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Phytase-active yeasts from grain-based food and beer

2011; Oxford University Press; Volume: 110; Issue: 6 Linguagem: Inglês

10.1111/j.1365-2672.2011.04988.x

1365-2672

Lina Nuobariene, Åse Hansen, Lene Jespersen, Nils Arneborg,

Pineapple and bromelain studies

Journal of Applied MicrobiologyVolume 110, Issue 6 p. 1370-1380 ORIGINAL ARTICLEFree Access Phytase-active yeasts from grain-based food and beer L. Nuobariene, L. Nuobariene Department of Food Science, University of Copenhagen, Frederiksberg, DenmarkSearch for more papers by this authorA.S. Hansen, A.S. Hansen Department of Food Science, University of Copenhagen, Frederiksberg, DenmarkSearch for more papers by this authorL. Jespersen, L. Jespersen Department of Food Science, University of Copenhagen, Frederiksberg, DenmarkSearch for more papers by this authorN. Arneborg, N. Arneborg Department of Food Science, University of Copenhagen, Frederiksberg, DenmarkSearch for more papers by this author L. Nuobariene, L. Nuobariene Department of Food Science, University of Copenhagen, Frederiksberg, DenmarkSearch for more papers by this authorA.S. Hansen, A.S. Hansen Department of Food Science, University of Copenhagen, Frederiksberg, DenmarkSearch for more papers by this authorL. Jespersen, L. Jespersen Department of Food Science, University of Copenhagen, Frederiksberg, DenmarkSearch for more papers by this authorN. Arneborg, N. Arneborg Department of Food Science, University of Copenhagen, Frederiksberg, DenmarkSearch for more papers by this author First published: 25 February 2011 https://doi.org/10.1111/j.1365-2672.2011.04988.xCitations: 30 Aase S. Hansen, Department of Food Science, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark.E-mail: aah@life.ku.dk AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: To screen yeast strains isolated from grain-based food and beer for phytase activity to identify high phytase-active strains. Methods and Results: The screening of phytase-positive strains was carried out at conditions optimal for leavening of bread dough (pH 5·5 and 30°C), in order to identify strains that could be used for the baking industry. Two growth-based tests were used for the initial testing of phytase-active strains. Tested strains belonged to six species: Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Kazachstania exigua (former name Saccharomyces exiguus), Candida krusei (teleomorph Issachenkia orientalis) and Arxula adeninivorans. On the basis of initial testing results, 14 strains were selected for the further determination of extracellular and intracellular (cytoplasmic and/or cell-wall bound) phytase activities. The most prominent strains for extracellular phytase production were found to be S. pastorianus KVL008 (a lager beer strain), followed by S. cerevisiae KVL015 (an ale beer strain) and C. krusei P2 (isolated from sorghum beer). Intracellular phytase activities were relatively low in all tested strains. Conclusions: Herein, for the first time, beer-related strains of S. pastorianus and S. cerevisiae are reported as phytase-positive strains. Significance and Impact of the Study: The high level of extracellular phytase activity by the strains mentioned previously suggests them to be strains for the production of wholemeal bread with high content of bioavailable minerals. Introduction Increased consumption of wholegrain products is recommended because several epidemiological studies find that intake of wholegrain food is protective against cardiovascular disease, certain type of cancers, type 