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Development and application of a real-time PCR-based assay to enumerate total yeasts and Pichia anomala, Pichia guillermondii and Pichia kluyveri in fermented table olives
Food Control 23 (2012) 356e362
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Development and application of a real-time PCR-based assay to enumerate total yeasts and Pichia anomala, Pichia guillermondii and Pichia kluyveri in fermented table olives Rosanna Tofalo, Maria Schirone, Giorgia Perpetuini, Giovanna Suzzi, Aldo Corsetti* Dipartimento Scienze degli Alimenti, Università degli Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Sant’Angelo (TE), Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2011 Received in revised form 20 July 2011 Accepted 26 July 2011
The objective of this work was to develop a real-time quantitative PCR (qPCR) assay to directly detect and quantify the total number of yeasts besides three species (Pichia anomala, Pichia guillermondii and Pichia kluyveri) associated to table olives. For this purpose, a real-time PCR protocol targeted to the region of the internal transcribed spacers ITS (rDNA) and the conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, respectively, was developed. This assay allowed to unequivocally distinguish the three species from other yeasts and lactic acid bacteria generally associated to table olives fermentation. qPCR performed well both with purified DNA and DNA extracted from olive brines. A reverse transcription-qPCR (RT-qPCR) assay from rRNA aimed to viable yeast quantification was also performed. To evaluate the effectiveness of the technique, the qPCR results were compared with those obtained by a plate count approach in synthetic medium. The small standard errors provided this assay reproducible and robust. qPCR efficiently enumerated cells at concentrations of as low as 102 CFU ml1 when standard curve was derived both from cells growing in a syntetic medium and in brines. The quantification method for total yeasts and P. anomala, P. guillermondii, P. kluyveri in table olive brines applied in this work was specific, reproducible, sensitive and fast. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Yeast quantification Pichia spp. real-time PCR Fermented table olives
1. Introduction Table olives are important components of the Mediterranean diet and their consumption is spread all around the world. They are generally produced by three main methods: Spanish-style green olives in brine, California-style black olives in brine and Greek-style naturally black olives in brine. The main purpose of these methods is the removal of fruit bitterness by hydrolysis of some phenolic compounds, especially oleuropein. In the first process, bitterness is removed by adding lye; in the Greek and Californian style processes, fruits are placed directly in brine and in an acidified solution, respectively, and oleuropein removal is slow and only partial (Aponte et al., 2010; Garrido-Fernández, Fernández Díez, & Adams, 1997). However, there are many aspects of traditional table olive fermentation still unexplored (Abriouel, Benomar, Lucas, & Gálvez, 2011), which could open new international market for unknown local products and constitute niche products (Panagou, Tassou, & Katsaboxakis, 2003). According to the last data published by the * Corresponding author. Tel.: þ39 0 861266896; fax: þ39 0 861 266940. E-mail address: [email protected] (A. Corsetti). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.07.032
International Olive Oil Council (IOOC, 2008), the annual production of table olives is 1.3e1.8 million tonnes. Notwithstanding table olive fermentation is an empirical process, in which the natural fermentation results from competitive activities of the indigenous microbiota, together with a variety of contaminating microorganisms present in raw material and the processing environment (Abriouel et al., 2011). This practice may lead to fluctuations of the final characteristics of the product, such as quality and flavor, and may determine its spoilage (Aponte et al., 2010; Lanciotti, Corbo, Sinigaglia, & Guerzoni, 1999; Panagou et al., 2003). Olive-fruit is an important microhabitat for a variety of yeast species, lactic acid bacteria (LAB) and filamentosus fungi. Both yeasts and LAB play a very important role in table olive fermentation because they contribute to the final product characteristics (Campaniello et al., 2005). The main LAB species involved in this process are lactobacilli, such as Lactobacillus plantarum and Lactobacillus pentosus (Hurtado, Reguant, Esteve-Zarvoso, Bordons, & Rozès, 2008; Panagou, Schillinger, Franz, & Nyckas, 2008). The presence of few other LAB species such as Enterococcus faecium has been reported, (Randazzo, Restuccia, Romano, & Caggia, 2004), but always as minority populations.
R. Tofalo et al. / Food Control 23 (2012) 356e362
Yeasts can play a double role, since they can produce volatile compounds and metabolites that improve the flavor properties of the final product and enhance LAB growth by the release of nutritive compounds. On the other hand, yeasts may cause gas-pocket formation, softening of the olive tissue or, package bulging, clouding of the brines and production of off flavors and odors. Pichia anomala (recently classified as Wickerhamomyces anomalus), Pichia membranifaciens, Candida boidinii, Debaryomyces hansenii, Rhodotorula glutinis and Saccharomyces cerevisiae are the species isolated with a higher frequency from diverse olive processes (ArroyoLópez, Durán Quintana, Ruiz Barba, Querol, & Garrido-Fernández, 2006; Coton, Coton, Levert, Casaregola, & Sohier, 2006). Moreover, the presence of Candida parapsilosis, Pichia kluyveri and Pichia guillermondi has been revealed during the entire fermentation period of four green Sicilian table olive (Aponte et al., 2010). The identification of yeast biota in table olive production by using molecular methods is still poor applied (Hurtado, Reguant, EsteveZarvoso, Bordons, & Rozès, 2008; Nisiotou, Chorianopoulus, Nychas, & Panagou, 2010). Actually, molecular methods widely applied to extimate yeast biodiversity are based on the variability of the ribosomal genes 5.8S,18S and 26S (Arroyo-López et al., 2006; Cai, Roberts, & Collins, 1996; Coton et al., 2006; Hurtado, Reguant, Esteve-Zarvoso, Bordons, & Rozès, 2008; James, Collins, & Roberts,1996; Nisiotou et al., 2010). Although these methods are rapid and accurate, culture isolation is necessary. Moreover, the presence of viable but non cultivable (VBNC) cells makes still more difficult to detect all the metabolically active yeast cells in fermented foods (McDougald, Rice, Weichart, & Kjelleberg, 2006). Then, these methods cannot allow the determination of yeast cells number present in the sample, an important parameter to control spoilage. Compared with other PCRbased methods, quantitative real-time PCR (qPCR) provides significant advantages. It allows DNA to be extracted directly from products without prior culture isolation (Wilhelm & Pingoud, 2003) and provides detection and enumeration of a number of fungi and foodborne pathogens (Mills, Johannsen, & Cocolin, 2002). In fact, the main advantage of qPCR is the low detection level, often as low as one cell per mL (Bleve, Rizzotti, Dellaglio, & Torriani, 2003; Delaherche, Claisse, & Lonvaud-Funel, 2004; Phister & Mills, 2003). This method has been widely applied to detect viable yeasts in different products such as pasteurized products (Bleve et al., 2003; Hierro, EsteveZarzoso, González, Mas, & Guillamón, 2006), orange juice, wine (Heras-Vazquez, Mingorance-Cazorla, Clemente-Jimenez, & Rodrizguez-Vico, 2003; López, Fernández-Espinar, Barrio, Ramón, & Querol, 2003), cheese (Vasdinyei & Deak, 2003), ‘‘alpeorujo’’ (Giannoutsou, Meintanis, & Karagouni, 2004), a residue from olive oil production, functional foods’, as well as yeasts of public health interest in dairy products (Makino, Fujimoto, & Watanabe, 2010). In the present work, we report on the development of a qPCR assay to directly detect and quantify the total number of yeasts as well as three main dominant species, P. anomala, P. guillermondii, and P. kluyveri in fermented table olives. Morever, viable and VBNC yeasts were detected by reverse transcription-PCR (RT-qPCR). 2. Materials and methods 2.1. Reference strains and culture conditions The references strains used in the present study are listed in Table 1 and represent the species commonly found in fermented table olives. Yeasts and LAB were stored at 80 C in YPD medium (1% yeast extract, 2% peptone, 2% glucose) and Man Rogosa Sharpe (MRS) (Oxoid, Milan, Italy), respectively, containing 20% (v/v) glycerol. Before experimental use, yeasts strains were grown in YPD for 16e18 h under aerobic conditions at 25 C, whereas LAB were propagated in MRS broth at 28 C in microaerophilic condition.
