Transcript Slide 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
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)
Role of Phenolic Compounds in Plants
Pigments
Defence systems
preformed
Attractants
induced
Protection
against
pests
I don‘t like it !!!
Protection
against
UV irradiation
Diversity of Plant Polyphenols
C6
C6
COOH
C6-C1 Compounds
CH
CH
COOH
C6-C3 Compounds
Phenolic Acids
O
C6
C6
C
CH
C6
C6
CH
C
C6
O
C6
O
C
C
C6-C1-C6 Compounds
C6-C2-C6 Compounds
C6-C3-C6 Compounds
Xanthones
Stilbenes
Flavonoids
Antimicrobial compounds from plants:
an overview
Class of compounds
Active compounds
(examples)
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)
Other biological activities
COMP. REV. FOOD SCI. FOOD SAFETY 8: 157
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
Antimicrobially
active fractions
Hydrolyzable
Tannins
mAU
10
9
1000
800
15
600
11 13
18
21
19
12 14
400
17
16
20
6 7
1
200
5
2
8
3 4
0
10
Food Chem 71, 61
20
30
40
50
60
Rapid Commun Mass Spectrom 18, 2208
70
min
Tannins
• Subclass of polyphenols
• (to) tan = precipitate proteins
• Condensed and hydrolyzable
tannins:
• Polyol core esterified with
phenolic acids
Penta-O-galloylglucose
Dried mango kernels
Purification
of tannins
Extract with hexane
Extract with
aqueous acetone
Liquid-liquid extraction
Ethyl acetate Dichloromethane
phase
phase
Low Pressure
Liquid Chromatography
Aqueous
phase
Purification of fractions
with antimicrobial activity

Fractions 1 to 3 Fractions 4.6 and 5.3
Semi-preparative HPLC
Penta-O-galloylglucose
Hexa-O-galloylglucose
Hepta-O-galloylglucose
Engels et al., J Agric Food Chem 57:7712
Antimicrobial activity of purified
gallotannins
Table 1. Antimicrobial activity of the hydrolyzable tannins isolated from mango
kernels.
Penta-O-
Hexa-O-
Hepta-O-
galloylglucose
galloylglucose
galloylglucose
(41.2 g/L)a,b
(83.7 g/L)a,b
(17.0 g/L)a,b
Bacillus subtilis
0.9  0.0
0.9  0.0
+/-
Bacillus amyloliquefaciens
0.8  0.1
0.9  0.1
n. d.
Escherichia coli AW 1.7
-
-
-
Escherichia coli GGG 10
-
n. d.
n. d.
Listeria monocytogenes
1.1  0.1
1.5  0.1
0.8  0.0
Pediococcus acidilactici
-
-
-
Staphylococcus aureus
0.8  0.1
1.1  0.0
-
Staphylococcus warneri
-
-
-
a)
Diameter of zone of inhibition (cm); b) -, no inhibition
a
Concentration was determined gravimetrically.
b
Diameters of the inhibition zones (n. d. = not determined, +/-, partial growth
Gallotannin-Iron complex visualised by
CAS agar diffusion assay
Gallic acid
EDTA
Mangiferin
Tannic acid
Mango kernel extract
Effect of iron on antimicrobial activity of mango
kernel extract
Indicator strain: B. subtilis.
[MIC of mango kernel extract] (g L -1)
10
Fe2+
Fe3+
1
0.1
0
1
2
3
[Fe II or Fe III] (mM)
4
5
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!
Dried mango kernels
Extract with hexane
Extract with
aqueous acetone
Liquid-liquid extraction
Ethyl acetate
phase
Dichloromethane
phase
Aqueous
phase
Low Pressure
Liquid Chromatography
Fractions 1 to 3
Fractions 4.6 and 5.3
HSCCC
Semi-preparative HPLC
Penta-O-galloylglucose
Hexa-O-galloylglucose
Hepta-O-galloylglucose
High Speed Counter Current Chromatography:
Principle of Separation
Component A
Component B
High Speed Counter Current Chromatography:
Principle of Separation
Component A
Component B
High Speed Counter Current Chromatography:
Principle of Separation
HSCCC
Separation of mango gallotannins by HSCCC
B
C
D
E
A
F
G
Identity and purity of gallotannins: LC/MS
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
Antimicrobial activities of gallotannins:
Structure-function relationships
0.25
MIC
Concentration to
withdraw iron from
CAS*Fe complex
Iron bindig capacity (g/L)
0.04
0.03
0.20
0.15
0.02
0.10
0.01
0.05
0.00
0.00
tetra
penta
hexa
hepta
octa
Degree of galloylation
nona
deca
MIC (g/L)
0.05
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?
