2013 ajev

Glucose and Ethanol Tolerant Enzymes Produced

by Pichia (Wickerhamomyces) Isolates from Enological Ecosystems Tania Madrigal,1 Sergi Maicas,1 and José J. Mateo Tolosa1* Abstract: A total of 17 Pichia (Wickerhamomyces) isolates obtained from enological ecosystems in the UtielRequena Spanish region were characterized by physiological (using API 20C AUX strips and ID Yeast Plus System miniaturized identification systems) and molecular (PCR-RFLP and sequencing) techniques as belonging to the species P. fermentans, P. membranifaciens, and W. anomalus. Data support the reclassification of P. anomala as Wickerhamomyces anomalus. In order to characterize their enzymatic abilities, xylanase, β-glucosidase, lipase, esterase, protease, and pectinase qualitative and quantitative assays were made. Wickerhamomyces anomalus and P. membranifaciens were the most interesting species as a source of enzymes for the winemaking industry. Glycosidase enzymes had a high degree of tolerance to high levels of glucose and ethanol, making them of great interest for enological use.

Key words: Pichia, Wickerhamomyces, must, wine, glycosidases, ethanol, glucose

It is well established that wine fermentations, as conducted by traditional methods (without inoculation), are not the result of the action of a single species or a single strain of yeast.

Rather, the final products result from the combined actions of several yeast species that grow more or less in succession throughout the fermentation process. Previous studies performed in various countries have described the isolation and identification of yeasts from grape surfaces, and quantitative data on the ecology of grape yeasts have shown that the isolation process of the total yeast population from the grapes is dependent on many factors (Esteve et al. 2001).

Fermentations are initiated by the growth of various species of Candida, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, and Torulaspora genera.

Their growth is generally limited to the first two or three days of fermentation, after which they die off. Subsequently, the most strongly fermenting and more ethanol-tolerant species of Saccharomyces take over the fermentation (Fleet and Heard 1993). It is believed that during the first step of the fermentation, low-fermentative yeasts produce some important reactions in must that improve the final flavor of wines (Gil et al.

1996, Lambrechts and Pretorius 2000).

Although yeasts can produce spoilage of the fruits (Arroyo et al. 2008), these microorganisms also possess many interesting technological properties for food processing (Charoenchai et al. 1997, Strauss et al. 2001, Maicas and Mateo 2005). Previous works have studied esterases, glycosidases, lipases, proteases, catalase, and killer activities of different yeast species related with different ecosystems (Mateo et al.

2011). These studies must be accompanied by other assays to assess a correct and unambiguous identification of the yeast species. Molecular methods have been used to explore this yeast biodiversity (Arroyo et al. 2008), as they confer a high degree of accuracy in the final identification.

Enzymes are used at various stages of winemaking, depending on grape variety and processing technology. They increase the juice yield, aroma, and color extraction and improve the clarification and sedimentation of grape must (Maicas and Mateo 2005). The action of the enzymes begins during the ripening and harvesting of grapes and continues through alcoholic and malolactic fermentation, clarification, and aging. Winemakers often supplement naturally occurring grape enzymes with commercial enzymes to increase production capacity of clear and stable wines with enhanced body, flavor, and bouquet (Mateo et al. 2011).

The ascomycetous yeasts Pichia ssp. and Wickerhamomyces anomalus are frequently associated with food and feed products, either as a production organism or as spoilage yeasts.

The ability to grow in preserved food and feed environments is due to their capacity to grow under low pH, high osmotic pressure, and low oxygen tension. They are also frequently isolated from wineries, generally associated with the production of volatile compounds involved in wine aroma (Charoenchai et al. 1997, Manzanares et al. 1999). This positive contribution to the wine aroma is focused on the production of volatile compounds, mainly ethyl acetate, and on the production of glycosidases and xylosidases (Manzanares et al. 1999). However,

Department of Microbiology and Ecology, Universitat de València, Dr. Moliner 50, E-46100-Burjassot, Valencia, Spain. *Corresponding author (email: Jose.J.Mateo@uv.es; tel: + 34 96 354 30 08; fax: + 34 96 354 45 70) Acknowledgments: This work was supported by RM2007-00001-00-00 from Instituto Nacional de Investigaciones Agrarias, Spain and INV-AE112-66049/ UV, Universitat de València, Spain.

