International Journal of Food Microbiology 205 (2015) 81–89

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains Anna Greppi a,⁎, Łukasz Krych b, Antonella Costantini a, Kalliopi Rantsiou a, D. Joseph Hounhouigan c, Nils Arneborg b, Luca Cocolin a, Lene Jespersen b a b c

Università di Torino, Dipartimento di Scienze Agrarie, Forestali e Alimentari, Grugliasco, Torino, Italy Department of Food Science, Food Microbiology, Faculty of Science, University of Copenhagen, Denmark Département de Nutrition et Sciences Alimentaires, Faculté des Sciences Agronomiques, Université d'Abomey-Calavi, Benin

a r t i c l e

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Article history: Received 1 December 2014 Received in revised form 19 March 2015 Accepted 6 April 2015 Available online 9 April 2015 Keywords: Phytate Phytase RT-qPCR Pichia kudriavzevii African fermented food products

a b s t r a c t Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninetythree yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase. Significant differences in gene expression were detected among the strains as well as between the media. A correlation was found between the PHYPk expression and phytase extracellular activity. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Phytate is the salt of myo-inositol hexakisphopshate (phytic acid, InsP6), generally regarded as an anti-nutritional factor due to its ability to chelate cations such as Fe3+, Zn2+, Ca2+ and Mg2+. Phytatecomplexes are not available for absorption in the human intestine unless digested by phytases, a class of phosphatases that catalyze the hydrolysis of phosphate from phytate (Raboy, 2003). Phytase is an enzyme of constantly growing attention, mostly as an animal but also human nutrition supplement. Phytases can be classified based on their active site motifs into histidine acid phosphatases (HAPs), β-propeller phytases (BPP) and purple acid phosphatases (PAP). HAP phytases are the most widely studied and they share a conserved N-terminal heptapeptide active site (RHGXRXP) and C-terminal catalytically active dipeptide, phosphohistidine (HD). The enzyme is naturally synthetized in plants and some microorganisms. Yeasts have been reported as useful microorganisms for phytase production (Wykoff and O'Shea, 2001; Andlid et al., 2004; Nuobariene et al., 2011; Olstorpe et al., 2009; Sandberg and Andlid, 2002; Segueilha et al., 1992; Ushasree et al., 2014; Fonseca-Maldonado et al., 2014). The ⁎ Corresponding author at: Largo Braccini 2, 10095, Grugliasco, Torino, Italy. Tel.: +39 011 670 8553; fax: +39 011 670 8549. E-mail address: [email protected] (A. Greppi).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.04.011 0168-1605/© 2015 Elsevier B.V. All rights reserved.

synthesis of phytases by yeasts is regulated by an external phosphate concentration and other factors such as: pH, temperature, carbon sources and occurrence of metal cations. The enzyme secretion can be intracellular, periplasmatic, directly to the culture medium or as bound to the cell wall. The genes encoding yeast phytases have so far been described for Debaryomyces castelli (PHYDc), Kodamaea ohmeri (PHY1), Hansenula fabianii (Hfphytase), Pichia anomala (PPHY), Schwanniomyces occidentalis, Pichia stipitis (PHO5), Pichia guilliermondii (PGUG), Kluyveromyces marxianus, and Saccharomyces cerevisiae (PHO3, PHO5, PHO11, PHO12). In general, the identified ORFs contain the consensus motif (RHGXRXP) and encode a 451–467 amino acid protein. Instead of using supplementation, fortification and synthetic addition of enzymes, phytate content in foods could be reduced by using high phytase-active microorganisms, in addition to food phytases (Fischer et al., 2014). Reduction of phytates by yeast phytases has been observed in a traditional food in Senegal (Antai and Nkwelang, 1999). Pichia kudriavzevii (formerly known as Candida krusei) is reported to be involved in the fermentation of several traditional African foods (Greppi et al., 2013a,b; Jespersen et al., 1994, 2005; Pedersen et al., 2012). The species has also been reported to produce phytase (Nuobariene et al., 2011; Quan et al., 2002). Quan et al. (2002) have studied biochemical properties of a cell-bound phytase produced by P. kudriavzevii WZ-001. However, to our knowledge the gene encoding the P. kudriavzevii phytase has not been neither identified nor studied yet.