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
Acknowledgements
Collaborators
• Dr. Reinhold Carle, U Hohenheim, Germany
Financial support
• NSERC and Research Chairs of Canada
…..for your attention
Absorbance at 280 nm [mV]
1 0 0 0
800
400
0
5 0 0
0
m A U
1 0
500
5 0 0
400
4 0 0
2 0
3 0
4 0
5 0
6 0
7 0
m in
Characterisation of fractions by LC/MS
300
3 0 0
200
2 0 0
100
1 0 0
0
0
m A U
1 0
6 0
7 0
m in
2 0
3 0
4 0
5 0
1 0
2 0
3 0
4 0
5 0
6 0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
m in
1 0
2 0
3 0
4 0
5 0
6 0
7 0
m in
3 5 0
300
3 0 0
2 5 0
E
200
2 0 0
1 5 0
100
UV Absorbance
1 0 0
5 0
0
0
m A U
600
7 0
m in
6 0 0
5 0 0
F
400
4 0 0
3 0 0
200
2 0 0
1 0 0
0
0
m A U
500
5 0 0
400
G
4 0 0
300
3 0 0
200
2 0 0
100
1 0 0
0
0
20
40
Retention time [min]
60
Separation of mango gallotannins by HSCCC
5 GG
6 GG
7 GG
4 GG
8 GG
9 GG
10 GG
Structural isomers of gallotannins
Meta-depsidic
bonding
C6-C1 Compounds: Hydroxybenzoic Acids
OH
OH
OH
OH
HO
OH
OH
COOH
Salicylic
acid
Willow barks
COOH
COOH
COOH
4-Hydroxybenzoic
acid
Protocatechuic
acid
Gallic
acid
Berries
Onion/potato
skins
Mango peels,
grapes
Preservatives
Volatile formation from lipid peroxides by
Lipoxygenase
-Injury of plant tissue disrupts separation of enzyme and substrate!
O
Linoleic acid
OH
LOX
O
OOH
13-LOOH
OH
O
O
O
hexanal + 12-oxo-9-cis-dodecenoic acid
OH
µV
550,000
penta I 10 yL 50 times diltution 10 yL 50 times diltution4.DATA
500,000
Max. 5236.5 counts.
469.1685
5237
Penta-O-galloy lglucose
5000
450,000
4500
400,000
4000
Dried mango
kernels and peels
350,000
300,000
Intensity, counts
250,000
3500
200,000
150,000
3000
2500
50,000
Penta-O-galloylglucose
Characterization
2000
Extract with
aqueous acetone
100,000
939.3364
1500
1000
134.8961 377.1760
500
0
RT [min]
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
Liquid-liquid-partitioning
650,000 µV
0
Analytical HPLC
• puglkg
200
75
400
600,000
Aqueous
remains
500,000
450,000
1600
1400
1200
1400
1600
1800
2000
Max. 1777.6 counts.
1091.3798
1200
400,000
Intensity, counts
350,000
1000
545.1867
1778
550,000
800
m/z, Da
hexa 10 yL 50 times diltution6.DATA
Ethyl acetate Dichloromethane
phase
phase
600
Hexa-O-galloy lglucose
Low Pressure
Liquid Chromatography
300,000
250,000
200,000
1000
800
Hexa-O-galloylglucose
600
150,000
469.1615
400
100,000
113.0110
Fraction 1 to 3 Fraction 4.1 to 5.8
50,000
200
0
RT [min]
0
5
10
15
20
30
35
40
45
50
55
60
65
70
HPLC
75
0
200
400
600
800
AB
1000
1200
1400
hepta 10 yL 50 times dilution 7.DATA
MS
500,000
1600
1800
2000
m/z, Da
Semi-prep HPLC
µV
550,000
25
Max. 872.8 counts.
621.2081
873
AntiOx
800
450,000
700
Penta-O-galloylglucose
400,000
350,000
600
Intensity, counts
300,000
500
Hexa-O-galloylglucose
250,000
200,000
300
150,000
255.2924
400
Hepta-O-galloylglucose
100,000
200
Hepta-O-galloylglucose
113.0106
Antioxidant and antimicrobial activities of mango
(Mangifera indica L.) by-products
1243.4275
283.3311
Hepta-O-galloy lglucose
545.1848
50,000
632.1988
Christina Engels – September 17, 2008
100
0
RT [min]
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
0
200
400
600
800
1000
1200
m/z, Da
1400
1600
1800
2000