Deciphering the microbiota of Greek table olives - A metagenomics approach
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1 1 st International Olive Conference Table Olives: Pursuing Innovation - Exploring Trends Thessaloniki, Greece, May 2018 Deciphering the microbiota of Greek table olives - A metagenomics approach Effie Tsakalidou Professor of Food Biochemistry Department of Food Science and Human Nutrition Agricultural University of Athens Iera Odos 75, Athens, Greece 1
2 Introduction 2
3 From Goddess Athena to table 3
4 Major varieties of Greek table olives Chalkidikis Trend Trend Production (tons) Production (tons) Producers Producers Trees Trees Kalamon Other Trend Trend Production (tons) Production (tons) Producers Producers Trees Trees
5 Overall production is Greece is increasing 5
6 Table Olives Microbiota Table olive fermentation occurs spontaneously, driven by the indigenous microbiota of the olives, without adding starter cultures The fermentation process is carried out by Lactic Acid Bacteria (e.g. Lb. plantarum) and Yeasts (e.g. Pichia sp., Candida sp.) Lactic Acid Bacteria are mainly responsible for the brine acidification providing microbiological stability and extended shelf life to the final product Yeasts mainly shape aroma and taste, degrade phenolic compounds and enhance the growth of Lactic Acid Bacteria Bonatsou et al. (2017) 6
7 Microbial biogeography Is the study of microbial diversity over time and space in an ecosystem, among others, in fermented foods It reveals the association of food microbial communities with the raw material environmental conditions processing technology High-throughput sequencing (HTS) technologies Metagenomics (diversity assay and shotgun) the study of the metagenome (the collective genome of microorganisms from a sample) that provides information on the microbial diversity & ecology of a specific ecosystem 7
8 Abundance of HTS studies of fermented foods and beverages grouped according to the food matrix De Filippis et al. (2017) 8
9 Pros and cons of shotgun metagenomics De Filippis et al. (2017) 9
10 Here we discuss the microbial ecology of Greek table olives prepared from 3 olive varieties in 3 different geographical regions of Greece using conventional microbiological analysis a metagenomics approach 10
11 Experimental set-up 11
12 Samples Kerasoelia (Mount Athos) (Amfissa region) Kalamon 12
13 Classical microbiological analysis 1. Total mesophilic microflora 2. Lactic acid bacteria isolation of colonies 3. Yeasts isolation of colonies 4. Propionic acid bacteria 5. Enterobacteriaceae 6. Pseudomonas 13
14 Identification of isolates A. Clustering Rep-PCR Bacteria BOXA1R primer Yeast (GTG) 5 primer B. Identification at the species level Bacteria 16S rdna 16S (F) / 16S (R) primers Yeasts ITS DNA ITS1 / ITS4 primers 14
15 Metagenomic analysis 1. Total DNA extraction in house protocol 2. DNA quality electrophoretically 3. Presence of Bacteria 16S rdna primers 27 (F) / 519 (R) Yeasts ITS DNA primers ITS1 (F) / ITS2 (R) Plant Actin Actin1 (F) / Actin2 (R) primers 4. Diversity Assay Illumina ΜiSeq platform 15
16 Results 16
17 log cfu/ml Microbial counts of lactic acid bacteria & yeasts Kerasoelia (Mount Athos) Kalamon (Amfissa Region) lactic acid bacteria yeast 17
18 rep-pcr 16S rrna gene sequencing M3: Pediococcus ethanolidurans BACTERIA M20: Pediococcus ethanolidurans M21: Pediococcus ethanolidurans M5: Pediococcus ethanolidurans M35: Lactobacillus plantarum M28: Lactobacillus plantarum M32: Lactobacillus plantarum M33: Lactobacillus plantarum M13: Lactobacillus plantarum M36: Lactobacillus plantarum M37: Lactobacillus plantarum M18: Lactobacillus plantarum 18
19 : Pichia membranifaciens : P. membranifaciens YEASTS : P. membranifaciens : P. membranifaciens : P. membranifaciens rep-pcr and ITS DNA sequencing : P. membranifaciens : P. membranifaciens : Groenewaldozyma auringiensis : G. auringiensis : Saccharomyces cerevisiae Candida cellae 87% Candida etchellsii 86% Candida apicola 85% Candida floris 83% Starmerella meliponinorum 83% C. cellae 87% C. etchellsii 86% C. apicola 85% C. floris 83% S. meliponinorum 82% : S. cerevisiae 19
20 Isolates Kerasoelia (Mount Athos) Kalamon (Amfissa Region) Total Lactic acid bacteria Lactobacillus plantarum/pentosus Lactobacillus casei/paracasei 7 7 Lactobacillus brevis 6 6 Pediococcus parvulus 9 9 Pediococcus ethanolidurans Yeasts Pichia membranifaciens Saccharomyces cerevisiae Groenewaldozyma auringiensis Total
