Phytase-active yeasts from grain-based food and beer

Authors


Aase S. Hansen, Department of Food Science, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark.
E-mail: aah@life.ku.dk

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 (= 39) used in this study, their isolation source and country of origin
SpeciesStrainsIsolation sourceCountry of origin
  1. *Kenkey, fermented maize dough from two local production sites in Accra, Ghana. Sampling was performed during the fermentation (Jespersen et al. 1994).

  2. †Pito, top-fermented sorghum beer from Tamale, northern Ghana. Sampling was performed on the dried yeast used for inoculation.

Arxula adeninivoransCBS7377Garden soilSouth Africa
CBS8335SoilItaly
Candida kruseiK3Kenkey*Ghana
K21KenkeyGhana
K131KenkeyGhana
K132KenkeyGhana
K168KenkeyGhana
K190KenkeyGhana
K191KenkeyGhana
K204KenkeyGhana
P1Pito†Ghana
P2PitoGhana
P9PitoGhana
P11PitoGhana
Kazachstania exiguaCBS7901SourdoughItaly
Saccharomyces bayanusCBS380Turbid beerDenmark
Saccharomyces cerevisiaeATCC26108Laboratory strain 
DGI342Baker’s yeastDenmark
CBS1171Brewer’s top yeastthe Netherlands
CBS1234Baker’s yeastthe Netherlands
CBS1236Baker’s yeastFrance
P3PitoGhana
P4PitoGhana
P5PitoGhana
P6PitoGhana
P10PitoGhana
KVL013Ale beerDenmark
KVL014Ale beerDenmark
KVL015Ale beerDenmark
Saccharomyces pastorianusKVL001Lager beerDenmark
KVL004Lager beerDenmark
KVL005Lager beerDenmark
KVL006Lager beerDenmark
KVL007Lager beerDenmark
KVL008Lager beerDenmark
KVL009Lager beerDenmark
KVL010Lager beerDenmark
KVL016Lager beerDenmark
KVL017Lager beerDenmark

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)
ComponentMedium composition, mg l−1
Delft + PDelft − PDelft + Phy
  1. NI, not included.

d-Glucose20 00020 00020 000
Phytic acid dipotassium saltNINI1850
KH2PO43510NINI
KCLNI20001700
(NH4)2SO4750075007500
MgSO4·7H20740740740
Trace metals
 EDTA30·0030·0030·00
 CaCl2·6H2O8·308·308·30
 ZnSO4·7H2O9·009·009·00
 FeSO4·7H2O6·006·006·00
 H3BO33·003·003·00
 MnCl2·4H2O1·501·501·50
 Na2MoO4·2H2O0·770·770·77
 CoCl2·6H2O0·850·850·85
 CuSO4·5H2O0·590·590·59
 KI0·200·200·20
Vitamins
 d-Biotin0·050·050·05
 p-Aminobenzoic acid0·200·200·20
 Nicotinic acid1·001·001·00
 Calcium pantothenate1·001·001·00
 Pyridoxine HCl1·001·001·00
 Thiamine HCl1·001·001·00
 myo-Inositol25·0025·0025·00

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 (μphyP) in the respiro-fermentative and respiratory growth phases, was calculated (see Fig. 1).

Figure 1.

 Growth curve of Saccharomyces cerevisiae KVL013 in Delft + P (phosphate) medium, ◆; Delft + Phy (phytic acid dipotassium salt) medium, •; and Delft − P (phosphate free) medium, bsl00066. 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.

image(1)

where Vt is the total sample volume (2 ml); V, volume of extra- or intracellular enzyme extract (0·2 ml); inline image, 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].

image(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).

image(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 2.

 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 μPhyP) 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 3.

 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 μPhyP 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)
StrainsλP*, hλPhy†, hRespiro-fermentative phaseRespiratory phase
μP‡, h−1μPhy§, h−1μPhyPμP, h−1μPhy, h−1μPhyP
  1. Results are expressed as the mean of three replicated measurements and standard deviation (±SD).

  2. *Duration of the lag phase for the yeast strains grown in Delft + P.

  3. †Duration of the lag phase for the yeast strains grown in Delft + Phy.

  4. ‡The maximum specific growth rate for the yeast strains grown in Delft + P.

  5. §The maximum specific growth rate for the yeast strains grown in Delft + Phy.

  6. ¶The maximum specific growth rate ratio calculated from the mean values.

  7. **No lag phase was observed.

