Canada Research Chair in Food Microbiology and Probiotics

Department of Agricultural, Food and Nutritional ScienceEdmonton, Alberta, Canadawww.ualberta.ca

Antimicrobial compounds from plantsMichael G. Gänzle, Christina Engels, and Andreas Schieber

Role of Lipoxygenases in Plants

Inhibition of microbial growth after injuryCell signalling after wounding and pathogen attackSynthesis of attractors to enemies of herbivoresLipid mobilisation during germinationRegulation of vegetative growth

Parasitic wasp (Cotesia marginiventris)

Beet Armyworm

Role of Phenolic Compounds in Plants

Pigments

Attractants

Protectionagainst

pests

Protectionagainst

UV irradiation

I don‘t like it !!!I don‘t like it !!!

Defence systemspreformed induced

COOH CH CH COOH

C

O

O

CH

CH

CC

CO

C6-C1 Compounds C6-C3 Compounds

CC66 CC66

Phenolic Acids

C6-C1-C6 Compounds

Xanthones

C6-C2-C6 Compounds

Stilbenes

C6-C3-C6 Compounds

Flavonoids

CC66 CC66CC66

CC66

CC66

CC66

Diversity of Plant Polyphenols

COMP. REV. FOOD SCI. FOOD SAFETY 8: 157

Class of compounds Active compounds (examples) Other biological activities

Organic acids Citric acid, fumatic acid Taste, flavour

Essential oils Carvacrol, eugenol Flavour

Allyl sulfides Allyl sulfides, allyl disulfide Flavour (garlic, onion)

Phenolic acids Hydroxybenzoic acid, cinnamic acid Taste, flavour

Polyphenols Gallocatechin, epigallocatechin

Taste, colour, tanning activity, iron complexation, antinutritive factors (protein and starch digestion)

Lipid oxidation products Hexanal Flavour (rancidity)

Antimicrobial compounds from plants:an overview

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?

Antimicrobial activity of mango polyphenols

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

HPLC Profile of Phenolic Compounds From Mango Kernel Extracts

Food Chem 71, 61

AntimicrobiallyAntimicrobiallyactive fractionsactive fractions

2

3

1

4

5

6 7

9 10

11

12

13

8

15

16

17 18

19

21

20

14

min 10 20 30 40 50 60 70

mAU

0

200

400

600

800

1000

HydrolyzableHydrolyzableTanninsTannins

Rapid Commun Mass Spectrom 18, 2208

Tannins

• Subclass of polyphenols

• (to) tan = precipitate proteins

• Condensed and hydrolyzable tannins:

• Polyol core esterified with phenolic acids

Penta-O-galloylglucose

Purificationof tanninsExtract with hexane

Extract withaqueous acetone

Liquid-liquid extraction

Ethyl acetatephase

Aqueousphase

Dichloromethanephase

Low Pressure Liquid Chromatography

Fractions 1 to 3 Fractions 4.6 and 5.3

Semi-preparative HPLC

Penta-O-galloylglucose

Hepta-O-galloylglucose

Hexa-O-galloylglucose

Dried mango kernels

Purification of fractions with antimicrobial activity

Engels et al., J Agric Food Chem 57:7712

Antimicrobial activity of purified gallotannins

a) Diameter of zone of inhibition (cm); b) -, no inhibition

Gallotannin-Iron complex visualised by CAS agar diffusion assay

Gallic acid Mangiferin Tannic acid

EDTA Mango kernel extract

[Fe II or Fe III] (mM)

0 1 2 3 4 5

[MIC

of m

ango

ker

nel e

xtra

ct] (

g L-1

)

0.1

1

10

Effect of iron on antimicrobial activity of mango kernel extract

Fe2+

Fe3+

Indicator strain: B. subtilis.

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!

High Speed Counter Current Chromatography

• High-speed counter current chromatography

• Isolation and purification of compounds

• Larger sample injections for preparative purification!

HSCCC

Extract with hexane

Extract withaqueous acetone

Liquid-liquid extraction

Ethyl acetatephase

Aqueousphase

Dichloromethanephase

Low Pressure Liquid Chromatography

Fractions 1 to 3 Fractions 4.6 and 5.3

Semi-preparative HPLC

Penta-O-galloylglucose

Hepta-O-galloylglucose

Hexa-O-galloylglucose

Dried mango kernels

X

Component A

Component B

High Speed Counter Current Chromatography: Principle of Separation

Component A

Component B

High Speed Counter Current Chromatography: Principle of Separation

High Speed Counter Current Chromatography: Principle of Separation

HSCCC

GF

E

D

C

A

Separation of mango gallotannins by HSCCC

B

Fraction Compound and purity m/z values A Tetra-O-galloyglucose (4 GG), 72% [M-H]- = 787; [M-2H]2- = 393