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In the present study we report on phytase-producing yeasts from traditional products commonly consumed in Benin (West Africa). The aims of the study were to identify the phytase-producing ability of autochthonous yeasts to improve our understanding about the phytase gene expression and enzymatic activity. Initially, several yeast strains were screened for phytase activity using growth-based liquid and solid tests. Subsequently, a phytase coding gene of P. kudriavzevii was identified and its level of expression in two growth media was investigated and compared with the corresponding enzyme activity measured during time. 2. Materials and methods 2.1. Yeast strains Ninety-three yeast isolates from mawè, gowé, ogi and tchoukoutou, previously identified (Greppi et al., 2013a,b), were investigated in this study. They belong to nine species namely: P. kudriavzevii (n = 44), S. cerevisiae (n = 20), Clavispora lusitaniae (n = 13), K. marxianus (n = 3), Millerozyma farinosa (n = 3), Candida glabrata (n = 3), Wickerhamomyces anomalus (n = 2), Hanseniaspora guilliermondii (n = 3) and Debaryomyces nepalensis (n = 2). The isolate names were given as follows: the first letter represents the sample (M-G-O-T) followed by the progressive number of isolation. A genetically modified strain able to produce phytases constitutively was used as positive control (S. cerevisiae BY80, Euroscarf Acc. No. YO1692). All the isolates were stored at − 80 °C in YPD medium (1% yeast extract (BD, Brøndby, Denmark), 2% glucose (Merk, Darmstadt, Germany), 2% bacto-peptone (BD), 15 g/l bacto agar (BD)) with 30% (v/v) glycerol. 2.2. Screening of phytase producing isolates The ability of the isolates to grow in the presence of phytate as a unique phosphate source was tested on solid and liquid media as described by Nuobariene et al. (2011). Briefly, three different defined media were prepared: Delft Phy, a medium containing phytic acid dipotassium salt (1850 mg/l; Sigma P5681) as a unique phosphate source, Delft +, a medium containing phosphate (3510 mg/l) but no phytate, used as positive control, and Delft−, a phosphate-free medium, used as negative control. The protocols for preparation and composition of each medium were reported by Nuobariene et al. (2011). Few yeast colonies were inoculated in 10 ml sterile YPD medium and cultivated overnight in a water bath at 30 °C with shaking (170 rpm/min). Yeasts cultures, OD600 level set at 0.1, were transferred in 50 ml of

YPD medium and cultivated for 10 h in a water bath at 30 °C with shaking (170 rpm/min). Subsequently, cells were spun down (Hermle Z216 MK, Germany) at 5000 ×g for 10 min, 4 °C, and the cell pellet was washed three times with 20 ml of sterile ultrapure water. The cell concentration was thereafter adjusted to an initial OD600 = 1.0. Two microliters of the 10−3 dilution was spotted in triplicates on Delft Phy, Delft+ and Delft− agar plates. The plates were incubated at 25 °C for 72 h and photographed afterwards. The growth was estimated by measuring the diameter of the colonies. Considering the same diameter range on Delft+ medium, results on Delft Phy plates were considered: negative (−), weak growth (+) when colony diameter was lower than 0.5 cm, normal growth (++) when diameter crossed 0.5 cm, high growth (+++) for all colonies reaching 1 cm and intense growth (++++) for colonies exceeding 1 cm. For liquid growth tests, 96-well microtiter plates (92096, TPP), wells were filled in triplicate with 200 μl of corresponding medium, inoculated with 2 μl of prepared yeast inocula and cultivated at 25 °C for 48 h. Yeast growth was monitored at OD600 using a Microplate reader (Varioskan Flash, Thermo Scientific, MA, USA). Measurements were taken every 2 h and 3 s shaking (300 rpm) was applied prior to data acquisition. For each medium, ln(ODtx / ODt = 0) for each time point was determined and standard deviation of three measurements was calculated. For each strain, OD600 measured at 48 h in Delft Phy was related to the OD600 in Delft+ medium. S. cerevisiae BY80 was used as positive control for all the growth-based tests. 2.3. P. kudriavzevii phytase gene expression 2.3.1. Yeast strains and growth conditions Based on the results from the screening tests, five P. kudriavzevii strains were selected for further analysis. Four of them showed a high growth rate in Delft Phy (G6, G5, M31 and M30) while one failed to grow in Delft Phy medium (M16). Yeast inoculum was prepared as described above. Cells in exponential phase were used for inoculation of Delft Phy (100 ml) and Delft+ to an initial OD600 = 1.0. For extraction of total RNA, cells were spun down (1000 ×g for 1 min, 4 °C) at 0, 2, 4, 6, 8, 10, 14, 24 and 48 h. All pellets were frozen at −20 °C in the presence of RNAlater (50 μl) and stored until RNA extraction. 2.3.2. RNA extraction, quantification and reverse transcription Total RNA was isolated using Total RNA Mini Kit (Qiagen, Hilden, Germany) according to the supplier's manual. Cell lysis was performed by a step of bead-beating (3 × 45 s, 4.5 m/s) in β-mercaptoethanol (600 μl) (Fast Prep, FP120 BIO 101, Savant, Santa Ana, CA, USA).