2 diabetes and obesity (Slavin 2003). Wholegrain bread also provides an important source of minerals in diet, such as zinc, iron, magnesium, potassium (Spiegel et al. 2009); however, they contain absorption inhibitors of minerals, such as phytic acid [IP6] (myo-inositol hexaphosphate), the main storage form for phosphorus in plants (Febles et al. 2002; Kumar et al. 2010). Phytic acid is highly charged with six phosphate groups extending from the central myo-inositol ring and acts as a strong chelator of cations and binds minerals, such as Zn2+, Fe2+, Ca2+, Mg2+ (Raboy 2003). These phytate complexes are insoluble at physiological pH, and, therefore, minerals and phosphate are unavailable for absorption in the human intestine (Brune et al. 1992; Iqbal et al. 1994; Lopez et al. 2002). The absorption of iron and zinc from a given food in the human's intestine depends on its amount of phytate. A higher amount of phytate leads to smaller amounts of bioavailable minerals (Navert et al. 1985; Brune et al. 1992; Hurrell et al. 1992). To increase the bioavailability of minerals, enzymatic degradation of phytate and its dephosphorylated isomer IP5 (inositol pentaphosphate) is needed (Sandberg et al. 1999). Characterized phytases are enzymes that catalyse the stepwise dephosphorylation of phytate to lower phosphoric esters of myo-inositol and phosphoric acid via penta- to monophosphates. This enzymatic activity produces available phosphate and nonchelated minerals for human absorption (Konietzny and Greiner 2002). Phytases are widespread in nature and present in plants, such as cereals and legumes (Eeckhout and DePaepe 1994; Viveros et al. 2000; Steiner et al. 2007), and in micro-organisms, such as bacteria and fungi (Howson and Davis 1983; Ullah and Gibson 1987; Lambrechts et al. 1992; Berka et al. 1997, 1998; Nakamura et al. 2000; Olstorpe et al. 2009). Cereal phytase varies in activity depending on source. For wheat, reported phytase activities range from 900 to 2886 U kg−1 dry matter, for rye from 4100 to 6100 U kg−1 dry matter and for barley from 400 to 2323 U kg−1 dry matter (Eeckhout and DePaepe 1994; Greiner and Egli 2003; Steiner et al. 2007). However, this activity in wheat is not enough to improve mineral bioavailability sufficiently in wheat bread (Harland and Harland 1980; Harland and Frolich 1989; Turk and Sandberg 1992; Turk et al. 1996). On the other hand, phytate is fully degraded during commercial rye bread production (Nielsen et al. 2007). Mineral bioavailability in bread can be increased, using high phytase-active yeasts, in addition to cereal phytase. A few studies have shown phytase activities in a large number of yeast strains ranging from 14 to 566 mU ml−1 (Lambrechts et al. 1992; Sano et al. 1999; Nakamura et al. 2000; Olstorpe et al. 2009). Several studies were carried out on phytase activity from baker's yeast (Saccharomyces cerevisiae) during leavening of bread dough (Harland and Harland 1980; Harland and Frolich 1989; Turk et al. 1996, 2000; Andlid et al. 2004). Phytase from baker's yeast was first extracted by Nayini and Markakis (1984) and they found an optimum pH to be 4·6 and optimum temperature of 45°C. On the other hand, In et al. (2009) found optimum pH for S. cerevisiae phytase to be 3·6 at 40°C and this is in accordance with Turk et al. (1996) who found a similar optimum pH of 3·5 at 30°C. Hellstrom et al. (2010) isolated nine yeast species from Tanzania togwa and showed that Issatchenkia orientalis (anamorph Candida krusei) and Hanseniaspora guilliermondii completely degraded all IP6 in YPD medium, supplemented