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2.2. Primer design P. anomala, P. guillermondii and P. kluyveri specific-primers as well as universal primers were designed from the region of the internal transcribed spacers ITS (rDNA) and from conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, respectively. Sequences available at the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov.) were retrieved and aligned using CLUSTAL X multiple-sequence alignment (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997). Appropriate primers were designed using Beacon Designer 3 program (Bio-Rad, Milan, Italy). The BLAST search (Basic Alignment Search Tool, http:// www.ebi.ac.uk/blastall/nucleotide.html) was used to check the specificity of each primer set. Furthermore, the properties of each primer were verified by Primer Tool Sigma Aldrich (www.sigmagenosys.com/calc/DNACalc.asp). The primer sequences besides amplicons size are shown in Table 2. These primers were used both for conventional PCR and qPCR. 2.3. Genomic DNA extraction Genomic DNA was isolated from yeasts by the procedure of Querol, Barrio, Huerta, and Ramón (1992), modified as follows: cells were resuspended in 500 ml of lysis buffer (25 mg of Rhizoctonia solani lytic enzyme/ml, 1 M sorbitol and 0.1 M EDTA pH 7.5) and incubated for 2 h at 45 C. Bacterial genomic DNAs were extracted from 1.5 ml of cultures in late-exponential-growth phase as reported by de Los Reyes-Gavillàn, Limsowtin, Tailliez, Sèchaud, & Accolas (1992). The concentration and purity of DNA were assessed by O.D. at 260 and 280 nm, as described by Sambrook, Fritschi, and Maniatis (1989). 2.4. Specificity of PCR assays All PCR amplifications were carried out in 25 ml final volumes containing 3 ml of DNA template (between 10 and 100 ng), 1x PCR buffer without MgCl2, 1.5 mM MgCl2, 0.5 mM of each primer, 100 mM dNTPs, 0.02 U/ml Taq Polymerase (Invitrogen, Italia). The thermal cycling program and primer sequences are reported in Table 2. All amplifications were performed in a GeneAmp PCR system 2700 (Applied Biosystems, Foster City, CA). The PCR products were analyzed by electrophoresis on a 1% (wt/v) agarose gel in 1x TAE buffer (40 mM Tris-acetate,1 mM EDTA, pH 8.3). The gels were stained with ethidium bromide (0.5 mg/ml) and visualized under UV-light. DNA Ladder 1 Kb plus (Invitrogen) was used as the size standard. 2.5. qPCR assays Each reaction mixture (25 ml) contained 12.5 ml 2XIQ SYBR Green PCR SupermixÔ (Bio-Rad), 5 pmol of each primer (Invitrogen), 1 ml DNA template (10 ng) and ultra pure water. Fluorescence emission was measured at the last step of each cycle. The qPCR program was common for each primer set (Table 2). The cycle threshold (Ct) was determined automatically by the instrument and was used for quantitative analyses. Melting temperature analysis of the PCR products was performed to determine the specificity of the PCR reaction. All samples were processed in triplicate. 2.6. Standard curves and efficiency Different mixed cultures containing known populations of S. cerevisiae, D. hansenii, C. boidinii and P. anomala were incubated for 48 h in YPD and synthetic brines (0.5% (v/v) lactic acid, 7% (wt/v) NaCl, 50 mM glucose, 10 mM sucrose, and 6% NaCl).
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Table 1 Yeast and bacterial strains used in this study and specific PCR results. Species
Strain designation
Candida boidinii Candida diddensiae Candida krusei Candida milleri Candida parapsilosis Candida rugosa Candida tropicalis Candida vini Candida zemplinina Debaryomyces hansenii
Hanseniaspora guilliermondii Issatchenkia orientalis Kloeckera apiculata Kluyveromyces lactis Kluyveromyces marxianus Pichia anomala Pichia guilliermondii Pichia kluyveri Pichia membranaefaciens Rhodotorula glutinis Rhodotorula minuta Rhodotorula mucilaginosa Saccharomyces bayanus Saccharomyces cerevisiae Saccharomyces paradoxus Saccharomyces pastorianus Yarrovia lipolytica Zygosaccharomyces mirakii Lactobacillus casei Lactobacillus fermentum Lactobacillus paraplantarum Lactobacillus pentosus Lactobacillus plantarum Leuconostoc pseudomesenteroides Weissella confusa
C1-4, LU-1, LU-3 ATCC 15541 ATCC 6258 MUCL 38021T ATCC 22019 ATCC 10571 DSM 5991 DBVPG 3227T CZ-3 MUCL 30242T CBS 767T H9, H13 DSM 3432 FG-1 CBS 104T C1-10 CBS 834T MUCL 28639T CBS 2030T, FOR-15 ATCC 9768 MUCL 29895T ATCC 2527 ATCC 32769 MUCL 30403T CBS 380T CBS 1171 CBS 432T CBS 1538T DBVPG 6132T C2-10 ATCC 393 ATCC 14931 DSM 10667T 1MO MO3f DSM 20193T DSM 20196
Origin
Fermented olives Shrimp Leaves Sourdough Leaves Human feces Rumen of cow Wine Wine Cherry Grape Pecorino abruzzese Diseased nail Fermented olives Grape Fermented olives Kefir Leaf Insects Fermented olives Wine Maize (Zea mays), leaves Intestine Orange Beer Beer Tree Grape Margarine Fermented olives Cheese Unknown Beer Fermented olives Sourdough Cane juice Sugar cane
Yeast-specific qPCR (152 bp)
Species-specific qPCR P. anomala (217 bp)
P. guilliermondii (228 bp)
Pichia kluyveri (216 bp)
þ þ þ e þ þ e e e
e e e e e e e e e e e e e e e e e þ e
e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e
e e e e e e e e e e e e e e e e e e e þ e e e e e e e e e e e e e e e e e
þ e þ e þ þ þ þ þ þ þ þ þ e þ e e e þ e e e e e e e
e e e e e e e e e e e e e e e e e
MUCL, Agro-Industrial Fungi and Yeast Collection, Universite’ Catholique de Louvain, Louvain-La-Neuve, Belgium; CBS, Centraalbureau voor Schimmelcultures, Baarn, The Netherlands; ATCC, American Type Culture Collection, Manassas, Va.; DSM; Deutsche Veterinarmedizinische Gesellschaft, Giessen, Germany, DBVPG Industrial Yeasts Collection, Dipartimento di Biologia Applicata, University of Perugia.
From YPD and synthetic brine cultures, 10-fold serial dilutions were performed and DNA from each dilution was extracted and quantified by qPCR using universal primer sets to determine Ct values. Type strains of P. anomala, P. guillermondii and P. kluyveri (Table 1) were analyzed as reported above using species-specific primers. Moreover, the same samples were serially diluted and plated on YPD media for viable counts. Standard curves were created by plotting the Ct values of the qPCRs performed on dilution series of DNA against the log of cells per milliliters, as determined by plate counts. The qPCR efficiencies were calculated in exponential phase from the given slopes and according to the equation
Table 2 Primer sequences and PCR conditions developed for qPCR. Species
Primer
Sequence (50 -30 )
PCR conditions
P. anomala
Anom2F AGK-R Guill 2F AGK-R Kluy2F AGK-R Oli-F Oli-R
GTT AAA ACC TTT AAC CAA TA AAA TGA CGC TCA AAC A CAA AAC ACA ATT TAA TTA TTT AAA TGA CGC TCA AAC A CAC CAA ACA CCT AAA AT AAA TGA CGC TCA AAC A CGT CAT AGA GGG TGA GAA TCC ACT TGT TCG CTA TCG GTC TC
5 min at 94 C; 1 min at 94 C, 30 s at 51 C, 1 min at 72 C for 30 cycles
P. guilliermondii P. kluyverii Total yeasts
E ¼ 10[1/slope]-1 (Rasmussen, 2001). In all PCR runs, negative controls (sterilized water), positive control and samples were run in triplicate. Sensitivity of qPCR assays was evaluated with reference to other reports (Hierro, Esteve-Zarvoso, González, Mas, & Guillamón, 2006; Martorell, Querol, & Fernández-Espinar, 2005). The results were analyzed using statistical software STATISTICA for Windows (STAT. version 8.0, StatSoft Inc. Tulsa, OK). All the Ct values are averages of at least three repetitions. 2.7. RT-PCR assay For reverse transcription-PCR (RT-qPCR), RNA was obtained using the RNA PowerSoilÒ Total RNA Isolation Kit (Mobio Laboratories, Inc) according to the manufacturer’s instructions. RNA from pure culture and mixed cultures, obtained as described above, was isolated and quantified by qPCR to enumerate total yeast populations. A 10-fold dilution series of isolated RNA was prepared. cDNAs were synthesized from each RNA dilutions according to Hierro et al. (2006) in order to construct standard curves. All the experiments were repeated five times in duplicate to ascertain the reliability and repeatability of the real-time assays. Three replicates of positive and negative controls were processed in each PCR run: a positive control with DNA from Pichia spp. and a negative control without template.