CBS73771·03·50·30 ± 0·030·24 ± 0·080·800·15 ± 0·030·16 ± 0·011·07
CBS83352·03·00·29 ± 0·010·25 ± 0·020·860·06 ± 0·010·08 ± 0·021·33
K3–**2·00·20 ± 0·010·18 ± 0·020·900·22 ± 0·010·02 ± 0·000·09
K210·53·00·23 ± 0·050·21 ± 0·030·910·09 ± 0·000·12 ± 0·011·33
K1311·03·00·49 ± 0·020·46 ± 0·010·940·16 ± 0·020·20 ± 0·031·25
K1321·02·00·30 ± 0·020·32 ± 0·051·070·09 ± 0·030·11 ± 0·021·22
K1681·01·50·26 ± 0·020·26 ± 0·011·000·18 ± 0·010·21 ± 0·021·17
K1900·27 ± 0·020·29 ± 0·011·070·11 ± 0·010·14 ± 0·011·27
K1911·02·00·29 ± 0·040·20 ± 0·030·690·11 ± 0·010·17 ± 0·011·55
K2041·03·50·30 ± 0·020·29 ± 0·010·970·12 ± 0·010·09 ± 0·000·75
P12·02·50·33 ± 0·020·31 ± 0·020·940·10 ± 0·010·10 ± 0·011·00
P20·53·50·29 ± 0·030·34 ± 0·051·170·09 ± 0·000·07 ± 0·000·78
P91·03·50·25 ± 0·010·33 ± 0·001·320·11 ± 0·010·13 ± 0·011·18
P113·00·26 ± 0·020·36 ± 0·021·390·11 ± 0·010·13 ± 0·021·18
CBS790110·00·20 ± 0·030·25 ± 0·011·250·22 ± 0·010·06 ± 0·010·27
CBS3803·04·50·35 ± 0·040·30 ± 0·030·860·13 ± 0·030·11 ± 0·030·85
ATCC261081·03·00·46 ± 0·120·37 ± 0·140·800·12 ± 0·010·18 ± 0·031·50
DGI3421·02·50·53 ± 0·060·38 ± 0·100·720·12 ± 0·010·14 ± 0·041·17
CBS11710·15 ± 0·020·10 ± 0·010·670·22 ± 0·090·32 ± 0·031·45
CBS12342·00·18 ± 0·010·16 ± 0·020·890·18 ± 0·010·18 ± 0·011·00
CBS12360·51·50·38 ± 0·140·22 ± 0·000·580·19 ± 0·040·19 ± 0·021·00
P32·50·27 ± 0·010·17 ± 0·010·630·17 ± 0·010·06 ± 0·010·35
P43·07·00·08 ± 0·010·08 ± 0·021·000·19 ± 0·010·18 ± 0·010·95
P53·00·20 ± 0·000·21 ± 0·021·050·15 ± 0·020·02 ± 0·000·13
P61·01·50·35 ± 0·020·26 ± 0·010·740·14 ± 0·010·05 ± 0·000·36
P101·00·34 ± 0·030·33 ± 0·130·970·13 ± 0·010·12 ± 0·020·92
KVL0132·00·19 ± 0·040·18 ± 0·070·950·17 ± 0·010·08 ± 0·010·47
KVL0140·24 ± 0·020·24 ± 0·011·000·14 ± 0·030·15 ± 0·011·07
KVL0150·15 ± 0·010·11 ± 0·010·730·21 ± 0·050·20 ± 0·010·95
KVL0013·00·23 ± 0·040·22 ± 0·080·960·12 ± 0·020·02 ± 0·010·17
KVL0041·00·33 ± 0·020·12 ± 0·020·360·11 ± 0·010·18 ± 0·021·50
KVL0050·15 ± 0·010·16 ± 0·011·070·18 ± 0·040·14 ± 0·020·78
KVL0060·20 ± 0·010·14 ± 0·020·700·26 ± 0·000·18 ± 0·030·69
KVL0073·00·18 ± 0·020·25 ± 0·021·340·16 ± 0·010·17 ± 0·021·06
KVL0085·014·00·23 ± 0·040·27 ± 0·011·180·09 ± 0·020·09 ± 0·011·00
KVL0090·23 ± 0·050·19 ± 0·040·820·16 ± 0·010·14 ± 0·040·86
KVL0102·00·23 ± 0·030·09 ± 0·010·390·12 ± 0·020·20 ± 0·021·67
KVL0161·00·25 ± 0·010·29 ± 0·021·160·12 ± 0·010·12 ± 0·011·00
KVL0170·16 ± 0·010·14 ± 0·010·860·30 ± 0·030·13 ± 0·020·43

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 μphyP 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 phytic acid dipotassium salt, to activate phytase production (Andlid et al. 2004; Veide and Andlid 2006; In et al. 2008). There is no internationally recognized unit defining phytase activity, which depends on assay conditions, e.g. substrate source and concentration, assay temperature and pH, reaction time (Selle and Ravindran 2007). In this study, one phytase activity unit is defined as the amount of enzyme that liberates 1 μmol ml−1 inorganic orthophosphate per minute from 3 mmol l−1 K-phytate at pH 5·5 and a temperature of 30°C. Two activities were calculated – volumetric and specific.

