1 Biodiversity in Traditional Polish Cheese Oscypek Determined by

2 Culture-dependent and -independent Approaches

3

4 Ángel Alegría1,2#, Pawel Szczesny1,3, Baltasar Mayo2, Jacek Bardowski1, Magdalena Kowalczyk1#*

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6 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland 1

7 Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (CSIC),

8 Villaviciosa, Asturias, Spain 2

9 Faculty of Biology, University of Warsaw, Warsaw, Poland 3

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11 * Corresponding author. Mailing address: Institute of Biochemistry and Biophysics, PAN, Pawinskiego

12 5A, 02-106 Warsaw, Poland. Phone: 48-22-5921222. Fax: 48-22-6584636. E-mail: [email protected].

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14 # M. Kowalczyk and Á. Alegría contributed equally to this work

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16 Journal section: Food Microbiology.

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17 ABSTRACT

18 Oscypek is a traditional Polish, scalded-smoked cheese, with a protected designation of origin

19 (PDO) status manufactured from raw sheep’s milk without starter cultures in the Tatra Mountains

20 region of Poland. This study was undertaken in order to get some insight on the microbiota which

21 develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use

22 of both culturing and the culture-independent methods of PCR-DGGE and pyrosequencing of 16S

23 rRNA gene amplicons. Culturing and culture-independent techniques are known to give

24 complementary results on the diversity and dynamics of microbial populations, thus providing a better

25 description of the cheese ecosystem of traditional Oscypek. Culture-dependent technique and PCR-

26 DGGE fingerprinting detected the predominant microorganisms in the traditional Oscypek whereas the

27 next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity.

28 Besides members of the most abundant bacterial genera in dairy products, e.g. Lactococcus,

29 Lactobacillus, Leuconostoc, Streptococcus and Enterococcus, identified by all three methods, other

30 subdominant belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly

31 Enhydrobacter) and various minor bacteria were identified by pyrosequencing. In addition to bacteria,

32 a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing

33 methods enabled the determination of a number of viable microorganisms from different microbial

34 groups and their isolation for potential future applications in specific cheese starter cultures.

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36 INTRODUCTION

37 Protected Designation of Origin (PDO) labels are granted to traditional agricultural products and

38 foodstuffs originating in a specific area whose quality or characteristics are essentially or exclusively

39 due to particular geographic environments (with its inherent natural and human factors), and whose

40 production, processing and preparation processes take place in a defined PDO region (European

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41 Commission, 2006). The quality of PDO cheeses is based on particular species and herds, grazing

42 pastures, and ancient technologies developed and maintained in a shared manner by the human

43 communities within the PDO area. All these factors select for specific microorganisms which have

44 evolved through the ages, whose activity plays a pivotal role in the sensorial, safety and preservative

45 properties of the products (Guinane et al., 2005; Smit et al., 2005). Therefore, identification, typing and

46 characterization of these microorganisms is essential for designing specific starters to control the

47 fermentation while preserving the original sensory profiles.

48 Oscypek is a traditional Polish, scalded-smoked cheese, which enjoys a PDO status since 2008.

49 The cheese has the shape of a spindle with a beautiful characteristic pattern imprinted by the carved

50 wooden moulds which give the cheese its final form. It is manufactured from raw sheep’s milk without

51 starter cultures in the Tatra Mountains region of Poland, according to the diagram flow chart depicted

52 in Figure 1. The traditional manufacturing process maintained throughout centuries may have selected

53 appropriate lactic acid bacteria (LAB) species and/or strains that could be used as specific starters. The

54 new cultures can also be used to complement or replace the starters currently used in large-scale dairy

55 industry (Ayad et al., 2001). In addition, traditional cheese ecosystems may harbor lactic acid bacteria

56 (LAB) strains showing unique flavor-forming capabilities (Ayad et al., 1999), enhanced bacteriophage

57 resistance (Madera et al., 2003) or production of new, broad-range antimicrobial agents (Alegría et al.,

58 2010).

59 Besides conventional microbial characterization, a vast array of culture-independent molecular

60 techniques is now available to address the diversity and evolution of the microbial populations

61 throughout cheese manufacture and ripening (Jany and Barbier, 2008). Among others, techniques such

62 as denaturing gradient gel electrophoresis (DGGE) (Randazzo et al., 2002), single-strand conformation

63 polymorphism (SSCP) (Duthoit, 2007), fluorescent in situ hybridization (FISH) (Ercolini et al., 2003),

64 length-heterogeneity-PCR (LH-PCR) (Lazzi et al., 2004), quantitative real-time PCR (qPCR)

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65 (Friedrich and Lenke, 2007), terminal-restriction fragment length polymorphism (TR-FLP) (Arteau et

66 al., 2010), complement classic culturing techniques, thus providing a more complete picture of the

67 cheese ecosystem. Next generation sequencing techniques, such as pyrosequencing (Margulies et al.,

68 2005), have recently begun to be applied to study the diversity and dynamics of the microbial

69 populations in natural food fermentations (Humblot and Guyot, 2009; Roh et al., 2010; Jung et al.,

70 2011). This method enables rapid insight into population structure, dynamics, gene content, and

71 metabolic potential of microbial communities.

