Microorganisms of Table Olives. Contribution to Safety and Beneficial Activity

Transcription

1 Microorganisms of Table Olives. Contribution to Safety and Beneficial Activity Dr. Chrysoula Tassou Biologist-Food Microbiologist Research Director Hellenic Agricultural Organisation DEMETER Institute of Technology of Agricultural Products (ITAP) S. Venizelou 1, Lycovrissi, Attica, Greece tel: , fax:

2 Since ancient times olives have been a key element in the nutrition of the people of the Mediterranean basin

3 Today olives are the most economically important, fermented product in the Western world. Table olives in the Mediterranean diet Table olives & olive oil

4 Raw olive fruits are not edible and need processing Important commercial types of olives after processing: Natural olives in brine (Greek type olives) Green olives in brine (Spanish type) In both types, the fermentation is the critical processing step

5 Natural ripe (black) olives Harvesting FERMENTATION in brine Gradual debittering - ripening

6 Green Spanish style Harvesting Debittering with ΝaΟΗ Washing with H 2 O green natural olives FERMENTATION in brine

7 Table olive fermentation Basic process for the preparation of natural black or green table olives. The microorganisms of raw olives are responsible for the fermentation.

8 Microorganisms of raw olive fruits Yeasts-fungi 5.4 (log 10 cfu/g) Lactic acid bacteria 4.0 Enterobacteriaceae 2.5 Pseudomonas spp. 3.5 putida cepacia fluorescens luteola Tassou, C. (1993) Microbiology of olives with emphasis on the antimicrobial activity of phenolic compounds. PhD Thesis Univ. of Bath, UK

9 Microorganisms of raw olive fruits* Yeasts Moulds Bacteria Pichia Rhodotorula Candida Saccharomyces Trichosporon Debaryomyces Aspergillus Fusarium Penicillium Alternaria Rhizopus Cladosporium Geotrichum Pullularia Aerobacter Achromobacter Pseudomonas Serratia Escherichia Micrococcus Bacillus * isolated from stored olives or oviposition sites of Dacus oleae Based on Vaughn (1949), Balatsouras & Vaughn (1958), Gonzalez Cancho (1957), Borbolla y Alcala et al. (1958)

10 Microorganisms of raw olive fruits Yeasts genera Brettanomyces Cryptococcus Candida Rhodotorula Saccharomyces Pichia Debaryomyces Hanseniaspora Hansenula Schizosaccharomyces Kloeckera Kluyveromyces Sporobolomyces Trichosporon Lactic acid bacteria Lactobacillus delbrueckii leichmannii helveticus acidophilus casei subsp. alactosus casei subsp. rhamnosus xylosus plantarum curvatus coryniformis fermentum brevis higardii Based on Pelagatti ( ).

11 Microorganisms of raw olive fruits (yeasts)* Olive fruits* Pichia holstii Kluyveromyces thermotolerans Crushed olives* Pichia caribbica Pichia mississippiensis Lachancea sp. Lachancea thermotolerans Kluyveromyces thermotolerans Zygosaccharomyces fermentati Pichia holstii Candida thermophila Olive fruits Pichia guilliermondii Candida maris Candida humicola Klyveromyces marxianus Cryptococcus laurentii Rhodotorula glutinis * Identified by molecular techniques Based on Hernandez et. al. 2007, Romo-Sanchez et al. (2010).

12 Table olive fermentation By placing in the brine, part of the microorganisms migrates to the brine and ferments the sugars derived from the olive flesh. The anaerobic conditions, the salt and the gradual reduction of ph, exert selective action on microorganisms. Under normal conditions, lactic acid bacteria and yeasts dominate. Basic metabolic products: lactic acid, acetic acid, ethanol. The temperature, the nutrient availability and the phenolic compounds, the salt and the ph are critical factors for the course of fermentation

