Microbiological and immunological aspects of TF

Transcription

1 BaSeFood - Sustainable exploitation of bioactive components from the Black Sea Area traditional foods (FP7-KBBE ) Microbiological and immunological aspects of TF Nadiya Boyko a,b,c a Uzhhorod National University, Uzhhorod, Ukraine. b nadiya.boyko@gmail.com. c On behalf of the representatives of the beneficiaries of the BaSeFood project and their colleagues who conducted this collaborative research (details at Cesena, ITALY; TFI-2012, 4-5, October, 2012

2 ASPECT(s) of TF Microbial Key microorganisms in TF Safety and Quality of TF Beneficial microorganisms in TF Immune Bioactive components and gut microbes Systemic and local host defence Cellular vs. humoral response Cesena, ITALY; TFI-2012, 4-5, October, 2012

3 TFs PLANTS are tested for persistent risk-relevant (SAFETY/QUALITY ISSUES) AND BENEFICIAL bacteria/microorganisms including microscopic fungi and anaerobes) within groups: A, B1, B2 and B3 during the food processing = 91 Bulgaria, Georgia, Ukraine, Russia, Romania and Turkey

4 (1) Plants samples (2) Ready-to-eat meals Both categories from THREE different sources home made street-market restaurants city-market Field-private yards Original recipes in villages

5 ANAERO and ENTERO-tests Latex-tests Chromogenic media MALDI VITEK 2 API-test systems

6 Family/Genera 1. Entero bacteriaceae 2. Vibrionaceae 3. Micrococcaceae Beneficial (A) (B2) Contaminants (B2) E. coli (EPEC), Salmonella enterica, Shigella dysenteriae, S. flexneri, E. coli O157:H7 Staphylococcus aureus, S. pyogenes (B1) Contaminants (B1) Citrobacter spp., Klebsiella spp., Enterobacter spp., Enterococcus spp., Proteus spp. Pseudomonas spp. Staphylococcus spp., Streptococcus spp. 4. LAB 5. Bacillus 6. Fungi 7. Facultative/ strong anaerobes 8. Other Gram- positive Lactobacillus acidophilus, L. salivarius, L. fermentum, etc. Bacillus subtilis Saccharomyces boulardii, S. cerevisiae, etc. Bifidobacterium bifidum, etc., Bacteroides distasonis, Clostridium buturicum Clostridium perfringens, botulinum, tetani, etc. except Clostridium buturicum Listeria spp. Campylobacter jejuni Candida spp.

7 Plant Components Dishes Plant Components Dishes Dishes Barley Carrot Flour Good king Henry roots Hot pepper Mint Onion Pumpkin Rose flowers Savoury Smilyan beans Tomato Walnuts Wheat Boza Pumpkin pie Rose jam Smilyan bean soup Sunflowers seeds Tahan halva Barley Carrot Flour Good king Henry roots Hot pepper Mint Onion Pumpkin Rose flowers Savoury Smilyan beans Tomato Walnuts Wheat Boza Pumpkin pie Rose jam Smilyan bean soup Sunflowers seeds Tahan halva 10 1 Bacterial concentration, CFU/ml 0 Plant Components Bacterial concentration, CFU/ml 10 3 Acinetobacter baumannii Aerococcus viridans / Pediococcus spp. Bacillus subtilis Corynebacterium fascians Enterobacter cloaceae E. coli (lactose -) E. faecalis Klebsiella oxytoca Pantoea agglomerans Staphylococcus aureus Staphylococcus auriculalris Xanthomonas campestris 0 Enterobacter cloaceae Enterobacter sakazakii Klebsiella oxytoca Serratia adorifera (biogroup 1) Barley Carrot Flour Good king Henry roots Hot pepper Mint Onion Pumpkin Rose flowers Savoury Smilyan beans Tomato Walnuts Wheat Boza Pumpkin pie Rose jam Smilyan bean soup Sunflowers seeds Tahan halva Bacterial concentration, CFU/ml 0 Actinomyces israeli / L. fermentium Bacillus subtilis Lactobacillus casei Streptococcus lactis A/B1/B2, Bulgarian samples