Manuscript submitted May 2012, revised Sept 2012, accepted Oct 2012. Publication costs of this article defrayed in part by page fees.

Copyright © 2013 by the American Society for Enology and Viticulture. All rights reserved. doi: 10.5344/ajev.2012.12077 1

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these yeasts can also act as wine spoilage microorganisms.

These yeasts are very interesting from both fundamental and applied points of view (Arroyo et al. 2005, Mateo et al. 2011).

The aim of our work has been to study the potential of Pichia species to be used as a source of enzymes in the winemaking industry and in other biotechnological processes.

Materials and Methods Yeast isolates and miniaturized identification systems.

The 17 isolates belonging to genus Pichia were previously isolated from the Utiel-Requena region (eastern Spain) (Mateo et al. 2011) and identified following morphological and physiological characteristics (Barnett 1990). Miniaturized identification systems used for this purpose were API 20C AUX strips (Bio Merieux S.A., La Balme-les-Grottes, France) and Rapid ID Yeast Plus System (Remel, Lenexa, KS). All processes were made according to manufacturer’s instructions.

Yeast typing by molecular techniques. DNA extraction.

Pure cultures of yeasts were grown on 10 mL of YEPD medium (1% yeast extract, 2% mycological peptone, 2% glucose) at 28°C for 24 to 48 hr on an orbital shaker. Small-scale preparation of chromosomal DNA was performed by using an Ultraclean Microbial DNA Isolation Kit (MoBio, Carlsbad, CA).

The quality of the extracted DNA was checked by electrophoresis in 0.8% (w/v) agarose mini-gels using TBE (45 mM Tris borate, 1 mM EDTA, pH 8.0) buffer with added ethidium bromide at a final concentration of 0.5 mg/mL. These gels were used to estimate the approximate DNA concentration.

An amount of 10% of the preparation was compared to digested λDNA cleaved with EcoRI and HindIII (Boehringer Mannheim GmbH, Mannheim, Germany).

PCR amplification. The region between the genes 18S rRNA and 28S rRNA was amplified using internal transcribed spacers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) as primers (Boehringer Mannheim) (White et al. 1990). The amplification reaction was performed in a Primus 25 thermocycler (MWG, Ebersberg, Germany) under the following conditions: a 50 µL reaction mixture was prepared with 1.5 U Taq DNA polymerase (Boehringer Mannheim), 0.5 µM of each primer, 0.2 mM of each dNTP, 10 mM Tris-HCl, 1.5 mM MgCl 2, 50 mM KCl (buffer), and 10 to 20 ng yeast DNA. The mixture was subjected to an initial denaturing step of 5 min at 95°C, followed by 35 cycles of 1 min at 95°C, 2 min at 52°C, and 2 min at 72°C, and a final extension step of 10 min at 72°C. Amplification products were separated by electrophoresis in a 0.8% (w/v) agarose gel. A 25 bp DNA ladder commercial standard (Takara, Shiga, Japan) was used. PCR amplifications were purified and washed with a high-pure PCR product amplification kit (Boehringer Mannheim).

Restriction analysis. PCR products were digested without subsequent purification with the following restriction enzymes: CfoI, HaeIII, Hinf I, and MspI for differentiation of yeasts to species or strain level. The final digestion volume was 20 µL, containing 12 µL amplified DNA solution, 2 µL restriction enzyme (10 U/µL) (Boehringer Mannheim), 2 µL restriction buffer (Boehringer Mannheim), and 4 µL purified

water. The mixture was incubated at 37°C for 8 hr. The restriction fragments were checked by electrophoresis in 1.2% (w/v) agarose gel. The restriction fragment sizes measured as base pairs were calculated in comparison with a 100 bp DNA ladder commercial standard (Takara). Profiles were compared with the www.yeast-id.org database (CECT, Paterna, Spain).

Sequence analysis of D1/D2 domains of 26S rDNA gene.