The extracellular specific and volumetric phytase activities differed dramatically between the two S. pastorianus strains (Table 4). Both activities were larger in S. pastorianus KVL008 than in S. pastorianus KVL016. Strain KVL016 had 1% of specific activity and 4% of volumetric activity of activities for strain KVL008. The extracellular phytase activities also differed among S. cerevisiae strains. The lowest specific activity was observed in strain CBS1236, which had 4% activity of the strain with the highest activity (KVL015). The lowest volumetric activity was observed in two S. cerevisiae strains: CBS1236 and KVL013. They had 22% activity of strain KVL015. Five strains of C. krusei could be grouped according to their extracellular specific activities into two groups: first group with activity of c. 110 mU (1010 CFU)−1 (strains K204 and P1) and second group with activity of c. 521 mU (1010 CFU)−1 (strains P2, K132 and P11). There were no differences in specific activities between the two C. krusei strains in the first group. Specific activities among C. krusei strains in the second group were almost equal and similar to those observed for A. adeninivorans (Table 4). Surprisingly, our results show that the strain with the highest extracellular specific and volumetric phytase activity was the negative control S. pastorianus KVL008 (a lager beer strain), followed by S. cerevisiae KVL015 (an ale beer strain) and C. krusei P2 (isolated from sorghum beer).

Table 4.   Volumetric (mU ml−1) and specific extracellular [mU (1010 CFU)−1] and intracellular (mU mg−1 total protein) phytase activities from yeast strains of Saccharomyces cerevisiae, Saccharomyces pastorianus, Candida krusei and Arxula adeninivorans
SpeciesStrainsPhytase activities*
ExtracellularIntracellular
mU ml−1mU (1010 CFU)−1mU ml−1mU mg−1 total protein
  1. *Results are expressed as the mean of three replicated measurements and standard error of the mean (±SEM).

S. cerevisiaeATCC261089 ± 1281 ± 226 ± 14 ± 1
DGI34210 ± 162 ± 76 ± 13 ± 1
CBS12363 ± 128 ± 220 ± 312 ± 1
P1028 ± 1457 ± 5214 ± 28 ± 1
KVL0133 ± 183 ± 2830 ± 517 ± 3
KVL01567 ± 1732 ± 836 ± 122 ± 3
S. pastorianusKVL00876 ± 61981 ± 2014 ± 28 ± 1
KVL0163 ± 126 ± 319 ± 311 ± 0
C. kruseiK13230 ± 5509 ± 6218 ± 111 ± 2
K20420 ± 3111 ± 1817 ± 210 ± 2
P114 ± 2110 ± 546 ± 227 ± 3
P250 ± 14595 ± 5819 ± 511 ± 2
P1135 ± 3460 ± 5315 ± 19 ± 2
A. adeninivoransCBS737761 ± 5519 ± 386 ± 13 ± 1

The intracellular specific and volumetric phytase activities within two species, S. pastorianus and C. krusei, were almost equal, except C. krusei strain P1, which had threefold higher specific and volumetric activities than other strains. The intracellular specific and volumetric activities, however, differed among S. cerevisiae strains (Table 4).

For all of the tested yeast strains, the extracellular specific phytase activities were significantly higher than the intracellular phytase-specific activities (Table 4). The extracellular volumetric activities, however, were higher than the intracellular volumetric activities for ten strains of 14 tested strains. The intracellular volumetric phytase activities were higher than the measured extracellular volumetric phytase activities for the following strains: S. cerevisiae KVL013 (10-fold), S. cerevisiae CBS1236 and S. pastorianus KVL016 (sevenfold) and C. krusei P1 (threefold).

Discussion

Yeast strains with high extracellular phytase activity might be used directly in bread making for the production of bread with high content of bioavailable minerals because of the activity of phytase during dough fermentation. Although phytase activity of baker’s yeast has been widely documented, both under bread dough leavening conditions (Harland and Harland 1980; Harland and Frolich 1989; Turk et al. 1996, 2000; Andlid et al. 2004) and other conditions (Nakamura et al. 2000; Haraldsson et al. 2005; Veide and Andlid 2006; In et al. 2008, 2009), there seems to be no high phytase-active yeasts available for bread industry today. Our objective therefore was to study the extra- and intracellular phytase activities of yeast strains, isolated from various grain-based food and beers, under conditions optimal for bread dough leavening in order to identify strains that could be used in baking industry to increase the bioavailability of minerals in bread.