with 5 mmol l−1 IP6, within 24 h of fermentation at 30°C and pH 6·5. During the first 3 h of fermentation, only marginal amount of IP6 was degraded. This is in agreement with the results obtained by Turk et al. (2000) from fermentations with baker's yeast in synthetic medium supplemented with 0·88 mg ml−1 IP6 at 30°C and pH 5·3 (conditions relevant for leavening of bread dough). To our knowledge, there are no high phytase-active yeasts available for bread industry today, so the potential of identification of yeasts to be used for bread making with high content of bioavailable minerals is of outstanding importance. The objective of this study was therefore to screen different yeast strains isolated from grain-based food and beer for phytase activity under conditions optimal for leavening of bread dough (30°C and pH 5·5). Materials and methods Yeast strains Yeast strains (n = 39) used in this study were isolated from grain-based food and beer, and their isolation source and country of origin are listed in Table 1. Yeast strains belong to the species S. cerevisiae (n = 13), Saccharomyces pastorianus (n = 10), Saccharomyces bayanus (n = 1), Kazachstania exigua (former name S. exiguus) (n = 1) and C. krusei (teleomorph I. orientalis) (n = 12). Additionally, two reference yeast strains (CBS7377 and CBS8335), belonging to Arxula adeninivorans, were used (Sano et al. 1999; Olstorpe et al. 2009). Table 1. Yeast strains (n = 39) used in this study, their isolation source and country of origin Species Strains Isolation source Country of origin Arxula adeninivorans CBS7377 Garden soil South Africa CBS8335 Soil Italy Candida krusei K3 Kenkey* Ghana K21 Kenkey Ghana K131 Kenkey Ghana K132 Kenkey Ghana K168 Kenkey Ghana K190 Kenkey Ghana K191 Kenkey Ghana K204 Kenkey Ghana P1 Pito† Ghana P2 Pito Ghana P9 Pito Ghana P11 Pito Ghana Kazachstania exigua CBS7901 Sourdough Italy Saccharomyces bayanus CBS380 Turbid beer Denmark Saccharomyces cerevisiae ATCC26108 Laboratory strain DGI342 Baker's yeast Denmark CBS1171 Brewer's top yeast the Netherlands CBS1234 Baker's yeast the Netherlands CBS1236 Baker's yeast France P3 Pito Ghana P4 Pito Ghana P5 Pito Ghana P6 Pito Ghana P10 Pito Ghana KVL013 Ale beer Denmark KVL014 Ale beer Denmark KVL015 Ale beer Denmark Saccharomyces pastorianus KVL001 Lager beer Denmark KVL004 Lager beer Denmark KVL005 Lager beer Denmark KVL006 Lager beer Denmark KVL007 Lager beer Denmark KVL008 Lager beer Denmark KVL009 Lager beer Denmark KVL010 Lager beer Denmark KVL016 Lager beer Denmark KVL017 Lager beer Denmark *Kenkey, fermented maize dough from two local production sites in Accra, Ghana. Sampling was performed during the fermentation (Jespersen et al. 1994). †Pito, top-fermented sorghum beer from Tamale, northern Ghana. Sampling was performed on the dried yeast used for inoculation. Yeast strains were stored at −80°C in YPG medium (d-(+)-glucose, 10 g l−1; bacto-yeast extract, 3 g l−1; bacto-peptone, 5 g l−1; pH 5·5) containing 20% glycerol. Growth media and preparation of yeast inocula Three different defined minimal media, modified from Albers et al. (1996), were used to investigate the ability of the strains to grow on media with phytate as the only phosphor source: Delft + P (positive control; phosphate-containing medium), Delft − P (negative control; phosphate-free medium) and Delft + Phy (phytate medium; phytic acid dipotassium salt as phosphate source) (Table 2). Table 2. Modified defined minimal medium used in the experiment: Delft + P (positive control; phosphate-containing medium), Delft − P (negative control; phosphate-free