This procedure was used to confirm the identifications previously carried out by RFLP analysis of the 5.8S-ITS rDNA region. PCR amplification for the D1/D2 domain of 26S rDNA was basically performed according to Kurtzman and Robnett (1998). The PCR product was purified using an UltraClean PCR Clean Up Kit (MoBio) according to the manufacturer’s instructions. Direct sequencing of the purified PCR products was performed by ABI Prism BigDye Terminator Cycle Sequence Ready Reaction Kit (Applied Biosystems, Foster City, CA). The sequences were aligned using the BLAST program, with complete or nearly complete 26S rDNA gene sequences retrieved from the EMBL nucleotide sequence data libraries.

Qualitative screening of biochemical activities. The biochemical activities listed below were assayed in duplicate and results qualitatively expressed as “–” (no activity), “+” (weak activity), and “++” (strong activity). A positive control was used in all assays.

Protease activity. Exocellular protease production was determined according to Strauss et al. (2001) by spreading yeast colonies onto YPD agar plates containing 20 g/L casein.

Plates were incubated at 28°C for 7 days. A clear zone around the colony is indicative of protease activity. β-Glucosidase activity. The basal medium consisted on 1.7 g/L Yeast Nitrogen Base (Difco, Lawrence, KS), 5 g/L ammonium sulphate, 5 g/L glucose, and 20 g/L agar. After autoclave, 2 mL sterile 1% (w/v) 4-methylumbelliferyl-β- d glucopyranoside (Sigma, St. Louis, MO) was added to 100 mL melted medium (Manzanares et al. 1999). The medium was then poured into Petri dishes and inoculated with 24-hrold yeast cultures. Plates were incubated at 28°C for 3 days.

The presence of the enzymatic activity was visualized as a fluorescent halo surrounding yeast growth by plate exposition to UV light.

Lipase and esterase activities. Yeast isolates were used to determine esterase and lipase activities on tributyrin and rhodamine olive-oil agar media, respectively, according to the procedures described by Hernandez et al. (2007). After 48 hr of incubation at 28°C in the media, colonies were analyzed.

For detection of lipase activity, they were irradiated with UV light at 350 nm; lipase activity was detected by an orange fluorescent halo around colonies. Esterase activity was detected by the formation of a clear transparent halo around colonies.

Pectinase activity. The assays were carried out in a medium of 1 g/L yeast extract, 1 g/L ammonium sulfate, 6 g/L NaHPO4, 3 g/L KH 2PO4, 5 g/L pectin, and 15 g/L agar. After streaking 48-hr-old yeast cultures onto the surface of the medium, the plates were incubated at 28°C for 5 days and then revealed by addition of a solution of 1 g/L hexadecyltrimethylammonium bromide. Activity was evidenced by the presence of a clear halo around the colonies.

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Xylanase activity. The ability of yeasts to produce an extracellular xylanase was measured according to the method described by Hernandez et al. (2007). The assays were carried out on yeast extract agar containing 5 g/L xylan, 5 g/L peptone, and 5 g/L NaCl. After streaking 48-hr-old yeast cultures onto the surface of the medium, the plates were incubated at 28°C for 7 days. Xylanase activity was evidenced by the presence of a clear halo around the colonies.

Quantitative activity assays. Yeast isolates that showed promising technological characteristics in the first screening were then subjected to quantitative estimation of their biochemical activities. Substrates and chemicals were obtained from Sigma. Experiments were carried out in duplicate and results were compared against a negative strain (Hanseniaspora occidentalis CECT 11329), which did not show any activity.

Xylanase and β-glucosidase activities were determined by adding 106 cells/mL of 24-hr-old cultures of the different isolates to an induction medium (0.1% yeast extract, 1% mycological peptone, 4% xylan). After incubating 2 days at 28°C, 2 mL yeast culture was centrifuged and the pellet resuspended in 750 µL 0.1 M pH 5.0 citrate phosphate buffer. Next, 250 µL p-nitrophenyl-β- d -glucopyranoside (for β-glucosidase assay) or p-nitrophenyl-β- d -xylopyranoside (for xylanase determination) solution (1 mg/mL) was added and the mixture was incubated at 40°C for 90 min. To stop the reaction, 1.0 mL 0.2 M Na 2 CO 3 was added. Yellow color released from the substrate was measured at 404 nm and activity was expressed as nanokatals (1 nkat = 1 nmol of pNP liberated in 1 min by 106 yeast).