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2.8. Quantification of total yeast, P. anomala, P. guilliermondii and P. kluyveri in fermented table olives and brines Twelve samples of table olives (cultivars Tonda di Cagliari and Pizz’e Carroga) were supplied from “Cooperativa Olivicoltori” located in Parteolla (Sardinia). All samples were stored for 7 months in a solution containing 9% (wt/v) NaCl and 1% (wt/v) lactic acid. Nucleic acids were isolated according to the protocol of Querol et al. (1992) with some modifications at the beginning of the process. In particular, 10 ml of brine (or 10 g of pitted olives) were used to collect cells. Samples were centrifuged at 10.000 rpm for 5 min and pellets were washed twice with sterile water. The extracted DNA was used for qPCR. RNA was obtained using the RNA PowerSoilÒ and cDNA was synthesized as reported above. Total yeast, P. anomala, P. guilliermondii and P. kluyveri were determined by qPCR. The unknown samples were run alongside a set of known standards in order to determine their CFU/ml. A standard curve was generated by plotting the Ct values of the known samples against the CFU/ml to generate a standard curve from which the unknown CFU/ml was calculated.
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3.2. qPCR detection limits To determine the sensitivity and detection limits of the qPCR assay, DNA obtained from pure and mixed cultures with known concentrations was serially diluted 10-fold and quantified by qPCR using the corresponding primer sets (Table 2). Then, the effectiveness of the quantification for this method was tested correlating the cell concentrations estimated by plate count and qPCR. Each DNA dilution besides plate count value was used to construct standard curves (Fig. 1). The Ct values obtained for concentrations in cell number ranging from 10 to 108 showed good linearity for triplicate
3. Results and discussion 3.1. Primer design and specificity In order to detect all the yeasts present in fermented table olives, universal primers (Oli-F/Oli-R) from conserved sequences of the variable D1/D2 domains of the 26S rRNA gene were designed, as reported in Table 2. The sequences of the variable D1/D2 domains of the 26S rRNA gene determined by Kurtzman and Robnett (1998) facilitated the design and the test of the assay for yeast species specificity. Only a DNA fragment of the expected size (152 bp) was found in presence of the yeast species considered here, whereas amplicons were not obtained for yeasts generally not associated with fermented table olives or for bacteria which could be detected in fermented olives and brines (Table 1). In particular, the above DNA fragment was not observed in presence of fermented table olive bacteria such as LAB. Moreover, Candida vini and Candida zemplinina were used as negative control to check the procedure. They were chosen since they are normally absent in fermented table olives, and they didn’t generate any PCR products further highlighting the primers specificity. Then, primers aimed to the quantification of P. anomala, P. kluyveri and P. guilliermondii were designed from the region of the internal transcribed spacers ITS (rDNA). The forward primer was common for all the three species and was homologous to a conserved region of the above gene. To evaluate the primer specificity, conventional PCR was performed using purified DNA from yeasts and bacteria listed in Table 1. One fragment of the expected size was obtained for each type strain: P. anomala (217 bp), P. kluyveri (216 bp) and P. guilliermondii (228 bp). The ITS regions are less evolutionarily conserved than the rRNA-coding genes and are therefore valuable regions for finding interspecific differences (Guillamón, Sabaté, Barrio, Cano, & Querol, 1998; Hierro, EsteveZarzoso, Mas, & Guillamon, 2007) being so useful to design yeast species-specific primers. Then, primers specificity was analyzed by qPCR using the optimized reaction conditions and purified DNA as described in material and methods. For all primer sets, a unique peak was observed in the melting curve of amplicon, suggesting the specificity of the amplification, i.e. each primer pair amplified a unique locus targeted on the genome (data not shown). To develop a real-time PCR method for yeasts quantification in which SYBR green I real-time PCR is used, it is necessary to design primers that do not amplify DNA sequences from any other microorganism that can be present in fermented food under study (Postollec et al., 2011).
Fig. 1. Standard curves obtained from 10-fold serial dilutions of DNA isolated from P. anomala, P. kluyveri, P. guilliermondii and mixed culture yeasts grown in YPD medium , and incubated in synthetic brine B. Ct value are the average of three repetitions. Error bars represent standard errors.
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Table 3 Correlation coefficients, slopes and efficiencies of standard curves obtained from 10-fold dilution of yeast strains grown in synthetic brine and YPD medium (mean S.D.). Assay
P. anomala P. kluyveri P. guilliermondii Total yeasts a b
Strain code
MUCL 28639T DBVPG 6901T CBS 2030T Mixed culture yeastsb
Synthetic brine R2
Slope
0.992 0.991 0.992 0.996
3.440 3.420 3.571 3.524
YPD
0.88 0.55 0.91 0.45
Efficiencya (%)
R2
Slope
95.29 96.06 90.56 92.20
0.992 0.990 0.990 0.994
3.344 3.353 3.359 3.454
Efficiencya (%)
0.52 0.38 0.71 0.63
99.08 98.71 98.47 94.76
Efficiency was estimated by the formula 101/slope1. S. cerevisiae, D. hansenii, C. boidinii and P. anomala.
samples of P. anomala. On the contrary, P. guillermondi and P. kluyveri were detected starting from 102 cell/ml and the lowest cell quantification for total yeast was 103 cell/ml. The correlation coefficients, slope and efficiencies of the amplification for the standard curves are shown in Table 3. In all the qPCR assays the yeast populations were effectively enumerated and good correlation with enumeration by culturing system was found (R2 0.990) (Table 3). Similar detection limits were detected using specific primer for yeast in other fermented food, demonstrating quantification ability of qPCR assays. In particular, Makino et al. (2010) developed a qPCR assay to directly detect and quantify nine emerging opportunistic yeast species in dairy product samples. Their detection limit using universal yeasts primers was between 102 and 103 cells/ml. Because some compounds found in table olives, such as salt and polyphenols, are known to inhibit PCR reaction, a comparison between the Ct values of DNAs standard curves obtained from the synthetic brines and YPD plate counts was made. The qPCR assay showed successful amplification in all brine samples artificially contaminated with yeasts. In fact, DNA fragments of expected size were found in all samples (data not shown). The standard curves obtained in both YPD and synthetic brines showed the same linearity allowing quantification in a range from 10 to 108 cell/reaction, with similar efficiencies as obtained for pure cultures (Fig. 1). These data demonstrated that the above mentioned compounds didn’t affect the amplification reaction. Inhibitory effect on PCR reaction by intrinsic factors has been reported in foods by many authors (Bleve et al., 2003; Delaherche et al., 2004; Hierro, Esteve-Zarvoso, González, Mas, & Guillamón, 2006; Hierro, Esteve-Zarvoso, Mas, & Guillamón, 2007; Martorell et al., 2005). Moreover, other authors found differences when PCR was performed with DNA extracted from pure cultures or directly from foods. Hierro et al. (2006) detected higher values of S. cerevisiae in must and wine by qPCR rather than plate count values. These data were confirmed by RT-qPCR, because RNA is strictly related to cell viability. In fact, the amplification of DNA allowed to detect live, death and metabolically active but not cultivable cells. To prevent the detection of dead microorganisms is necessary to study gene expression as marker for cell viability (Bleve et al., 2003).
correlations were 0.991 and 0.995 in the standard curves obtained from YPD and brines, respectively. The efficiencies were 97.43% and 96.41%, respectively (Table 4). These efficiency values were better than those obtained using DNA as the template (data not shown). Similar results were obtained by Hierro et al. (2006). 3.4. RT-qPCR analysis of fermented table olives In order to obtain a quantification of yeast populations in fermented table olives, a real-time RT-qPCR assay was optimized. The quantitative capability of the qPCR was evaluated determining the relationship between the amounts of detected yeast by qPCR and the plate count in YPD. Table 5 shows the values of CFU/ml obtained by qPCR, RT-qPCR and plate counts. Since the successful application of PCR-based techniques depends on the quality of the template good protocols for DNA extraction are needed. In this study a kit generally used for soil, grape and must, that are very complex matrices because of the presence of PCR inhibitors, was applied with some modifications. In particular, to obtain the complete lysis of cells a storage at 20 C for 20 min was applied. Moreover, since foods could contain various PCR inhibitors such as polyphenols, tannins and polysaccharides (Hierro, Esteve-Zarvoso, González, Mas, & Guillamón, 2006; Martorell et al., 2006; Phister & Mills, 2003) standard curves by the dilution of cells incubated in brines were constructed. With regards to yeast count in brines, count values ranged from 104 to 105 CFU/ml and only one sample (S21) with a value of 106 CFU/ml; while the values obtained by qPCR ranged from 106 to 107 CFU/ml, except for S9 and S3 which were of 104 and 105 CFU/ml, respectively. The higher total yeast counts observed by qPCR and RT-qPCR with respect to plate count may be due to the large presence of VBNC populations. The values of total yeast population showed similar results with all enumeration methods for only three samples (S3, S9, S21). qPCR revealed also the presence of the three species of Pichia tested, with P. anomala and P. guilliermondii present in the majority of the analyzed samples (Table 6). In particular, P. anomala was detected in 7 out 12 samples: S1, S3, S11, S15, S17, S21 and S23, ranging from 102-105 CFU/ml.