Table 1 Growth screening of 93 yeast strains in liquid Delft Phy (phytate-containing media) at 48 h. For each species, strains have been grouped according to the range of the actual OD600 values measured at 48 h in Delft Phy (max–min value). In the last column, the ratio OD600 Delft Phy/OD600 Delft+ (phosphate-containing media) is presented for each group as max–min value. Yeast growth at 48 h in liquid Delft Phy medium Yeast species

No of strains (TOT)

No of strains (groups)

Strain ID

OD600 Delft Phy

OD600 Delft Phy/Delft+

Pichia kudriavzevii

44

43

2.56–1.99

1.16–0.90

Saccharomyces cerevisiae

20

G5, G6, G4, O13, T42, O12, O11, M32, M31, O10, M30, T41, M29, T40, T39, T38, G3, T37, M28, M27, M26, M25, T36, O9, T35, M24, M23, M22, M21, M20, O8, M19, T34, M18, T33, O7, O6, O5, T32, O4, O3, M17, T31 M16 T30, T29, T28, T27, O2, T26, M15 M14, T25, T24, M13, T23, T22, T21, T20, T19, T18 T17, T16, T15 M4, M3 M10, M9, M8 M7, M6 M5 M2, G1, M1 M12, G2, O1, T14, T13 T12, T11, T10, T9, T8, T7, M11, T6 T5, T4, T3 T2, T1 BY80

0.61 2.51–2.01 1.75–1.28 0.59–0.51 2.31–2.07 2.29–1.50 2.27–1.43 0.35 2.17–1.43 2.12–1.70 1.58–1.31 1.66–1.61 1.15–1.11 2.50

0.30 1.01–0.94 1.06–0.95 0.36–0.33 1.01 0.99–0.84 1.01–0.97 0.20 1.02–0.95 1.03–0.96 1.03–0.76 1.05–1.04 0.96–0.89 1.00

Wickerhamomyces anomalus Millerozyma farinosa Candida glabrata Kluyveromyces marxianus Clavispora lusitaniae Hanseniaspora guilliermondii Debaryomyces nepalensis Positive strain

2 3 3 3 13 3 2 1

1 7 10 3 – – 2 1 – 5 8 – – –

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Concentration of total RNA was estimated by measuring the absorbance at 260 nm (NanoDrop 1000, Thermo Scientific). The RNA quality was verified with an agarose gel (0.8%) electrophoresis. Prior to reverse transcription (RT) all RNA samples were treated with DNAase for 3 h at 37 °C (AMBION, Copenhagen, Denmark) that was thereafter deactivated. Success rate of chromosomal DNA elimination was verified with PCR. Total RNA (500 ng) was reverse transcribed using GeneAmp RNA PCR Kit (Applied Biosystems, Foster City, CA, USA) in a final volume of 100 μl containing 1× Buffer, 5.5 mM MgCl2, 0.5 mM of each dNTP, 2.5 μM random hexamers, 40 U RNase inhibitor and 125 U of Multiscribe Reverse Transcriptase. The following program was used for the RT: 25 °C for 10 min, 48 °C for 30 min and 95 °C for 5 min (SureCycler 8800, Agilent Technologies, Santa Clara, CA, USA). The cDNA was stored at −80 °C. 2.3.3. Primer design and selection of target sequence for qPCR Two reference genes were selected for the RT-qPCR: ACT1 (actin 1, GeneBank # AJ389086) and LSC2 (β subunit of succinyl-CoA ligase, GeneBank # ALNQ01000029). LSC2 was identified by alignment with

