplasmic membrane, hydrophobic proteins of the mitochondrial membrane, nuclear membrane, cytoplasm proteins, and cell wall are all specic targets of ethanol (Chow and Palecek 2004; Mauricio and Salmon 1992; Cartwright et al. 1986; Casey and Ingledew 1982). The yeast cell wall is the main determinant of cellular strength, and plays an important role in cell morphogenesis and cell growth (Cabib et al. 2001; Kollar et al. 1995). The cell wall is the rst structure in direct contact with the surroundings, provides physical and osmotic protection during fermentation and limits cell exposure to foreign proteins such as proteases (Chow and Palecek 2004). It is not a rigid structure and endures all the changes that the cell undergoes during division, morphogenesis and differentiation. Under a normal growth situation, the yeast Saccharamyces cerevisiae cell wall amounts for approximately 20% of the cell dry mass (Cabib et al. 2001). It is composed of cross-linked b-1,3 and b-1,6-glucans surrounding and connected to a thin shell of chitin and mannoproteins (Klis et al. 2002). The glucans and chitin are responsible for the mechanical rigidity of the cell wall. The mannoproteins provide a negative charge at physiologic pH and a hydrophilic environment (Lipke and Ovalle 1998; Kapteyn et al. 1999). Susceptibility of S. cerevisiae to lysis by glucanaseprotease mixtures has been qualitative measured to com n et al. 2003; pare the state of cell walls (Codo Ovalle et al. 1998; Lim et al. 1995; Douglas et al. 1994). In this study, the effect of the A. tequilana juice on cell wall composition and cell wall integrity was studied using YPD media as a control, employing three S. cerevisiae strains, two isolated from different agave musts (tequila and mezcal) and one bakery yeast strain.

Materials and methods Three S. cerevisiae yeast strains belonging to the xico), AR5, SLM and CIATEJ collection (Me LP, isolated from the mezcal, tequila, and bakery industry, respectively, were used. Fermentation in Agave tequilana must and YPD Fermentations in A. tequilana must were performed in 500-ml Erlenmeyer asks containing 200 ml of Agave tequilana juice sterilized at 120C for 15 min. The A. tequilana must was prepared by ltering concentrated A. tequilana juice provided by a tequila factory with a concentration of 30 g l1 of agave sugar and adding 1 g l1 of ammonium sulfate. The laboratory medium YPD (2% w/w peptone; 2% w/w yeast extract), containing 30 g l1 of glucose was used as a control. For both agave-based musts and the YPD medium, fermentations were carried out at 30C and pH 5. The initial yeast population inoculated was 2 106 cell ml1. Samples were taken at 0, 6, 12, and 24 h of fermentation for sugar consumption and mineral quantication. Sugar consumption and minerals quantication Reducing sugars were determined by DNS method as described by Miller (1959). The level of calcium, iron, manganese, and phosphorous were determined by using a inductively coupled plasma spectrometer Perkin Elmer (ICP Optima 3200 RL), with a method according with a Mexican norm (NOM-117-SSA1-1994). Extraction and quantication of cell wall polysaccharide Cells were collected during the early exponential phase, i.e., OD 600~ 1 (or 0.4 mg cell dry mass), based on the consideration that yeast cells are under non-limiting nutritional conditions at this growth phase. Culture samples (50 ml) were harvested at 4C by centrifugation (5 min at 4,500 g) and washed twice with ice-cold water. The pellet was resuspended in 0.5 ml of


Antonie van Leeuwenhoek (2007) 91:151157


ice-cold TrisHCl 10 mM/EDTA 1 mM at pH 7.5. Preparation and acid hydrolysis of the cell wall, as well as quantication of glucose and mannose released from H2SO4 hydrolysis of b-glucan and mannan were performed by highperformance liquid chromatography (HPLC), using an aminex column HPS87C (Bio-Rad), isothermal at 80C, with a mobile phase of water at 0.6 ml min1, and refraction index (IR) detector. Susceptibility to lytic action by b(1,3)-glucanase Susceptibility of S. cerevisiae to lysis by b(1,3)glucanase was investigated out following the procedure of Ovalle et al. (1998), with some modications. The assay was performed by resuspending 2 107 cells in a 3 ml solution of 10 mM TrisHCl and 1 mM EDTA, at pH 7.5 and 30C. The change of optical density at 660 nm was monitored every 10 min for the rst 2 h, and then every 30 min after the addition of 0.4 U b-1,3-Dglucanase from Helix pomatia (BioChemika). Determination of total carbohydrate The total carbohydrate content of the cell walls was determined using the phenolsulphuric acid method according to Dubois et al. (1956) with a glucose/mannose mixture (60:40) for the calibration curve.