8 92.99% A B1a B1b B2 BULGARIA 0.01% 0.03% 6.97% 8.34% A B1a B1b B % 64.95% % GEORGIA

9 BULGARIAN samples Xantomonas campestris Aerococcus viridans Pantoea agglomerans Escherichia coli (lactose + and lactose ) Pedicococcus cerevisae Pediococcus damnosus Pedicococcus pentosaceus Corynebacterium fascians Klebsiella oxytoca K.ozaenae, K. pneumoniae Acinetobacter baumannii Enterococcus faecalis Enterobacter cloacae Staphylococcus auriculalris S. aureus B1a/b and B2 GEORGIAN samples Escherichia coli (lactose + and lactose ) Erwinia herbicola Pantoae agglomerans Pseudomonas fluorescens Enterobacter cloacae Serratia odorifera Citrobacter freundii Bacillus cereus Sacharomyces spp. Aspergillus spp. Cryptococcus spp. Staphylococcus saprophyticus S. aureus Listeria monocytogenes QUALITY AND SAFETY

10 ROMANIA 48.14% A B1a B1b B2 RUSSIA 62.8% A B1a B1b B % 15.16% 0.64% 0.33% 23.98% 36.23% TURKEY 62.8% A B1a B1b B2 UKRAINE 74.81% A B1a B1b B2 0.64% 0.33% 0% 36.23% 11.53% 13.67%

11 ROMANIAN samples 1.Lactobacillus casei 2.Bifidobacterium longum 3.Bacillus subtilis 4.B. licheniformis 5.Candida famata (Torulopsis candida) / 6.E. coli TURKEY samples 1.B. subtilis 2.Lactobacillus delbrueckii 3.Actinomyces israelii 4.Bifidobacterium dentium 5.Staphylococcus epidemidis 6.S. aureus 7.Serratia odorifera biogroup 1 8.Eikenella corrodens A / B2/B3 RUSSIAN samples 1.Lactobacillus plantarum 2.B. sublilis 3.L. galinarum 4. L. acidophilus 5.B. subtilis 6. L. plantarum 7.Actinomyces israelii 8.israelii / 9.E. coli (lactose positive and negative) 10.Pantoea agglomerans 11.Streptococcus viridans 12.Streprococcus epedermidis 13.Proteus vulgaris QUALITY AND SAFETY

12

13 Cesena, ITALY; TFI-2012, 4-5, October, 2012

14 Plants Country Bacteria Carrot Bulgaria Russia Ukraine Bacillus subtilis, Staphylococcus aureus, Enterobacter cloacae, Streptococcus lactis, Pseudomonas spp. Bacillus subtilis, Erwinia herbicola, Enterobacter cloacae, Pseudomonas spp. Onion Bulgaria Bacillus subtilis, Escherichia coli (lac-) Ukraine Bacillus subtilis Tomato Bulgaria Bacillus subtilis, Pantoea agglomerans, Enterobacter sakazaki, Klebsiella oxytoca Ukraine Russia Bacillus spp., Pantoea agglomerans, Aerococcus viridans

15 10 6 E. faecalis Plant Components Dishes Barley Carrot Flour Good King Henry root Hot pepper Mint Onion Pumpkin Rose flowers Savoury Smilyan beans Tomato Walnuts Wheat Boza Pumpkin pie Rose jam Smilyan bean soup Sunflowers seeds Tahan halva Bacterial concentration, CFU/ml

16 CТX АMK АМP АМК АМП Eikenella corrodens Providencia rettgeri АМК АН CTX Pantoea agglomerans

17 24 h 48h 72 h 1, CFU/ml of Pantoea agglomerans, per os

18 Anaerobes 1. Not detected in leaves (green parts of plants) 2. Bacteroides ovatus had been isolated from garlic and tomato purchased in INDUSTRIAL AND street market AND supplement with Candida spp. 3. Clostridium butyricum had been found in parsley (root) and carrot taken from street market 4. Clostridium carnis contaminated beet from Rysyno, street market as well as beans from city market 5. Eubacterium lentum was detected as only the isolate from onion (street market) Plant contaminations with obligatory pathogenic anaerobes were not observed; in the same time some of them: Bacteroides ovatus (predominant commensal intestinal microbe which can cause systemic antibody response in IBD), and Eubacterium lentum (Eggerthella lenta) is found in the human colon and feces and have been implicated as a cause of ulcerative colitis, liver and anal abscesses and systemic bacteremia species can be rather treated as potentially pathogenic than saprophytic species