72 In this study, culturing and the culture-independent methods of DGGE and pyrosequencing of

73 segments of the 16S rRNA gene were used to type major and indicator microbial populations in

74 traditional Oscypek cheeses. This allowed identification of the dominant cultivable bacterial species

75 and assessment of the microbial diversity and dynamics during the manufacture and ripening of several

76 cheese batches.

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78 MATERIALS AND METHODS

79 Cheese samples and sampling conditions. Cheeses manufactured by four independent cheese

80 makers from the Tatra Mountains in Poland were sampled in September 2009. Microbiological and

81 culture-independent molecular analyses were performed on four batches representing traditional

82 Oscypek cheeses produced mostly from unpasteurized ewe’s milk in shepherds’ huts. Curd, fresh (1

83 day) and smoked (3 day) cheeses were sampled according to FIL-IDF standard 50 B, and kept under

84 refrigeration until analysis.

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86 Microbial analysis by culturing. Twenty gram samples of curd or cheese were mixed with 180 ml

87 of PS (0.9% sodium chloride solution) at 37ºC and homogenized in a laboratory homogenizer (H500

88 Pol-Eko-Aparatura, Wodzisław Śląski, Poland) for 5 min at 24,000 rpm. Serial 10-fold dilutions in PS

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89 were then plated in duplicate onto seven general and selective media, as follows.

90 Total mesophilic bacteria. Mesophilic bacteria were counted on plate count agar supplemented

91 with 0.1% skimmed milk (PCMA; Merck, Darmstadt, Germany) after 72 h of incubation under both

92 aerobic and anaerobic (in 2.5 l jars with AnaeroGen; Oxoid Ltd., Basingstoke Hampshire, UK)

93 conditions at 30ºC. In addition, BHI (Oxoid Ltd., Basingstoke Hampshire, UK) agar plates

94 supplemented with 0.2% of cysteine (BHIAC) were used to count total anaerobic bacteria after

95 incubation at 37ºC for 72 h in anaerobiosis (with AnaeroGen).

96 Lactococci. Lactococci were grown on M17 (Oxoid Ltd., Basingstoke Hampshire, UK) agar

97 supplemented with lactose 10 g/l (LM17A) and enumerated after 48 h of incubation at 30ºC.

98 Lactobacilli. Lactobacilli were grown on de Man, Rogosa and Sharpe agar (MRSA; Merck),

99 adjusted to pH 5.4 and counted after 72 h incubation under aerobic and anaerobic conditions at 37ºC

100 (AnaeroGen).

101 Leuconostoc spp. Dextran-producing leuconostoc were grown on sucrose enriched agar

102 supplemented with vancomycin (tryptone 10 g/l, yeast extract 5 g/l, sucrose 10 g/l, CaCO3 20g/l, agar

103 15 g/l, and vancomycin 200 µg/ml), and counted after incubation for five days at 21ºC.

104 Enterobacteria and coliforms. These were grown on MacConkey agar (MCA; Merck), and counted

105 after 48 h of incubation at 37ºC.

106 Yeasts and moulds. Dilutions of the samples were plated on yeast-extract glucose chloramphenicol

107 agar (YGCA; yeast extract 5 g/l, glucose 20 g/l, agar 15 g/l, and chloramphenicol 0.1 g/l). Yeasts and

108 filamentous moulds were independently counted after five days of incubation at 28º C.

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110 Molecular identification of lactic acid bacteria. After incubation, plates were photographed and

111 colonies of different morphologies were counted and selected for identification. Isolates were purified

112 by subculturing and stored at -80ºC in fresh medium with 15% (v/v) of glycerol.