medium) and Delft + Phy (phytate medium; phytic acid dipotassium salt as phosphate source) Component Medium composition, mg l−1 Delft + P Delft − P Delft + Phy d-Glucose 20 000 20 000 20 000 Phytic acid dipotassium salt NI NI 1850 KH2PO4 3510 NI NI KCL NI 2000 1700 (NH4)2SO4 7500 7500 7500 MgSO4·7H20 740 740 740 Trace metals EDTA 30·00 30·00 30·00 CaCl2·6H2O 8·30 8·30 8·30 ZnSO4·7H2O 9·00 9·00 9·00 FeSO4·7H2O 6·00 6·00 6·00 H3BO3 3·00 3·00 3·00 MnCl2·4H2O 1·50 1·50 1·50 Na2MoO4·2H2O 0·77 0·77 0·77 CoCl2·6H2O 0·85 0·85 0·85 CuSO4·5H2O 0·59 0·59 0·59 KI 0·20 0·20 0·20 Vitamins d-Biotin 0·05 0·05 0·05 p-Aminobenzoic acid 0·20 0·20 0·20 Nicotinic acid 1·00 1·00 1·00 Calcium pantothenate 1·00 1·00 1·00 Pyridoxine HCl 1·00 1·00 1·00 Thiamine HCl 1·00 1·00 1·00 myo-Inositol 25·00 25·00 25·00 NI, not included. Trace minerals and vitamins were prepared separately as 100-fold concentrated stock solution, filter sterilized (0·2-μm Minisart filters; Bie & Bernzten, Herlev, Denmark) and stored at 4°C. Potassium, ammonium and magnesium salts were prepared separately as 50-fold concentrated stock solution, autoclaved at 121°C for 15 min and stored at 4°C. Heat-sensitive phytic acid dipotassium salt (Bae et al. 1999; Fredrikson et al. 2002) was freshly prepared as 50-fold concentrated stock solution, filter sterilized (0·2-μm Minisart filters) and added after autoclaving and cooling to 45°C. To stabilize the pH during yeast cultivation, 50 mmol l−1 succinic acid/NaOH buffer, pH 5·5, was used (Turk et al. 2000; Andlid et al. 2004). For solid medium, bacto-agar (20 g l−1) (Becton Dickinson, Broendby, Denmark) was suspended in corresponding medium (Delft + P, Delft − P and Delft + Phy) and autoclaved at 121°C for 15 min. After cooling to 45°C, 100-fold concentrated solutions of vitamins and trace minerals and 50-fold phytic acid dipotassium salt solution was added. Yeast inocula were prepared in two steps. First, few yeast colonies were resuspended in 10-ml sterile YPG medium and cultivated in a shaking water bath (170 rev min−1) at 30°C overnight. Second, yeast culture was resuspended in 250-ml Erlenmeyer flasks, containing 50 ml of YPG medium, and cultivated in a shaking water bath (170 rev min−1) at 30°C overnight. The inoculation level was set to OD600 = 0·1. Subsequently, cells were harvested by centrifugation (Sorvall RT6000D; Buch & Holm, Herlev, Denmark) at 5000 g for 10 min, 4°C, washed three times with 20-ml sterile ultrapure water and diluted to an initial OD600 = 1. Prepared yeast inocula were used for growth test on agar plates, growth test on microtitre plates and phytase extraction. Growth test on agar plates Sterile, 90-mm-diameter Petri dishes were used for growth test on solid medium. Prepared yeast inocula were resuspended in sterile NaCl solution (8·5 g l−1) to an initial OD600 = 0·1 and serial diluted to obtain 103- and 104-fold dilutions. From each dilution, 2 μl was inoculated on Delft + P, Delft − P and Delft + Phy agar plates, cultivated at 25°C for 72 h and, thereafter, photographed. Growth test in microtitre plates Sterile flat-bottom, 96-well tissue culture plates (92096, TPP) were used for growth test in liquid medium. Microtitre plate wells were filled in triplicate with 200 μl of corresponding medium (Delft + P, Delft − P or Delft + Phy), inoculated with 2 μl of prepared yeast inocula and cultivated at 25°C for 48 h. Yeast growth was monitored as optical density at 600 nm (OD600) using a Microplate reader (M965 AcuuReader; Metertech; Food Diagnostics, Grenaa, Denmark). Before OD measurement, the plate was agitated for 