19 Row material, INDUSTRIAL species of MICROSCOPIC FUNGI: Sporothrix cyanescens, Aspergillus nidulans Sporothrix shenckii, Aspergillus flavus, Paecilomyces lilacinus, Scopulariopsis candida, Zygosporium masonii. Only the one of the samples tomatoes was contaminated by Candida spp. The greatest biodiversity of microscopic fungi had been observed in samples purchased at street-markets (personnel and/or agricultural): Sporothrix cyanescens, Aspergillus nidulans, Gymnoascus dankaliensis, Paecilomyces lilacinus, Aspergillus niger, Sporothrix shenckii, Scopulariopsis candida, Aspergillus terreus, Fusarium chlamidosporum. Most of the samples taken from this place were contaminated by Candida spp. Samples obtained from the private yard/fields were mostly contaminated by microscopic fungi of next dominant species: Aspergillus nidulans, Fusarium moniliforme, Sporothrix cyanescens, only in very low amount of tested samples was isolates belong to the different species representatives of the genus Candida spp.

20 Summary 1. Samples from street market are more contaminated than city market and yard 2. Ratio and species of isolated and identified bacteria (A/B1/B2) are different in each country, but mainly amount of beneficial bacteria significantly less, dominating group is potentially pathogenic bacteria when foodborne pathogens are rarely found 3. Isolated microbial species are often plant-specific 4. Food safety in most cases within required demands 5. Food quality should be an important issue

21 The only good microbe is a dead microbe... The most common type of prebiotic is from the soluble dietary fibre inulin Inulin is common in many plants containing fructan Many of these plants are frequently eaten as vegetables asparagus, garlic, leek, onion, artichoke and are an excellent source of inulin As the need for functional foods (N-TF???) rises, prebiotics are being added to many every day food choices such as cereals, biscuits, breads, table spreads, drinks, and yoghurts.

22 Immuno-modulation functions???? Health? Inflammation? Homeostasis at mucosal sites X1 microbiota originated from diet/plant X2 gut microbiota X3 microbiota contamination - external from environment and. X4 Food Ag

23 Beneficial (A) Fermented product Environmental, (B1) L. acidophilus Boza Candida pelliculosae L. casei Boza Kvass Sauerkraut Candida famata L. fermentum Boza Candida intermidia B. breve Boza Trichosporon species B. longum Boza Kvass Lactobacillus spp. Sautéed pickled green beans L. delbrueckii Kvass Sauerkraut Candida norvegensis Candida krusei Candida albicans L. plantarum Kvass Еnterococcus faecalis L. delbrueckii Kvass Sauerkraut Bifidobacterium dentinum Sauerkraut Sautéed pickled green beans Fermented product Boza Boza Sautéed pickled green beans Sauerkraut Potentially pathogenic, (B2) Clostridium buturicum Fermented product Boza Socata E. coli Socata Socata A. israeli Boza Kvass Socata Sautéed pickled green beans Sautéed pickled green beans Sautéed pickled green beans Sauerkraut (supermarkets) Staphylococcus epidermidis Sautéed pickled green beans

24 Bacterial concentration, CFU/ml Candida dubliniensis/famata Enterococcus faecium Cryptococcus humicola Lactobacillus casei Bifidobacterium dentium Lactobacillus delbrueckii Det. Limit Days

25 Antibacterial properties (inhibition zones, mm) of strains isolated from TF L. delbrueckiiand L. casei Bacteria (clinical strais) L. casei (Kvass) L. delbrueckii (Sauerkraut) L. casei and L. delbrueckii (Sauerkraut) 1 Str. mitis 30 ±2.2 2 S. enterica 26±1.8 3 S.aureu(MRSA) 22 ± ± E. cloacae 36 ± K. pneumoniae 24 ± EPEC E. coli 22 ± 2.7