the b-glucans and mannan in all the yeast strains. These changes in cell wall composition modied the b(1,3)-glucanase sensitivity which was lower following growth in A. tequilana juice, but only for the AR5 and SLM yeast. Strain LP showed the same b(1,3)-glucanase sensitivity in both growth media (Table 1). Effect of growth media on glucan/mannan synthesis response Comparing the glucan/mannan ratio in the two media, a difference in response was observed among strains studied in A. tequilana juice. The AR5 yeast strain increased glucan synthesis, while the SLM and LP yeast strains increased mannan synthesis (Fig. 1). It can be also observed that in both media, the glucan/mannan ratio was higher than 1 (g g1) for the AR5 yeast strain and was less than 1 (g g1) for SLM and LP yeast strains (Fig. 1). Therefore, the AR5 yeast strain had more glucan in its structure than the SLM and LP strains. Effect of growth media on cell wall integrity Differences in cell wall structure as well as in glucan and mannan synthesis were observed depending upon growth media. The susceptibility of cells to lytic action by b(1,3)-glucanase was additionally evaluated as a means to estimate cell wall integrity. For the yeasts isolated from agave, the activity was higher in YPD media than in A. tequilana juice (Fig. 2), while in the case of the LP yeast strain, the activity of the enzyme was similar in both growth media, as expected from the different responses in cell wall structure and glucan/mannan ratios in different media (Table 1 and Fig. 1). Effect of growth media on sugar and minerals consumption Sugar consumption rate was determined for the three yeasts in both media. For the agave isolated strains (AR5 and SLM), the sugar consumption rate increased in YPD medium (Fig. 3), while the sugar consumption rate for the bakery yeast (LP) was similar in both medium (Fig. 3). Comparing

Results Effect of growth medium on cell wall composition A difference in cell wall composition was observed between the bakery yeast (LP) and the two agave isolated yeast (AR5 and SLM) strains (Table 1). In general, the yeast strain LP had a higher cell wall mass, b-glucans and mannan content than the agave isolated yeast strains, in both YPD and A. tequilana juice. In regards to the effect of the growth media on cell wall composition, it was shown that A. tequilana juice decreased the cell wall mass, and the quantity of


154 Table 1 Effects of media on cell wall composition and cell wall sensitivity Strain and growth condition AR5 (YPD) AR5 (ATJ) SLM (YPD) SLM (ATJ) LP (YPD) LP (ATJ) Cell wall (dry mass %) 12.5 11.7 8.8 8.4 19.5 15.3 2.6 2.3 2.5 2.3 2.5 2.4 b-glucans (lg mg1) 52.4 36.4 48.7 36.2 79.3 56.4 1.8 2.4 1.7 2.3 2.2 2.3

Antonie van Leeuwenhoek (2007) 91:151157

Mannan (lg mg1) 44.1 26.2 53.4 41.6 91.4 72.1 3.2 2.8 2.5 3.2 3.3 2.1

b(1,3)-glucanase sensitivity (MLR) ( 102 min1) 0.21 0.074 0.20 0.058 0.09 0.09

ATJ, Agave tequilana Juice; YPD, Yeast extractPeptoneDextrose

B-glucan/mannan ratio (g/g)

1.5 1.2 0.9 0.6 0.3 0 AR5 SLM


not shown). Phosphorous concentration decreased at 24 h because of consumption by yeasts (Table 2), while the calcium concentration was the same at 0 h and 24 h (Table 2).


Fig. 1 b-Glucan/mannan (lg lg ) ratio in yeasts isolated from tequila, mezcal, and bakery (AR5, SLM, and LP, respectively) in two different media; YPD (n) and ATJ (h)

the initial levels of minerals, it can be seen that calcium concentration is considerably higher in A. tequilana juice than in YPD medium (Table 2), while the concentration of phosphorous was similar in both medium (Table 2). Iron and manganese concentrations were less than 0.3 mg l1, the other minerals were in traces (data

Maximum sugar consumption rate (g/l.h)

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 50 100 150 200 Time (min) 250 300

A. tequilana juice is the most commonly used must for tequila fermentation. However, this medium contains several minerals and organic compounds that are toxic to yeasts like furans and pez 2002; vanillin (Mancilla-Margali and Lo Palmqvist et al. 1999; Fitzgerald et al. 2004). It was reported that Saccharomyces and nonSaccharomyces yeasts isolated from agaves musts possess stress tolerance to some conditions during fermentation due possibly to their cell wall structure (Fiore et al. 2005; Arrizon et al. 2006). In this study, a comparison of the polysaccharides in the yeast cell wall was carried out in different media (A. tequilana juice and YPD) with two Saccharomyces yeasts isolated from two different agave musts (AR5 and SLM, from tequila and