3.3. Quantification of total yeast populations by RT-qPCR In order to obtain a more realistic quantification of total viable yeasts, an RT-qPCR assay was developed. In the first step, a mixed culture of selected yeast was diluted from 108 to 10 cell/ml. Total RNA was isolated in triplicate from each dilution and subjected to RT-qPCR. cDNA from each RNA dilution was synthesized as described in material and methods. Then, the synthesized cDNAs were subjected to RT-qPCR assay. The same procedure was carried out with a culture of the same yeasts incubated for 24 h in synthetic brines. A non-reverse-transcribed control was used to verify that RNA samples were DNA free. No amplification was obtained in nonreverse-transcribed control sample. The detection limit of the RTqPCR assay was of 103 CFU/ml (Fig. 2), and the coefficient
Fig. 2. Total yeast standard curves obtained from serially diluted RNA isolated from mixed culture yeasts grown in YPD medium , and inoculated in synthetic brine B. Ct value are averages of results of three replicates. Error bars represent standard errors.
R. Tofalo et al. / Food Control 23 (2012) 356e362 Table 4 Correlation coefficients, slopes and efficiencies of standard curves obtained from serial dilutions of RNA isolated from mixed culture yeasts grown in YPD medium and synthetic brine (mean S.D.). Yeast cell assay
R2
Slope
Efficiencya (%)
Synthetic brine YPD
0.995 0.991
3.411 0.45 3.385 0.63
96.41 97.43
a
Efficiency was estimated by the formula 101/slope1.
P. anomala is one of the most abundant yeast throughout the fermentation process in all kinds of olives (Coton et al., 2006; Durán Quintana, García-García, & Garrido-Fernández, 2003; Hernández, Martín, Aranda, Pérez-Nevado, & Córdoba, 2007; Marquina et al., 1992). It can play a double role during olive fermentation, in fact it can cause softening and gas-pocket formation (Aponte et al., 2010; Asehraou, Peres, Brito, Faid, & Serhrouchni, 2000; Faid, Akhartouf, & Asenhraon, 1994). Moreover this species together with S. cerevisiae has been related to “alambrado” (bloater) spoilage in spontaneous fermentation in black olives (Durán Quitana, González, & Garrido, 1979). On the other hand P. anomala produces killer toxins. It has been successfully used for the biocontrol of Penicillium (Boysen, Björneholm, & Schnürer, 2000) and other yeasts from Dekkera/Brettanomyces genera (Comitini, De Ingeniis, Pepe, Mannazzu, & Ciani, 2004). Moreover, it has antioxidant activity in fact it could produce bioactive antioxidants, retarding oxidative degeneration of fatty substances and improving human health (Arroyo-López, Querol, Bautista-Gallego, & GarridoFernández, 2008). The relevance of the species P. anomala in table olive fermentation has been pointed out by several authors, proposing its use as starter culture (Hernández et al., 2007). The species belonging to Pichia genus commonly isolated from olive fermentation in Spain, Portugal and Italy are P. anomala and P. membranifaciens (Aponte et al., 2010; Fernández González, Gárcia, Garrido Fernández, & Durán Quintana, 1993; Lanciotti et al., 1999; Marquina et al., 1992). Only the sample S13 was characterized by the presence of P. kluyvery. This microorganism is also commonly associated to table olive fermentation. For instance Hurtado, Reguant, Esteve-Zarzoso, Bordons, and Rozès (2008) found P. kluyveri, during processing of Arbequina table olives. In particular these authors studied the yeast population dynamic during the processing of Arbequina table olives and reported that at the beginning of the fermentation, C. diddensiae was the most abundant species in brine. After the disappearance of this species, yeast population was mainly composed of P. kluyveri and C. boidinii. Moreover, P. kluyveri is well known as killer toxin producing yeast (Sangorrín, Lopes, Jofré, Querol, & Caballero, 2007). It produces toxins against other yeast
Table 5 Quantification of total yeasts in brine samples by plating, qPCR and RT-qPCR (mean S.D.). Samples
CFU ml1 (plating on YPD agar)
Cells ml1 qPCR
RT-qPCR
S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 S21 S23
(11.4 0.2) 104 (3.9 0.1) 105 (2.5 0.5) 104 (2.4 0.1) 104 (3 0.6) 104 (2.8 0.3) 104 (7.4 0.7) 105 (5.1 0.9) 105 (2.5 0.5) 105 (5.6 1.2) 105 (9.1 0.6) 106 (4.6 0.7) 105
(9.8 0.4) 106 (11.4 0.1) 105 (1.6 0.5) 106 (2.4 0.8) 106 (7.7 0.2) 104 (1.5 0.6) 107 (1.1 0.8) 107 (6.7 0.9) 106 (2.3 0.8) 106 (1.4 0.3) 106 (1.5 0.2) 106 (5.3 0.1) 106
(7.5 0.2) 106 (12.3 0.5) 105 (2.8 0.3) 106 (1.5 0.2) 106 (5.9 0.5) 104 (2.9 0.7) 107 (1.3 0.2) 107 (5.1 0.4) 106 (1.6 0.2) 106 (2.1 0.9) 106 (1.2 0.8) 106 (3.6 0.5) 106
361
Table 6 Quantification by qPCR of P. anomala, P. guilliermondii and P. kluyveri in brine samples supplied from “Cooperativa Olivicoltori”by qPCR (mean S.D.). Brine samples S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 S21 S23
Cells ml1 P. anomala
P. guilliermondii
P. kluyveri
(1.5 0.3) 104 (3 0.1) 105 <102 <102 <102 (1.2 0.2) 105 <102 (6.1 0.1) 103 (9.4 0.2) 102 <102 (2.2 0.3) 104 (6.7 0.2) 103
<102 <102 <102 (7.5 0.8) 102 <102 <102 <102 <102 <102 (3.1 0.5) 103 <102 (2.6 0.7) 102
<102 <102 <102 <102 <102 <102 (1.6 0.6) 102 <102 <102 <102 <102 <102
genera including killer-sensitive strains of S. cerevisiae (Marquina et al., 1992). P. guilliermondii was present in only three samples, S7, S19 and S23, at concentration of 102e103 CFU/ml. It is a species related to high volatile phenol production (Barata, Lobre, Correira, MalfeitoFerreira, & Loureiro, 2006; Martorell et al., 2006). Aponte et al. (2010) revealed the presence of this yeast, together with C. parapsilosis and P. kluyveri, during the entire fermentation period of green Sicilian table olive. P. guilliermondii showed pectolytic and/or xylanolytic capabilities (agents of softening and gas-pocket formation), but also b-glucosidase activity related to the debittering process of olives. Like P. anomala and P. kluyveri, P. guilliermondii exhibited a broad killer spectrum against yeasts that likely cause spoilage of table olives (Hernández et al., 2007). 4. Conclusion Food industries need rapid and sensitive methods to detect microorganisms present in fermented foods, to better control the fermentation process and prevent food spoilage. In particular, accurate quantification is of prime importance for most applications in food microbiology sector. Many studies showed that real-time PCR is a useful method for quantification of a range of microorganisms of medical, alimentary and environmental importance (Postollec et al., 2011). The qRT-PCR assays developed in this study provides a simple and specific tool for monitoring table olives fermentation, in order to better understand the relationship among yeast populations. Moreover a qPCR assay was developed to directly detect and quantify three species commonly associated to table olives, such as P. anomala, P. guillermondii and P. kluyveri. The applied assay is specific, reproducible, sensitive and fast and could be extended to other food items and to a variety of food-monitoring approaches. Thus, it could offer several advantages in routine food analysis in both industrial and quality control settings and it should be of great importance for technological purposes. In fact any modification in the fermentation process of table olives in general is not straightforward and requires a previous exhaustive study of relative changes in the diverse microbial populations and on product stability. Reformulations inevitably change the intrinsic physicochemical properties of food, which may, in turn, support the growth of foodborne pathogens and, ultimately, cause disease (Bautista Gallego, Arroyo-López, Durán Quintana, & Garrido-Fernández, 2010). Acknowledgments G. Perpetuini is beneficiary of a grant financed by the European Social Fund (FSI). D. Presutti is acknowledged for his technical
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support. We are grateful to Cooperativa Olivicoltori from Parteolla of Sardinia for their kindly cooperation. References Abriouel, H., Benomar, N., Lucas, R., & Gálvez, A. (2011). Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Aloreña green table olives. International Journal of Food Microbioliogy, 144, 487e496. Aponte, M., Ventorino, V., Blaiotta, G., Volpe, G., Farina, V., Avellone, G., et al. (2010). Study of green Sicilian table olive fermentations through microbiological, chemical and sensory analyses. Food Microbiology, 27, 162e170. Arroyo-López, F. N., Durán Quintana, M. C., Ruiz Barba, J. L., Querol, A., & GarridoFernández, A. (2006). Use of molecular methods for the identification of yeast associated with table olives. Food Microbiology, 23, 791e796. Arroyo-López, F. N., Querol, A., Bautista-Gallego, J., & Garrido-Fernández, A. (2008). Role of yeasts in table olive production. 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Food Control journal homepage: www.elsevier.com/locate/foodcont