26 Commensally (beneficial) Opportunistic pathogens Food borne pathogens 1. Escherichia coli 058 (human) E. coli (EPEC) Listeria monocytogenes 2. Escherichia coli Schaedler Pseudomonas aeruginosa Staphylococcus aureus (murine) 3. Escherichia coli АТСС Klebsiella oxytoca Salmonella enterica 4. Saccharomyces cerevisiae 5. Lactobacillus salivarius 6. Lactobacillus delbrueckii 7. Lactobacillus casei 8. Lactobacillus acidophilus 9. Lactobacillus fermentium 10. Bacteroides distasonis 11. Enterococcus faecalis / E. faecium 12. Bacillus subtilis 090/ B. subtilis Morganella morganii Klebsiella pneumoniae Klebsiella pllanticola Enterobacter spp. Proteus mirabilis MRSA (Methicillin resistant Staphylococcus aureus) Streptococcus pyogenes Streptococcus pneumoniae Candida albicans Staphylococcus saprophyticus Staphylococcus epidermidis Shigella dysenteriae Shigella sonnei

27 Bacillus subtilis Enterococcus faecium Lactobacillus acidophilus Pseudomonas aeruginosa 2500 CFU/ml, x Hours X2 gut microbiota + X4 Food Ag

28 Plant Bacteria 1 Nettle Schaedler s Escherichia coli 2 Carrot Escherichia coli 058 Enterobacter cloaceae 3 Beet Enterobacter cloaceae 4 Onion Escherichia coli Garlic Staphylococcus aureus ATCC MRSA Enterococcus faecalis Pseudomonas aeruginosa ATCC Staphyloccus epidermidis Morganella morganii 6 Cucumber Bacillus subtilis Tomato (local) 8 Pepper sweet (local) Bacillus subtilis 8130 B. subtilis 3, B. licheniformis 31 Lactobacillus salivarius Escherichia coli 058 Enteropath. E. coli (EPEC) Salmonella enteritidis Shigella sonnei Listeria monocytogenes Bacillus subtilis 8130 Klebsiella oxytoca Proteus mirabilis Morganella morganii 9 Grape MRSA Escherichia coli Melon juice Shigella sonnei Plant Bacteria 11 Cherry (local) Enteropath. E. coli (EPEC) Enterobacter cloaceae Salmonella enteritidis Shigella sonnei Listeria monocytogenes Bacillus subtilis 090 Bacillus subtilis 8130 B. subtilis 3, B. licheniformis 31 Lactobacillus salivarius Enterococcus faecalis Enterococcus fecium Streptococcus pyogenes Staphylococcus saprophyticus MRSA Klebsiella oxytoca Proteus mirabilis 12 Pomegra nate Morganella morganii B. subtilis 3, B. licheniformis 31 Lactobacillus salivarius Staphylococcus epidermidis 13 Currants Escherichia coli ATCC Schaedler s Escherichia coli Escherichia coli 058 Enteropath. E. coli (EPEC) Salmonella enteritidis Shigella sonnei Listeria monocytogenes Bacillus subtilis 090 Bacillus subtilis 8130 B. subtilis 3, B. licheniformis 31