DO (660 nm)


Fig. 2 Cell wall susceptibility in different media; dark (YPD) and white (ATJ) with the three yeasts isolated from tequila, mezcal, and bakery; AR5 (n and h), SLM (m and n) and LP (r and e), respectively

Fig. 3 Maximum sugar consumption rate in Agave tequilana juice (JA) and yeast peptone dextrose medium (YPD) for AR5 (n), SLM (n) and LP (h) yeast strains


Antonie van Leeuwenhoek (2007) 91:151157 Table 2 Calcium and phosphorous concentration in Agave tequilana juice and YPD fermentations Yeast strains Time (h) Agave tequilana juice Ca (mg l1) AR5 SLM LP 0 24 0 24 0 24 137.1 136.2 136.5 137.2 137.2 136.7 1.2 1.4 0.9 1.1 0.8 1.2 P (mg l1) 30.1 19.8 29.8 17.9 30.3 20.1 0.4 0.5 0.3 0.6 0.5 0.3 YPD Ca (mg l1) 3.5 3.6 3.4 2.9 3.5 3.4 0.2 0.3 0.4 0.2 0.2 0.3


P (mg l1) 32.1 19.7 31.5 18.2 31.2 24.1 0.5 0.6 0.3 0.4 0.5 0.4

Ca, Calcium; P, Phosphorous

mezcal, respectively) and compared to a bakery derived yeast (LP). Cell wall synthesis and degradation is highly adaptable to the changes in environment and cell division. Also, the cell wall responds to genetic defects that limit synthesis of one component by up regulating production of other components. For example, disrupting genes that code for glucan synthases increases chitin and mannoproa-Rodr guez et al. 2000; tein production (Garc Popolo et al. 1997), and it is well known that the over-expression of the EXG1 gene of exob(1,6)glucanase produces a tolerance to the killer K1 toxin and a decrease in the yeast b(1,6)-glucan content (Montijn et al. 1999; Jiang et al. 1995). The yeasts tested in this study have differences in their cell wall structures, irrespective of the media used. AR5 has more glucan in its cell wall structure, whereas the strains SLM and LP have more mannan. The level of these polysaccharides was increased in the yeasts that were grown in A. tequilana juice. Therefore, it is possible that in A. tequilana juice, the expression for genes for mannan synthesis could be increased in the SLM and LP yeast strains, while possibly the genes expression for glucan synthesis was increased in the AR5 strain. As a result, differences in the cell wall structure were observed as an adaptation to stress caused by A. tequilana juice, and this response to stress is also observed in a reduction of the sugar consumption rate of the agave isolated yeasts (AR5 and SLM) compared to when they were grown in YPD media. It was also observed that the activity of the b(1,3)glucanase enzyme decreased in strains grown in A. tequilana juice, specically in the AR5 and SLM strains. Aguilar-Uscanga and Francois

(2003), reported an increase in the b(1,6)-glucan synthesis which produces high cell wall lysis resistance. The AR5 and SLM strains showed a specic adaptation to the stress conditions associated with A. tequilana juice by an unknown mechanism. It is possible that A. tequilana juice induce expression of the genes of b(1,6)-glucan synthesis, and as a consequence decreased activity of b(1,3)-glucanase. Lagorce et al. (2002) reported that a decrease of b-glucan and mannan levels is associated with an increase in chitin content. Our results showed a signicant decrease in b-glucan and mannan, therefore it would be interesting to evaluate chitin level in the cell wall of the studied strains. Additionally, as the synthesis of mannan and glucan vary between the strains in response to A. tequilana juice composition, it would be interesting to investigate the gene expression directing the synthesis of these polysaccharides during the fermentation in A. tequilana juice and other agave juices involved in different Mexican beverages. It was observed that the level of calcium concentration was very high in A. tequilana juice and it was not consumed during fermentation. Thus, it could be possible that this mineral and other compounds produce the stress conditions in A. tequilana juice fermentation.

Conclusions The synthesis of mannan and glucan vary between strains in response to A. tequilana juice composition. Possibly, the cell wall composition of yeast changes in order to respond to the stress



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conditions of A. tequilana juice. The two yeasts isolated from agave musts were better adapted to the A. tequilana juice fermentation and as a consequence they were more resistant to the b(1,3)-glucanase activity. As the concentration of calcium is considerable in A. tequilana juice, future studies have to be performed in order to study the role of the concentration of this mineral in directing glucan and mannan synthesis gene expression and in the stress response of yeasts.
pez for the HPLC Aknowledgements We thank Zaira Lo technical support and Anaberta Cardador for a critical reading of the manuscript.

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