Development and application of a real-time PCR-based assay to enumerate total yeasts and Pichia anomala, Pichia guillermondii and Pichia kluyveri in fermented table olives Rosanna Tofalo, Maria Schirone, Giorgia Perpetuini, Giovanna Suzzi, Aldo Corsetti* Dipartimento Scienze degli Alimenti, Università degli Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Sant’Angelo (TE), Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2011 Received in revised form 20 July 2011 Accepted 26 July 2011
The objective of this work was to develop a real-time quantitative PCR (qPCR) assay to directly detect and quantify the total number of yeasts besides three species (Pichia anomala, Pichia guillermondii and Pichia kluyveri) associated to table olives. For this purpose, a real-time PCR protocol targeted to the region of the internal transcribed spacers ITS (rDNA) and the conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, respectively, was developed. This assay allowed to unequivocally distinguish the three species from other yeasts and lactic acid bacteria generally associated to table olives fermentation. qPCR performed well both with purified DNA and DNA extracted from olive brines. A reverse transcription-qPCR (RT-qPCR) assay from rRNA aimed to viable yeast quantification was also performed. To evaluate the effectiveness of the technique, the qPCR results were compared with those obtained by a plate count approach in synthetic medium. The small standard errors provided this assay reproducible and robust. qPCR efficiently enumerated cells at concentrations of as low as 102 CFU ml1 when standard curve was derived both from cells growing in a syntetic medium and in brines. The quantification method for total yeasts and P. anomala, P. guillermondii, P. kluyveri in table olive brines applied in this work was specific, reproducible, sensitive and fast. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Yeast quantification Pichia spp. real-time PCR Fermented table olives
1. Introduction Table olives are important components of the Mediterranean diet and their consumption is spread all around the world. They are generally produced by three main methods: Spanish-style green olives in brine, California-style black olives in brine and Greek-style naturally black olives in brine. The main purpose of these methods is the removal of fruit bitterness by hydrolysis of some phenolic compounds, especially oleuropein. In the first process, bitterness is removed by adding lye; in the Greek and Californian style processes, fruits are placed directly in brine and in an acidified solution, respectively, and oleuropein removal is slow and only partial (Aponte et al., 2010; Garrido-Fernández, Fernández Díez, & Adams, 1997). However, there are many aspects of traditional table olive fermentation still unexplored (Abriouel, Benomar, Lucas, & Gálvez, 2011), which could open new international market for unknown local products and constitute niche products (Panagou, Tassou, & Katsaboxakis, 2003). According to the last data published by the * Corresponding author. Tel.: þ39 0 861266896; fax: þ39 0 861 266940. E-mail address: [email protected] (A. Corsetti). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.07.032
International Olive Oil Council (IOOC, 2008), the annual production of table olives is 1.3e1.8 million tonnes. Notwithstanding table olive fermentation is an empirical process, in which the natural fermentation results from competitive activities of the indigenous microbiota, together with a variety of contaminating microorganisms present in raw material and the processing environment (Abriouel et al., 2011). This practice may lead to fluctuations of the final characteristics of the product, such as quality and flavor, and may determine its spoilage (Aponte et al., 2010; Lanciotti, Corbo, Sinigaglia, & Guerzoni, 1999; Panagou et al., 2003). Olive-fruit is an important microhabitat for a variety of yeast species, lactic acid bacteria (LAB) and filamentosus fungi. Both yeasts and LAB play a very important role in table olive fermentation because they contribute to the final product characteristics (Campaniello et al., 2005). The main LAB species involved in this process are lactobacilli, such as Lactobacillus plantarum and Lactobacillus pentosus (Hurtado, Reguant, Esteve-Zarvoso, Bordons, & Rozès, 2008; Panagou, Schillinger, Franz, & Nyckas, 2008). The presence of few other LAB species such as Enterococcus faecium has been reported, (Randazzo, Restuccia, Romano, & Caggia, 2004), but always as minority populations.
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Yeasts can play a double role, since they can produce volatile compounds and metabolites that improve the flavor properties of the final product and enhance LAB growth by the release of nutritive compounds. On the other hand, yeasts may cause gas-pocket formation, softening of the olive tissue or, package bulging, clouding of the brines and production of off flavors and odors. Pichia anomala (recently classified as Wickerhamomyces anomalus), Pichia membranifaciens, Candida boidinii, Debaryomyces hansenii, Rhodotorula glutinis and Saccharomyces cerevisiae are the species isolated with a higher frequency from diverse olive processes (ArroyoLópez, Durán Quintana, Ruiz Barba, Querol, & Garrido-Fernández, 2006; Coton, Coton, Levert, Casaregola, & Sohier, 2006). Moreover, the presence of Candida parapsilosis, Pichia kluyveri and Pichia guillermondi has been revealed during the entire fermentation period of four green Sicilian table olive (Aponte et al., 2010). The identification of yeast biota in table olive production by using molecular methods is still poor applied (Hurtado, Reguant, EsteveZarvoso, Bordons, & Rozès, 2008; Nisiotou, Chorianopoulus, Nychas, & Panagou, 2010). Actually, molecular methods widely applied to extimate yeast biodiversity are based on the variability of the ribosomal genes 5.8S,18S and 26S (Arroyo-López et al., 2006; Cai, Roberts, & Collins, 1996; Coton et al., 2006; Hurtado, Reguant, Esteve-Zarvoso, Bordons, & Rozès, 2008; James, Collins, & Roberts,1996; Nisiotou et al., 2010). Although these methods are rapid and accurate, culture isolation is necessary. Moreover, the presence of viable but non cultivable (VBNC) cells makes still more difficult to detect all the metabolically active yeast cells in fermented foods (McDougald, Rice, Weichart, & Kjelleberg, 2006). Then, these methods cannot allow the determination of yeast cells number present in the sample, an important parameter to control spoilage. Compared with other PCRbased methods, quantitative real-time PCR (qPCR) provides significant advantages. It allows DNA to be extracted directly from products without prior culture isolation (Wilhelm & Pingoud, 2003) and provides detection and enumeration of a number of fungi and foodborne pathogens (Mills, Johannsen, & Cocolin, 2002). In fact, the main advantage of qPCR is the low detection level, often as low as one cell per mL (Bleve, Rizzotti, Dellaglio, & Torriani, 2003; Delaherche, Claisse, & Lonvaud-Funel, 2004; Phister & Mills, 2003). This method has been widely applied to detect viable yeasts in different products such as pasteurized products (Bleve et al., 2003; Hierro, EsteveZarzoso, González, Mas, & Guillamón, 2006), orange juice, wine (Heras-Vazquez, Mingorance-Cazorla, Clemente-Jimenez, & Rodrizguez-Vico, 2003; López, Fernández-Espinar, Barrio, Ramón, & Querol, 2003), cheese (Vasdinyei & Deak, 2003), ‘‘alpeorujo’’ (Giannoutsou, Meintanis, & Karagouni, 2004), a residue from olive oil production, functional foods’, as well as yeasts of public health interest in dairy products (Makino, Fujimoto, & Watanabe, 2010). In the present work, we report on the development of a qPCR assay to directly detect and quantify the total number of yeasts as well as three main dominant species, P. anomala, P. guillermondii, and P. kluyveri in fermented table olives. Morever, viable and VBNC yeasts were detected by reverse transcription-PCR (RT-qPCR). 2. Materials and methods 2.1. Reference strains and culture conditions The references strains used in the present study are listed in Table 1 and represent the species commonly found in fermented table olives. Yeasts and LAB were stored at 80 C in YPD medium (1% yeast extract, 2% peptone, 2% glucose) and Man Rogosa Sharpe (MRS) (Oxoid, Milan, Italy), respectively, containing 20% (v/v) glycerol. Before experimental use, yeasts strains were grown in YPD for 16e18 h under aerobic conditions at 25 C, whereas LAB were propagated in MRS broth at 28 C in microaerophilic condition.