13 Microbiology of table olive fermentation Stages of fermentation Stage Ι (48-72 hours) Enterobacter cloacae, Citrobacter freudii, Enterobacter aerogenes, Escherichia coli, Aeromonas hydrophila, Flavobacterium diffusum, F. balustinum, Pseudomonas spp. (Gram -) Bacillus spp., Micrococcus spp., Clostridium spp. (Gram +) Stage ΙI (14-15 days) Gradual dominance of Pediococcus spp. & Leuconostoc spp. (cocci) Reduction of Gram (-) bacteria Stage ΙΙΙ (main fermentation stage) Dominance of Lactobacillus spp and specifically of L. plantarum, L. pentosus Other species: L. brevis, L. fermentum, L. cellobiosus, L. casei End of stage: ph 4,0 or lower

14 Microbial evolution during naturally black olive fermentation at 25 C - - lactic acid bacteria, -O- yeasts, - - enterobacteria - pseudomonads Tassou, C.C., Panagou, E.Z. and Katsaboxakis, K.Z. (2002), Food Microbiology 19:

15 Microbiology of table olive fermentation Nychas, G., Panagou E.Z.,.. Tassou C. (2002) Microbial colonization of naturally black olives during fermentation and associated biochemical activities in the cover brine. Lett. Appl. Microb. 34:

16 Microbiology of table olive fermentation Nychas, G., Panagou E.Z.,.. Tassou C. (2002) Microbial colonization of naturally black olives during fermentation and associated biochemical activities in the cover brine. Lett. Appl. Microb. 34:

17 Microbiology of table olive fermentation The fermentation is considered complete and successful when: The desired microbiota, the physicochemical characteristics (ph, acidity, salt) and the organoleptic characteristics of olives have been developed.

18 Microbiology of fermentation -Lactic acid bacteria Bonatsou S., Tassou C., Panagou E. Nychas GJ. (2017) Microorganisms 5: 30

19 Microbiology of fermentation -Yeasts Bonatsou S., Tassou C., Panagou E. Nychas GJ. (2017) Microorganisms 5: 30

20 Microbiology of fermentation -Yeasts Bonatsou S., Tassou C., Panagou E. Nychas GJ. (2017) Microorganisms 5: 30

21 Microbiology of fermentation Lactic acid bacteria Lb. pentosus 606 Ln. mesenteroides B275 Ln. mesenteroides B276 Ln. mesenteroides B265 Ln. mesenteroides B267 Ln. mesenteroides B273 Ln. mesenteroides B259 Ln. mesenteroides B269 Ln. mesenteroides B274 Ln. mesenteroides B260 Ln. mesenteroides B264 Ln. mesenteroides B271 Ln. mesenteroides B266 Ln. mesenteroides B268 Ln. mesenteroides B263 Ln. mesenteroides B262 Ln. mesenteroides B270 Lb. pentosus B279 Lb. pentosus E129 Lb. pentosus E83 Lb. pentosus E84 Lb. pentosus E182 Lb. pentosus 612 Lb. pentosus E141 Lb. plantarum E45 Lb. pentosus E43 Lb. plantarum E10 Lb. plantarum E50 Lb. pentosus E130 Lb. plantarum E68 Lb. plantarum E79 Lb. plantarum E73 Lb. plantarum E66 Lb. plantarum E77 Lb. plantarum E4 Lb. pentosus E128 Lb. pentosus E139 Lb. pentosus E108 Lb. pentosus E111 Lb. pentosus E121 Lb. plantarum E131 Lb. pentosus E105 Lb. pentosus E101 Lb. plantarum E69 Lb. pentosus 609 Lb. pentosus 632 Lb. pentosus E96 Ln. mesenteroides B261 Lb. pentosus 390A Lb. pentosus 625A Lb. pentosus E100 Lb. pentosus B283 Lb. plantarum E71 Lb. plantarum B282 Lb. pentosus E119 Lb. pentosus E120 Lb. pentosus 637 Lb. pentosus E110 Lb. pentosus E97 Lb. pentosus E106B Lb. pentosus E89 Lb. pentosus E104 Lb. pentosus B278 Lb. pentosus B281 Lb. paraplantarum B280 Lb. pentosus B284 Lb. pentosus B285 Ln. pseudomesenteroides B277 Lb. pentosus E95 Lb. casei group E93 Lb. casei group E94 71 different strains of lactic acid bacteria isolated from fermented olives of Greek varieties and identified using molecular tools 13 Lactobacillus plantarum 37 Lb. pentosus 1 Lb. paraplantarum 2 Lb. casei group (Lb. casei, Lb. paracasei) 17 Leuconostoc mesenteroides 1 Ln. pseudomesenteroides Cluster analysis of PFGE ApaI digestion fragments of the different lactic acid bacteria strains recovered from olives and brine calculated by the unweighted average pair grouping method. The distance between the pattern of each strain is indicated by the mean correlation coefficient (r%).

