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Canada Research Chair in Food Microbiology and Probiotics

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  1. Antimicrobial compounds from plants Michael G. Gänzle, Christina Engels, and Andreas Schieber Canada Research Chair in Food Microbiology and Probiotics Department of Agricultural, Food and Nutritional Science Edmonton, Alberta, Canada www.ualberta.ca

  2. Role of Lipoxygenases in Plants Inhibition of microbial growth after injury Cell signalling after wounding and pathogen attack Synthesis of attractors to enemies of herbivores Lipid mobilisation during germination Regulation of vegetative growth Beet Armyworm Parasitic wasp (Cotesia marginiventris)

  3. Role of Phenolic Compounds in Plants Pigments Attractants Defence systems preformed induced Protection against pests I don‘t like it !!! Protection against UV irradiation

  4. Diversity of Plant Polyphenols C6 C6 C6-C1 Compounds C6-C3 Compounds Phenolic Acids C6 C6 C6 C6 C6 C6 C6-C1-C6 Compounds Xanthones C6-C2-C6 Compounds Stilbenes C6-C3-C6 Compounds Flavonoids

  5. Antimicrobial compounds from plants:an overview COMP. REV. FOOD SCI. FOOD SAFETY 8: 157

  6. Antimicrobial polyphenols from plants:Limitations for food applications - Much of the literature data is based on complex mixture of compounds => are Canadian berries the same as Finnish berries? - Compounds have potent biological and technological activities other than antimicrobial activity => blue beef with citrus flavour? - Limited data on mode of action of polyphenols => Interactions with other hurdles applied in food processing?

  7. Antimicrobial activity of mango polyphenols • Mangifera indica L. • World production: 30 Mio t./year • 35 – 60% peels and kernels • Recovery of valuable compounds?

  8. HPLC Profile of Phenolic Compounds From Mango Kernel Extracts Antimicrobially active fractions Hydrolyzable Tannins Food Chem 71, 61 Rapid Commun Mass Spectrom 18, 2208

  9. Tannins • Subclass of polyphenols • (to) tan = precipitate proteins • Condensed and hydrolyzable tannins: • Polyol core esterified with phenolic acids Penta-O-galloylglucose

  10. Purificationof tannins Dried mango kernels Extract with hexane Extract with aqueous acetone Liquid-liquid extraction Ethyl acetate phase Dichloromethane phase Aqueous phase Low Pressure Liquid Chromatography • Purification of fractions with antimicrobial activity Fractions 4.6 and 5.3 Fractions 1 to 3 Semi-preparative HPLC Penta-O-galloylglucose Hexa-O-galloylglucose Hepta-O-galloylglucose Engels et al., J Agric Food Chem 57:7712

  11. Antimicrobial activity of purified gallotannins a) Diameter of zone of inhibition (cm); b) -, no inhibition

  12. Gallotannin-Iron complex visualised by CAS agar diffusion assay Gallic acid Mangiferin Tannic acid EDTA Mango kernel extract

  13. Effect of iron on antimicrobial activity of mango kernel extract Indicator strain: B. subtilis. Fe2+ Fe3+

  14. Antimicrobial activity of mango gallotannins- interim summary - - Gallotannins from mango exhibit highly selective antibacterial activity - Gallotannins have tanning and iron-complexing activities Further studies to determine minimum inhibitory activities and the mode of action require higher concentrations of purified compounds!

  15. High Speed Counter Current Chromatography • High-speed counter current chromatography • Isolation and purification of compounds • Larger sample injections for preparative purification! Dried mango kernels Extract with hexane Extract with aqueous acetone Liquid-liquid extraction X Ethyl acetate phase Dichloromethane phase Aqueous phase Low Pressure Liquid Chromatography HSCCC Fractions 4.6 and 5.3 Fractions 1 to 3 Semi-preparative HPLC Penta-O-galloylglucose Hexa-O-galloylglucose Hepta-O-galloylglucose

  16. High Speed Counter Current Chromatography: Principle of Separation Component A Component B

  17. High Speed Counter Current Chromatography: Principle of Separation Component A Component B

  18. High Speed Counter Current Chromatography: Principle of Separation

  19. HSCCC

  20. Separation of mango gallotannins by HSCCC C B D E A F G

  21. Identity and purity of gallotannins: LC/MS

  22. Antimicrobial activities of gallotannins: Structure-function relationships MIC Concentration to withdraw iron from CAS*Fecomplex

  23. Antimicrobial activities of gallotannins: Inhibitory spectrum Food Chem 71, 61, J Agric Food Chem 57:7712

  24. Mode of action of gallotannins Resistance of lactic acid bacteria and bifidobacteria: the iron anomaly Lactic acid bacteria and bifidobacteria do not require iron for growth as their metabolism depends neither on Fe-S enzymes of the respiratory chain (all aerobes), nor on Fe-S enzymes involved in metabolic pathways of strict anaerobes. Resistance of Enterobacteriaceae and plant pathogens: - permeability barrier of the outer membrane? - production of siderophores for iron sequestration?

  25. Food and Feed applications of gallotannins- perspectives - Highly selective antimicrobial activity - selective inhibition of pathogens, coupled with stimulation of protective lactic acid bacteria on food? - beneficial shifts of intestinal microbiota in feed applications? Role of cations in antimicrobial activity - Do other divalent cations (Ca2+, Mg2+) mitigate antimicrobial activity of gallotannins? - Does tanning activity alter food qualitiy? - reduced iron availability in food and feed?

  26. Antimicrobial compounds from plants: Limiations and perspectives - Most literature data is based on complex mixture of compounds - Compounds have potent biological and technological activities other than antimicrobial activity - Very limited data on mode of action Exploration of the diversity of plant bioactives by => Preparative purification of active compounds => Determination of structure-function relationships => Mode of action and relationship of antimicrobial activity to other biological activities

  27. Acknowledgements • Collaborators • Dr. Reinhold Carle, U Hohenheim, Germany • Financial support • NSERC and Research Chairs of Canada • …..for your attention

  28. Characterisation of fractions by LC/MS E F UV Absorbance G

  29. Separation of mango gallotannins by HSCCC 6 GG 5 GG 7 GG 8 GG 4 GG 9 GG 10 GG

  30. Structural isomers of gallotannins Meta-depsidic bonding

  31. C6-C1 Compounds: Hydroxybenzoic Acids 4-Hydroxy- benzoic acid Proto- catechuic acid Salicylic acid Gallic acid Willow barks Onion/potato skins Mango peels, grapes Berries Preservatives

  32. Volatile formation from lipid peroxides by Lipoxygenase -Injury of plant tissue disrupts separation of enzyme and substrate! Linoleic acid LOX 13-LOOH hexanal + 12-oxo-9-cis-dodecenoic acid

  33. Dried mango kernels and peels Characterization Penta-O-galloylglucose Extract with aqueous acetone • Analytical HPLC • puglkg Liquid-liquid-partitioning Ethyl acetate phase Dichloromethane phase Aqueous remains Low Pressure Liquid Chromatography Hexa-O-galloylglucose Fraction 1 to 3 Fraction 4.1 to 5.8 HPLC AB Semi-prep HPLC MS AntiOx Penta-O-galloylglucose Hexa-O-galloylglucose Hepta-O-galloylglucose Hepta-O-galloylglucose Antioxidant and antimicrobial activities of mango (Mangifera indica L.) by-products Christina Engels – September 17, 2008