Strains belonging to S. cerevisiae, K. exigua and C. krusei have often been isolated from bread leavens (Hansen 2006). In this study, one K. exigua strain CBS7901 isolated from sourdough, eight C. krusei strains isolated from kenkey and three baker’s yeast strains (S. cerevisiae) were tested. Furthermore, 24 yeast strains isolated from beer and belonging to S. cerevisiae, S. bayanus, S. pastorianus and C. krusei were also tested.

Inorganic phosphate is an important nutrient required in millimolar concentrations in yeast for the synthesis of phospholipids, nucleic acids and cellular metabolites (Wykoff and O’Shea 2001). As phosphate is often present in only low amounts in the environment, some yeast cells may produce phytase. Phytase is an inducible enzyme in most micro-organisms and its expression is subjected to a complex regulation (Greiner 2005).

To investigate whether the yeast species and strains in our study produce phytase, the simple and rapid, growth-based test on solid Delft + Phy medium was used (Howson and Davis 1983; Lambrechts et al. 1992; Bae et al. 1999). However, because of the fact that some micro-organisms may grow on solid phytate medium but not in liquid medium and vice versa (Tseng et al. 2000), growth test in liquid Delft + Phy medium in parallel with the test on solid medium was performed. Intensive growth of A. adeninivorans on Delft + Phy plates after 3 days of cultivation is in agreement with previous results describing this yeast species as phytase positive (Sano et al. 1999). Interestingly, growth of S. cerevisiae P10 (isolated from pito) was conspicuously intensive in comparison with other S. cerevisiae strains, and the growth curve was similar to that of C. krusei (data not shown), which has been described as the most prominent species for phytase production (Quan et al. 2001; Hellstrom et al. 2010). Expectedly, very slight growth of yeasts colonies was observed on Delft − P plates, except for A. adeninivorans, indicating that this yeast species may possess an unusual large storage pool of phosphate in the form of e.g. polyphosphate in the vacuoles (Shirahama et al. 1996).

Yeast phytases have been found to be mostly extracellular (Nayini and Markakis 1984; Sano et al. 1999; Nakamura et al. 2000), with the exception of C. krusei (Quan et al. 2001), Pichia anomala (Vohra and Satyanarayana 2001), Schwanniomyces castellii (Lambrechts et al. 1992), which are cell-wall bound. Our results show that, for all the tested strains, the extracellular phytase activity is higher than the intracellular phytase activity. In fact, the intracellular phytase activity of almost all the strains is very low. From an industrial point of view, the extracellular phytase activity would be more important for bread making than the intracellular phytase activity, because the yeast cells should be intact in the dough in order to ensure a good fermentation. In this case, the intracellular phytase will not have access to phytate in the dough.

Our results demonstrate that the phytase activity of S. cerevisiae and C. krusei is strain dependent. These data agree with those of Turk et al. (2000) and Hellstrom et al. (2010), respectively, and they stress the fact that yeast should be considered at a strain level, and not only at a species level, when selecting high phytase-active yeasts for bread making. Interestingly, among the six S. cerevisiae strains and five C. krusei strains tested, the highest extra- and intracellular phytase activities are found for KVL015 (an ale beer strain) and P2 (isolated from sorghum beer), respectively.

To the best of our knowledge, lager beer yeast (S. pastorianus) has never been described as phytase positive. In this study, we have screened ten strains of S. pastorianus for phytase production. After the preliminary screening, we chose two of them for further investigations. Saccharomyces pastorianus KVL016 shows very low extracellular specific phytase activity. Unexpectedly, S. pastorianus KVL008, which was chosen as a negative control, shows the highest extracellular specific and volumetric phytase activities among all the strains screened in this study. In fact, the specific extracellular phytase activity of S. pastorianus KVL008 is c. 3-times higher than the activity of the other screened strains. At present, we do not have a clear explanation for this phenomenon. However, this finding shows that slow yeast growth in liquid medium, supplemented with phytate, does not necessarily indicate low phytase activity.

In conclusion, the three yeast strains, i.e. KVL008, KVL015 and P2, with the highest extracellular specific and volumetric phytase activities are beer strains. Saccharomyces pastorianus KVL008 is a lager beer strain, S. cerevisiae KVL015 is an ale beer strain and C. krusei P2 is isolated from sorghum beer. In our days, brewer’s yeast was not used for bread making. However, before the commercial production of baker’s yeast began, ale-barm or brewer’s yeast was used for this purpose (Davidson 1999). The high phytase activities of the yeast strains mentioned earlier, observed under conditions optimal for bread dough leavening (i.e. pH 5·5 and 30°C), suggest that these yeast strains may be a particularly interesting source of phytase for the production of wholegrain bread with high content of bioavailable minerals.

Acknowledgements

This PhD project was financed by a scholarship from Faculty of LIFE Science, University of Copenhagen, Denmark.

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