29 Plant Bacteria 13 Currants Lactobacillus salivarius Enterococcus faecalis Enterococcus faecium Streptococcus pyogenes Staphylococcus aureus ATCC Staphylococcus epidermidis Staphylococcus saprophyticus MRSA Klebsiella pneumoniae Klebsiella oxytoca Proteus mirabilis Morganella morganii 14 Viburnum opulus (Cranberry) 16 Blue berries local (yafyny), fresh Escherichia coli ATCC Schaedler s Escherichia coli Escherichia coli 058 Enteropath. E. coli (EPEC) Enterobacter cloaceae Salmonella enteritidis Bacillus subtilis 090 B. subtilis 3, B. licheniformis 31 Salmonella enteritidis Shigella sonnei Pseudomonas aeruginosa ATCC Enterococcus faecalis Enterococcus faecium Staphylococcus epidermidis Staphylococcus saprophyticus MRSA Plant Bacteria 17 Flowers of elderberry Enteropath. E. coli (EPEC) Salmonella enteritidis Shigella sonnei Pseudomonas aeruginosa Lactobacillus salivarius Lactobacillus acidophilus Streptococcus pyogenes Staphylococcus epidermidis Saccharomyces boulardii Candida albicans 18 Lemon Schaedler s Escherichia coli Escherichia coli 058 Enteropath. E. coli (EPEC) Enterobacter cloaceae Salmonella enteritidis Listeria monocytogenes Bacillus subtilis 090 Bacillus subtilis 8130 B. subtilis 3, B. licheniformis 31 Pseudomonas aeruginosa Lactobacillus salivarius Lactobacillus acidophilus Enterococcus faecalis Enterococcus faecium Streptococcus pyogenes Staphylococcus aureus ATCC Staphylococcus epidermidis Staphylococcus saprophyticus MRSA

30 Plant Bacteria 18 Lemon Klebsiella pneumoniae Klebsiella oxytoca Proteus mirabilis Morganella morganii 19 Okroshka (Russian) 20 Bread Kvass (Russia) 21 Churchkhela cherry 22 Churchkhela pomegranate (Georgia) 23 Nettle with walnut (Georgia) Enteropath. E. coli (EPEC) Klebsiella pneumoniae Morganella morganii Escherichia coli ATCC Schaedler s Escherichia coli Enterobacter cloaceae Salmonella enteritidis Shigella sonnei Listeria monocytogenes Bacillus subtilis 090 Bacillus subtilis 8130 B. subtilis 3, B. licheniformis 31 Staphylococcus saprophyticus MRSA Klebsiella oxytoca Proteus mirabilis Morganella morganii Pseudomonas aeruginosa ATCC Salmonella enteritidis Bacillus subtilis 8130 B. subtilis 3, B. licheniformis 31 Salmonella enteritidis Plant Bacteria 24 Long loaf bread (Georgia) Staphylococcus saprophyticus 25 Tsiteli doli bread with makhobeli (Georgia) 26 Plum sauce green (Georgia) 27 Plum sauce red (Georgia) Enteropath. E. coli (EPEC) Salmonella enteritidis Listeria monocytogenes Streptococcus pyogenes MRSA Klebsiella pneumoniae Klebsiella oxytoca Morganella morganii Enteropath. E. coli (EPEC) Salmonella enteritidis Bacillus subtilis 090 Bacillus subtilis 8130 Lactobacillus salivarius Proteus mirabilis Morganella morganii Enterococcus faecalis Enteropath. E. coli (EPEC) Salmonella enteritidis Shigella sonnei Bacillus subtilis 090 Bacillus subtilis 8130 Lactobacillus salivarius Proteus mirabilis Enterococcus faecalis Morganella morganii

31 Plant Bacteria 28 Black tea (Georgia) Escherichia coli ATCC Salmonella enteritidis Pseudomonas aeruginosa ATCC Enterococcus faecalis Enterococcus faecium 29 Pomazanka (UzhNU) 30 Sauerkraut (ONAF) 31 Socata (local) Streptococcus pyogenes Morganella Klebsiella oxytoca morganii Morganella morganii Enteropath. E. coli (EPEC) Shigella sonnei Lactobacillus salivarius Escherichia coli ATCC Schaedler s Escherichia coli Escherichia coli 058 Enteropath. E. coli (EPEC) Enterobactercloaceae Salmonella enteritidis Shigella sonnei Listeria monocytogenes Pseudomonas aeruginosa ATCC Enterococcus faecalis Enterococcus faecium Streptococcus pyogenes Staphylococcus aureus ATCC Staphylococcus epidermidis MRSA Klebsiella pneumoniae Klebsiella oxytoca Proteus mirabilis Morganella morganii Plant Bacteria 32 Smilyan bean soup (Bulgaria) Salmonella enteritidis Shigella sonnei 33 Cereal Boza (Bulgaria) 34 Sunflower seed thini halva 35 Barley (Bulgaria) 36 Tomato (Bulgaria) Escherichia coli ATCC Schaedler s Escherichia coli Enteropath.E. coli (EPEC) Enterobacter cloaceae Salmonella enteritidis Shigella sonnei Bacillus subtilis 8130 B. subtilis 3, B. licheniformis 31 Staphylococcus aureus ATCC Staphylococcus epidermidis Staphylococcus saprophyticus MRSA Klebsiella pneumoniae Klebsiella oxytoca Proteus mirabilis Lactobacillus acidophilus Escherichia coli ATCC Schaedler s Escherichia coli Escherichia coli 058 Enteropath. E. coli (EPEC) Enterobacter cloaceae Shigella sonnei Staphylococcus epidermidis Staphylococcus saprophyticus Proteus mirabilis