B Penta-O-galloyglucose (5 GG) 90% [M-H]- = 939; [M-2H]2- = 469

C Hexa-O-galloyglucose (6 GG) 94% M-H]- = 1091; [M-2H]2- = 545

D Hepta-O-galloyglucose (7 GG) 90% M-H]- = 1243; [M-2H]2- = 621

E Octa-O-galloyglucose (8 GG) 100% M-H]- = 1395; [M-2H]2- = 697

F Nona-O-galloyglucose (9 GG) 86% [M-H]- = 1547; [M-2H]2- = 773

G Deca-O-galloyglucose (10 GG) 87% [M-H]- = 1699; [M-2H]2- = 849

Identity and purity of gallotannins: LC/MS

Antimicrobial activities of gallotannins: Structure-function relationships

MIC Concentration to withdraw iron from

CAS*Fe complex

Antimicrobial activities of gallotannins: Inhibitory spectrum

Sensitive organisms Gram-positive Bacillus spp. Listeria spp. Staphylococcus spp. Clostridium spp. Gram-negative Campylobacter spp., Yersinia spp., some Escherichia coli and Pseudomonas spp.

Resistant organisms Gram-positive: Lactic acid bacteria, bifidobacteria Gram-negative: Most E. coli and Salmonella enterica, Erwinia spp., some Pseudomonas spp.

Food Chem 71, 61, J Agric Food Chem 57:7712

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?

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?

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

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

Acknowledgements

Collaborators• Dr. Reinhold Carle, U Hohenheim, Germany

Financial support• NSERC and Research Chairs of Canada

…..for your attention

UV

Abs

orba

nce

G

F

E

Characterisation of fractions by LC/MS

10 GG9 GG

8 GG

7 GG

6 GG5 GG

Separation of mango gallotannins by HSCCC

4 GG

Meta-depsidic

bonding

Structural isomers of gallotannins

Salicylicacid

4-Hydroxy-benzoic

acid

Proto-catechuic

acid

Gallicacid

Willow barksWillow barks BerriesBerries Onion/potatoOnion/potatoskinsskins

Mango peelsMango peels,,grapesgrapes

Preservatives

C6-C1 Compounds: Hydroxybenzoic Acids

COOH

OH

COOH

OH

COOH

OH

OH

COOH

OH

OHHO

Volatile formation from lipid peroxides by Lipoxygenase

O

OH

OOH

O

O

OHO

O

OH

Linoleic acid

13-LOOH

hexanal + 12-oxo-9-cis-dodecenoic acid

-Injury of plant tissue disrupts separation of enzyme and substrate!

LOX

Antioxidant and antimicrobial activities of mango (Mangifera indica L.) by-productsChristina Engels – September 17, 2008

CharacterizationAnalytical HPLC• puglkg

Dried mango kernels and peels

Extract with aqueous acetone

Liquid-liquid-partitioning

Ethyl acetatephase

Aqueousremains

Dichloromethanephase

Low Pressure Liquid Chromatography

Fraction 1 to 3 Fraction 4.1 to 5.8

Semi-prep HPLC

Penta-O-galloylglucose

Hepta-O-galloylglucose

Hexa-O-galloylglucose

HPLC

MS

AB

AntiOx

Max. 5236.5 counts.

200 400 600 800 1000 1200 1400 1600 1800 2000m/z, Da

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5237

Intensity, counts

469.1685

939.3364

134.8961 377.1760

Max. 5236.5 counts.

200 400 600 800 1000 1200 1400 1600 1800 2000m/z, Da

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5237

Intensity, counts

469.1685

939.3364

134.8961 377.1760

Max. 1777.6 counts.

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z, Da

0

200

400

600

800

1000

1200

1400

1600

1778

Intensity, counts

545.1867

1091.3798

469.1615

113.0110

Max. 1777.6 counts.

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z, Da

0

200

400

600

800

1000

1200

1400

1600

1778

Intensity, counts

545.1867

1091.3798

469.1615

113.0110

Max. 872.8 counts.

200 400 600 800 1000 1200 1400 1600 1800 2000m/z, Da

0

100

200

300

400

500

600

700

800

873

Intensity, counts

621.2081

255.2924

113.0106

1243.4275283.3311

545.1848 632.1988

Max. 872.8 counts.

200 400 600 800 1000 1200 1400 1600 1800 2000m/z, Da

0

100

200

300

400

500

600

700

800

873

Intensity, counts

621.2081

255.2924

113.0106

1243.4275283.3311

545.1848 632.1988

Penta-O-galloylglucose

Hexa-O-galloylglucose

Hepta-O-galloylglucose

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100,000

50,000

0

Hexa-O-galloy lglucose

RT [min]

hexa 10 yL 50 times diltution6.DATAµV

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Hepta-O-galloy lglucose

RT [min]

hepta 10 yL 50 times dilution 7.DATAµV

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50,000

0

Penta-O-galloy lglucose

RT [min]

penta I 10 yL 50 times diltution 10 yL 50 times diltution4.DATAµV