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homologous gene sequences of S. cerevisiae and Candida albicans (Nailis et al., 2006). A phytase gene of P. kudriavzevii was identified by screening for the conserved N and C- terminal motifs found in the histidine acid phosphatases in all contigs of a recently published draft genome of P. kudriavzevii (GeneBank # ALNQ00000000.1, Chan et al., 2012). This was performed by reverse translation of the conserved protein domains by using CLC Main Workbench 6. The identified sequence named as PHYPk was found on contig 396 (GeneBank # ALNQ01000395). Primers specific for PHYPk (fTGTCCCTTCCACTAGTTT- rAACTTACCGATAACCAGC), for ACT1 (f ACCATGTTCCCAGGTATTGC- rCACATTTGTTGGAAGGTGGA) and for LSC2 (fAATGGTTTCTGGAGTTGCAGC- rGATGAAGTTGTTGCCTCTAAA) were designed using CLC Main Workbench 6 (CLC Bio, Qiagen, Arhus, Denmark) and their efficiency was evaluated using NCBI database. PCR conditions for all primer sets were tuned using a gradient-PCR (SureCycler 8800, Agilent Technologies). The amplification efficiency (E) was estimated by quantitative PCR (qPCR) (7500 Fast Real-time PCR System, Applied Biosystems, CA, USA) using serial dilutions of pooled cDNA samples as standards (Rasmussen, 2001).

Fig. 1. Growth profiles of P. kudriavzevii strains G6, G5, M31, M30 and M16 in Delft Phy (gray squares), Delft+ (black circles) and Delft− (black triangles) media. Growth was monitored as OD600 for 48 h. Standard deviations for triplicate measurements are indicated.

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2.3.4. Quantitative PCR qPCR was carried out using the 7500 Fast Real-time PCR System (Applied Biosystems). The expression of the target gene was analyzed in triplicates in a presence of both reference genes and a non-template control. The reaction mix (20 μl) was composed of 10 μl of 1 × Fast SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA), 1 μl of each primer (10 pmol/μl), 5 μl of cDNA and nuclease-free water up to 20 μl. The qPCR temperature profile was as follows: 10 min at 95 °C followed by 45 cycles of 10 s at 95 °C, 30 s at 57 °C and 30 s at 72 °C. Subsequent to the amplification, a melting curve analysis was performed. A sample was considered to be below the detection limit when the fluorescence threshold was not reached within 45 cycles. All reactions were performed in duplicate using two independent RNA isolations.

2.3.5. Data analysis The Ct data (average of three replicates) were transformed into copy numbers considering the relative efficiencies using the GeneEx software

(ver. 4.1.7; MultiD Analyses AB, Goteborg, Sweden). The expression of the target genes was analyzed by normalizing the copy number values of the target gene with the geometric mean of the two reference genes. Data collected for each strain in a given medium were normalized to the expression level at t0. Results were expressed as ratio copy number to t0, for all time points (referred as CN). Changes in phytase gene expression in Delft Phy and Delft + was also studied relative to the expression level at t0 using the 2− ΔΔCt method (Livak and Schmittgen, 2001), for each reference gene (referred as LSC2 and ACT). The ΔΔCt was calculated using the following equation:  ΔΔCt ¼ Ct

target gene −Ct reference gene



Delft



tx −

Ct

target gene −Ct reference gene

 Delft

t0:

Before the use of the comparative method, it was verified that the PCR efficiencies of reference and target genes were approximately equal. Results were expressed as a fold increase (or decrease) of the target gene during the growth, normalized to the expression of the reference genes.

Fig. 2. Partial nucleotide sequence of PHYPk and its deduced amino acid sequence that is shown below the corresponding codons. In bold, methionine (M), RHGXRXP and HD motifs. Arrows indicate the primer binding sites.

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Fig. 3. Changes in PHYPk gene expression during growth of P. kudriavzevii G6, G5, M31 and M30 in Delft Phy and Delft+ (positive control). A: Copy number ratio in Delft Phy (Black bars) and B: Delft+ (white-striped bars) at tx/t0. Different letters indicate significant differences (p b 0.05): a, b, and c referred to Delft Phy while x, y, and z referred to Delft+.