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2.2. Primer design P. anomala, P. guillermondii and P. kluyveri specific-primers as well as universal primers were designed from the region of the internal transcribed spacers ITS (rDNA) and from conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, respectively. Sequences available at the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov.) were retrieved and aligned using CLUSTAL X multiple-sequence alignment (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997). Appropriate primers were designed using Beacon Designer 3 program (Bio-Rad, Milan, Italy). The BLAST search (Basic Alignment Search Tool, http:// www.ebi.ac.uk/blastall/nucleotide.html) was used to check the specificity of each primer set. Furthermore, the properties of each primer were verified by Primer Tool Sigma Aldrich (www.sigmagenosys.com/calc/DNACalc.asp). The primer sequences besides amplicons size are shown in Table 2. These primers were used both for conventional PCR and qPCR. 2.3. Genomic DNA extraction Genomic DNA was isolated from yeasts by the procedure of Querol, Barrio, Huerta, and Ramón (1992), modified as follows: cells were resuspended in 500 ml of lysis buffer (25 mg of Rhizoctonia solani lytic enzyme/ml, 1 M sorbitol and 0.1 M EDTA pH 7.5) and incubated for 2 h at 45 C. Bacterial genomic DNAs were extracted from 1.5 ml of cultures in late-exponential-growth phase as reported by de Los Reyes-Gavillàn, Limsowtin, Tailliez, Sèchaud, & Accolas (1992). The concentration and purity of DNA were assessed by O.D. at 260 and 280 nm, as described by Sambrook, Fritschi, and Maniatis (1989). 2.4. Specificity of PCR assays All PCR amplifications were carried out in 25 ml final volumes containing 3 ml of DNA template (between 10 and 100 ng), 1x PCR buffer without MgCl2, 1.5 mM MgCl2, 0.5 mM of each primer, 100 mM dNTPs, 0.02 U/ml Taq Polymerase (Invitrogen, Italia). The thermal cycling program and primer sequences are reported in Table 2. All amplifications were performed in a GeneAmp PCR system 2700 (Applied Biosystems, Foster City, CA). The PCR products were analyzed by electrophoresis on a 1% (wt/v) agarose gel in 1x TAE buffer (40 mM Tris-acetate,1 mM EDTA, pH 8.3). The gels were stained with ethidium bromide (0.5 mg/ml) and visualized under UV-light. DNA Ladder 1 Kb plus (Invitrogen) was used as the size standard. 2.5. qPCR assays Each reaction mixture (25 ml) contained 12.5 ml 2XIQ SYBR Green PCR SupermixÔ (Bio-Rad), 5 pmol of each primer (Invitrogen), 1 ml DNA template (10 ng) and ultra pure water. Fluorescence emission was measured at the last step of each cycle. The qPCR program was common for each primer set (Table 2). The cycle threshold (Ct) was determined automatically by the instrument and was used for quantitative analyses. Melting temperature analysis of the PCR products was performed to determine the specificity of the PCR reaction. All samples were processed in triplicate. 2.6. Standard curves and efficiency Different mixed cultures containing known populations of S. cerevisiae, D. hansenii, C. boidinii and P. anomala were incubated for 48 h in YPD and synthetic brines (0.5% (v/v) lactic acid, 7% (wt/v) NaCl, 50 mM glucose, 10 mM sucrose, and 6% NaCl).
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Table 1 Yeast and bacterial strains used in this study and specific PCR results. Species
Strain designation
Candida boidinii Candida diddensiae Candida krusei Candida milleri Candida parapsilosis Candida rugosa Candida tropicalis Candida vini Candida zemplinina Debaryomyces hansenii
Hanseniaspora guilliermondii Issatchenkia orientalis Kloeckera apiculata Kluyveromyces lactis Kluyveromyces marxianus Pichia anomala Pichia guilliermondii Pichia kluyveri Pichia membranaefaciens Rhodotorula glutinis Rhodotorula minuta Rhodotorula mucilaginosa Saccharomyces bayanus Saccharomyces cerevisiae Saccharomyces paradoxus Saccharomyces pastorianus Yarrovia lipolytica Zygosaccharomyces mirakii Lactobacillus casei Lactobacillus fermentum Lactobacillus paraplantarum Lactobacillus pentosus Lactobacillus plantarum Leuconostoc pseudomesenteroides Weissella confusa
C1-4, LU-1, LU-3 ATCC 15541 ATCC 6258 MUCL 38021T ATCC 22019 ATCC 10571 DSM 5991 DBVPG 3227T CZ-3 MUCL 30242T CBS 767T H9, H13 DSM 3432 FG-1 CBS 104T C1-10 CBS 834T MUCL 28639T CBS 2030T, FOR-15 ATCC 9768 MUCL 29895T ATCC 2527 ATCC 32769 MUCL 30403T CBS 380T CBS 1171 CBS 432T CBS 1538T DBVPG 6132T C2-10 ATCC 393 ATCC 14931 DSM 10667T 1MO MO3f DSM 20193T DSM 20196
Origin
Fermented olives Shrimp Leaves Sourdough Leaves Human feces Rumen of cow Wine Wine Cherry Grape Pecorino abruzzese Diseased nail Fermented olives Grape Fermented olives Kefir Leaf Insects Fermented olives Wine Maize (Zea mays), leaves Intestine Orange Beer Beer Tree Grape Margarine Fermented olives Cheese Unknown Beer Fermented olives Sourdough Cane juice Sugar cane
Yeast-specific qPCR (152 bp)
Species-specific qPCR P. anomala (217 bp)
P. guilliermondii (228 bp)
Pichia kluyveri (216 bp)
þ þ þ e þ þ e e e
e e e e e e e e e e e e e e e e e þ e
e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e
e e e e e e e e e e e e e e e e e e e þ e e e e e e e e e e e e e e e e e
þ e þ e þ þ þ þ þ þ þ þ þ e þ e e e þ e e e e e e e
e e e e e e e e e e e e e e e e e
MUCL, Agro-Industrial Fungi and Yeast Collection, Universite’ Catholique de Louvain, Louvain-La-Neuve, Belgium; CBS, Centraalbureau voor Schimmelcultures, Baarn, The Netherlands; ATCC, American Type Culture Collection, Manassas, Va.; DSM; Deutsche Veterinarmedizinische Gesellschaft, Giessen, Germany, DBVPG Industrial Yeasts Collection, Dipartimento di Biologia Applicata, University of Perugia.
From YPD and synthetic brine cultures, 10-fold serial dilutions were performed and DNA from each dilution was extracted and quantified by qPCR using universal primer sets to determine Ct values. Type strains of P. anomala, P. guillermondii and P. kluyveri (Table 1) were analyzed as reported above using species-specific primers. Moreover, the same samples were serially diluted and plated on YPD media for viable counts. Standard curves were created by plotting the Ct values of the qPCRs performed on dilution series of DNA against the log of cells per milliliters, as determined by plate counts. The qPCR efficiencies were calculated in exponential phase from the given slopes and according to the equation
Table 2 Primer sequences and PCR conditions developed for qPCR. Species
Primer
Sequence (50 -30 )
PCR conditions
P. anomala
Anom2F AGK-R Guill 2F AGK-R Kluy2F AGK-R Oli-F Oli-R
GTT AAA ACC TTT AAC CAA TA AAA TGA CGC TCA AAC A CAA AAC ACA ATT TAA TTA TTT AAA TGA CGC TCA AAC A CAC CAA ACA CCT AAA AT AAA TGA CGC TCA AAC A CGT CAT AGA GGG TGA GAA TCC ACT TGT TCG CTA TCG GTC TC
5 min at 94 C; 1 min at 94 C, 30 s at 51 C, 1 min at 72 C for 30 cycles
P. guilliermondii P. kluyverii Total yeasts
E ¼ 10[1/slope]-1 (Rasmussen, 2001). In all PCR runs, negative controls (sterilized water), positive control and samples were run in triplicate. Sensitivity of qPCR assays was evaluated with reference to other reports (Hierro, Esteve-Zarvoso, González, Mas, & Guillamón, 2006; Martorell, Querol, & Fernández-Espinar, 2005). The results were analyzed using statistical software STATISTICA for Windows (STAT. version 8.0, StatSoft Inc. Tulsa, OK). All the Ct values are averages of at least three repetitions. 2.7. RT-PCR assay For reverse transcription-PCR (RT-qPCR), RNA was obtained using the RNA PowerSoilÒ Total RNA Isolation Kit (Mobio Laboratories, Inc) according to the manufacturer’s instructions. RNA from pure culture and mixed cultures, obtained as described above, was isolated and quantified by qPCR to enumerate total yeast populations. A 10-fold dilution series of isolated RNA was prepared. cDNAs were synthesized from each RNA dilutions according to Hierro et al. (2006) in order to construct standard curves. All the experiments were repeated five times in duplicate to ascertain the reliability and repeatability of the real-time assays. Three replicates of positive and negative controls were processed in each PCR run: a positive control with DNA from Pichia spp. and a negative control without template.