Lipase and esterase activities were determined by using 106 cells/mL of 24-hr-old cultures in YEPD. After centrifugation, cells were resuspended in 900 µL 0.1 M Tris HCl pH 8.0 buffer and then added to 100 µL p-nitrophenyl palmitate (for lipase determination) or 1 mg/mL p-nitrophenyl butyrate (for esterase assay) solution. The mixture was incubated at 37°C for 60 min and then stopped with addition of 1.0 mL 0.2 M Na 2CO3. Yellow color released from the substrate was measured at 404 nm and activity was expressed as nanokatals (1 nkat = 1 nmol of pNP liberated in 1 min by 10 6 yeast).

6 Pectinase activity was determined by adding 10 cells/mL of 24-hr-old cultures of the different isolates to YEPD medium containing 1% pectin prepared in sodium acetate buffer (0.05 M pH 5.5). Cultures were incubated at 28°C for 7 days.

The reaction was stopped with 1.0 mL dinitrosalicylic acid solution (Miller 1959), after which the mixture was boiled for 10 min and cooled. The color was read at 540 nm. The amount of reducing sugar released was quantified using galacturonic acid as standard. The enzyme activity was calculated as nanokatals (1 nkat = 1 nmol of galacturonic acid liberated in 1 6 min by 10 yeast).

The protease assay procedure was previously described by Tremacoldi and Carmona (2005). Briefly, yeasts were inoculated in YPD-casein medium and incubated at 26°C for 6 days 6 to induce protease activity. After adding 10 cells/mL to a solution containing 500 µL 50 mM Tris HCl pH 7.5 buffer and 500 µL 1% (w/v) casein in the same buffer, the mixture was incubated 12 hr at 30°C and 1 mL 3% (w/v) TCA was added.

Tubes were maintained on ice for 30 min and centrifugated.

Liberated tyrosine was determined by absorbance at 280 nm.

The protease activity was calculated as nanokatals (1 nkat = 1 nmol of tyrosine liberated in 1 hr by 106 yeast).

Influence of environmental conditions on enzymatic activities. The effect of various levels of glucose and ethanol on enzymatic activities was determined by alteration of these parameters in the assay mixture, prepared as described in previous section and added with 1 to 20% (w/v) glucose and 1 to 20% (v/v) ethanol. Blanks were prepared with no addition of any compound. The pH optimum was determined in citrate-phosphate buffer covering a pH range from 3.0 to 8.0, at 40°C for 90 min. The temperature optimum was measured from 4°C to 60°C for 90 min of incubation in the same buffer, at pH 6.0. Assays were performed in triplicate.

Statistical analysis. Dendrograms were generated using InforBIO V5.28-J4 software, setting the UPGMA method of clustering and Euclidean similarity. InforBIO (Informationbase for Biology) is an e-Workbench for the database, classification, and identification of microbes.

Results Yeasts population. The non-Saccharomyces yeasts isolated, belonging to 12 different species, were isolated from Bobal musts obtained from the Utiel-Requena region by using lysine agar as differential medium. Main genera detected were Hanseniaspora and Pichia (including P. anomala, recently named as Wickerhamomyces anomalus) (35% of each genus) and other species (Mateo et al. 2011). A total of 17 Pichia and Wickerhamomyces isolates were used in this study.

Physiological characterization of Pichia isolates. Pichia isolates gave negative results for trehalose, lipase, α-galactosidase, β-galactosidase, urease, and n-acetylglucosamine by using the Rapid ID system. Statistical analysis of the results allowed the differentiation of three groups among Pichia isolates (Figure 1A): one group included all yeasts with positive results for α-glucosidase, β-glucosidase, and β-fucosidase, the second group included yeasts with negative results for all the tests except for prolyn-arylamidase and histidyl-arylamidase, and the third group included two isolates that could be clearly differentiated from the other two groups.

All isolates showed negative results for arabinose and galactose by using the API20C AUX miniaturized system.

Isolates could be classified in the same three groups (Figure 1B) previously described by using the Rapid ID system.