32 The pro- and anti-bacterial effect of plants/foods is Strain but not Species specific 1 hour 24 hours 48 hours 1 hour 24 hours 48 hours Schadler`s Escherichia coli Escherichia coli 058 Bacterial concentration, CFU/ml < 1000 Bacterial concentration, CFU/ml < :1 1:10 1:25 1:125 1:625 Control Dilutions of PLUM SAUCE RED 1:1 1:10 1:25 1:125 1:625 Control Dilutions of PLUM SAUCE RED

33 Results: Stimulatory Plant Bacteria 1 Cabbage Lactobacillus salivarius 2 Pumpkin Lactobacillus salivarius 3 Cumin tea Lactobacillus salivarius 4 Cherry (local) 5 Buckwheat porridge crumby (Russia) Saccharomyces boulardii Candida albicans Morganella morganii

34 L. salivariuswas stimulated by the extracts of fresh white cabbage, melon, pumpkin and cumin tea. Our local cherries and traditional Russian Buckwheat porridge crumby similarly affected the Sascharomyces cerevisae (but also Candida) and Morganella morganii. The Georgian plum dressing (green and red), sauerkraut (a traditional Ukrainian fermented food), Socata and Boza (Bulgarian and Romanian fermented drinks) had strong inhibitory activity onthe growth of most tested pathogenic bacteria, including Enteropathogenic E. coli(epec), Shigella sonnei, Salmonella enteritidis, Proteus mirabilis, Enterococcus faecalisand others. The other registered effects proved less desirable, however not less important (inhibition of the growth of the tested strains of lactobacilli, bacilli, enterococci and other representatives of the commensal microbiota).

35 Only positive effect we observed in cases of: Fresh extracts pure juices: Beet, Garlic, and Blue berries And foods: Okroshka(Russian); Churchkhela(Georgian); Nettle with walnut (Georgian) all types of Georgian bread (Long loaf bread and Tsiteli doli bread with makhobeli), Black tea (Georgian), Smilyan bean soup (Bulgaria), Sunflower seed thini halva (Bulgaria), and Pomazanka(Ukraine) Slightly negative effect had been detected in cases when pure plant extracts of: Nettle, Cucumber, Tomato and Onion(local) and Barley(Bulgaria) were tested Carrot, Currants, Cherries, and all fermented dishes Socata (Romania), Boza(Bulgaria) and Sauerkraut(Ukraine) demonstrated complex effect

36 Bifidobacterium spp. Candida spp. Enterococcus faecalis/faecium Escherichia coli Klebsiella pneumoniae Lactobacillus spp. Candida spp. Bifidobacterium spp. Lactobacillus spp. Enterococcus faecalis/faecium Escherichia coli Klebsiella pneumoniae Bacterial concentration, CFU/mg Det.Limit Dill Days Bacterial concentration, CFU/mg Det.Limit Kale Days Bifidobacterium spp. Candida spp. Enterococcus faecalis/faecium Escherichia coli Klebsiella pneumoniae Lactobacillus spp. Candida spp. Bifidobacterium spp. Lactobacillus spp. Enterococcus faecalis/faecium Escherichia coli Klebsiella pneumoniae Bacterial concentration, CFU/mg Det.Limit Nettle Days Bacterial concentration, CFU/mg Det.Limit Control Days