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2.4. Measurements of phytase activity 2.4.1. Enzyme extraction Yeast strains were grown in flasks containing 100 ml of Delft Phy medium. The medium was inoculated with yeast inocula as previously described (OD600 = 0.1), and cultivated at 30 °C for 48 h. Each strain was inoculated in triplicates. Samples of extracellular and intracellular enzyme extracts were collected after 2, 8, 24 and 48 h after inoculation. Enzyme extracts were prepared as described by Nuobariene et al. (2011). To confirm that the enzyme is repressed by inorganic phosphate, the same protocol was followed for yeast strains grown on Delft+ medium. 2.4.2. Phytase activity Phytase activity was measured as previously described (Nuobariene et al., 2011). Briefly, 0.2 ml of enzyme extract was added to 0.8 ml of phytic acid dipotassium solution (3 mmol/l phytic acid dipotassium

salt in 0.2 mol/l sodium acetate/HCl buffer, pH 5.5) pre-incubated at 30 °C per 5 min. The solution was mixed and incubated at 30 °C. Samples were measured at different time intervals, every time the reaction was stopped by adding trichloroacetic acid (TCA) to the sample. Blank was measured with sodium acetate buffer and TCA, together with enzyme extract. To measure the liberated inorganic phosphate, 0.4 ml of the sample was added to 3.2 ml of freshly prepared acid molybdate reagent (1 volume of 10 mmol/l ammonium molybdate, 1 volume of 2.5 mol/l sulfuric acid and 2 volume of acetone). Absorbance of the yellow at 355 nm was measured in a spectrophotometer, using sodium acetate buffer with TCA as blank. A standard curve was prepared with KH2PO4 in 0.2 mol/l sodium acetate buffer/HCl buffer (pH 5.5). 2.4.3. Activity calculation One unit of phytase activity is defined as the amount of protein that releases 1 μmol of inorganic phosphate within 1 min. Volumetric

Fig. 4. PHYPk expression (black squares; primary axes) and related extracellular (grey circles; secondary axes) and intracellular (grey triangles; secondary axes) volumetric enzyme activity of P. kudriavzevii strains G6, G5, M31, M30, and M16 at 2, 8, 24 and 48 h in Delf Phy medium. Stars (*) indicate a correlation (Pearson's correlation coefficient) N 0.95 between mRNA expression and enzyme activity.

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phytase activities were calculated for both extra- and intracellular phytases. The activity was measured at four different time points (0, 15, 30 and 45 min) and ΔCPO4 was calculated (Olstorpe et al., 2009). Enzyme activity was expressed as milliUnit per ml of enzyme extract (mU/ml). All assays were performed in triplicates and the mean and standard errors of the mean calculated. 2.5. Statistics The significance of the variations was determined by ANOVA (XLStat, Addinsoft SARL, Paris, France). Tukey's honestly significant difference post-hoc comparison was performed and a p value of b 0.05 was considered significant. Correlation coefficients were calculated by using Pearson correlation. 3. Results 3.1. Growth on liquid and solid phytate or phosphate-media Results from the liquid-based screening at 48 h are presented in Table 1. No isolate grew on Delft− liquid medium whereas all the tested isolates grew on Delft + medium (data not shown). P. kudriavzevii isolates, except one strain (M16), grew better than any other species in liquid medium, reaching an actual OD600 between 1.99 and 2.56 within 48 h and an OD600 ratio Delft Phy/Delft + between 1.16 and 0.90 (Table 1). Results from the solid-medium confirmed the observations from the liquid-medium, except for some strains of S. cerevisiae and Cl. lusitaniae, which did not grow on plates as expected from the results of the liquid assay (Supplementary material, Table S1). Fig. 1 presents the results performed in liquid media for the five strains selected for the PHYPk expression study i.e. G6, G5, M31, M30 and M16. As seen in Fig. 1, although the OD600 of the four positive strains at t48 was almost equal, their growth curves in Delft Phy and Delft+ differed. Strains G6 and G5 started growing in Delft Phy and Delft+ at t12 while M31 at t8 and M30 at t10. The growth curve of M30 in Delft Phy liquid medium differed from G6, G5 and M31. The strain chosen as negative control (M16) was not able to grow in the Delft Phy medium while it starts growing in Delft+ at t14 (Fig. 1).