The first group exhibited positive results for methyl-α-Dglucopiranoside, cellibiose, lactose, maltose, sucrose, trehalose, melizitose, and raffinose, while the second group included isolates that only showed positive results for glucose.

The third group included two isolates that could be clearly differentiated from the other two groups.

Molecular identification of isolates. Isolated strains were subjected to a PCR-RFLP analysis of the 5.8S-ITS rDNA region. The PCR-amplified products of the 5.8S-ITS region showed variations in length, ranging from 470 to 600 bp. The profile comparison of these isolates (Table 1) with the yeast-id

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database and published papers (Bautista et al. 2011), complemented with physiological data, allowed identification to the specie level. Profiles corresponding to the species Wickerhamomyces anomalus (formerly Pichia anomala) (Wa1–Wa8), P. fermentans (Pf1 and Pf2), and P. membranifaciens (Pm1–Pm7) were found. These results were confirmed with those obtained by sequence analysis of the D1/D2 domains of the 26S rDNA gene. There is a clear correspondence between these data and

those obtained by using identification miniaturized systems.

The first group includes isolates identified as W. anomalus by molecular techniques, the second includes P. membranifaciens isolates and the third one includes two isolates belonging to P. fermentans species.

Qualitative detection of enzyme activities. Biochemical activities were qualitatively determined for all yeast isolates (Table 2). As usually occurs in studies of this nature, activities were species-dependent (Fernandez et al. 2000, Arevalo et al. 2005). Briefly, only five isolates from W. anomalus and one isolate from P. fermentans showed a weak protease activity, while considerable β-glucosidase activity was detected for all isolates from W. anomalus species, as other authors have previously published (Bautista et al. 2011). Lipase activity was a more widespread property but only half of the isolates of P. fermentans and P. membranifaciens showed this activity. Esterase activity seemed to be a characteristic of W. anomalus, while pectinase and xylanase were present in all species tested.

Qualitative results obtained for esterase and xylanase activities do not agree with those described by other authors (Hernandez et al. 2007, Bautista et al. 2011). Instead of a clear halo around colonies, both activities were evidenced by the

Table 2 Qualitative assays carried out for Pichia/ Wickerhamomyces isolates, expressed as “–” (no activity), “±” (very weak activity), “+” (weak activity), and “++” (strong activity).

Activity Isolate β-Glucosidase Protease Lipase Esterase Pectinase Xylanase

Figure 1 Cluster analysis of yeast isolates based on results obtained by miniaturized systems: (A) Rapid ID Yeast Plus System, (B) API 20C AUX strips.

Wa1 Wa2 Wa3 Wa4 Wa5 Wa6 Wa7 Wa8 Pf1 Pf2 Pm1 Pm2 Pm3 Pm4 Pm5 Pm6 Pm7

++ ++ ++ ++ ++ ++ ++ ++ + – – – – – – – +

– – + – + + + + – – + – – – – – –

++ ++ + + ++ + ++ + – ++ – – – – + ++ +

+ + + + + + + + – – – – – – – – +

++ + + + + + ++ ++ + + – – – – – + –

+ + + + + + + – – – – – – – – – +

Table 1 Restriction analysis of the 5.8S-ITS PCR products and sequence information for the D1/D2 domains of the 26S rDNA gene of the yeasts isolated from wine.

Profile I II III a

Isolates (n) 8 2 7

PCR product (bp) 690 450 460

Restriction fragments (bp) CfoI 630 170, 100, 100, 80 220, 130, 110

HinfI 320, 270 250, 200 260, 200

HaeIII 630 340, 90 290, 170

MspI 690 250, 200 220, 130, 110

Identity (%)a 99.8 99.3 99.6

Closest relative sp.

W. anomalus P. fermentans P. membranifaciens

Sequence identity in the D1/D2 region of isolates of the 26S ribosomal gene and the closest relative species in the NCBI GenBank database.

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growth of the microorganism on the surface of the plate; the lack of the enzymatic activity was proved by the absence of the colony. Our results were confirmed by the quantitative study of the assayed activities.