R. Tofalo et al. / Food Control 23 (2012) 356e362
2.8. Quantification of total yeast, P. anomala, P. guilliermondii and P. kluyveri in fermented table olives and brines Twelve samples of table olives (cultivars Tonda di Cagliari and Pizz’e Carroga) were supplied from “Cooperativa Olivicoltori” located in Parteolla (Sardinia). All samples were stored for 7 months in a solution containing 9% (wt/v) NaCl and 1% (wt/v) lactic acid. Nucleic acids were isolated according to the protocol of Querol et al. (1992) with some modifications at the beginning of the process. In particular, 10 ml of brine (or 10 g of pitted olives) were used to collect cells. Samples were centrifuged at 10.000 rpm for 5 min and pellets were washed twice with sterile water. The extracted DNA was used for qPCR. RNA was obtained using the RNA PowerSoilÒ and cDNA was synthesized as reported above. Total yeast, P. anomala, P. guilliermondii and P. kluyveri were determined by qPCR. The unknown samples were run alongside a set of known standards in order to determine their CFU/ml. A standard curve was generated by plotting the Ct values of the known samples against the CFU/ml to generate a standard curve from which the unknown CFU/ml was calculated.
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3.2. qPCR detection limits To determine the sensitivity and detection limits of the qPCR assay, DNA obtained from pure and mixed cultures with known concentrations was serially diluted 10-fold and quantified by qPCR using the corresponding primer sets (Table 2). Then, the effectiveness of the quantification for this method was tested correlating the cell concentrations estimated by plate count and qPCR. Each DNA dilution besides plate count value was used to construct standard curves (Fig. 1). The Ct values obtained for concentrations in cell number ranging from 10 to 108 showed good linearity for triplicate
3. Results and discussion 3.1. Primer design and specificity In order to detect all the yeasts present in fermented table olives, universal primers (Oli-F/Oli-R) from conserved sequences of the variable D1/D2 domains of the 26S rRNA gene were designed, as reported in Table 2. The sequences of the variable D1/D2 domains of the 26S rRNA gene determined by Kurtzman and Robnett (1998) facilitated the design and the test of the assay for yeast species specificity. Only a DNA fragment of the expected size (152 bp) was found in presence of the yeast species considered here, whereas amplicons were not obtained for yeasts generally not associated with fermented table olives or for bacteria which could be detected in fermented olives and brines (Table 1). In particular, the above DNA fragment was not observed in presence of fermented table olive bacteria such as LAB. Moreover, Candida vini and Candida zemplinina were used as negative control to check the procedure. They were chosen since they are normally absent in fermented table olives, and they didn’t generate any PCR products further highlighting the primers specificity. Then, primers aimed to the quantification of P. anomala, P. kluyveri and P. guilliermondii were designed from the region of the internal transcribed spacers ITS (rDNA). The forward primer was common for all the three species and was homologous to a conserved region of the above gene. To evaluate the primer specificity, conventional PCR was performed using purified DNA from yeasts and bacteria listed in Table 1. One fragment of the expected size was obtained for each type strain: P. anomala (217 bp), P. kluyveri (216 bp) and P. guilliermondii (228 bp). The ITS regions are less evolutionarily conserved than the rRNA-coding genes and are therefore valuable regions for finding interspecific differences (Guillamón, Sabaté, Barrio, Cano, & Querol, 1998; Hierro, EsteveZarzoso, Mas, & Guillamon, 2007) being so useful to design yeast species-specific primers. Then, primers specificity was analyzed by qPCR using the optimized reaction conditions and purified DNA as described in material and methods. For all primer sets, a unique peak was observed in the melting curve of amplicon, suggesting the specificity of the amplification, i.e. each primer pair amplified a unique locus targeted on the genome (data not shown). To develop a real-time PCR method for yeasts quantification in which SYBR green I real-time PCR is used, it is necessary to design primers that do not amplify DNA sequences from any other microorganism that can be present in fermented food under study (Postollec et al., 2011).
Fig. 1. Standard curves obtained from 10-fold serial dilutions of DNA isolated from P. anomala, P. kluyveri, P. guilliermondii and mixed culture yeasts grown in YPD medium , and incubated in synthetic brine B. Ct value are the average of three repetitions. Error bars represent standard errors.
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Table 3 Correlation coefficients, slopes and efficiencies of standard curves obtained from 10-fold dilution of yeast strains grown in synthetic brine and YPD medium (mean S.D.). Assay
P. anomala P. kluyveri P. guilliermondii Total yeasts a b
Strain code
MUCL 28639T DBVPG 6901T CBS 2030T Mixed culture yeastsb
Synthetic brine R2
Slope
0.992 0.991 0.992 0.996
3.440 3.420 3.571 3.524
YPD
0.88 0.55 0.91 0.45
Efficiencya (%)
R2
Slope
95.29 96.06 90.56 92.20
0.992 0.990 0.990 0.994
3.344 3.353 3.359 3.454
Efficiencya (%)
0.52 0.38 0.71 0.63
99.08 98.71 98.47 94.76
Efficiency was estimated by the formula 101/slope1. S. cerevisiae, D. hansenii, C. boidinii and P. anomala.
samples of P. anomala. On the contrary, P. guillermondi and P. kluyveri were detected starting from 102 cell/ml and the lowest cell quantification for total yeast was 103 cell/ml. The correlation coefficients, slope and efficiencies of the amplification for the standard curves are shown in Table 3. In all the qPCR assays the yeast populations were effectively enumerated and good correlation with enumeration by culturing system was found (R2 0.990) (Table 3). Similar detection limits were detected using specific primer for yeast in other fermented food, demonstrating quantification ability of qPCR assays. In particular, Makino et al. (2010) developed a qPCR assay to directly detect and quantify nine emerging opportunistic yeast species in dairy product samples. Their detection limit using universal yeasts primers was between 102 and 103 cells/ml. Because some compounds found in table olives, such as salt and polyphenols, are known to inhibit PCR reaction, a comparison between the Ct values of DNAs standard curves obtained from the synthetic brines and YPD plate counts was made. The qPCR assay showed successful amplification in all brine samples artificially contaminated with yeasts. In fact, DNA fragments of expected size were found in all samples (data not shown). The standard curves obtained in both YPD and synthetic brines showed the same linearity allowing quantification in a range from 10 to 108 cell/reaction, with similar efficiencies as obtained for pure cultures (Fig. 1). These data demonstrated that the above mentioned compounds didn’t affect the amplification reaction. Inhibitory effect on PCR reaction by intrinsic factors has been reported in foods by many authors (Bleve et al., 2003; Delaherche et al., 2004; Hierro, Esteve-Zarvoso, González, Mas, & Guillamón, 2006; Hierro, Esteve-Zarvoso, Mas, & Guillamón, 2007; Martorell et al., 2005). Moreover, other authors found differences when PCR was performed with DNA extracted from pure cultures or directly from foods. Hierro et al. (2006) detected higher values of S. cerevisiae in must and wine by qPCR rather than plate count values. These data were confirmed by RT-qPCR, because RNA is strictly related to cell viability. In fact, the amplification of DNA allowed to detect live, death and metabolically active but not cultivable cells. To prevent the detection of dead microorganisms is necessary to study gene expression as marker for cell viability (Bleve et al., 2003).
correlations were 0.991 and 0.995 in the standard curves obtained from YPD and brines, respectively. The efficiencies were 97.43% and 96.41%, respectively (Table 4). These efficiency values were better than those obtained using DNA as the template (data not shown). Similar results were obtained by Hierro et al. (2006). 3.4. RT-qPCR analysis of fermented table olives In order to obtain a quantification of yeast populations in fermented table olives, a real-time RT-qPCR assay was optimized. The quantitative capability of the qPCR was evaluated determining the relationship between the amounts of detected yeast by qPCR and the plate count in YPD. Table 5 shows the values of CFU/ml obtained by qPCR, RT-qPCR and plate counts. Since the successful application of PCR-based techniques depends on the quality of the template good protocols for DNA extraction are needed. In this study a kit generally used for soil, grape and must, that are very complex matrices because of the presence of PCR inhibitors, was applied with some modifications. In particular, to obtain the complete lysis of cells a storage at 20 C for 20 min was applied. Moreover, since foods could contain various PCR inhibitors such as polyphenols, tannins and polysaccharides (Hierro, Esteve-Zarvoso, González, Mas, & Guillamón, 2006; Martorell et al., 2006; Phister & Mills, 2003) standard curves by the dilution of cells incubated in brines were constructed. With regards to yeast count in brines, count values ranged from 104 to 105 CFU/ml and only one sample (S21) with a value of 106 CFU/ml; while the values obtained by qPCR ranged from 106 to 107 CFU/ml, except for S9 and S3 which were of 104 and 105 CFU/ml, respectively. The higher total yeast counts observed by qPCR and RT-qPCR with respect to plate count may be due to the large presence of VBNC populations. The values of total yeast population showed similar results with all enumeration methods for only three samples (S3, S9, S21). qPCR revealed also the presence of the three species of Pichia tested, with P. anomala and P. guilliermondii present in the majority of the analyzed samples (Table 6). In particular, P. anomala was detected in 7 out 12 samples: S1, S3, S11, S15, S17, S21 and S23, ranging from 102-105 CFU/ml.