37 Bifidobacterium spp. Candida spp. Enterococcus faecalis/faecium Escherichia coli Klebsiella pneumoniae Lactobacillus spp. Candida spp. Escherichia coli Klebsiella pneumoniae Lactobacillus spp. Bifidobacterium spp. Enterococcus faecalis/faecium Det.Limit Persimmon Days Det.Limit Pomegranate Days Bacterial concentration, CFU/mg Bacterial concentration, CFU/mg Candida spp. Bifidobacterium spp. Enterococcus faecalis/faecium Escherichia coli Klebsiella pneumoniae Lactobacillus spp Bacterial concentration, CFU/mg Det.Limit Sideritis Days

38 M Protein Molecular Weight Marker (Fermentas) 1 nettle, mouse 1; 2 nettle, mouse 2; 3 sidiritis, mouse 1; 4 sidiritis, mouse 2; 5 kale, mouse 1; 6 control mouse

39 CD markers FACS assay Mice: PEC cells, Spleen, MLNs, PPs CD45/CD3/CD4/CD8 (T4/T8) CD4/CD45RB/CD25 (Treg and T activated) CD220/IgM/IgD/CD19 (B1/B2) CD56/CD80/CD86 (NK/DCs) CD11a/CD11b/CD11c Kappa/IgAs DCs CD1a/CD80/CD83 IFR hot methanol extracts of plants 1.Nettle 2.Sideritis 3.Dill 4.Kale 5.Persimmon 6.Pomegranate More than 2600 samples of FACS (1) Spleen, Pec, MLNs and PPs (2) 3, 7, 14, 21 and 28 th days 6 extracts and 3 mice per each time point worked up separately only for the BALB/c mice

40 IgA, IgM, IgG1 level, mkg/ml 2,5 2,0 1,5 1,0 0,5 0,0 2,5 2,0 1,5 1,0 0,5 0,0 2,5 2,0 1,5 1,0 0,5 0,0 Day 30 x 20 Day 14 x 20 Day 7 x 20 IgA IgM IgG1 x 20 x 20 x 20 x 20 x 20 x 20 x 20 x 20 x 20 IgA, IgM, IgG1 secretion in different compartments of SI from BALB/c mice in dynamic 2,5 2,0 Day 3 1,5 1,0 0,5 x 20 x 20 x 20 x 20 0,0 Duodenum Jejunum Ileum PPs

41 Modulation of host immune response by strains isolated from Bulgarian Bosa and Romanian Socata X1 microbiota originated from diet/plant L. casei / L. plantarum, L. fermentium, L. acidophilus, B. longum, A. israeli / L.delbrueckii, B. breve / L. acidophilus, B. longum, C. pelliculosae

42 The ratio of CD1a + dendritic cells is associated to higher production of pro- inflammatoric cytokines 1. Selected Gram-negative bacteria of the gut microbiota such as Escherichia coli Schaedler and Morganella morganii activate DC as verified by increased CD83 expression 2. Escherichia coli Schaedler modified the ratio of tolerogenic (CD1a - ) and inflammatory (CD1a + ) human DC subsets 3. The increased ratio of CD1a + DC correlated with the concentration of the pro-inflammatory cytokine IL-6 4. Increasing numbers of Escherichia coli Schaedler resulted in higher proinflammatoric cytokine (TNF-α, IL-1ß, IL-6, IL-12) and chemokine (CXCL8/IL-8, CXCL10/IP10) production. Morganella morganii and Escherichia coli 058 did not induce TNF-α and IL-12 production

43 24h 6h CD1-CD80+ CD1 APC CD1-CD80+ CD1 APC Pomegranate 3,9 ± 0, ,8 ± 785,7 Pomegranate 2,6 ± 0,29** 10282,5 ± 415,6 9037,5 ± Dill 0,1 ± 0** 340,1** Dill 0,1 ± 0,04** 11387,5 ± 988,9** Nettle 0 ± 0** 39402,5 ± 340,7** Nettle 0 ± 0** 35885,0 ± 1048,9** Kale 0 ± 0** 6685 ± 355,6 Kale 0 ± 0** 9775,0 ± 470,9 Persimmon 4,7 ± 0, ,0 ± 515,5 Persimmon 2,4 ± 0,19** 11112,5 ± 532,0** 5795,0 ± Sideritis 5,0 ± 0,63* 611,3* Sideritis 2,4 ± 0,15** 11392,5 ± 148,2** Control 4,2 ± 0, ,5 ± 641,6 Control 3,5 ± 0, ,0 ± 1327,0 1. Nettle significantly (p< 0.01) effects on expression of pro-inflammatory CD1a+ cells compare to the control 2. Dill and Kale demonstrated similar but less strong influence 3. Sideritis is statistically lowered the expression of CD1a+ on 6h but not on 24 h of incubation Cesena, ITALY; TFI-2012, 4-5, October, 2012

