Characterization of microbiota in Plaisentif cheese by high-throughput sequencing

Abstract High-throughput DNA sequencing (HTS) was used in this study to investigate the microbiota of Plaisentif production, an artisanal antique cheese fabricated in the Italian Alps during the violet's blooming season. The dynamics of the microbiota was described in four production points for nine different producers. The bacteria present in all samples correspond to four phyla: Proteobacteria, Firmicutes, Bacteroidetes, and Acinetobacteria. Of these, Proteobacteria and Firmicutes were the most abundant in milk and curd whereas Firmicutes dominated in cheese samples. The results showed a higher bacterial diversity in the initial steps of cheese making (milk, curd), while the final product presented a lower number of genera mainly represented by lactic acid bacteria. In ripened cheeses, core bacterial community was composed by the genera Lactococcus, Lactobacillus and Streptococcus. Although most of the reads from the final ripened cheese correspond to few LAB, it is still possible to observe some variability between the producers. The HTS revealed that some producers used starters, even if it is not considered by the Plaisentif production's technical policy. The obtained results highlight the great potential of the HTS methodologies in the dairy industry not only from the scientific point of view but also from practical approach.

[1]  Marc W Allard,et al.  Characterization of microflora in Latin-style cheeses by next-generation sequencing technology , 2012, BMC Microbiology.

[2]  A. Majhenič,et al.  Phenotypic and Genotypic Characterization of Indigenous Lactobacillus Community from Traditional Istrian Ewe's Cheese , 2012 .

[3]  L. Cocolin,et al.  Microbial diversity, dynamics and activity throughout manufacturing and ripening of Castelmagno PDO cheese. , 2010, International journal of food microbiology.

[4]  C. Delbès,et al.  Application of SSCP-PCR fingerprinting to profile the yeast community in raw milk Salers cheeses. , 2006, Systematic and applied microbiology.

[5]  S. Delgado,et al.  Diversity and evolution of the microbial populations during manufacture and ripening of Casín, a traditional Spanish, starter-free cheese made from cow's milk. , 2009, International journal of food microbiology.

[6]  A. Chao,et al.  Estimating the Number of Classes via Sample Coverage , 1992 .

[7]  Robert C. Edgar,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2001 .

[8]  Susan M. Huse,et al.  Microbial diversity in the deep sea and the underexplored “rare biosphere” , 2006, Proceedings of the National Academy of Sciences.

[9]  Michele Iacono,et al.  “Remake” by High-Throughput Sequencing of the Microbiota Involved in the Production of Water Buffalo Mozzarella Cheese , 2012, Applied and Environmental Microbiology.

[10]  S. Sørensen,et al.  The fate of indigenous microbiota, starter cultures, Escherichia coli, Listeria innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by pyrosequencing and quantitative real time (qRT)-PCR. , 2012, International journal of food microbiology.

[11]  G. Casella,et al.  Pyrosequencing enumerates and contrasts soil microbial diversity , 2007, The ISME Journal.

[12]  Haluk Dogan,et al.  Metagenomic analysis of the microbial community in kefir grains. , 2014, Food microbiology.

[13]  G. Barbier,et al.  Culture-independent methods for identifying microbial communities in cheese. , 2008, Food microbiology.

[14]  Jasmina Havranek,et al.  Dynamics of Bacterial Communities during the Ripening Process of Different Croatian Cheese Types Derived from Raw Ewe's Milk Cheeses , 2013, PloS one.

[15]  Orla O'Sullivan,et al.  The complex microbiota of raw milk. , 2013, FEMS microbiology reviews.

[16]  Pablo Vinuesa,et al.  Characterization of the bacterial biodiversity in Pico cheese (an artisanal Azorean food). , 2015, International journal of food microbiology.

[17]  John Bunge,et al.  Estimating the Number of Species in a Stochastic Abundance Model , 2002, Biometrics.

[18]  I. Good THE POPULATION FREQUENCIES OF SPECIES AND THE ESTIMATION OF POPULATION PARAMETERS , 1953 .

[19]  William A. Walters,et al.  QIIME allows analysis of high-throughput community sequencing data , 2010, Nature Methods.

[20]  M. Halpern,et al.  Culturable Psychrotrophic Bacterial Communities in Raw Milk and Their Proteolytic and Lipolytic Traits , 2007, Applied and Environmental Microbiology.