22 NAGREF NATIONAL AGRICULTURAL RESEARCH FOUNDATION National and European Research projects on table olive fermentation implemented in NAGREF-ITAP since Study of the microbiology of olive fruit (GSRT) ( ) 2. Ιmprovement of texture characteristics of some European olive fruit varieties suitable for table olive purposes (ΕU, FAIR-CT ) ( ) 3. Biocontrol of olive fermentation; Microbiological and organoleptic studies for the improvement of safety, quality and acceptance of the final product (ΕU, FAIR-9526) ( ) 4. Technological improvement of the fermentation and preservation of green olives (GSRT-PAVE 99) ( ) 5. Technological improvement of the fermentation and preservation of natural black olives and new product development (GSRT-PAVE 99) ( ) 6. Natural fermentation of green olives with selected strains of lactic acid bacteria resistant to phenolic substances (NAGREF-British Council) ( ) 7. Setting-up a network of Technology Dissemination Centres to optimise SME s in the olive and olive oil sector (TDC- OLIVE) (ΕU, FOOD-CT ) ( ) 8. Improvement of green olive fermentation using probiotic lactic acid bacteria as starters (GSRT-PENED) ( ) 9. Green olive fermentation with probiotic lactic acid bacteria (NAGREF-Tunisia) ( ) 10. Study of the parameters related to processing, preservation and distribution of table olives. Application of improved techniques at industrial level for the quality improvement and minimization of environmental impact Reg. (ΕC) 2080/05 (12/06-3/08) 11. Preservation of green Kalamata table olives with P. Stathopoulos company. 12. Training of the sensory panel on the organoleptic characteristics of table olives according to the method of Intern.l Olive Council (COI/OT/MO No1/Rev Sensory analysis of table olives ) Reg.(ΕC)867/08 (4/2012-3/2015) 13. Table olive fermentation with selected probiotic bacteria. Towards a functional food PROBIOLIVES (EU, FP7-SME-2008) ( )

23 Microbiology of fermentation Lb. pentosus 606 Ln. mesenteroides B275 Lactic acid bacteria Ln. mesenteroides B276 Ln. mesenteroides B265 Ln. mesenteroides B267 Ln. mesenteroides B273 Ln. mesenteroides B259 Ln. mesenteroides B269 Ln. mesenteroides B274 Ln. mesenteroides B260 Ln. mesenteroides B264 Ln. mesenteroides B271 Ln. mesenteroides B266 Ln. mesenteroides B different strains of lactic acid bacteria isolated from fermented olives of Greek varieties Ln. mesenteroides B263 Ln. mesenteroides B262 Ln. mesenteroides B270 Lb. pentosus B279 Lb. pentosus E129 Lb. pentosus E83 Lb. pentosus E84 Lb. pentosus E182 Lb. pentosus 612 Lb. pentosus E141 Lb. plantarum E45 Lb. pentosus E43 Lb. plantarum E10 Lb. plantarum E50 Lb. pentosus E Lactobacillus plantarum Lb. plantarum E68 Lb. plantarum E79 Lb. plantarum E73 Lb. plantarum E66 Lb. plantarum E77 37 Lb. pentosus Lb. plantarum E4 Lb. pentosus E128 Lb. pentosus E139 Lb. pentosus E108 Lb. pentosus E111 1 Lb. paraplantarum Lb. pentosus E121 Lb. plantarum E131 Lb. pentosus E105 Lb. pentosus E101 Lb. plantarum E69 Lb. pentosus 609 Lb. pentosus 632 Lb. pentosus E96 Ln. mesenteroides B261 2 Lb. casei group (Lb. casei, Lb. paracasei) Lb. pentosus 390A Lb. pentosus 625A Lb. pentosus E100 Lb. pentosus B283 Lb. plantarum E71 17 Leuconostoc mesenteroides Lb. plantarum B282 Lb. pentosus E119 Lb. pentosus E120 Lb. pentosus 637 Lb. pentosus E110 1 Ln. pseudomesenteroides Lb. pentosus E97 Lb. pentosus E106B Lb. pentosus E89 Lb. pentosus E104 Lb. pentosus B278 Lb. pentosus B281 Lb. paraplantarum B280 Lb. pentosus B284 Lb. pentosus B285 Ln. pseudomesenteroides B277 Lb. pentosus E95 Lb. casei group E93 Lb. casei group E94 9 of them showed PROBIOTIC POTENTIAL IN VITRO Cluster analysis of PFGE ApaI digestion fragments of the different lactic acid bacteria strains recovered from olives and brine calculated by the unweighted average pair grouping method. The distance between the pattern of each strain is indicated by the mean correlation coefficient (r%).