Quantification of enzymatic activities. Quantification of the different enzymatic activities showed that P. fermentans isolates had no β-glucosidase activities (Table 3). Changes in W. anomalus isolates were very high, ranging from 0.52 to 1.89 nkat. β-Glucosidase activities in P. membranifaciens isolates were very homogeneous (~0.65 nkat) with the exception of Pm7, which showed the highest activity. Xylanase activity showed very low changes among Wa isolates, ranging from 0.28 to 0.37 nkat. Only one of the P. fermentans showed this activity, but Pm isolates showed the highest xylanase activities, with special attention on the Pm7 isolate. Esterase and lipase activities were very low in all isolates assayed; slight differences in lipase activity were found but W. anomalus gave the highest values. Pectinase activities were low in P. fermentans and P. membranifaciens isolates, as highest values were reached when W. anomalus were assayed, mainly Wa7 and Wa8 isolates.

These data agree with those shown in Table 2 and confirm W. anomalus and P. membranifaciens Pm7 are the most interesting yeasts to be used as a source of enzymes for enology, confirming previous published results (Bautista et al. 2011, Mateo et al. 2011). The procedure allowed for ease in the quantification of the activities, but it is difficult to compare the data published by different researchers because there is not a unique mode to define units for enzymatic activity. An effort should be made to use a standard unit of measure.

Effect of environmental conditions on enzymatic activities. Only glycosidase activities (β-glucosidase and xylanase) maintain their activity in the presence of glucose or ethanol

concentrations present in must or wine, respectively, showing these are the most interesting enological activities produced by these yeasts. Taking into account the low activities

by P. fermentans, the influence of ethanol and glucose was only studied in W. anomalus and P. membranifaciens isolates. Figure 2 shows data for Wa6 and Pm7 isolates. In both yeasts, β-glucosidase remained ~20% at 10% (w/v) glucose and was not changed at higher levels of glucose. The enzyme retained ~50% of its maximal xylanase activity, even in the presence of 20% (w/v) glucose (Figure 2A). These results are in agreement with other Pichia species (Manzanares et al. 1999) regarding their high tolerance to glucose. When ethanol was added to the reaction mixture, the W. anomalus and P. membranifaciens isolates showed a similar behavior (Figure 2B). The activities slightly decreased at the highest ethanol content, remaining 80% of activity at 20% (v/v) ethanol. All enzymes had an optimum pH at 6.0 with minimum at pH 3.0 (40% of the maximum activity) and pH 8.0 (60% of the activity) (Figure 2C). β-Glucosidase from Wa6 isolate showed some differences regarding its dependence on pH and is more stable in the pH range studied. β-Glucosidase from both isolates showed the same dependence on temperature, with a maximum at 40ºC (Figure 2D). Xylanase from the Pm7 isolate had the same optimum temperature, but was less active at low temperatures; nevertheless, xylanase from Wa6 isolate showed a higher optimum temperature (50°C) and maintained 20% activity atl low temperatures. All enzymes showed thermal inactivation at 60°C.

Discussion Pichia is a ubiquitous yeast that can be isolated from diverse agronomical ecosystems (Strauss et al. 2001, Arroyo et al. 2005, Hernández et al. 2007). The original definition of

Table 3 Quantitative activity for yeast isolates. (Units are described in the text.)

Isolate Wa1 Wa2 Wa3 Wa4 Wa5 Wa6 Wa7 Wa8 Pf1 Pf2 Pm1 Pm2 Pm3 Pm4 Pm5 Pm6 Pm7 CECT 11329 a

β-Glucosidase 0.89 0.56 0.52 0.61 0.93 1.51 0.95 0.67

a

± 0.09 ± 0.06 ± 0.08 ± 0.07 ± 0.06 ± 0.10 ± 0.08 ± 0.08 nd nd 0.76 ± 0.04 0.63 ± 0.05 0.60 ± 0.04 0.64 ± 0.08 0.61 ± 0.07 0.63 ± 0.09 2.12 ± 0.12 nd

Xylanase 0.37 0.36 0.33 0.35 0.36 0.28 0.36 0.35 0.38

± 0.06 ± 0.05 ± 0.04 ± 0.06 ± 0.05 ± 0.03 ± 0.02 ± 0.04 ± 0.06 nd 0.69 ± 0.08 0.54 ± 0.07 0.55 ± 0.07 0.62 ± 0.06 0.58 ± 0.09 0.70 ± 0.08 0.86 ± 0.08 nd