21 Extraction of total DNA No Sample DNA ng/well 260/280 1 Kalamon (Amfissa Region) S. macedonicus (+) No Sample DNA ng/well 260/280 1 Kerasoelia (Mount Athos) S. macedonicus (+) : MM ladder 21
22 Confirmation of bacteria and yeast DNA * * * * * Bacteria Yeast Plant Bacteria Yeast Plant No Sample No Sample No Sample No Sample No Sample No Sample 1 blank 2 3 Kalamon 6 blank 7 8 Kalamon 12 blank Kalamon 1 Kerasoelia (Mount Athos) 2 S. macedonicus (+) 3 blank 4 Kerasoelia (Mount Athos) 5 S. pombe (+) 6 blank 7 Kerasoelia (Mount Athos) 8 S. pombe (-) 9 blank 4 (Amfissa Region) 5 S. macedonicus (+) 9 (Amfissa Region) 10 S. pombe (+) 11 S. macedonicus (-) 15 (Amfissa Region) 16 S. pombe (-) 17 S. macedonicus (-) Olive samples (+) control 18 Koroneiki (+) *: MM ladder 22
23 Bacteria genera Kerasoelia (Mount Athos) Raoultella (2%) Cellulosimicrobium (17%) Citrobacter (2%) Leuconostoc (2%) Lactobacillus (74%) Kalamon Bacteroides (3%) Cellulosimicrobium (90%) Pseudomonas (2%) Lactobacillus (4%) Cellulosimicrobium (90%) Pediococcus (3%) (Amfissa Region) Cellulosimicrobium (39%) Lactobacillus (12%) Idiomarina (13%) Halanaerobium (10%) Salinicola (17%) 23
24 Kerasoelia (Mount Athos) Yeast genera Marasmius (5%) Sporobolomyces (6%) Debaryomyces (5%) Saccharomyces (50%) Taphrina (6%) Kalamon Kluyveromyces (3%) Debaryomyces (7%) Kluyveromyces (3%) Debaryomyces (6%) Kluyveromyces (2%) (Amfissa Region) Schwanniomyces (11%) Debaryomyces (9%) Pichia (80%) Pichia (86%) Pichia (69%) 24
25 Differences at dominant bacteria genera level Genus (%) Kerasoelia (Mount Athos) Kalamon (Amfissa Region) Cellulosimicrobium Lactobacillus Differences at dominant yeast genera level Genus (%) Kerasoelia (Mount Athos) Kalamon (Amfissa Region) Pichia Saccharomyces
26 Kerasoelia (Mount Athos) leptolyngbya spp. streptococcus thermophilus Bacteria species Lactobacillus brevis (17%) Citrobacter freundii (2%) Lactobacillus sp. (39%) Cellulosimicrobium cellulans (17%) Lactobacillus paracasei (6%) Pseudomonas fragi (1%) Kalamon (Evoia Olive.Kalamon Island) Bacteroides sp. (3%) Carnobacterium maltaromaticum (2%) Cellulosimicrobium cellulans (90%) Lactococcus lactis (1%) (Evoia Olive. Island) Lactobacillus buchneri (3%) Pediococcus parvulus (3%) Cellulosimicrobium cellulans (90%) Lactobacillus plantarum (11%) Olive.Amfissa (Amfissa Region) Salinicola halomonas salaria (16%) Lactobacillus plantarum (6%) Idiomarina sp. (13%) Halanaerobium salsuginis (10%) Cellulosimicrobium cellulans (39%) 26
27 Kerasoelia (Mount Athos) Yeast species Taphrina kruchii (5%) Debaryomyces hansenii (5%) Saccharomyces bayanus (50%) Sporobolomyces singularis (6%) Marasmius menieri (5%) Kalamon Olive.Kalamon Debaryomyces hansenii (7%) Olive. Debaryomyces hansenii (6%) Pichia manshurica (15%) Olive.Amfissa (Amfissa Region) Debaryomyces hansenii (9%) Schwanniomyces etchellsii (11%) Pichia manshurica (46%) Pichia membranifaciens (33%) Pichia membranifaciens (71%) Pichia manshurica (28%) Pichia membranifaciens (40%) 27
28 Differences at dominant bacteria species level Species (%) Kerasoelia (Mount Athos) Kalamon (Amfissa Region) Cellulosimicrobium cellulans Lactobacillus brevis Differences at dominant yeast species level Species (%) Kerasoelia (Mount Athos) Kalamon (Amfissa Region) Saccharomyces bayanus Pichia manshurica Pichia membranifaciens
29 Conclusions The present work is one of the first metagenomics approaches of naturally fermented Greek table olives It clearly reveals the impact of the cultivar and the environmental conditions on the table olives microbiota However, a more thorough study is needed, including samples from all Greek varieties and with a much broader geographical dispersion It highlights the need of a polyphasic and holistic fingerprinting approach in a food ecosystem 29
30 The group Kostas Papadimitriou, PhD Maria Kazou, PhD student Voula Alexandraki, PhD student & Marina Georgalaki, PhD Rania Anastasiou, PhD Georgia Zoumpopoulou, PhD Eugenia Manolopoulou, MSc PAVET ( ) Developing methodologies for complete quality control-characterization of bioactive molecules of extra virgin olive oil and table olives, using modern techniques and chemometric analysis 30
31 Thank you for your attention! 31
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