3 s. Measurements were taken every 2 h. For experiments performed in the Delft + P and Delft + Phy media, the duration of the lag phase (λ), as well as the maximum specific growth rate (μ) and the maximum specific growth rate ratio (μphy/μP) in the respiro-fermentative and respiratory growth phases, was calculated (see Fig. 1). Figure 1Open in figure viewerPowerPoint Growth curve of Saccharomyces cerevisiae KVL013 in Delft + P (phosphate) medium, ◆; Delft + Phy (phytic acid dipotassium salt) medium, •; and Delft − P (phosphate free) medium, . Cell concentration is measured every 2 h by optical density (OD) at 600 nm. Cell growth is represented by Ln(OD/OD0), where OD0 is the initial OD. The maximum slope (solid line, in respiro-fermentative phase; dashed line, in respiratory phase) represents the maximum specific growth rate (μ). The interaction of the maximal slope with the x-axis represents the lag time (λ). Phytase extraction Each yeast culture was prepared in a 500-ml Erlenmeyer flask, containing 100 ml of Delft + Phy medium. Medium was inoculated with prepared yeast inocula and cultivated in a shaking water bath (170 rev min−1) at 30°C for 48 h. Inoculation level was set to OD600 = 0·1. Each yeast strain was cultured in triplicate. The number of culturable cells after cultivation was determined in triplicate, by counting colony-forming units (CFU) on YPG agar plates after 48-h incubation at 25°C. Extracellular enzyme extract was prepared as follows: After cultivation, cells were harvested by centrifugation (5000 g for 10 min, 4°C), supernatants were collected, filtered (0·2-μm Minisart filters) and kept on ice (max 1 h) until activity measurements were performed. Intracellular (cytoplasmic and/or cell-wall bound) enzyme extract was prepared as follows: The pellets were washed three times with 20-ml sterile ultrapure water and resuspended in 5-ml ice-cold buffer (0·2 mol l−1 sodium acetate/HCl, pH 5·5). One gram of glass beads (425–600 μm; Sigma) was added and vortexed for 1 min, then held on ice for 1 min, a total of five times. Homogenate was centrifuged at 5000 g for 20 min, 4°C, filtered (0·2-μm Minisart filters) and kept on ice (max 20 min) until activity measurements were performed. Determination of phytase activity Phytase activity was assayed combining the methods of Quan et al. (2002) and Olstorpe et al. (2009): 0·8 ml of phytic acid dipotassium solution (3 mmol l−1 phytic acid dipotassium (P5681; Sigma-Aldrich, Broendby, Denmark) in 0·2 mol l−1 sodium acetate/HCl buffer, pH 5·5) was preincubated at 30°C for 5 min, and 0·2 ml of enzyme extract was added, mixed and incubated at 30°C. Samples were taken at different intervals (0, 15, 30 and 45 min) during assaying, and the reaction was stopped immediately by adding 1 ml of 10% trichloroacetic acid (TCA). Enzyme blank was prepared from sodium acetate buffer mixed with enzyme extract and TCA. Measurements of the liberated inorganic phosphate from the phytic acid dipotassium salt was modified according to Heinonen and Lahti (1981). To the assay tubes, containing 0·4 ml of sample, 3·2 ml of freshly prepared acid molybdate reagent (1 volume of 10 mmol l−1 ammonium molybdate, 1 volume of 2·5 mol l−1 sulfuric acid and 2 volume of acetone) was added. Absorbance of the yellow colour was measured at 355 nm in a spectrophotometer (Agilent 8453; Agilent Technologies, Horsholm, Denmark), using sodium acetate buffer with TCA as a blank. Phosphate standard curve was prepared with KH2PO4 (Sigma-Aldrich, P5655), dissolved in 0·2 mol l−1 sodium acetate/HCl buffer (pH 5·5) and measured under