Activity (nkat) Lipase Esterase 0.49 0.59 0.72 0.71 0.62 0.63 0.45 0.52 0.43 0.44

± 0.06 ± 0.07 ± 0.07 ± 0.09 ± 0.08 ± 0.07 ± 0.03 ± 0.04 ± 0.05 ± 0.04 nd 0.55 ± 0.07 0.50 ± 0.08 0.44 ± 0.04 0.42 ± 0.05 0.41 ± 0.03 0.70 ± 0.08 nd

0.17 ± 0.03 nd 0.20 ± 0.04 0.19 ± 0.05 0.17 ± 0.04 0.19 ± 0.03 0.17 ± 0.02 0.16 ± 0.02 0.24 ± 0.03 0.16 ± 0.02 nd 0.23 ± 0.03 nd nd nd nd 0.18 ± 0.02 nd

Values are means ± SD (n = 3); nd: not detected.

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Pectinase 0.39 0.27 0.23 0.17 0.21 0.20 0.52 0.42 0.21 0.24

± 0.03 ± 0.02 ± 0.02 ± 0.01 ± 0.02 ± 0.01 ± 0.16 ± 0.02 ± 0.02 ± 0.02 nd nd nd 0.12 ± 0.01 0.11 ± 0.01 0.19 ± 0.01 0.15 ± 0.01 nd

Protease nd nd 0.17 ± 0.02 nd 0.20 ± 0.04 0.17 ± 0.02 0.15 ± 0.02 0.22 ± 0.03 nd nd 0.11 ± 0.01 nd nd nd nd nd nd nd

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genus included yeasts with very different physiological and molecular characteristics. The placement of P. anomala in the genus Wickerhamomyces by Kurtzman et al. (2008) following multigene phylogenetic analysis has raised concern whether these results argued for a new genus. Results here support the need to define the new Wickerhamomyces genus

Figure 2 Effect of (A) glucose concentration (% w/v), (B) ethanol concentration (% v/v), (C) pH, and (D) temperature on β-glucosidase and xylanase activities from Wa6 and Pm7. Maximum values are those reported in Table 2. Assays were performed in triplicate.

that includes the old P. anomala as a new species named W. anomalus (Kurtzman et al. 2008). Additional efforts should be made to establish a definitive taxonomy in the yeast world, mainly regarding those of industrial interest.

The studies of β-glucosidase enzymes must address the terpene compounds that contribute to the varietal character of wines (Mateo and Jimenez 2000). There are some differences between qualitative and quantitative data for this activity obtained in our work, probably due to the different substrates used in the assays. All W. anomalus isolates produced β-glucosidase in both assays, but P. membranifaciens Pm7 isolate, which shows the highest activity, was the only one from this species showing activity in the qualitative assay. An extensive review of 317 strains from 20 wine yeast species indicated that yeasts of the Candida, Debaryomyces, Hanseniaspora, Kloeckera, Kluyveromyces, Metschnikowia, Pichia anomala (now W. anomalus), Saccharomycodes, Schizosaccharomyces, and Zygosaccharomyces genera carry out β-glucosidase activities (Rosi et al. 1994). No data has been previously published regarding the production of β-glucosidase by P. membranifaciens. In a screening of 48 yeast strains of the genera Candida, Kluyveromyces, Debaryomyces, and Pichia anomala for production of extracellular glucose tolerant β-glucosidase activity, all yeast strains tested produced extracellular β-glucosidase activity, but enzymes from only 15 yeasts showed high glucose tolerance (Saha and Bothast 1996). All isolates studied in the present work produced β-glucosidase glucose tolerant, indicating the importance of this genus as a source of this enzyme.