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113 Cryoprotected cultures were recovered in the isolation medium, and colonies were used as a source

114 of DNA in amplifications by polymerase chain reaction (PCR). Cell extracts were obtained by a

115 mechanical procedure in a Mini-Beadbeater apparatus (BioSpec Products, Inc., Bartlesville, OK, USA),

116 after suspending a single colonies in 100 µl of sterile water and mixing with 50 mg of sterile glass

117 beads (Sigma-Aldrich, Inc., Saint Louis, MISS, USA). Cell extracts were separated from cellular debris

118 by centrifugation at 13,000×g for 10 min. Supernatants were used as a source of template DNA for

119 PCR amplification of a large segment of 16S rRNA gene with the universal bacterial primer 27F (5’-

120 AGAGTTTGATYMTGGCTCAG-3’) and the universal prokaryotic primer 1492R (5’-

121 GGTTACCTTGTTACGACTT-3’).

122 Amplicons were purified using GenElute PCR Clean-Up columns (Sigma-Aldrich) and sequenced

123 with an ABI 373 DNA sequencer (Applied Biosystems, Foster City, Ca., USA) using primer 27F. On

124 average, 850 bp were obtained and compared with those in the GenBank database, using the online

125 BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/), and in the Ribosomal Database Project

126 (http://rdp.cme.msu.edu/index.jsp). Sequences were assigned to a given species if they shared a

127 similarity equal or higher than 97% to those of that species (Stackebrandt and Goebel, 1994; Palys et

128 al., 1997).

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130 Denaturing gradient gel electrophoresis (DGGE) analysis. DGGE analysis involved the

131 following steps: isolation of total DNA from curd and cheese samples, PCR amplification with DGGE

132 primers, electrophoresis and identification of bands in gels.

133 Isolation of total microbial DNA. Curd and cheese samples were homogenized in PS. 2-ml

134 samples of homogenates were centrifuged at 9,000×g for 1 min. For the isolation of bacterial DNA the

135 pellets were resuspended in 300 µl of TES buffer (25 mM Tris, 10 mM EDTA, 50 mM sucrose)

136 containing 20 mg/ml of lysozyme (cat. no. 62971, Fluka, Sigma-Aldrich) and 15 µl of mutanolysin 1

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137 U/μl (cat. no. M9901, Sigma-Aldrich). Then samples were incubated for 1 h at 37°C. After

138 centrifugation at 9,000×g for 1 min pellets were resuspended in 100 µl of Tris buffer (10 mM Tris HCl,

139 pH 8.5) and DNA isolation was performed by using the commercial Genomic Mini DNA Purification

140 kit (A&A Biotechnology, Gdynia, Poland) following the manufacturer’s recommendations. Isolation of

141 DNA from fungi was based on the method of DNA isolation for Saccharomyces cerevisiae (Sherman et

142 al., 1986).

143 PCR amplification. Purified DNA was used as a template for PCR amplification of the V3 variable

144 region of the prokaryotic 16S rRNA gene with primers F357 (5’-TACGGGAGGCAGCAG-3’), to

145 which a 39-bp GC sequence was linked to give rise to GC-F357, and R518 (5’-

146 ATTACCGCGGCTGCTGG-3’) (Muyzer et al., 1993). Group specific amplification of 16S rRNA for

147 the lactococci-enterococci-streptococci group and the lactobacilli-leuconostoc-pediococci group was

148 performed with the primer pairs LB2 GC-(5’-GATTYCACCGCTACACATG-3’) with LAC3 (5’-

149 AGCAGTAGGGAATCTTCGG-3’) and LB2-GC with LB1 (5’-AGCAGTAGGGAATCTTCCA-3’),

150 respectively (Endo and Okada, 2005). Finally, amplification of the D1 domain of the 26S rRNA gene of

151 fungi was obtained with primers NL1 GC-(5’-GCCATATCAATAAGCGGAGGAAAAG-3’) and LS2

152 (5’-ATTCCCAAACAACTCGACTC-3’) (Cocolin et al., 2002). PCR amplification conditions were as

153 reported by various authors.

154 Electrophoresis conditions. Amplicons were analyzed electrophoretically in 8% polyacrilamide gels

155 with 40-60% and 30-50% formamide denaturing gradients for bacteria and fungi, respectively, using a

156 DCode device (Bio-Rad, Richmond, Ca., USA). Electrophoresis proceeded at 75 V for 17 h for bacteria

157 and at 130 V for 4.5 h for yeasts and moulds.

158 Identification of DGGE bands. Bands corresponding to the most common bacterial species were

159 identified as previously reported (Flórez and Mayo, 2006) by migration in comparison to that of a

160 control ladder of known strains. Bands migrating at positions other than the control strains were

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161 identified by DNA isolation, reamplification with the same primers without the GC clamps, sequencing

162 and comparison of the sequences against the databases.

163

164 Pyrosequencing analysis of Oscypek. In order to address the bacterial diversity, three samples of the

165 same cheese batch were analyzed by pyrosequencing.

166 Amplicon preparation. Primers for the target area were selected to span a region between 250 and

167 500 bp in length (a combination of average mean read length and maximum recommended amplicon

168 size of a 454 Life Sciences, Roche Applied Science GS FLX amplicon sequencing run). A 294-

169 nucleotide sequence of V5 and V6 region of the 16S rRNA gene (with respect to Escherichia coli 16S

170 rDNA position 786 to 1079) was amplified by PCR from the isolated DNA.