3.3. Quantification of total yeast populations by RT-qPCR In order to obtain a more realistic quantification of total viable yeasts, an RT-qPCR assay was developed. In the first step, a mixed culture of selected yeast was diluted from 108 to 10 cell/ml. Total RNA was isolated in triplicate from each dilution and subjected to RT-qPCR. cDNA from each RNA dilution was synthesized as described in material and methods. Then, the synthesized cDNAs were subjected to RT-qPCR assay. The same procedure was carried out with a culture of the same yeasts incubated for 24 h in synthetic brines. A non-reverse-transcribed control was used to verify that RNA samples were DNA free. No amplification was obtained in nonreverse-transcribed control sample. The detection limit of the RTqPCR assay was of 103 CFU/ml (Fig. 2), and the coefficient
Fig. 2. Total yeast standard curves obtained from serially diluted RNA isolated from mixed culture yeasts grown in YPD medium , and inoculated in synthetic brine B. Ct value are averages of results of three replicates. Error bars represent standard errors.
R. Tofalo et al. / Food Control 23 (2012) 356e362 Table 4 Correlation coefficients, slopes and efficiencies of standard curves obtained from serial dilutions of RNA isolated from mixed culture yeasts grown in YPD medium and synthetic brine (mean S.D.). Yeast cell assay
R2
Slope
Efficiencya (%)
Synthetic brine YPD
0.995 0.991
3.411 0.45 3.385 0.63
96.41 97.43
a
Efficiency was estimated by the formula 101/slope1.
P. anomala is one of the most abundant yeast throughout the fermentation process in all kinds of olives (Coton et al., 2006; Durán Quintana, García-García, & Garrido-Fernández, 2003; Hernández, Martín, Aranda, Pérez-Nevado, & Córdoba, 2007; Marquina et al., 1992). It can play a double role during olive fermentation, in fact it can cause softening and gas-pocket formation (Aponte et al., 2010; Asehraou, Peres, Brito, Faid, & Serhrouchni, 2000; Faid, Akhartouf, & Asenhraon, 1994). Moreover this species together with S. cerevisiae has been related to “alambrado” (bloater) spoilage in spontaneous fermentation in black olives (Durán Quitana, González, & Garrido, 1979). On the other hand P. anomala produces killer toxins. It has been successfully used for the biocontrol of Penicillium (Boysen, Björneholm, & Schnürer, 2000) and other yeasts from Dekkera/Brettanomyces genera (Comitini, De Ingeniis, Pepe, Mannazzu, & Ciani, 2004). Moreover, it has antioxidant activity in fact it could produce bioactive antioxidants, retarding oxidative degeneration of fatty substances and improving human health (Arroyo-López, Querol, Bautista-Gallego, & GarridoFernández, 2008). The relevance of the species P. anomala in table olive fermentation has been pointed out by several authors, proposing its use as starter culture (Hernández et al., 2007). The species belonging to Pichia genus commonly isolated from olive fermentation in Spain, Portugal and Italy are P. anomala and P. membranifaciens (Aponte et al., 2010; Fernández González, Gárcia, Garrido Fernández, & Durán Quintana, 1993; Lanciotti et al., 1999; Marquina et al., 1992). Only the sample S13 was characterized by the presence of P. kluyvery. This microorganism is also commonly associated to table olive fermentation. For instance Hurtado, Reguant, Esteve-Zarzoso, Bordons, and Rozès (2008) found P. kluyveri, during processing of Arbequina table olives. In particular these authors studied the yeast population dynamic during the processing of Arbequina table olives and reported that at the beginning of the fermentation, C. diddensiae was the most abundant species in brine. After the disappearance of this species, yeast population was mainly composed of P. kluyveri and C. boidinii. Moreover, P. kluyveri is well known as killer toxin producing yeast (Sangorrín, Lopes, Jofré, Querol, & Caballero, 2007). It produces toxins against other yeast
Table 5 Quantification of total yeasts in brine samples by plating, qPCR and RT-qPCR (mean S.D.). Samples
CFU ml1 (plating on YPD agar)
Cells ml1 qPCR
RT-qPCR
S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 S21 S23
(11.4 0.2) 104 (3.9 0.1) 105 (2.5 0.5) 104 (2.4 0.1) 104 (3 0.6) 104 (2.8 0.3) 104 (7.4 0.7) 105 (5.1 0.9) 105 (2.5 0.5) 105 (5.6 1.2) 105 (9.1 0.6) 106 (4.6 0.7) 105
(9.8 0.4) 106 (11.4 0.1) 105 (1.6 0.5) 106 (2.4 0.8) 106 (7.7 0.2) 104 (1.5 0.6) 107 (1.1 0.8) 107 (6.7 0.9) 106 (2.3 0.8) 106 (1.4 0.3) 106 (1.5 0.2) 106 (5.3 0.1) 106
(7.5 0.2) 106 (12.3 0.5) 105 (2.8 0.3) 106 (1.5 0.2) 106 (5.9 0.5) 104 (2.9 0.7) 107 (1.3 0.2) 107 (5.1 0.4) 106 (1.6 0.2) 106 (2.1 0.9) 106 (1.2 0.8) 106 (3.6 0.5) 106
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Table 6 Quantification by qPCR of P. anomala, P. guilliermondii and P. kluyveri in brine samples supplied from “Cooperativa Olivicoltori”by qPCR (mean S.D.). Brine samples S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 S21 S23
Cells ml1 P. anomala
P. guilliermondii
P. kluyveri
(1.5 0.3) 104 (3 0.1) 105 <102 <102 <102 (1.2 0.2) 105 <102 (6.1 0.1) 103 (9.4 0.2) 102 <102 (2.2 0.3) 104 (6.7 0.2) 103
<102 <102 <102 (7.5 0.8) 102 <102 <102 <102 <102 <102 (3.1 0.5) 103 <102 (2.6 0.7) 102
<102 <102 <102 <102 <102 <102 (1.6 0.6) 102 <102 <102 <102 <102 <102
genera including killer-sensitive strains of S. cerevisiae (Marquina et al., 1992). P. guilliermondii was present in only three samples, S7, S19 and S23, at concentration of 102e103 CFU/ml. It is a species related to high volatile phenol production (Barata, Lobre, Correira, MalfeitoFerreira, & Loureiro, 2006; Martorell et al., 2006). Aponte et al. (2010) revealed the presence of this yeast, together with C. parapsilosis and P. kluyveri, during the entire fermentation period of green Sicilian table olive. P. guilliermondii showed pectolytic and/or xylanolytic capabilities (agents of softening and gas-pocket formation), but also b-glucosidase activity related to the debittering process of olives. Like P. anomala and P. kluyveri, P. guilliermondii exhibited a broad killer spectrum against yeasts that likely cause spoilage of table olives (Hernández et al., 2007). 4. Conclusion Food industries need rapid and sensitive methods to detect microorganisms present in fermented foods, to better control the fermentation process and prevent food spoilage. In particular, accurate quantification is of prime importance for most applications in food microbiology sector. Many studies showed that real-time PCR is a useful method for quantification of a range of microorganisms of medical, alimentary and environmental importance (Postollec et al., 2011). The qRT-PCR assays developed in this study provides a simple and specific tool for monitoring table olives fermentation, in order to better understand the relationship among yeast populations. Moreover a qPCR assay was developed to directly detect and quantify three species commonly associated to table olives, such as P. anomala, P. guillermondii and P. kluyveri. The applied assay is specific, reproducible, sensitive and fast and could be extended to other food items and to a variety of food-monitoring approaches. Thus, it could offer several advantages in routine food analysis in both industrial and quality control settings and it should be of great importance for technological purposes. In fact any modification in the fermentation process of table olives in general is not straightforward and requires a previous exhaustive study of relative changes in the diverse microbial populations and on product stability. Reformulations inevitably change the intrinsic physicochemical properties of food, which may, in turn, support the growth of foodborne pathogens and, ultimately, cause disease (Bautista Gallego, Arroyo-López, Durán Quintana, & Garrido-Fernández, 2010). Acknowledgments G. Perpetuini is beneficiary of a grant financed by the European Social Fund (FSI). D. Presutti is acknowledged for his technical
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