Data from our work allow including Pichia and Wickerhamomyces isolates as interesting yeasts to be used as xylanase source. Xylanases lead to an improved yield of juice by liquefaction of fruit and vegetables; stabilization of the fruit pulp; increased recovery of aromas, essential oils, vitamins, mineral salts, edible dyes, and pigments; reduction of viscosity; and hydrolysis of substances that hinder the physical or chemical clearing of the grape juice or that may cause cloudiness in the concentrate (Wong and Saddler 1993). The main desirable properties for xylanases for use in the food industry are high stability and optimum activity at acid pH. Ganga and Martinez (2004) showed that six of the nine species identified as non-Saccharomyces secreted this activity at pH values studied. These species corresponded to K. thermotolerans/Zygosaccharomyces cidri/Z. fermentans, M. pulcherrima, Candida ssp., Dekkera ssp., T. delbruekki, and Z. microellipsoides. Our results show that Pichia and Wickerhamomyces isolates maintain 40 to 80% of maximum activity at pH 3.0.

The ability of yeasts to release proteases because of their potential to degrade haze proteins in wine and to generate nutrient sources for microorganisms has been observed by researchers (Lagace and Bisson 1990). Yeast protease may liberate amino acids and peptides from grape protein during fermentation, which can benefit growth of microorganisms during or after alcoholic fermentation. Another aspect is that yeast cells may release nitrogen-containing metabolites to the media. The composition of amino acids peptides and proteins in wine is based on grape-related compounds transferred and

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transformed during the winemaking process and breakdown products through the protease activity from yeasts and compounds released by yeasts (Alexandre et al. 2001). Results here indicate that protease activity in Pichia and Wickerhamomyces isolates was very low, according with results obtained by other authors (Charoenchai et al. 1997, Strauss et al. 2001); other data obtained in our laboratory suggested that Hanseniaspora isolates could be a more interesting yeast to obtain this enzymatic activity.

The wine yeasts Candida stellata, C. pulcherrima, C. krusei, and Torulasporaspora delbrueckii/C. colliculosa were shown to have the potential to produce extracellular lipolytic activity (Charoenchai et al. 1997). These enzymes could degrade lipids originating from the grape or any autolytic reactions of yeasts, releasing free fatty acids into the juice or wine that may potentially impact on wine quality.

Moreover, the liberation of medium-chain fatty acids, such as decanoic acid or octanoic acid, could inhibit the growth of S. cerevisiae and malolactic bacteria (Edwards et al. 1990). Our work showed that Pichia or Wickerhamomyces isolates have low lipase and esterase activities and they are not suitable for relevant enzymatic production.

The ethanol had low inhibitory effect on glycolytic activities and even showed a stimulatory effect. The change in polarity of the medium induced by ethanol could alter enzyme conformation and, consequently, its active site, thus reducing its activity. Nevertheless, the enzymatic activities were unaffected by such phenomenon; this may be due to the intrinsic structural characteristics of the enzyme and/or active site and a probable protective mechanism that reduces the unfolding rate may be due to immobilization of the enzyme on the yeast cell membranes (Barbagallo et al. 2004). Alternatively, higher ethanol concentrations may have altered membrane permeability, thereby allowing easier access between the intracellular enzyme and substrate (Pemberton et al. 1980).

The low inhibition rate by glucose and ethanol indicates that these enzymes are good candidates for use in enological processes, including xylan degradation, where combined stability is appreciated. Taking into account data obtained in this work, both enzymes show ~50% activity at must or wine pH. Nevertheless, activity of both enzymes is very low at fermentation temperatures, mainly regarding white wines.

Data shown in this work conclude the interest of Pichia and Wickerhamomyces isolates to be used as a source of enzymes for enology. Nevertheless, more extensive studies should be made to isolate, purify, and determine the optimal conditions for using these enzymes and determine their compatibility with the technological processes used in winemaking industry. Future research focusing on the activity of these enzymes in wine fermentation and the physiological and metabolic features of non-Saccharomyces yeasts is required.

Conclusion This work supports the new classification of Pichia anomala yeasts as a new species called Wickerhamomyces anomalus; physiological and molecular data support this reclassification.

One isolate, P. membranifaciens Pm7, has shown the most

interesting data regarding enzymatic activities, mainly for glycosidase enzymes. Both β-glucosidase and xylanase activities had the highest values and had high tolerance to glucose, ethanol, and acid pH; therefore, this isolate is a promising source of enzymes for used in enology and other food industries.

Moreover, the low activity at fermentation temperatures makes them more useful in wine than in must conditions.

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