171 Fusion primers were designed in which a proprietary primer sequence (Adaptor) of the Roche GS

172 FLX sequencing technology and a sample-specific 10-nucleotide key sequence (Multiplex Identifier -

173 MID) was included, to differentiate between distinct samples. The forward primer was 5’-

174 CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGATTAGATACCCTGGTAGT-3’, where the

175 underlined sequence corresponded to the forward primer E786F (Coloqhoun, 1997) and the reverse

176 primer was 5’-

177 CCATCTCATCCCTGCGTGTCTCCGACTCAGATATCGCGAGTCACGACACGAGCTGACGAC-

178 3’, where the underlined sequence is the universal primer equivalent of E. coli position 1061 to 1079 in

179 L. lactis IL1403.

180 For each sample, a 50-µl PCR mix was prepared containing 1× PCR buffer, 200 µM

181 deoxynucleoside triphosphate mix (Fermentas, St. Leon-Rot, Germany), 0.4 µM of each primer, and

182 1.25 U of Ex Taq polymerase (Takara Bio Inc., Otsu, Shiga, Japan). To each reaction mixture, 1 µl of

183 the extracted template DNA was added. The PCR conditions used were 95°C for 5 min and 30 cycles

184 of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by one cycle at 72°C for 7 min.

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185 Amplicons quality check and quantity measurement. Amplicon samples were purified using

186 Ampure Beads XP (Beckman Coulter Inc., High Wycombe, UK) and run on Agilent Bioanalyzer 2100

187 to check sample quality. Amplicon DNA quantity was estimated using QuantIT Pico Green Assay

188 (Invitrogen, Carlsbad, CAL, USA). Prior to sequencing the samples were diluted to the same

189 concentration and pooled.

190 Ultra-deep amplicon sequencing on 454 GS FLX. DNA amplicons were sequenced on a GS FLX

191 (454) pyrosequencer (Roche). Samples were clonally amplified with emulsion PCR. DNA carrying

192 beads were loaded on the medium region of PTP plate (1/16) and sequenced in one direction on GS

193 FLX Titanium instrument.

194 Sequence reads for each amplicon were extracted from the resulting SFF files (standard flowgram

195 files) into FASTA format and split into separate pools based on the MIDs using the sfffile command.

196 Sequences obtained from pyrosequencing were then processed with Mothur software, version 1.7.2

197 (Schloss et al., 2009) that has implemented the algorithms cited below. In the first step reads were

198 trimmed to analyze only regions with average score over 50 bases, window of at least 35. Then, reads

199 shorter than 150 bases or with ambiguous base call (an “N”) or containing homopolymeric track longer

200 than 8 bases were removed. Afterwards, unique reads were aligned to SILVA-compatible alignment

201 database and based on this alignment potential chimeric sequences were removed using the

202 ChimeraSlayer algorithm (Haas et al., 2011). Finally, the remaining sequences were clustered into

203 OTUs (Operational Taxonomic Units) and assigned to respective taxonomic branches.

204 Structures of communities of all samples were compared using weighted and unweighted Unifrac

205 algorithm (Lozupone et al., 2007) and Yue and Clayton measure (Yue and Clayton, 2005).

206 Trimmed sequences were also used as queries with the BLASTN program (Altschul et al, 1990)

207 against NCBI’s NT database. Results from those searches were then analyzed with MEGAN software

208 (Huson et al., 2007).

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209

210 RESULTS

211 Basic microbial analysis. Results of the count of majority and indicator microbial populations of

212 the different traditional Oscypek cheese samples analyzed in this work are summarized in Table 1.

213 Counts of total mesophilic bacteria under aerobic (in PCMA), and anaerobic (in PCMA and BHIAC)

214 conditions did not show statistical differences, similarly to counts of lactobacilli in aerobiosis and

215 anaerobiosis (both in MRSA). In general, mesophilic bacteria counts matched those obtained in

216 LM17A, which suggested that Lactococcus spp. was the dominant microorganism in all cheeses (at a

217 level of near 1.0×109 CFU/g). High numbers of Lactobacillus species were also observed in all cheese

218 samples, although at a level of one or two logarithmic units lower than those of lactococci. Dextran-

219 producing Leuconostoc were present in all samples at variable levels (ranging from 5.72 to 7.82

220 logarithmic units per gram). Yeasts and moulds were also present in all cheese samples in numbers

221 ranging from 5 to 6 logarithmic units per gram. Finally, enterobacteria populations were present at

222 similar levels as yeasts and mo