the same conditions as the enzyme sample. The sensitivity of the phytase assay was estimated to be from 0 to 2 μmol ml−1 of inorganic phosphate. One unit of phytase activity was defined as the amount of phytase that liberates 1 μmol ml−1 inorganic phosphate per minute from a 3 mmol l−1 K-phytate solution at pH 5·5 and a temperature of 30°C. This temperature and pH value were considered optimal for bread dough leavening (Haros et al. 2001). Two phytase activities were calculated: volumetric and specific. Volumetric activities of extra- (E) and intracellular (I) phytase were expressed as unit per ml of enzyme extract (U ml−1). Extracellular phytase activities were measured in culture supernatants, and intracellular (cytoplasmic and/or cell-wall bound) phytase activities were measured in crushed pellet supernatants. The phytase activity was measured at several time points (0, 15, 30 and 45 min) and calculated from the steepest part of the activity curve. (1) where Vt is the total sample volume (2 ml); V, volume of extra- or intracellular enzyme extract (0·2 ml); , measured changed phosphate concentration during time (μmol ml−1); Δt, reaction time (min). Specific activity of extracellular phytase (E) was expressed as unit per 1010 CFU [U (1010 CFU)−1]. (2) where cCFU is the concentration of CFU (n ml−1); Specific activity of intracellular phytase (I) was expressed as unit per milligram of total protein (U mg−1 total protein). (3) where cmg protein is the total protein concentration (mg ml−1). Protein determination Protein concentrations in intracellular enzyme extracts were determined by measuring the absorbance of intracellular enzyme extract at 600 nm using the Bio-Rad Colorimetric Protein assay, kit II (Bio-Rad Laboratories Inc., Sundbybergm, Sweden). Bovine serum albumin (BSA) was used as protein concentration standard. Results Yeast growth on solid Delft + Phy medium To check the ability of yeasts to grow on phytic acid as the only phosphorus source in a solid medium, yeast strains were cultivated on minimal Delft medium plates, supplemented with phytic acid dipotassium salt (Delft + Phy). As controls, yeast strains were cultivated on phosphate-containing (Delft + P, positive control) and phosphate-free (Delft − P, negative control) minimal Delft medium plates. All 39 tested yeast strains grew on Delft + Phy as well as on Delft + P plates after 72-h incubation at 25°C (data not shown). For most of the tested strains, no major differences were noted in colony sizes between the two different media (data not shown). However, smaller colonies were observed on Delft + Phy plates in comparison with colonies on Delft + P plates for S. cerevisiae strains P3, P4 (Fig. 2), P5, P6 and C. krusei strain K3 (data not shown). Differences in colony sizes and growth intensities were observed between species. With the exception of C. krusei K3, intensive growth and big colonies on Delft + Phy and Delft + P were species-specific traits for C. krusei. As an example, C. krusei K190 colony growth is shown in Fig. 2. Noticeably, slower growth and smaller colonies were observed for S. pastorianus and S. cerevisiae strains (Fig. 2). With the exception of A. adeninivorans, very slight growth of yeast colonies was observed on Delft − P plates (Fig. 2). Figure 2Open in figure viewerPowerPoint Growth of Candida krusei K190, Saccharomyces cerevisiae P4, Saccharomyces pastorianus KVL008 and Arxula adeninivorans CBS 7377 on Delft + P (phosphate) medium (top left); Delft + Phy (phytic acid dipotassium salt) medium (top right); and Delft − P (phosphate free) medium (bottom) plates after 72-h incubation. Yeast growth in liquid Delft + Phy medium Growth in liquid medium was quantified by measuring OD600 of the culture in microtitre plates over 48 h. The capabilities of the species to grow in medium, where the only source of phosphorus was phytate, were determined by comparing the growth parameters of the yeasts (i.e. final OD600, λ and μPhy/μP) in Delft + Phy with those of positive (Delft + P) and negative (Delft − P) controls (Olstorpe et al. 2009). All strains were able to grow in liquid Delft + Phy to varying extents (Fig. 3). Of the tested yeast strains, 37% grew very well in Delft + Phy and reached 93–98% of the final optical density in Delft + P. This intensive growth in Delft + Phy was a species-specific trait for C. krusei and A. adeninivorans (Fig. 3). For S. cerevisiae, the final optical density ranged from 19% (strain P3) to 95% (strain P10), for S. pastorianus from 40% (strain KVL008) to 80% (strain KVL009), for S. bayanus it was about 86% and for K. exigua it was 60% (Fig. 3). Minimal growth occurred for all strains in Delft − P with OD600 ranging from 0·01 to 0·1 (Fig. 3). Figure 3Open in figure viewerPowerPoint Optical density values (OD600, horizontal axis) of yeast cell growth at 25°C in Delft − P (black bars), Delft + Phy (grey bars) and Delft + P (white bars) medium after 48-h cultivation. Error bars represent standard error from three separate growth analyses. The data in Table 3 show that no lag phase was observed for 20 strains of 39 grown in Delft + P medium, while in Delft + Phy medium, only eight strains did not show a lag phase. The lag phase for all strains was longer in Delft + Phy medium except for three strains of S. cerevisiae, four strains of S. pastorianus and one strain of C. krusei, which did not show a lag phase when either grown in Delft + Phy or in Delft + P media (Table 3). Of all tested strains, S. pastorianus KVL008 had the longest lag phases in both media. The μPhy/μP was larger than one for c. 33 and 50% of the strains in the respiro-fermentative phase and in the respiratory phase, respectively (Table 3). These findings indicate that the growth of these yeast strains is more rapid in the Delft + Phy medium than in the Delft + P medium, especially in the respiratory phase. Table 3. Growth parameters for the yeast strains grown in modified defined minimal medium Delft + P (phosphate) and in Delft + Phy (phytic acid dipotassium salt) Strains λP*, h λPhy†, h Respiro-fermentative phase Respiratory phase μP‡, h−1 μPhy§, h−1 μPhy/μP¶ μP, h−1 μPhy, h−1 μPhy/μP CBS7377 1·0 3·5 0·30 ± 0·03 0·24 ± 0·08 0·80 0·15 ± 0·03 0·16 ± 0·01 1·07 CBS8335 2·0 3·0 0·29 ± 0·01 0·25 ± 0·02 0·86 0·06 ± 0·01 0·08 ± 0·02 1·33 K3 –** 2·0 0·20 ± 0·01 0·18 ± 0·02 0·90 0·22 ± 0·01 0·02 ± 0·00 0·09 K21 0·5 3·0 0·23 ± 0·05 0·21 ± 0·03 0·91 0·09 ± 0·00 0·12 ± 0·01 1·33 K131 1·0 3·0 0·49 ± 0·02 0·46 ± 0·01 0·94 0·16 ± 0·02 0·20 ± 0·03 1·25 K132 1·0 2·0 0·30 ± 0·02 0·32 ± 0·05 1·07 0·09 ± 0·03 0·11 ± 0·02 1·22 K168 1·0 1·5 0·26 ± 0·02 0·26 ± 0·01 1·00 0·18 ± 0·01 0·21 ± 0·02 1·17 K190 – – 0·27 ± 0·02 0·29 ± 0·01 1·07 0·11 ± 0·01 0·14 ± 0·01 1·27 K191 1·0 2·0 0·29 ± 0·04 0·20 ± 0·03 0·69 0·11 ± 0·01 0·17 ± 0·01 1·55 K204 1·0 3·5 0·30 ± 0·02 0·29 ± 0·01 0·97 0·12 ± 0·01 0·09 ± 0·00 0·75 P1 2·0 2·5 0·33 ± 0·02 0·31 ± 0·02 0·94 0·10 ± 0·01 0·10 ± 0·01 1·00 P2 0·5 3·5 0·29 ± 0·03 0·34 ± 0·05 1·17 0·09 ± 0·00 0·07 ± 0·00 0·78 P9 1·0 3·5 0·25 ± 0·01 0·33 ± 0·00 1·32 0·11 ± 0·01 0·13 ± 0·01 1·18 P11 – 3·0 0·26 ± 0·02 0·36 ± 0·02 1·39 0·11 ± 0·01 0·13 ± 0·02 1·18 CBS7901 – 10·0 0·20 ± 0·03 0·25 ± 0·01 1·25 