24 Assessment of probiotic potential by in vitro tests (Lb. casei Shirota & Lb. rhamnosus GG used as reference strains) Test Strains Low ph (SR%) a Bile salts (SR%) b Bile salts hydrolysis Haemolytic activity d Antibiotic resistance e Caco-2 (Adherence% ) Lb. pentosus B c α K, C, S Lb. pentosus E γ K, C, S Lb. pentosus E γ K, G Lb. pentosus E γ K, A Lb. plantarum B γ K, G, E Lb. plantarum E γ K, G Lb. plantarum E γ K, G Lb. paracasei subs. paracasei γ K, G, S E93 Lb. paracasei subs. paracasei γ K, G, S E94 Lb. casei Shirota γ S, E, P, T, C Lb. rhamnosus GG γ K, A, P a survival rate after 3h in low ph, b survival rate after 4h in bile salts, c 0, no hydγrolysis; 1, partial hydrolysis. d a-haemolysis, γ-haemolysis, e A: ampicillin, V: vancomycin, G: gentamycin, K: kanamycin, S: streptomycin, P: penicillin, E: erythromycin, T: tetracycline, C: chloramphenicol

25 Definition of probiotics live microorganisms which, when consumed in adequate amounts, confer a health benefit on the host (FAO/WHO, 2002) In the early 1930's, in Japan, Minoru Shirota made a probiotic yoghurt type product, Yakult, from fat-free fermented milk with the strain Lactobacillus casei shirota. The term "probiotic" was introduced in 1965 by Lilly & Stillwell. Pro- + bios = Life (promotes life)

26 Probiotic microorganisms They are mainly lactic acid bacteria but also yeasts. They are used mainly in fermented dairy foods but also as food supplements. To exert their beneficial action, consumption of at least cfu/g is needed

27 Known probiotic strains Lactobacillus species L. acidophilus L. plantarum L. casei subspecies rhamnosus L. brevis L. delbreuckii subspecies bulgaricus Bifidobacterium species B. adolescentis B. bifidum B. longum B. infantis B. breve

28 Other: Streptococcus salivarius ssp. thermophilus Lactococcus lactis ssp. lactis Lactococcus lactis s ssp. cremoris Enterococcus faecium Leuconostoc mesenteroides ssp. dextranicum Propionibacterium freudenreichii Pediococcus acidilactici Saccharomyces boulardii

29 Minimum requirements for characterization as probiotics Identification (genus, species, strain) In vitro tests for selection: eg. resistance to gastric acidity, digestive enzymes, bile salts, antimicrobial action on pathogens Safety test: that the probiotic strain is safe and free of contamination in the form administered In vivo studies to document action on health

30 Beneficial actions of probiotics Antimicrobial actions (inhibition of pathogenic microorganisms) Biochemical actions (relieving lactose intolerance, lowering cholesterol, antihypertensive action, etc.) Physiological actions (stimulation of the immune system, treatment of allergies, etc.)