[21]  S. Delgado,et al.  Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis. , 2012, Food microbiology.

[22]  Marie-Christine Montel,et al.  Traditional cheeses: rich and diverse microbiota with associated benefits. , 2014, International journal of food microbiology.

[23]  Eric P. Nawrocki,et al.  An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea , 2011, The ISME Journal.

[24]  M. T. Bottero,et al.  Simultaneous detection of cow and buffalo milk in mozzarella cheese by Real-Time PCR assay , 2011 .

[25]  C. Pedrós-Alió,et al.  Dipping into the Rare Biosphere , 2007, Science.

[26]  William A. Walters,et al.  Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms , 2012, The ISME Journal.

[27]  Y. Husimi,et al.  Temperature sweep gel electrophoresis: a simple method to detect point mutations. , 1991, Nucleic acids research.

[28]  R. Myers,et al.  Detection and localization of single base changes by denaturing gradient gel electrophoresis. , 1987, Methods in enzymology.

[29]  F. De Filippis,et al.  A Selected Core Microbiome Drives the Early Stages of Three Popular Italian Cheese Manufactures , 2014, PloS one.

[30]  E. Vaughan,et al.  Artisanal and experimental Pecorino Siciliano cheese: microbial dynamics during manufacture assessed by culturing and PCR-DGGE analyses. , 2006, International journal of food microbiology.

[31]  Baltasar Mayo,et al.  Diversity of thermophilic bacteria in raw, pasteurized and selectively-cultured milk, as assessed by culturing, PCR-DGGE and pyrosequencing. , 2013, Food microbiology.

[32]  Paul D. Cotter,et al.  High-Throughput Sequencing for Detection of Subpopulations of Bacteria Not Previously Associated with Artisanal Cheeses , 2012, Applied and Environmental Microbiology.

[33]  David A. Mills,et al.  Facility-Specific “House” Microbiome Drives Microbial Landscapes of Artisan Cheesemaking Plants , 2013, Applied and Environmental Microbiology.

[34]  C. Hill,et al.  High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain. , 2011, FEMS microbiology letters.

[35]  B. Taminiau,et al.  Microbiota characterization of a Belgian protected designation of origin cheese, Herve cheese, using metagenomic analysis. , 2014, Journal of dairy science.

[36]  J. Tiedje,et al.  Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy , 2007, Applied and Environmental Microbiology.

[37]  C. E. SHANNON,et al.  A mathematical theory of communication , 1948, MOCO.

[38]  W. D. de Vos,et al.  Comparative Analysis of Pyrosequencing and a Phylogenetic Microarray for Exploring Microbial Community Structures in the Human Distal Intestine , 2009, PloS one.

[39]  Søren Thue Lillevang,et al.  Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing , 2011 .

[40]  J. Ogier,et al.  Biodiversity of Bacterial Ecosystems in Traditional Egyptian Domiati Cheese , 2006, Applied and Environmental Microbiology.

[41]  Magdalena Kowalczyk,et al.  Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches , 2012, Applied and Environmental Microbiology.

[42]  R. P. Ross,et al.  The microbial content of raw and pasteurized cow milk as determined by molecular approaches. , 2013, Journal of dairy science.

[43]  G Corrieu,et al.  Does smearing inoculum reflect the bacterial composition of the smear at the end of the ripening of a French soft, red-smear cheese? , 2004, Journal of dairy science.

[44]  E. H. Simpson Measurement of Diversity , 1949, Nature.

[45]  P. McSweeney Biochemistry of cheese ripening: Introduction and overview , 2004 .

[46]  A. Gruss,et al.  Molecular Fingerprinting of Dairy Microbial Ecosystems by Use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis , 2004, Applied and Environmental Microbiology.

[47]  J. Bae,et al.  Investigation of archaeal and bacterial diversity in fermented seafood using barcoded pyrosequencing , 2010, The ISME Journal.

[48]  Danilo Ercolini,et al.  rRNA-based monitoring of the microbiota involved in Fontina PDO cheese production in relation to different stages of cow lactation. , 2014, International journal of food microbiology.

[49]  C. Delbès,et al.  Monitoring Bacterial Communities in Raw Milk and Cheese by Culture-Dependent and -Independent 16S rRNA Gene-Based Analyses , 2007, Applied and Environmental Microbiology.