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negative strain M16. The same strain did not show any significant differences in the level of PHYPk gene expression in any of the two media (data not shown). The results obtained from the analysis using 2−ΔΔCt method (data normalized on LSC2 and ACT1, respectively) matched with those obtained by copy number analysis (CN) (Supplementary material, Fig. S1). The trend of the expression of the gene was comparable and the only variation was the evaluation of the statistical differences. 3.3. Quantification of phytase activities Both extracellular and intracellular phytase activities were determined for strains of P. kudriavzevii at four different time points during the growth curve: t2, t8, t24 and t48 (Fig. 4). Values of volumetric extracellular activity ranged from 49.17 to 4.08 mU/ml. For strain G6 and M30 significant differences were found for the extracellular activity at t2. Higher values of extracellular activity were measured for strain G5 at t8 while for strain M31 at t2. Unexpectedly, for all the four strains the minimum extracellular activity values were detected at t48. By looking at the intracellular volumetric activity, the values ranged from 42.79 to 2.19 mU/ml. The intracellular activity was significantly higher at t2 for strain G6 and M30. Strain M31 showed a different pattern of intracellular activity (Fig. 4). The phytase activity of strain M16 (negative strain) was significantly lower both at extra and intracellular level compared to G6, G5, M31 and M30 P. kudriavzevii strains. 3.4. Correlation between RNA level and enzymatic activity The data obtained from the measurement of phytase activity were also correlated with RNA expression values. In general, a high correlation was found between the enzyme activity and the expression of PHYPk (Fig. 4). This was particularly true for the extracellular activity for the strains where correlation coefficients of 0.99, 0.82, 0.79 and 0.95 were found for strain G6, G5, M31 and M30, respectively. At intracellular level, no correlation was found for strain M31 while 0.95, 0.94 and 0.96 were the correlation values obtained for strains G6, G5 and M30, respectively. In general, when no mRNA expression was detected, phytase activity could be measured (Fig. 4).

3.2. Expression profiling of PHYPk gene 4. Discussion Based on the identified sequence of phytase gene, primers for PHYPk were developed. Fig. 2 reports the nucleotide and deduced amino acid sequence of PHYPk, together with the primer positions used in this study. The amino acid sequence contained the N-terminal and C-terminal motifs (RHGXRX and HD) and the N-linked glycosylation sites. A start codon, codifying a methionine, was identified as the beginning of the protein, but no stop-codon was found in suitable place inside contig 396. Primers were designed to amplify the 793 to 970 nucleotide position on contig 396, containing RHGXRXP motif (Fig. 2). Fig. 3 presents the results of the PHYPk expression profiling in Delft Phy (yA) and Delft + (yB), normalized to t0, for the four positive strains (G6, G5, M31 and M30). As seen in Fig. 3, the level of PHYPk expression was strain dependent and significantly lower in the Delft + medium than in the Delft Phy medium for all P. kudriavzevii strains. However, for strain G6 PHYPk was expressed significantly higher in the Delft+ than for the Delft Phy medium from t10. For growth in the Delft Phy medium, different PHYPk expression patterns were observed among the four strains of P. kudriavzevii. For strains G6, M31 and M30 PHYPk were strongly up-regulated at the very beginning of the growth phase i.e. t2, where after a down-regulation was observed. For strain G5 a transient response in PHYPk expression was observed with maximum expression at t8. For growth in the Delft + medium, PHYPk expression was upregulated at t10 for strain G6 and t48 for strain M31 whereas practically no up-regulation was observed for strains G5 and M30. Significant differences were detected between media for each strain, except for the