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34 Mechanisms of action Lactic acid production - ph reduction in the GIT and pathogen inhibition eg. Clostridium, Salmonella, Shigella, E. coli, etc. Reduction of toxic and carcinogenic metabolites production. Increased acidity in the intestine helps in the absorption of trace elements, especially calcium. Production of β-d-galactosidase enzyme for lactose degradation. Production of antimicrobials such as Bacteriocins and Vitamins (B & K vitamins) Inhibition of pathogen adhesion to the intestinal epithelium.

35 Suggested probiotic consumption Minimum intake: 100g of a probiotic food with 10 7 cfu/g. Most probiotics do not permanently adhere to the epithelium but exhibit their action with their increase and metabolism in their passage through the intestine. Daily intake of these bacteria is the best way for their activity and effectiveness.

36 Poly-probiotics 1. Lactobacillus gasseri PA16/8, Bifidobacterium bifidum MF20/5, Bifidobacterium longum SP07/3 2. Lactobacillus acidophilus, Bifidobacterium longum 3. Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum 4. Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus Casei, var. Rhamnosus, Enterococcus faecium

37 Food is the best way to take probiotics. There is a synergistic effect between the components of the food (prebiotics) and the probiotic cultures. The natural balancing of the acid environment in the stomach by the food also increases the stability of probiotics.

38 Traditional fermented foods as probiotic carriers The fermented foods provide: a traditional preservation method desired by consumers components valuable to health eg. antioxidants, vitamins, fibre present in fruits & vegetables the occurence of beneficial microorganisms and their enrichment with probiotic bacteria may add value to them.

39 Table olives as a functional probiotic food Table olives are a high nutritional food that provides essential fatty acids, fibers, vitamins and trace minerals, mainly calcium, iron, potassium, magnesium, phosphorus and iodine. They contain a very high percentage of unsaturated fatty acids, especially oleic acid. They also contain polyphenols and flavonoids that have anti-inflammatory action. Their enrichment with probiotic bacteria will add value to the product. The suggested daily intake for adults is g olives/day, or 5-7 olives.

40 Probiotics from and for olives Probiotic lactic acid bacteria isolated from olive microbiota, were used successfully as starters in lab and pilot scale green olive fermentations 1) Lb. pentosus B281 x2 2) Lb. plantarum B282 x2 3) Cocktail B281+B282 x2 a) 8% (w/v) NaCl b) 10% (w/v) NaCl

41 Log cfu/ml Log cfu/ml Log cfu/ml Log cfu/ml The probiotic lactic acid bacteria were maintained at high levels during fermentation (3 months) regardless of the salt level 10 9 Lb. pentosus B Lb. plantarum B AL 6 BL 5 AH AL 5 BH BL 4 AH 4 BH Time (d) Time (d) Lb. pentosus B281 & Lb. plantarum B Control SL SH SL 6 5 CL CH CL 4 SH 4 CH Time (d) Time (d) L: Low salt, H: High salt, : Brine,, : Olives

42 Survival of probiotics at the end of fermentation (PFGE) Inoculated Strain Fermentation time (d) Brine 8% NaCl Survival rate (PFGE) Fermentation time (d) Brine 10% NaCl Survival rate (PFGE) Lactobacillus pentosus B % 1 100% B B Lactobacillus plantarum B282 Mixed culture (B281+B282) % % % % 1 100% 1 100% % % % % %B281 / 53.33%B %B281 / 56.25%B %B %B281 / 57.14%B %B %B281

43 Packaging and storage 1) Autochthonous fermentation Control) 2) With Lb. pentosus B281 3) With Lb. plantarum B282 4) With Cocktail B281+B282 Packaging in MAP: 70% N 2 : 30% CO 2 or in brine Storage at 4 C and 20 C

44 Survival of probiotics at high levels ( cfu/g) after 6 months of storage log cfu/g log cfu/g : 20 C : 4 C log cfu/g log cfu/g 9 8 Lb. pentosus B Lb. plantarum B Time (d) Time (d) 9 8 Lb. pentosus B281 & Lb. plantarum B Control Time (d) Time (d)