0·22 ± 0·01 0·06 ± 0·01 0·27 CBS380 3·0 4·5 0·35 ± 0·04 0·30 ± 0·03 0·86 0·13 ± 0·03 0·11 ± 0·03 0·85 ATCC26108 1·0 3·0 0·46 ± 0·12 0·37 ± 0·14 0·80 0·12 ± 0·01 0·18 ± 0·03 1·50 DGI342 1·0 2·5 0·53 ± 0·06 0·38 ± 0·10 0·72 0·12 ± 0·01 0·14 ± 0·04 1·17 CBS1171 – – 0·15 ± 0·02 0·10 ± 0·01 0·67 0·22 ± 0·09 0·32 ± 0·03 1·45 CBS1234 – 2·0 0·18 ± 0·01 0·16 ± 0·02 0·89 0·18 ± 0·01 0·18 ± 0·01 1·00 CBS1236 0·5 1·5 0·38 ± 0·14 0·22 ± 0·00 0·58 0·19 ± 0·04 0·19 ± 0·02 1·00 P3 – 2·5 0·27 ± 0·01 0·17 ± 0·01 0·63 0·17 ± 0·01 0·06 ± 0·01 0·35 P4 3·0 7·0 0·08 ± 0·01 0·08 ± 0·02 1·00 0·19 ± 0·01 0·18 ± 0·01 0·95 P5 – 3·0 0·20 ± 0·00 0·21 ± 0·02 1·05 0·15 ± 0·02 0·02 ± 0·00 0·13 P6 1·0 1·5 0·35 ± 0·02 0·26 ± 0·01 0·74 0·14 ± 0·01 0·05 ± 0·00 0·36 P10 – 1·0 0·34 ± 0·03 0·33 ± 0·13 0·97 0·13 ± 0·01 0·12 ± 0·02 0·92 KVL013 – 2·0 0·19 ± 0·04 0·18 ± 0·07 0·95 0·17 ± 0·01 0·08 ± 0·01 0·47 KVL014 – – 0·24 ± 0·02 0·24 ± 0·01 1·00 0·14 ± 0·03 0·15 ± 0·01 1·07 KVL015 – – 0·15 ± 0·01 0·11 ± 0·01 0·73 0·21 ± 0·05 0·20 ± 0·01 0·95 KVL001 – 3·0 0·23 ± 0·04 0·22 ± 0·08 0·96 0·12 ± 0·02 0·02 ± 0·01 0·17 KVL004 – 1·0 0·33 ± 0·02 0·12 ± 0·02 0·36 0·11 ± 0·01 0·18 ± 0·02 1·50 KVL005 – – 0·15 ± 0·01 0·16 ± 0·01 1·07 0·18 ± 0·04 0·14 ± 0·02 0·78 KVL006 – – 0·20 ± 0·01 0·14 ± 0·02 0·70 0·26 ± 0·00 0·18 ± 0·03 0·69 KVL007 – 3·0 0·18 ± 0·02 0·25 ± 0·02 1·34 0·16 ± 0·01 0·17 ± 0·02 1·06 KVL008 5·0 14·0 0·23 ± 0·04 0·27 ± 0·01 1·18 0·09 ± 0·02 0·09 ± 0·01 1·00 KVL009 – – 0·23 ± 0·05 0·19 ± 0·04 0·82 0·16 ± 0·01 0·14 ± 0·04 0·86 KVL010 – 2·0 0·23 ± 0·03 0·09 ± 0·01 0·39 0·12 ± 0·02 0·20 ± 0·02 1·67 KVL016 – 1·0 0·25 ± 0·01 0·29 ± 0·02 1·16 0·12 ± 0·01 0·12 ± 0·01 1·00 KVL017 – – 0·16 ± 0·01 0·14 ± 0·01 0·86 0·30 ± 0·03 0·13 ± 0·02 0·43 Results are expressed as the mean of three replicated measurements and standard deviation (±SD). *Duration of the lag phase for the yeast strains grown in Delft + P. †Duration of the lag phase for the yeast strains grown in Delft + Phy. ‡The maximum specific growth rate for the yeast strains grown in Delft + P. §The maximum specific growth rate for the yeast strains grown in Delft + Phy. ¶The maximum specific growth rate ratio calculated from the mean values. **No lag phase was observed. Extra- and intracellular phytase activities On the basis of the results mentioned previously, 14 yeast strains were selected for further determination of extracellular and intracellular phytase activity. Four S. cerevisiae strains, i.e. ATCC26108, DGI342, CBS1236 and KVL015, and one C. krusei strain K132, were selected as representative strains within species with higher μphy/μP values in the respiratory phase than in the respiro-fermentative phase, and with values close to one (KVL015) or higher. The strains S. cerevisiae P10 and S. pastorianus KVL016 were selected as those with the highest final optical density within these two species. Four C. krusei strains, i.e. P1, P2, P11 and K204, were selected as representative strains within a species for their rapid growth in liquid Delft + Phy medium. Saccharomyces cerevisiae KVL013 and S. pastorianus KVL008 were chosen as negative controls for their very low final optical density in liquid Delft + Phy when compared with Delft + P. Moreover, KVL008 had the longest lag phase in both media in comparison with all other strains. Arxula adeninivorans CBS7377 was chosen as the positive control for general comparison, because this species is well known to exhibit high extra- and intracellular phytase activities (Sano et al. 1999; Olstorpe et al. 2009). The 14 selected strains were pregrown in Delft + Phy, containing 3 mmol l−1 ph