44 Gene-expression expression (mrna) levels of Nod-like receptors (NLRs) in DCs Nod2, Nalp1 and Nalp3 NLR-genes are activated by bacteria whereas Nod1 is down regulated

45 Duodenum Jejunum Ileum PPs MLNs Serum Day 3 Day 7 Day 14 Day 21 Day pg/ml IFN IL2 IL10 IL12 IL17A

46 Duodenum Jejunum Ileum PPs MLNs Serum Day 3 Day 7 Day 14 Day 21 Day pg/ml IL2 IL10 IL12 IL17A

47 Duodenum Jejunum Ileum PPs MLNs Serum Day 3 Day 7 Day 14 Day 21 Day pg/ml TNF IL2 IL10 IL12 IL17A

48 Duodenum Jejunum Ileum MLNs Serum Day 3 Day pg/ml IL2 Duodenum Jejunum Ileum PPs MLNs Serum Day 3 Day 7 Day 14 Day 21 Day 28 Day 3 Duodenum Jejunum PPs Day pg/ml pg/ml TNF IL2 0.0 IL2

49 A vegan or vegetarian diet substantially alters the human colonic faecal microbiota. J. Zimmer, B. Lange, J-S Frick, H. Sauer, K. Zimmermann, A. Schwiertz, K. Rusch, S. Klosterhalfen, and P. Enck. European Journal of Clinical Nutrition (2012) 66, Different plants possess different beneficial properties due to presence of bioactive compounds A blackberry extract was able to suppress lipid A-induced interleukin-12 release from mouse bone marrow-derived dendritic cells [1] Stinging nettle leaf extract prevented the maturation of dendritic cells [2] Echinacea purpurea was shown to be able to regulate immune responses (the properties are dose depended [3] 1.Dai, J., Patel, J. D., Mumper, R. J., Characterization of blackberry extract and its antiproliferative and anti-inflammatory properties. J Med Food, 10(2): Broer, J., Behnke, B., Immunosuppressant effect of IDS 30, a stinging nettle leaf extract, on myeloid dendritic cells in vitro. J Rheumatol., 29(4): Benson, J. M., Pokorny, A., J., Echinacea purpurea extracts modulate murine dendritic cell fate and function. Food Chem Toxicol, 48(5): Cesena, ITALY; TFI-2012, 4-5, October, 2012

50 Knowing the mechanisms of changes in gut microbial homeostasis in response to the TF or FF or other healthy diet, we will know the role of key bacteria/microorganisms in: - Initiation or Regulation of host metabolic of other misbalance via modulation of human/host immune response - Indication (diagnostic) of diseases Cesena, ITALY; TFI-2012, 4-5, October, 2012

51 Anzhela Dolgikh Andriy Pauk Larysa Buhyna Viktor Petrov Ivan Kutchak Vitaliya Dutova Yuriy Rusyn Cesena, ITALY; TFI-2012, 4-5, October, 2012

52 Mariam Jorjadze (Elkana, Georgia) Tana Sapundzhieva and Iordanka Alexieva (UFT, Bulgaria) Carmen Costea (ASE, Romania) Dmitry Karpenko (MSUFP, Russia) Osman Hayran and Bike Kocaoglu (YEDITEPE, Turkey) Helena Costa (INSA) and Paul Kroon (IFR) 1. Slovak Republic, Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, Kosice 2. Hungary, Institute of Immunology, University Cesena, of Debrecen ITALY; TFI-2012, 4-5, October, 2012