The aim of the present study was to test the ability of the yeasts isolated from traditional African foods of Benin to hydrolyse phytate. Assays using both liquid and solid media are mandatory as yeasts producing phytase on solid media may not produce the enzyme in liquid media or vice versa (Mukesh et al., 2004; Tseng et al., 2000). In general, we could not find qualitative differences between the liquid and solid assay. No growth was detected on negative medium, confirming the need of phosphate for yeast growth. A pronounced difference was seen between growth in phosphate-rich and phosphate-free medium, and this was also the case for the phytate medium. Our results demonstrate that growth differences are present at both the species and the strain level. This is in agreement with previous studies (Hellström et al., 2010; Nuobariene et al., 2011; Türk et al., 2000). P. kudriavzevii (formerly known as C. krusei) was the species that grew best in liquid and solid media, containing phytate as unique phosphate source. Strains of P. kudriavzevii are frequently isolated from African traditional foods (Greppi et al., 2013a,b; Jespersen et al., 1994, 2005) and this species has earlier been reported to produce phytase (Nuobariene et al., 2011; Quan et al., 2001, 2002). Quan et al. (2001) demonstrated the production of a cell-bound phytase by P. kudriavzevii WZ-001. The authors reported that enzyme synthesis is regulated by phosphate concentration in the medium. In particular, the synthesis of the enzyme is repressed by the presence of phosphate in the medium (Quan et al., 2001).

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By screening for the conserved terminal motifs found in the HAPs in a draft genome sequence of P. kudriavzevii published (Chan et al., 2012) we could identify and partially describe a phytase gene (referred to as PHYPk) on contig 396. A start codon was identified, however, unfortunately, no stop-codon was found in suitable place inside contig 396. As this was out of the scope for the present work, further molecular studies are necessary to find the end of the gene by e.g. RACE-PCR or chromosome walking technique, and describe the entire sequence as it was already done for other yeast phytases (Li et al., 2009; Ragon et al., 2008; Watanabe et al., 2009). LSC2 and ACT1 were chosen as reference genes, because in previous studies they were already used in gene expression analysis in C. albicans (Kofla and Ruhnke, 2007; Vandenbosch et al., 2012). The reference gene stability was checked and the expression of both genes was not influenced by the experimental conditions (data not shown). In general, PHYPk gene was expressed to a higher extent in the medium containing phytic acid as the only phosphate source, clearly underlining its role in this limiting condition of growth. However, the expression level was strain dependent. The strain chosen as negative control showed to some extent both intra- and extracellular activity which confirmed what already underlined by Nuobariene et al. (2011). Synthesis of phytase by yeast is governed by Pi concentration in the environment. Our findings suggest that in P. kudriavzevii the regulation occurs as previously described for S. cerevisiae (Andlid et al., 2004; Ogawa et al., 2000; Oshima, 1997; Veide and Andlid, 2006; Yoshida et al., 1989). For the majority of the strains we observed a strong reduction of the PHYPk expression in a phosphate-containing medium. At the same conditions, no phytase activity was measured for any of the strains (data not shown). PHYPk expression seemed to occur in response to phosphate starvation. It has previously been reported that a certain Pi concentration will repress yeast phytase production differently in different media and pH, and that the temperature has a strong effect of phytase activity (Andlid et al., 2004; Li et al., 2009; Ushasree et al., 2014). Our findings on P. kudriavzevii are not in agreement with Quan et al. (2001) which showed that phytase production by P. kudriavzevii occurs in the late stage of exponential growth phase. As previously mentioned, this might be due to different growth conditions of the strains. However, considering that the enzyme has been secreted during 48 h we expected the highest extracellular activity to be at the end of cultivation. At the moment, we do not have a clear explanation for that. Expressions of other genes than PHYPk also seem to play a role in the activity of P. kudriavzevii. Further studies are therefore necessary to better investigate the molecular basis of the phytase production in P. kudriavzevii, the presence and interactions with other phytase genes and a more detailed investigation of the genetic system responsible for transcriptional regulation of phytases. The identification and study of regulatory genes in P. kudriavzevii and the identification of appropriate mutants in the autochthonous strains may be an opportunity to improve even more phytate degradation in fermented foods. Considering the importance of this species in African traditional fermentation and the occurrence of micronutrient deficiencies in African countries, studying phytase production during yeast growth in fermented foods is of significant importance. In the present work, for the first time, a phytase-coding gene of P. kudriavzevii PHYPk was identified, its expression level quantified and correlated with enzyme activity. Furthermore, the phytase gene expression was for the first time investigated during growth. The identified gene PHYPk seemed to be involved in the hydrolysis of phytate allowing the yeast growth in the absence of free phosphate. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2015.04.011. Acknowledgment This work was partly financed by the Danida funded projects: “Capability Building for Research and Quality Assurance in Traditional

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Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains.

Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional Afr...
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