45 Survival of probiotic strains at the end of storage (detected by PFGE) Inoculated strain Fermentation time (d) L. pentosus B % 4 o C 20 o C Survival rate Survival rate 56 * 92.86% 93.33% % 20% L. plantarum B % 56 * 40% 46.66% % 10% Mixed inoculum (B281 & B282) 1 90% B281 / 0% B * 73.33% B281 / 0% B % B281/0% B282 40% B281/0% B282 60% B281/6.66% B282

46 Sensory evaluation end of fermentation

47 Distinctions The product Probiotic olives submitted by Agric. Univ. Athens, received the 2nd prize at the ECOTROPHELIA 2012 Innovative Food Competition Patent No «Functional table olives fermented with lactic acid bacteria with probiotic properties» PROBIOTIC OLIVES (OBI ) by PEMETE

48 Lactic acid bacteria as bioprotective cultures Bioprotection is the enrichment of a foodstuff with bio-protective cultures of bacteria capable of inhibiting the growth of spoilage and pathogenic microorganisms. It helps to extend the shelf life and maintain food safety. Bioprotective cultures can be used with the fermentation cultures or as culture adjuncts.

49 Lactic acid bacteria as bioprotective cultures

50 Lactic acid bacteria as bioprotective cultures Safety of table olives Τhe activity of lactic acid bacteria during fermentation, results in the production of a variety of metabolic compounds, in which lactate and acetate predominate, lowering the ph of the brine and decreasing thus the presence of pathogenic microorganisms. In addition, antimicrobial compounds (e.g. ethanol, bacteriocins) produced by certain strains of lactic acid bacteria contribute to a better preservation effect of the final product. For this reason, fermented foods have been generally considered less likely to cause foodborne infection or intoxication.

51 Safety of table olives A survey was carried out in Greece involving 69 different commercially available table olive preparations, including Spanish-style green olives, naturally black olives and dry-salted olives. No enterobacteriaceae, pseudomonads, B. cereus, or Clostridium perfringens were detected in any of the samples analyzed given the physicochemical characteristics found (average ph, ; salt content, ). Absence of Salmonella spp., L. monocytogenes, E. coli, Bacillus cereus and C. perfringens reported by Grounta, Nychas Panagou (2013) Int. J. Food Microb. 161: Similar observations in Spain and Italy

52 Safety of table olives Fermented Olives Removal of brine Fresh brine addition (NaCl 6% w/v) Packaging 70% N 2 :30% CO 2 Storage at 4 ο & 20 C Inoculation (10 7 cfu/ml) Escherichia coli O157:H7, Listeria monocytogenes Salmonella Enteritidis

53 Log cfu/ml Log cfu/ml Log cfu/ml Safety of table olives (a) (a) ημέρες ημέρες 14 ημέρες 22ημέρες Time (days) Time (days) 4 3 Escherichia coli O157:H7 Salmonella Enteritidis 8 (a) Listeria monocytogenes Time (days)

54 Safety of table olives Bacillus cereus Salmonella Enteritidis PT4 Panagou E., Tassou C.,.Nychas GJ. (2008) J. Food Protection 71: Panagou E., Nychas GJ. Sofos J. (2013) Food Control 29:32-41

55 Recently, Grounta et al. (2013) demonstrated also that Salmonella, E. coli O157:H7, L. monocytogenes and S. aureus did not survive on natural black olives stored under aerobic conditions.

56 Therefore the results indicate that: Fermented olives can be a probiotic, functional food of high added value, Fermented olives are safe, since they are not a favorable environment to support the growth of foodborne pathogens.

57 Organoleptic evaluation of table olives The first National Taste panel has been trained according to the method COI/T.20/Doc.No 6/Rev.1 (Sept.2007) by Dr. Tertivanidis and Assoc. Prof. Panagou at the laboratory of ITAP and is provided to interested companies.

58 NAGREF NATIONAL AGRICULTURAL RESEARCH FOUNDATION Thank you for your attention!